2010 - Management of Fungal Plant Pathogens PDF

Management of Fungal Plant Pathogens
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Management of Fungal
Plant Pathogens
Edited by
Arun Arya
Professor and Head, Department of Botany and
Coordinator Environment Science Programme, Faculty of Science
The Maharaja Sayajirao University of Baroda,
Vadodara, India
and
Analía Edith Perelló
Assistant Professor and Research Scientist, CONICET - CIDEFI,

and Coordinator MSc Vegetal Protection Programme, Plant Pathology,
Facultad de Ciencias Agrarias y Forestales, Universidad Nacional
de La Plata, Provincia de Buenos Aires, Argentina
CABI is a trading name of CAB International
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A catalogue record for this book is available from the British Library,
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Library of Congress Cataloging-in-Publication Data
Management of fungal plant pathogens / edited by Arun Arya,

Analía Edith Perelló.
p. cm.
Includes bibliographical references and index.
ISBN 978-1-84593-603-7 (alk. paper)
1. Fungal diseases of plants. 2. Phytopathogenic fungi–Control.
3. Plant-pathogen relationships. I. Arya, Arun. II. Perelló,
Analía Edith. III. title.
SB733.M36 2010
632'.4–dc22
2009023395
ISBN-13: 978 1 84593 603 7
Typeset by AMA Dataset, Preston, UK.

Printed and bound in the UK by the MPG Books Group.
Contents
Contributors viii
Preface xi
PART I: BOTANICALS IN FUNGAL PEST MANAGEMENT
1 Recent Advances in the Management of Fungal Pathogens of Fruit Crops 3

Arun Arya
2 Botanicals in Agricultural Pest Management 14

Ashok Kumar, Priyanka Singh and N.K. Dubey
3 Deleterious Effects of Fungi on Postharvest Crops and Their

Management Strategies 28
A.O. Ogaraku
4 Exploitation of Botanicals in the Management of Phytopathogenic

and Storage Fungi 36
Pramila Tripathi and A.K. Shukla
5 Use of Plant Extracts as Natural Fungicides in the Management of

Seedborne Diseases 51
Gustavo Dal Bello and Marina Sisterna
PART II: DISEASE CONTROL THROUGH RESISTANCE
6 Resistance to Septoria Leaf Blotch in Wheat 69

María R. Simón
7 Barley and Wheat Resistance Genes for Fusarium Head Blight 78

S.A. Stenglein and W.J. Rogers
v
vi Contents
8 Sustainable Management of Rice Blast (Magnaporthe grisea

(Hebert) Barr): 50 Years of Research Progress in Molecular Biology 92
S. Nandy, N. Mandal, P.K. Bhowmik, M.A. Khan and S.K. Basu
PART III: BIOLOGICAL CONTROL MECHANISMS
9 Postharvest Technology – Yeast as Biocontrol Agents: Progress,

Problems and Prospects 109
Neeta Sharma and Pallavi Awasthi
10 Biological Control of Plant Diseases: An Overview and

the Trichoderma System as Biocontrol Agents 121
Abhishek Tripathi, Neeta Sharma and Nidhi Tripathi
11 Physiological Specialization of Ustilaginales (Smut) of

Genera Bromus, Zea and Triticum in Argentina 138
Marta M. Astiz Gassó and María del C. Molina
PART IV: ENDOPHYTES IN PLANT DISEASE CONTROL
12 Status and Progress of Research in Endophytes from

Agricultural Crops in Argentina 149
Silvina Larrán and Cecilia Mónaco
13 Effect of Tillage Systems on the Arbuscular Mycorrhizal Fungi

Propagule Bank in Soils 162
Santiago Schalamuk and Marta N. Cabello
14 Mechanism of Action in Arbuscular Mycorrhizal Symbionts

to Control Fungal Diseases 171
Arun Arya, Chitra Arya and Renu Misra
15 Role of Fungal Endophytes in Plant Protection 183

S.K. Gond, V.C. Verma, A. Mishra, A. Kumar and R.N. Kharwar
PART V: MANAGING FUNGAL PATHOGENS CAUSING LEAF DAMAGE
16 The Rust Fungi: Systematics, Diseases and Their Management 201

M.S. Patil and Anjali Patil
17 Etiology, Epidemiology and Management of Fungal Diseases of Sugarcane 217

Ayman M.H. Esh
18 New and Emerging Fungal Pathogens Associated with Leaf Blight

Symptoms on Wheat (Triticum aestivum) in Argentina 231
19 Diseases of Fenugreek (Trigonella foenum-graecum L.) and Their Control

Measures, with Special Emphasis on Fungal Diseases 245
S.N. Acharya, J.E. Thomas, R. Prasad and S.K. Basu
Contents vii
20 Fungal Diseases of Oilseed Crops and Their Management 263

S.S. Adiver and Kumari
21 Occurrence of Pyrenophora tritici-repentis Causing Tan Spot in Argentina 275

M.V. Moreno and A.E. Perelló
22 Epidemiological Studies on Septoria Leaf Blotch of Wheat in Argentina 291

Cristina A. Cordo
PART VI: ALTERNATIVE CONTROL STRATEGIES
23 Review of Thecaphora amaranthicola M. Piepenbr., Causal Agent

of Smut on Amaranthus mantegazzianus Pass. 311
M.C.I. Noelting, M.C. Sandoval, M.M.A. Gassó and M.C. Molina
24 Population Biology and Management Strategies of Phytophthora sojae

Causing Phytophthora Root and Stem Rots of Soybean 318
Shuzhen Zhang and Allen G. Xue
25 Management of Fungal Pathogens – A Prerequisite for Maintenance of

Seed Quality During Storage 329
Anuja Gupta
26 Controlling Root and Butt Rot Diseases in Alpine European Forests 345
Paolo Gonthier
27 Some Important Fungal Diseases and Their Impact on Wheat Production 362
Aakash Goyal and Rajib Prasad
Index 375
The colour plate section can be found following page 50.

Contributors
Acharya, S.N., Agriculture and Agri-Food Canada Research Centre, Lethbridge, AB,
Canada T1J 4B1
Adiver, S.S., Oilseeds Scheme, Main Agricultural Research Station, University of Agricultural
Sciences, Dharwad 580 005, Karnataka, India (shivaputra_adiver@rediffmail.com)
Arya, Arun, Department of Botany, Faculty of Science, The Maharaja Sayajirao University
of Baroda, Vadodara 390002, India (aryaarunarya@rediffmail.com)
Arya, Chitra, Department of Botany, Faculty of Science, The Maharaja Sayajirao Univer-
sity of Baroda, Vadodara 390002, India (caarya@yahoo.co.in)
Astiz Gassó, Marta M., Instituto Fitotécnico Santa Catalina (IFSC), Facultad de Ciencias
Agrarias y Forestales, Universidad Nacional de La Plata, CC 4, 1836 Llavallol, Buenos
Aires, Argentina (astizgasso@yahoo.com.ar)
Awasthi, Pallavi, Mycology and Plant Pathology Division, Department of Botany, Univer-
sity of Lucknow, Lucknow 226007, India
Basu, S.K., Department of Biological Sciences, University of Lethbridge, Lethbridge, AB,
Canada T1K 3M4 (saikat.basu@uleth.ca)
Bhowmik, P.K., Bioproducts and Bioprocesses, Lethbridge Research Center, Agriculture
and Agri-Food Canada, Lethbridge, AB Canada T1J 4B1
Cabello, Marta N., Comisión de Investigaciones Científicas de la Provincia de Buenos Aires
(CICBA) – Instituto de Botánica Spegazzini, Calle 53 N° 577, 1900 La Plata, Argentina
(mcabello@museo.fcnym.unlp.edu.ar)
Cordo, Cristina A., Comisión de Investigaciones Científicas de la Provincia de Buenos
Aires, Centro de Investigaciones de Fitopatología (CIDEFI) – Facultad de Ciencias Agra-
rias y Forestales, 60 y 119, (1900) La Plata, Argentina (cristcordo@hotmail.com)
Dal Bello, Gustavo, Centro de Investigaciones de Fitopatología (CIDEFI), Facultad de Cien-
cias Agrarias y Forestales, Universidad Nacional de La Plata, 60 y 119, CC 31, 1900 La
Plata, Argentina (dalbello@speedy.com.ar)
Dubey, N.K., Centre of Advanced Study in Botany, Banaras Hindu University, Varanasi
221005, India (nkdubey2@rediffmail.com)
Esh, Ayman M.H., Biotechnology and Tissue Culture Laboratories, Sugar Crops Research
Institute, Agricultural Research Center, Giza, Egypt (aymanesh@gmail.com)
Gond, S.K., Mycopathology and Microbial Technology Laboratory, Centre of Advanced
Study in Botany, Banaras Hindu University, Varanasi 221005, India
viii
Contributors ix
Gonthier, Paolo, Department of Exploitation and Protection of Agricultural and Forestry

Resources (DIVAPRA), Plant and Forest Pathology, University of Torino, Via L. da Vinci,
44, I-10095 Grugliasco (TO), Italy (paolo.gonthier@unito.it)
Goyal, Aakash, Agriculture and Agri-Food Canada, Lethbridge Research Center, Lethbridge
AB-T1J4B1, Canada (akgroyal@gmail.com)
Gupta, Anuja, Indian Agricultural Research Institute, Regional Station, Karnal – 132 001,
Haryana, India (agupta_2005@yahoo.com)
Khan, M.A., Department of Weed Science, NWFP Agricultural University, Peshawar, NWFP,
Pakistan 25130
Kharwar, R.N., Mycopathology and Microbial Technology Laboratory, Centre of Advanced Study
in Botany, Banaras Hindu University, Varanasi 221005, India (rnkharwar@yahoo.com)
Kumar, Ashok, Centre of Advanced Study in Botany, Banaras Hindu University, Varanasi
221005, India
Kumari, Oilseeds Scheme, Main Agricultural Research Station, University of Agricultural
Sciences, Dharwad 580 005, Karnataka, India
Larrán, Silvina, Centro de Investigaciones de Fitopatología (CIDEFI), Facultad de Ciencias
Agrarias y Forestales, Universidad Nacional de La Plata, 60 y 119, CC 31, 1900 La Plata,
Argentina
Mandal N., Bidhan Chandra Krishi Vishavidalay, Nadia, WB, India 741252
Mishra, A., Mycopathology and Microbial Technology Laboratory, Centre of Advanced
Misra, Renu, Department of Botany, Faculty of Science, The Maharaja Sayajirao University
of Baroda, Vadodara 390002, India
Molina, María del C., Consejo de Investigaciones Científicas y Técnicas (CONICET),
Facultad de Ciencias Agrarias y Forestales, Universidad Nacional de La Plata, CC 4,
1836 Llavallol, Buenos Aires, Argentina
Mónaco, Cecilia, Centro de Investigaciones de Fitopatología (CIDEFI), Facultad de Cien-
cias Agrarias y Forestales, Universidad Nacional de La Plata, 60 y 119, CC 31, 1900 La
Plata, Argentina (cecilia.monaco7@gmail.com)
Moreno, M.V., CONICET – Facultad de Agronomía de Azul, Universidad Nacional del
Centro de la Provincia de Buenos Aires, República de Italia No. 780, Azul CP 7300, Bue-
nos Aires, Argentina (morevir@yahoo.com.ar)
Nandy, S., Bioproducts and Bioprocesses, Lethbridge Research Center, Agriculture and
Agri-Food Canada, Lethbridge, AB Canada T1J 4B1
Noelting, M.C.I., Instituto Fitotécnico de Santa Catalina, Facultad de Ciencias Agrarias y
Forestales, Universidad Nacional de La Plata, Garibaldi 3400, Llavallol 1836 CC 4 Bue-
nos Aires, Argentina (mcnoelting@hotmail.com)
Ogaraku, A.O., Plant Science and Biotechnology Unit, Department of Biological Sciences,
Nasarawa State University, PMB 1022, Keffi, Nasarawa State, Nigeria (ogara006@yahoo.
com)
Patil, Anjali, Department of Botany, Rajaram College, Kolhapur 416004 (M.S.), India
(dhirajanj@gmail.com)
Patil, M.S., Department of Botany, Shivaji University, Kolhapur (M.S.), India
Perelló, Analía Edith, CIDEFI (Centro de Investigaciones de Fitopatología) – CONICET
(Consejo Nacional de Investigaciones Científicas y Técnicas), Facultad de Ciencias
Agrarias y Forestales de la Universidad Nacional de La Plata, La Plata, Provincia de
Buenos Aires, Argentina (anaperello@yahoo.com.ar)
Prasad, Rajib, Agriculture and Agri-Food Canada, Lethbridge Research Center, Lethbridge
AB-T1J4B1, Canada
Rogers, W.J., Laboratorio de Biología Funcional y Biotecnología (BIOLAB), Facultad de
Agronomía, Universidad Nacional del Centro de la Provincia de Buenos Aires (UNICEN),
x Contributors
Av. República de Italia # 780 (CC 47), (7300) Azul, Buenos Aires, Argentina; FIBA –
Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), Argentina
Sandoval, M.C., Facultad de Ciencias Agrarias, UNLZ, Ruta 4 Km 2 Llavallol, Buenos Aires,
Argentina
Schalamuk, Santiago, CONICET – Centro de Investigaciones de Fitopatología (CIDEFI) y
Cerealicultura, Facultad de Ciencias Agrarias y Forestales, Universidad Nacional de La
Plata, 60 y 119, CC 31, 1900 La Plata, Argentina (sschala@yahoo.com.ar)
Sharma, Neeta, Mycology and Plant Pathology Division, Department of Botany, University
of Lucknow, Lucknow 226007, India (dr_neeta_sharma2003@yahoo.com)
Shukla, A.K., Department of Botany, Rajiv Gandhi University, Rono Hills, Itanagar 791
112, India
Singh, Priyanka, Centre of Advanced Study in Botany, Banaras Hindu University, Varanasi
221005, India
Simón, María R., Cerealicultura, Facultad de Ciencias Agrarias y Forestales, Universidad
Nacional de La Plata, 60 y 119, CC 31, 1900 La Plata, Argentina (mrsimon@agro.unlp.
edu.ar)
Sisterna, Marina, Centro de Investigaciones de Fitopatología (CIDEFI), Facultad de Ciencias
Agrarias y Forestales, Universidad Nacional de La Plata, 60 y 119, CC 31, 1900 La Plata,
Argentina (mnsisterna@infovia.com.ar)
Stenglein, S.A., Laboratorio de Biología Funcional y Biotecnología (BIOLAB), Facultad de
Agronomía, Universidad Nacional del Centro de la Provincia de Buenos Aires (UNICEN), Av.
República de Italia # 780 (CC 47), (7300) Azul, Buenos Aires, Argentina; FIBA – Consejo
Nacional de Investigaciones Científicas y Técnicas (CONICET), Argentina (stenglein@
faa.com.unicen.edu.ar)
Thomas, J.E., Department of Biological Sciences, University of Lethbridge, Lethbridge, AB,
Canada T1K 3M4
Tripathi, Abhishek, Department of Bioscience and Biotechnology, Banasthali University,
PO Banasthali Vidyapith, 304022 Rajasthan, India (abhitri77@yahoo.com)
Tripathi, Nidhi, Department of Bioscience and Biotechnology, Banasthali University, PO
Banasthali Vidyapith, 304022 Rajasthan, India
Tripathi, Pramila, Department of Botany, D.A.V.-P.G. College, Kanpur 208001 (U.P.), India
(pramilatripathi_bhu@rediffmail.com)
Verma, V.C., Mycopathology and Microbial Technology Laboratory, Centre of Advanced
Xue, Allen G., Eastern Cereal and Oilseed Research Centre, Agriculture and Agri-Food
Canada, 960 Carling Ave., Ottawa, Ontario, Canada, K1A 0C6 (axue@agr.gc.ca)
Zhang, Shuzhen, Soybean Research Institute, Key Laboratory of Soybean Biology of Chinese
Education Ministry, Northeast Agricultural University, Harbin, Heilongjiang, China, 150030
Preface
Oldest life forms have been reported from the North Pole Dome area of Western Australia,
which dates back 3556 million years. Non-septate mycelium remains of Eomycetopsis
robusta were recovered from late Precambrian chert of Australia. Having appeared first on
planet Earth, microbes have immense potential to influence all other life forms. Plant dis-
eases have caused epidemics and have had a profound influence on wars, famine and the
changing economy. Microbes including fungi need no introduction to common man; they
are progressive, ever changing and evolving in their own way, so they are capable of adapt-
ing to every condition of life. The French biochemist, Louis Pasteur, once said, ‘The role of
the infinitely small is infinitely large.’
Potentially immortal fungi spread their tentacles in 1845, when potato late blight fun-
gus caused havoc in Ireland. Soon after, Plasmopara viticola threatened the wine industry
in France. First reported in 1819 in Sweden, apple scab disease caused by Venturia inaequa-
lis threatened apple cultivation in the Kashmir Valley in India in 1973. Panama disease of
banana, wilt diseases of pigeon pea, castor and guava and smut and rust of cereals are some
other serious fungal diseases. The chance discovery of Bordeaux mixture by P.A. Millardet
in France paved the way to the chemical control of plant diseases. Phytopathologists are
confronted by a volley of challenges in the wake of a resurgence of new diseases and the
obligation to fulfil international trade agreements. We have to protect the environment and
at the same time ensure the safety and security of farmers in the field by making a concen-
trated effort to minimize crop losses due to fungi and other microbes.
This book provides an overview of our current knowledge of some plant–pathogen
interactions in economically important crops, emphasizing the importance of pathogenic
fungi on fruits, cereals, postharvest crops and the establishment of plant diseases and draw-
ing together fundamental new information on their management strategies based on con-
ventional and eco-friendly methods, with an emphasis on the use of microorganisms and
various biotechnological aspects of agriculture, which could lead to sustainability in mod-
ern agriculture.
The book examines the role of microbes in growth promotion, as bioprotectors and
bioremediators, and presents practical strategies for using microbes in sustainable agricul-
ture. In addition, the use of botanicals vis-à-vis chemical pesticides has also been reviewed.
Contributions on new research fields such as mycorrhizae and endophytes have been
xi
xii Preface
included. The book also examines in different chapters host–pathogen interactions in the
light of the new tools and techniques of molecular biology and genetics.
Dr Arya expresses his deep sense of indebtedness and admiration to the late Dr S.N.
Bhargava and to Professor Bihari Lal, ex Head of the Department of Botany, University of
Allahabad, who taught him his first lessons in plant pathology at the University of Allaha-
bad. He is grateful to his father, the late Shri O.P. Arya, for inspiring him to write about the
management of plant diseases and pests, which has proved most useful to plant growers.
He honours his grandfather, Baba Shankaranand, who fed him with sweet mangoes during
his childhood and who motivated him to love plants and to learn how to nurture them and
research into new and improved varieties.
We are grateful to the entire staff of our institutions and the cooperation and collabora-
tive efforts of the plant pathology experts of Argentina (Universidad Nacional de La Plata,
Universidad Nacional de Lomas de Zamora, Universidad Nacional del Centro) and India
(Botany Department, The Maharaja Sayajirao University of Baroda), who made this book
possible.
We thank all those who have contributed their valuable articles to this volume and are
sure that the present work, which consists of 27 different chapters written by learned
experts in the field, will be immensely useful to postgraduate students, researchers, aca-
demics, progressive farmers and practising horticulturists, as well as those involved in the
various agro-industries. We are hopeful that the available knowledge in the field, newer
technologies and disease-resistant varieties will be used in different parts of the world and
that ultimately the plant disease scenario will change. All appreciations and good wishes
are extended to the members of the CABI team, particularly Ms. Sarah Mellor, for helpful
discussions and skilled assistance in the reviewing of the manuscripts, and also for helping
us in various ways to accomplish this project satisfactorily in the stipulated time. And also
for the cooperation and collaborative effort of the Plant Pathology experts that made this
book possible.
Arun Arya
Analia Edith Perelló
Part I
Botanicals in Fungal
Pest Management
1 Recent Advances in the Management
of Fungal Pathogens of Fruit Crops
Arun Arya
Department of Botany, Faculty of Science, The Maharaja Sayajirao University
of Baroda, Vadodara, India
Abstract
Fruits constitute a rich source of sugars, vitamins, minerals and medicinally important compounds
like flavonoids, which prevent cancer and cardiovascular diseases. These are eaten as a dessert or
processed into jams, jellies, ice creams and drinks; grapes are dried to make raisins. The science of
protecting fruit crops began with the discovery of Bordeaux mixture by P.A. Millardet in France. But
still we have yet to find many new techniques and fungicide formulations to control diseases; such as
bunch rot of grapes (Botrytis cinerea), apple scab (Venturia inaequalis), wilt of guava (Fusarium
solani), Panama wilt of banana (F. cubense), mango malformation (F. moniliforme), blue mould of
citrus (Penicillium citrinum) and anthracnose of papaya (Colletotrichum papayae), etc. Losses from
postharvest fruit diseases range from 1 to 20% in the USA and from 10 to 40% in India. The pathogens
have developed resistance against various fungicides and the postharvest phase is minimized. Alterna-
tive strategies like the use of biocontrol methods and the application of botanicals have been tried. A
large number of plants are screened for the presence of effective secondary metabolites. Integrated pest
management, using improved cultural practices (pruning methods to control Botrytis bunch rot in
grapes), the use of solarization (in strawberries), the application of growth hormone (NAA in the case
of mango malformation), along with minimum dosage of fungicides, are recommended to control
various fruit diseases.
The world fruit market is expanding; we are more concerned about human nutrition now, but at
the same time serious enough to protect the environment from pollution. The economics of a success
story will have to revolve around the use of various cutting-edge technologies and, at the same time,
the use of simpler and more effective methods acceptable to fruit growers. Biotechnologists have tried
to enhance the activity of biocontrol agents; at the same time, efforts are being made for genetic trans-
formation involving molecular breeding. This technology involves intimate knowledge of the gene,
regulatory components and gene functional environment (i.e. the domain where the gene is located).
Once an understanding of the molecular basis of genes involved in resistance has been achieved, we
will be able to isolate the alleles of those genes and their inclusion will lead to transformed, disease-
free plants.
Introduction Taken either as a dessert or processed, the

nutritional value of fruits depends chiefly
Fruits constitute an important component on the quality and concentration of sugars,
of our daily diet. The use of dates, fig, mango vitamins and other essential minerals. Plants
and grapes is mentioned in ancient texts. suffer with a number of diseases and pests
 CAB International 2010. Management of Fungal Plant Pathogens

(eds A. Arya and A.E. Perelló) 3
4 A. Arya
during their growth phase. Fungi not only into those that penetrate the fruits while
blemish, disfigure or cause rot to a number still in the field, but develop in their tissues
of fruits but also reduce their market value only after harvest, during storage or market-
(Arya, 2004). Realizing the importance of ing, and those that initiate penetration during
postharvest diseases, Stevens and Stevens or after harvest. Symptoms in stylar end rot of
(1952) mentioned, ‘of all losses caused by guava caused by Phomopsis psidii become
plant disease those that occur after harvest more prominent during storage (Arya, 1983).
are the most costly, whether measured in Verhoeff (1974) describes how quiescent
monetary terms or in man hours’. India is infection is established in young fruits:
the second most important fruit producing
1. Shortage of adequate substances in
country of the world. It produces the high-
young fruits.
est quantity of mango, while the productiv-
2. The incapability of the pathogen to pro-
ity of grapes in India at 56 t/ha is a world
duce cell wall degrading enzymes in the
record. The export of mango increased in
young fruit.
the nineties from 25,000 to 44,000 t (a 25%
3. The presence of antifungal compounds.
share of world trade) (Neelam, 1993). Fruit
4. The accumulation of phytoalexins (Swin-
production is 49 Mt (Arya, 2004).
burne, 1983).
The fruit growing industry has devel-
oped a lot, overcoming the hurdles of biotic The first theory claims that young unripe
and abiotic stresses. The industry needs a fruit does not provide the pathogen with the
comprehensive strategy to face the chal- nutrition and energy required for its develop-
lenges and opportunities of a global econ- ment. The artificial increase of the sugar level
omy. Lewis (1985) stated that ‘a few key in apple was achieved by the use of a chemi-
discoveries have led to a breakthrough in cal such as 2,4-dinitrophenol on the fruit. It
our understanding of the biological genome accelerated the decay caused by Botryospha-
and our ability to alter it, which may equal eria ribis (Sitterly and Shay, 1960). It has been
in significance the development of nuclear found that antifungal compounds become
energy in the Physical Sciences.’ toxic in the presence of sugars.
The second theory suggests that the
unripe fruit does not supply the pathogen
with compounds that induce activity in cell
Fungal Infection of wall degrading pectolytic enzymes.
Fruits and Fruit Trees The third and fourth theories point to a
relation between the formation of antifungal
Brown rot of citrus fruit is caused in the compounds in the young tissues and the
orchard by Phytophthora citrophthora. Many creation of quiescent infections. Chemicals
fungi that penetrate the host in the field such as 3,4-dihydroxy benzaldehyde have
cause quiescent infection. The grey mould proven fungistatic activity in the green
in strawberries is caused by B. cinerea. The banana fruit. In unripe avocado fruit, a link
Botrytis spores with which the strawberry has been established between the presence of
field is filled during bloom can germinate in a diene and monoene antifungal compounds
a drop of water on the petal or other parts of in the fruit rind and the quiescent infection
the flower and later penetrate the senes- of C. gloeosporioides in such a fruit. The
cenced parts of the flower into the edge of reduction in the concentration of the diene
the receptacle of the strawberry, where they probably results from lipoxygenase enzy-
develop a dormant mycelium. During ripen- matic activity that increases as ripening
ing and storage, as the resistance of the fruit progresses and the fruit softens (Prusky
to the pathogen decreases, the preliminary et al., 1982, 1985). The dormant state of
mycelium enters an active stage and decay Alternaria alternata in young mango fruits
develops (Powelson, 1960; Jarvis, 1962). Post- has been attributed to the presence of two
harvest pathogens can be divided, according antifungal resorcinols in the unripe fruit
to the timing of their penetration of the host, rind (Droby et al., 1986).
Recent Advances in the Management of Fungal Pathogens of Fruit Crops 5
Recent Advances in the Management (Sarig et al., 1996). The tolerance of grapes
of Fungal Pathogens to sulphur dioxide is unique among fresh
fruits. It eradicates most of the postharvest
Cultural practices pathogens. However, the benefits of sulphur
dioxide disappear after a short period of
time. Hence, sodium bisulphate in packing
Initial infection of most temperate fruits is
cases reacts with the moisture in the air in
carried from the orchard; therefore, prehar-
grape containers. This treatment is used
vest cultural practices, if adopted, consider-
exclusively for the long distance transporta-
ably reduce postharvest diseases during
tion of grapes (Hedberg, 1977). Fumigation
transit and storage. Strict orchard hygiene
with acetic acid is effective in controlling
and maintenance of tree vigour is recom-
M. fructicola, R. stolonifer and Alternaria
mended to reduce losses from Botryospha-
species on peaches, nectarines, apricot and
eria rot of apple. Pezicula malicorticis and
cherries (Sholberg and Gaunce, 1996). Rela-
Nectria galligena infection in apple start
tively few fumigation treatments have been
from cankered portions. The removal of
developed for pome and stone fruits.
dead and senile plant parts and canker por-
tions helps to reduce the incidence of many
postharvest diseases. The incidence of many
rots may also be reduced if the rotted fruits Heat treatments
are frequently collected and dumped in a
deep trench and later covered with a thick Heat treatments may be applied by hot water
layer of soil to prevent the dissemination of dips or hot vapour exposure. Hot water is
their spores. If such rotted fruits are destroyed useful in controlling fungal infections, while
by burning some distance away from the exposure to hot vapour controls insects.
orchard, this also helps to reduce the inci- Postharvest decay of strawberries caused by
dence of many rots in temperate fruits. B. cinerea and R. stolonifer has been con-
Proper pruning can prevent Botrytis rot of trolled by exposing the fruits to humid air at
grapes (Philips et al., 1990). 44°C for 40–60 min (Couey and Follstad,
The influence of N, P, K, Ca and Mg 1966). Akamine and Arisumi (1953) have
nutrients on storage rots of apple and pear reported hot water treatments for fruit rot of
has been studied extensively (Sharples, 1980). papaya (48°C for 20 min). Two methods
Susceptibility to Gloeosporium rot was cor- have been suggested: one involves a short-
related negatively with fruit Ca, but corre- term heat treatment above 40°C (usually
lated positively with K/Ca ratios. Higher 44–55°C) for a few minutes to 1 h and in the
doses of nitrogen increase the incidence of other, the fruits are exposed to 38–46°C but
G. album (Montgomery and Wilkinson, 1962). for a longer duration (12 h to 4 days) (Fallik
Calcium sprays to control bitter pit in apples et al., 1996). The LD50 temperature for spo-
also confer resistance to P. expansum. rangiospores of R. stolonifer exposed to hot
water for 4 min was 49°C, whereas that for
germinating spores was only 39°C (Eckert
Fumigation and Sommer, 1967).
Safe fumigating agents that disappear after

a short time, such as the use of ozone and Ionizing radiation and UV illumination
sulphur dioxide and acetic acid, can be
recommended to reduce dependence on con- Ionizing radiation may harm the genetic
ventional fungicides. Ozone application to material of the living cell directly, leading
grapes (0.1 mg/g grapes) during 20 min expo- to mutagenesis and eventually to cell death.
sure reduced decay caused by Rhizopus Most studies are carried out with Co60 gamma
stolonifer and prolonged shelf life. This treat- rays. It has been seen that multicellular
ment was as effective as sulphur dioxide conidia of Alternaria and Stemphylium or
6 A. Arya
bicellular spores such as Cladosporium and Another treatment for extending the
Diplodia are more resistant to gamma radia- postharvest life of apple, pear and plum is
tion than the unicellular spores of other by coating the skin with a product called
fungal species (Sommer et al., 1964). Since ‘Prolong’, a mixture of sucrose esters of fatty
radiation can penetrate fruit tissues, it has a acids and polysaccharide (Banks and
therapeutic effect. Plant tissues can produce Harper, 1981). It alters the permeability of
phytoalexins (defence chemicals) in response fruits to gases in such a way that oxygen
to radiation effect. Low doses of UV-C light permeability is reduced considerably, while
(wavelength 190–280 nm) can induce resis- carbon dioxide permeability is little affected.
tance in a wide range of fruit and vegetables This coating had little effect on grapes and
(Barkai-Golan, 2001). UV light has a germi- strawberries.
cidal effect and, at the same time, it induces
activity of PAL and peroxidase enzymes
(Droby et al., 1993). Search for the antagonists:
criteria of selection
Chemically impregnated wrappers Various strains of antagonist must be com-

pared for effectiveness in controlling fruit
decay and for phenotypic characteristics
Wrapping grape clusters in tissue paper
that are useful in determining their com-
impregnated with sodium orthophenyl buty-
mercial potential; for example, the differen-
rate and sodium metabisulphate reduces
tiation criteria for decay control on apple
postharvest decay. Volatile fungal inhibi-
includes the biological control efficacy of
tors also provide effective control of grapes
the strains, spectrum of activity (pathogens
against A. niger and P. canescens (Sharma
to be tested, cultivar range, fruit maturity
and Vir, 1984). Potassium iodide wraps pro-
stages), ability to colonize wounded and
vide effective control of G. roseum on apples
sound fruit surfaces under various condi-
(Sharma and Kaul, 1988). Development of
tions, utilization of substrates occurring in
Botryodiplodia rot of apples was retarded
fruits, or growth at cold storage tempera-
by wrapping them in papers dipped in cul-
tures and at 37°C.
ture filtrate of Streptomyces thermoflavus
In addition, these antagonists must
(Gupta and Gupta, 1983).
meet strict regulations for safety as they are
being applied to consumable commodities,
i.e. fruits. Thus, in developing biocontrol
Fruit skin coatings systems for postharvest disease manage-
ment of fruits, the key requirements for suc-
cessful commercialization of an antagonist
Skin coatings can improve the keeping qual-
must be well defined and strain searches
ity of fruits by decreasing water loss and
should continue until adequate strains are
retarding ripening and rotting by various
found that meet all the safety requirements.
pathogens. Coating is generally done with
oils, waxes and colloidal solutions of car-
boxymethyl cellulose. Apples coated with
mustard oil, paraffin and castor oil checked Enhancement in biocontrol
the infection of a large number of pathogens activity of antagonists
(Sumbali and Mehrotra, 1980; Kaul and Mun-
jal, 1982; Sharma and Kaul, 1988). Applica- Postharvest environments are better defined
tion of hydrogenated groundnut oil provided than field conditions, wherein abiotic and
effective control of Alternaria rot of apple biotic factors can be determined with rela-
(Tak et al., 1985). Skin coating with neem tive ease and manipulated to the antago-
oil completely checked blue mould rot in nist’s advantage, although the mechanism(s)
apples (Kerni et al., 1983). of biocontrol have not yet been fully explained
and, to date, there have been only a few of many more biocontrol agents for posthar-
attempts to exploit these mechanisms to vest fruit rots.
improve postharvest biocontrol (Janisiew-
iez et al., 1992). The reports available on the
mechanism of the biocontrol of posthar- Biocontrol: an integrated approach
vested commodities suggest that competi-
tion for nutrients and space plays a major
Recently there has been an increased inter-
role in most cases (Wisniewski et al., 1991;
est in enhancing the efficacy of biocontrol
Calvente et al., 1999). In most of the systems
agents by adding some synthetic chemicals
where microbial communities are involved,
like calcium chloride or nitrogenous com-
interactions are density dependent and often
pounds or sugar analogues. For example, a
more than one type of interaction occurs at a
mixture of Cryptococcus laurentis and thi-
specific time which is dependent on the
abendazole has been observed to reduce
growth phase of different microorganisms,
95% of P. expansum infection in pear (Sugar
population density and species diversity.
et al., 1994). Enhancement of biocontrol activ-
Basically, three different types of interac-
ity of antagonists by the addition of nitrog-
tions, namely competition for nutrients,
enous (L-asparagine, and L-proline) and
competition for space and inhibition by sec-
carbohydrate (2-deoxy-D-glucose) compound
ondary metabolites, have been observed in
has been reported in apple and pear fruit
preharvest sprays of B. subtilis to control C.
(Janisiewiez, 1994). Similarly, a combina-
gloeosporioides on avocado (Korsten et al.,
tion of 2-deoxy-D-glucose and Candida
1997). The main approaches used to improve
saitoana is reported to be useful in reducing
biological control in postharvest systems
postharvest diseases (Wilson and El-Ghaouth,
are: (i) manipulation of the environment; (ii)
1997). Recently, a bioactive coating having a
use of mixed cultures of antagonists; (iii)
combination of C. saitoana and 0.2% gly-
physiological and genetic manipulation of
colchitosan has been found more effective in
antagonists; (iv) combining field and post-
controlling rot development caused by B.
harvest applications; (v) manipulation of
cinerea, P. digitatum and P. expansum in sev-
formulations; and (vi) integration with other
eral cultivars of apples, oranges and lemon
methods.
(El-Ghaouth et al., 2000a,b). The same group
In the case of the development of Bio-
of researchers showed that the application
Save, the effectiveness of the antagonist, a
of C. saitoana with 0.2% 2-deoxy-D-glucose,
saprophytic strain of P. syringae L-59-66, in
before inoculation of pathogens, was more
reducing blue mould and grey mould decay
effective in controlling the decay of apple,
on apples and pears in a laboratory setting
orange and lemon caused by B. cinerea, P.
was demonstrated to EcoScience Corp
expansum and P. digitatum than either C.
(Orlando, Florida, USA). The commercial
saitoana or 0.2% 2-deoxy-D-glucose alone.
setting of the test, the involvement of indus-
For the postharvest treatment of fruits,
try in conducting those tests and the encour-
stock of biocontrol agent is usually made in
aging results were the key factors in obtaining
lyophilized cultures, agar slant or spore sus-
a commitment to develop the antagonist for
pensions and is maintained at low tempera-
commercial use. EcoScience Corp then inves-
ture and at the same osmotic concentration
tigated the potential for registration and for-
in culture medium (Churchill, 1982).
mulation of the antagonist before making
this commitment. Mass production by fer-
mentation and the biomass yield of P. syrin-
gae strain L-59-66 was determined before Botanicals as Antifungal Agents in
scale-up experiments (Janisiewiez, 1998). Postharvest Disease Control of Fruits
Extensive technical support and quality con-
trol have been instrumental in the success of Fruits and vegetables have a number of con-
this product. Similar support and testing stituents and inducible volatile aromatic
need to be conducted for the development and flavour compounds (Tripathi, 2007).
8 A. Arya
These aromatic and flavour components are B. cinerea and C. gloeosporioides directly on
produced generally by fruits during ripening the fruit at 0.4 µl ml (Vaughn et al., 1993).
and provide resistance to the fruits at the Among the five compounds, benzaldehyde
postharvest stage. The flavour compounds was the most toxic to the fungi.
are secondary metabolites having unique
properties of volatility and low water solu-
bility. As potential fungicides, their natural Plant extracts
occurrence as part of the diet, their ephemeral
nature and their biodegradability suggest low
Fungitoxic activity of plant extracts can be
toxic residue problems. Such compounds
tested by the poisoned food technique (Gro-
could be extracted and applied to other
ver and Moore, 1962). Tripathi (2005) tested
harvested perishables. Some of the volatile
24 taxa belonging to 12 different families for
aromatic components, namely acetalde-
their antifungal activity against P. italicum.
hyde, six carbon (C6) aldehydes, benzalde-
Most of the plants showed either poor or
hyde, hexenel and hexanal, are of significant
moderate (50–100%) activity. Leaf extracts of
importance.
seven plants, namely Acacia nilotica (ethyl
Vapours of acetaldehyde have been
alcohol), Citrus aurantifolia (ethyl acetate),
used to control B. cinerea (Prasad and Sta-
Murraya koenigii (ethyl acetate), Nerium
delbacher, 1973). Avissar and Pesis (1991)
indicum (ethyl acetate), Ocimum gratissi-
reported acetaldehyde to be active against B.
mum (benzene, ethyl acetate), O. sanctum
cinerea and R. stolonifer causing rot to straw-
(petroleum ether), Prunus persica (ethyl ace-
berry fruits. The effect of trans-2-hexenel
tate) and bark extract of A. farnesiana and A.
on the control of blue mould disease (P.
nilotica (ethyl acetate extract) showed 100%
expansum) in the reduction of patulin con-
activity against test fungus. The leaves of
tent and on fruit quality improvement of
Achyranthes aspera and Hyptia suaveolens
‘Conference’ pears was evaluated and
showed poor activity.
greater reduction of decay was obtained by
Arya (1988) tried leaf extracts of Aegle
treatment at 12.5 µl/l at 20°C for 24 or 48 h
marmelos, O. sanctum, Azadirachta indica,
after inoculation (Neri et al., 2006).
Crataeva nurvala, Ephedra foliata (shoot),
Jasmonates are naturally occurring
Eucalyptus occidentalis, Lawsonia inermis
plant growth regulators that are widely dis-
and Strichnos nux vomica in three different
tributed in the plant kingdom and are
concentrations on two fruit rot pathogens,
known to regulate various aspects of plant
P. psidii and P. viticola. Extracts obtained
development and responses to environmen-
from Ephedra and Eucalyptus were most
tal stresses. The antifungal activity of six
effective at 25% concentration in the case of
glucosinolates has been tested on several
P. viticola, while a higher concentration
postharvest pathogens, namely B. cinerea,
(75%) leaf extract of ‘neem’ (A. indica) was
R. stolonifer, Monilinia laxa, Mucor piri-
most effective, causing 82.3% spore inhibi-
formis and P. expansum, both in vitro and
tion. Tulsi caused 76.4% inhibition. The
in vivo (Mari et al., 1996).
fungicidal nature of ‘neem’ and ‘tulsi’ was
Fumigation of apples with acetaldehyde,
reported earlier by Pandey et al. (1983)
a natural volatile compound produced by
against Pestalotia psidii.
various plant organs, inhibits P. expansum
development in the fruit (Stadelbacher and
Prasad, 1974), while fumigation of strawber-
ries with acetaldehyde considerably reduces Essential oils
decay caused by R. stolonifer and B. cinerea.
Evaluation of 15 volatile odour compounds, Volatile oils are sweet-smelling lipids synthe-
released from raspberries and strawberries sized and stored in various plant parts. These
during ripening, for their ability to inhibit oils are essentially mixtures of two classes
postharvest decay fungi showed that 5 of of terpenoids, i.e. the monoterpenes and the
them inhibited the growth of A. alternata, sesquiterpenes, the former predominating in
most cases. Among the 49 essential oils tested, Antibodies are produced in response to inva-
those of palmrosa (Cymbopogon martini) and sion of an antigen. The remarkable potential
red thyme (Thymus zygis) showed the great- of recombinant DNA technology has made
est inhibitory effect on B. cinerea spore ger- it possible for plants to express antibodies
mination at the lowest concentration. The against pathogen proteins, which in turn
next best inhibitors were essential oils of enable them to defend against the target
clove buds (Eugenia caryophyllata) and cin- pathogen. The expression of pathogen-
namon leaf (Cinnamomum zeylanicum). The specific antibody in plants is termed ‘planti-
most frequently occurring constituents in body’ (Smith, 1996; Gibbs, 1997). The
essential oils showing high antifungal activity plantibodies produced in the cell cytosol are
were: D-limonene, cineole, a-pinene, b-pinene, expected to interact with their targets, ren-
b-myrcene and camphor. The fungicidal dering them inactive (Zhang and Wu, 1998).
activity of the individual components, sin-
gly and in combination, is being studied
(Wilson et al., 1997). Essential oil derived Induced Resistance
from another species of Thymus, T. capita-
tus, reduced the development of B. cinerea
Induced resistance is a new concept proposed
markedly in inoculated mandarin fruits when
by the American phytopathologist, Joseph
applied as a vapour. Scanning electron micro-
Kuc (1995). According to Kuc, resistance in
scopic observations indicated a direct dam-
plant tissues can be enhanced by modulat-
aging effect of the thyme oil on fungal
ing their natural defence mechanisms. Vari-
hyphae (Arras and Piga, 1994).
ous physical, chemical and biological elicitors
can enhance resistance in plants. Use of chi-
tosan, a deacetylated derivative of chitin,
Gel and latex and salicylic acid can be made to offer a
possible alternative to synthetic pesticides.
Gel derived from Aloe vera has been found ASM (acibenzolar-s-methyle) is the first
to have antifungal activity against four com- commercially available product that acti-
mon postharvest pathogens, P. digitatum, P. vates a systemic acquired resistance (SAR)
expansum, B. cinerea and A. alternata. The in plants like other biological inducers.
natural gel suppressed both germination
and mycelial growth. Latex present in some
fruits is another natural fungicide which is Host Defence Through
effective against diseases of banana, papaya Gene Silencing
and other fruits (Adikaram et al., 1996).
Papaya latex contains proteases, glucosi-
Scientists working on Eutypa dieback dis-
dases, chitinases and lipases, while a cystein-
ease of grapevine in Switzerland (2008)
rich protein, hevien, was isolated from the
found the involvement of glutathion-s-
latex of rubber tree (Hevea brasiliensis). It
transferase in the detoxification of toxins, of
showed a strong antifungal activity in vitro
the jasmonic acid signalling path way, and
against B. cinerea and species of Fusarium
of several effector genes underlying a more
and Trichoderma (van Parijs et al., 1991).
general response where the toxins could be
recognized as an elicitor for the trunk patho-
gens. Grapevines were tested for infiltration
Use of Plantibodies for of double standard RNA into leaves for easy
Disease Control testing of genes. dsRNA were functional in
Puccinia striiformis to suppress recognition
Drawing a clue from the potential antibodies by host plants (Newton, 2002). Genes that
in combating human diseases, plant scien- encode for post-transcriptional gene silenc-
tists are now geared to extend this remark- ing have been characterized in plants and
able technology to plant disease control. fungi (Dalmay et al., 2000).
10 A. Arya
A variety of gene silencing phenomena are isolated from plant viruses, bacteria,
that have been discovered are: (i) the dupli- fungi or other plants and introduced in the
cated DNA sequence is inactivated by muta- plants. Genes have been transferred by sci-
tion in the meiotic phase, a process known entists in India from Amaranthus to potato
as repeat induced mutation (RIP) (Selker for improving protein quality and quantity,
et al., 1987); (ii) the duplicated DNA and from mangroves to annual crops for
sequence during the meiotic phase is inacti- imparting tolerance to salinity. Powell et al.
vated by methylation, methylation induced (1994) reported that transgenic tomato fruits
premeiotically (MIP) (Goyon and Faugeron, expressing the gene of fungal PG-inhibiting
1989); (iii) multiple copies of transgenes in glycoproteins of plants were more resistant
the vegetative phase are irreversely inacti- to B. cinerea than the control fruits. Scien-
vated and silencing is called ‘quelling’ tists have tried to prevent ethylene produc-
(Romano and Macino, 1992); and (iv) silenc- tion by plant tissue using an antisense gene.
ing is maintained even in the absence of The fruits would not ripen here until treated
transgenes (van West et al., 1999) or another exogenously with ethylene. PR protein genes
process called MSUD (Shiu et al., 2001). appear to be a very potential source for can-
didate genes providing fungal resistance.
These proteins may play a direct role in
defence by attacking and degrading patho-
Disease-resistant Transgenic Plants gen cell wall components.
The first specific fungal-resistant gene,
Newly developed techniques in plant breed- Hm1, has been isolated from maize, confer-
ing such as restriction fragment length poly- ring resistance to race 1 of the fungus Helm-
morphism techniques and gene transfer inthosporium carbonum (Johal and Briggs,
methods can be used to develop these cul- 1992). After fungal-resistance genes have
tivars. In contrast to conventional breed- been isolated, they can be transferred to pro-
ing, this later technology allows the transfer vide resistance to a specific race of fungal
of traits from one species into the genomes pathogens. Woloshuk et al. (1991) identified
of plants of other species with the preser- in tobacco a salt stress-inducible vacuolar
vation of the intrinsic properties of the protein with an inhibitory effect on the
acceptor plant (Cornelissen and Melchers, growth of P. infestans in vitro. It was sug-
1993). gested that this protein, described as Osmo-
A transgenic plant contains, within its tin, inhibited growth by interfering with the
genome, a foreign DNA that has been intro- fungal membrane, hence disturbing cellular
duced artificially via genetic engineering. The function. As with class I hydrolyses, the
creation of such plants involves the intro- protein could be arrested extracellularly by
duction of genes for resistance from unre- modification of the corresponding gene
lated plant species. Desirable target genes (Melchers et al., 1993).
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2 Botanicals in Agricultural
Pest Management
Ashok Kumar, Priyanka Singh and N.K. Dubey

Centre of Advanced Study in Botany, Banaras Hindu University, Varanasi, India
Abstract
The overzealous and indiscriminate use of most of the synthetic fungicides has created different types
of environmental and toxicological problems. The ultimate aim of recent research in this area has been
the development of alternative control strategies to reduce dependency on synthetic fungicides.
Recently, in different parts of the world, attention has been paid to the exploitation of higher plant
products as novel chemotherapeutants in plant protection because of their non-phytotoxicity, system-
icity and easy biodegradability. The exploitation of natural products to control fungal infestation and
prolong storage life of food commodities has received more attention. Biologically active natural prod-
ucts have the potential to replace synthetic fungicides. Currently, different plant products have been
formulated for large-scale application as botanical pesticides in the eco-friendly management of plant
pests and are being used as alternatives to synthetic pesticides in crop protection. This chapter deals
with the current status and future prospects of botanical pesticides in eco-friendly management of dif-
ferent plant pests.
Introduction the world is destroyed by various pests,

including bacteria, fungi, viruses, insects,
The constant growth of the world’s popula- rodents, nematodes, etc. Losses at times are
tion requires substantial resources for the so severe as to lead to famine in large areas
production of food. One of the greatest chal- of the world that are densely populated.
lenges of the world is to produce enough Considerable attention has been given to
food for the growing population. Produc- losses in the field caused by different pests,
tion as well as protection of food commodi- but research into postharvest losses of food
ties is necessary to nourish the ever-growing commodities is still required. So, priority
population. The situation is particularly should be given to postharvest studies, par-
critical in developing countries, where the ticularly in hot and humid tropical climates
rate of net food production is slowing down where at least half of the foodstuffs may be
in relation to population growth. The world lost between harvest and consumption. Con-
food situation is aggravated by the fact that siderable postharvest losses of food commodi-
in spite of the use of all available means of ties are brought about due to fungi, insects
plant protection, a major proportion of the and rodents. International agencies that moni-
yearly production of food commodities of tor world food resources have acknowledged

14 (eds A. Arya and A.E. Perelló)
Botanicals in Agricultural Pest Management 15
that one of the most feasible options for meet- of mycotoxins. Unseasonal rains and flash
ing future food needs is the reduction of post- floods are very common in India, which
harvest losses (Tripathi and Dubey, 2004). enhances the moisture content of the grains,
Fungi are significant destroyers of food- making them more vulnerable to fungal attack
stuffs during storage, rendering them unfit (Srivastava, 1987). Fungi can grow on simple
for human consumption by retarding their and complex food products and produce vari-
nutritive value. Many agricultural commod- ous metabolites (Khosravi et al., 2007). Up to
ities are vulnerable to attack by a group of now, more than 100,000 fungal species are
fungi that are able to produce toxic metabo- considered as natural contaminants of agri-
lites called mycotoxins. Production of myc- cultural and food products (Kacaniova, 2003).
otoxins by several fungi has added a new The quality and safety of food is of importance
dimension to the gravity of the problem. so that markets are not compromised by the
Fungal toxins are low molecular weight sale of low quality or unsafe food.
chemical compounds which are not detected
by the body’s antigens. Their effect is more
often chronic rather than acute; hence, they Control of Fungal Infestation
produce no obvious symptoms. Thus, myc- During Storage
otoxins are insidious poisons (Pitt, 2002).
Cereals and grains are major mycotoxin vec- Attempts to control postharvest diseases
tors because they are consumed by both have been carried out by different physical
humans and animals. According to FAO and chemical treatments.
estimates, 25% of the world food crops are
affected by mycotoxins each year. These tox-
ins can develop during production, harvest- Physical methods
ing, or storage of grains, nuts and other crops.
Mycotoxins are among the most potent muta-
Several techniques are used for the preser-
genic and carcinogenic substances known.
vation of food and feeds. Drying, freeze-
They pose chronic health risks: prolonged
drying, cold storage, modified atmosphere
exposure through diet has been linked to
storage and heat treatments are all physical
cancer and kidney, liver and immune sys-
methods of food preservation (Farkas, 2001)
tem disease (Srivastava et al., 2008). Among
(Table 2.1).
mycotoxins, aflatoxins chiefly produced by
strains of Aspergillus flavus are the most
Cold storage
dangerous and about 4.5 billion people in
underdeveloped countries are at risk of Low temperature inhibits the germination of
chronic exposure to aflatoxicosis through spore/conidia and pathogenicity significantly
contaminated foods (Williams et al., 2004; (Tian, 2001). It reduces the metabolic activi-
Srivastava et al., 2008). In most of the devel- ties of various microbes associated with food-
oping countries, total permissible aflatoxin stuffs, which would be helpful in enhancing
content in food has been set around 20 ppb the shelf life of edibles. However, cold storage
(Mishra and Das, 2003). Aflatoxins are potent has its limitations, such as unavailability in
toxic, carcinogenic, mutagenic, immuno- most developing countries and an inability to
suppressive agents, produced as secondary check psychrophilic microorganisms.
metabolites by the fungus Aspergillus, A.
parasiticus and A. nomius on a variety of Heat treatment
food products. In addition, aflatoxin inhib-
its seed germination, seedling growth, root High temperature plays a significant role
elongation, chlorophyll and carotenoid syn- in controlling the metabolic activities of
thesis, as well as protein, nucleic acid and organisms because it affects the enzymatic
some enzyme synthesis in seeds. activities in all organisms adversely (Lagu-
Climatic conditions in India are most nas and Castaigne, 2008; Moatsou et al.,
conducive to mould invasion and elaboration 2008). Heat treatment can check microbial
16 A. Kumar et al.
Table 2.1. Some physical and chemical methods used in the prevention of fungal contamination and
mycotoxin production.
Methods Fungi/mycotoxins References
Physical:
Sunlight Aspergillus flavus/aflatoxin Shantha and Sreenivasamurthy (1977)
Solar irradiation A. parasiticus/aflatoxin B1 Samarajeewa et al. (1985)
Electric light A. flavus/aflatoxin B1 Chourasia and Roy (1991)
UV light A. flavus/aflatoxin Shantha and Sreenivasamurthy (1977)
UV-C radiation Colletotrichum gloeosporioides Cia et al. (2007)
Infrared light Penicillium citrinum Qing et al. (2002)
γ Radiation Cryptococcus neoformans Dadachova et al. (2004)
α Radiation C. neoformans Martinez et al. (2006)
Autoclaving All types of moulds Coomes et al. (1966)
Cooking Food-spoiling moulds Rehana and Basappa (1990)
Roasting Food-spoiling moulds Ogunsanwo et al. (2004)
Dry heat Fusarium graminearum Clear et al. (2002)
Low temperature/ Some soil fungi Janna et al. (2005)
refrigeration
Chemical:
H2O2 A. flavus/aflatoxin Sreenivasamurthy et al. (1967)
Na-hypochlorite A. flavus/aflatoxin Shantha et al. (1986)
Azoxystrobin C. lupini Thomas et al. (2008)
Chlorothalonil C. lupini Thomas et al. (2008)
Copper oxychloride C. lupini Thomas et al. (2008)
Carbendazim A. carbonarius/ochratoxin Medina et al. (2007)
Mancozeb Penicillium sp., Trichoderma sp. Magarey et al. (1997)
Maneb F. graminearum/ZEN D’Mello et al. (1998)
Nitroimidazole Sclerophoma pityophila Olender et al. (2008)
Organotin C. gloeosporioides Rehman et al. (2008)
Blitox Aspergillus spp. Satish et al. (2008)
Captan Aspergillus spp. Satish et al. (2008)
Dithane M-45 Aspergillus spp. Satish et al. (2008)
Thiram Aspergillus spp. Satish et al. (2008)
SAAF A. flavus Kumar et al. (2008)
Bavistin A. flavus Kumar et al. (2008)
Wettasul-80 A. flavus Kumar et al. (2008)
Ceresan A. flavus Kumar et al. (2008)
Diphenylamine A. flavus Kumar et al. (2008)
growth efficiently but the technique is not is also efficient in checking microbial growth
suitable for long-term storage. and proliferation, as well as mycotoxin
production. The irradiation of food com-
Radiation modities during storage is unattainable in
developing countries.
Sun drying of food commodities (grains and
pulses) before storage is preferable in most
underdeveloped countries but the tech-
nique is unsuitable in the case of vegetable Chemical methods
crops. High-energy radiation like γ rays
(Petushkova et al., 1988), UV rays (Oteiza In order to minimize the losses caused by
et al., 2005), infrared (Qing et al., 2002), etc., moulds in the field and also during storage,
many synthetic fungicides have been intro- postharvest diseases of fruits, vegetables and
duced (Table 2.1). The discovery of Bor- other edibles as a viable alternative to the use
deaux mixture is significant in the history of of present day synthetic fungicides (Wilson
the chemical control of plant diseases. In the et al., 1999; Pang et al., 2002). Microbial
past few decades, various synthetic chemi- antagonists have been reported to protect a
cals have played a significant role in the variety of harvested perishable commodi-
management of such losses. Several chemi- ties against a number of postharvest patho-
cal additives also function as preservatives, gens (Wisniewski et al., 2001). However,
even though the exact mechanisms or tar- decreasing efficacy and lack of consistency
gets are often not known (Davidson, 2001. when applied as stand-alone treatments
The organic acids, acetic, lactic, propionic, under commercial conditions (Droby et al.,
sorbic and benzoic acids, are used as food 2001) are limiting their use. Hence, these
preservatives (Brul and Coote, 1999). Both drawbacks in alternative methods have
sorbic and benzoic acid have a broad spec- increased interest in developing further
trum of activity (Nielsen and De Boer, 2000; alternative control methods, particularly
Davidson, 2001). Benzoic acid and sodium those which are environmentally sound and
benzoate are used primarily as antifungal biodegradable.
agents (Davidson, 2001). Recently, some
technology like TiO2 photocatalytic ozona-
tion has been found to be efficient in con-
trolling postharvest spoilage of kiwifruit Botanicals as Fungitoxicants
(Hur et al., 2005).
The indiscriminate application of syn- Recently, in different parts of the world, atten-
thetic chemicals as antimicrobials has con- tion has been drawn towards the exploitation
tributed greatly to the management of losses of higher plant products as novel chemo-
caused by fungi, but these chemicals have therapeutants in plant protection. Because of
led to a number of ecological and health non-phytotoxicity, systemicity, easy biode-
problems due to their residual toxicity gradability and the stimulatory nature of
(Kneževi and Serdar, 2008), carcinogenicity, host metabolism, plant products possess the
teratogenicity, hormonal imbalance, sper- potential to be of value in pest management
matotoxicity, etc. (Pandey, 2003; Kumar (Mishra and Dubey, 1994). Higher plants con-
et al., 2007). History also shows that over- tain a wide spectrum of secondary metabo-
zealous use of synthetic pesticides has led lites such as phenols, flavonoids, quinones,
to numerous problems unforeseen at the tannins, essential oils, alkaloids, saponins
time of their introduction. Different types of and sterols. Such plant-derived chemicals
ecological problems have been reported may be exploited for their different biologi-
from time to time by these xenobiotics, such cal properties (Tripathi et al., 2004). Terres-
as acute and chronic poisoning of applica- trial plants produce a spectrum of natural
tors, farm workers, and even consumers, products, namely terpenoids, phenolics and
extensive groundwater contamination, resis- alkaloids. Many of these are thought to have
tance development in pests (Wilson et al., an ecological function for the plants pro-
1997), effect on non-target organisms (Wik- ducing them, serving to defend the plants
telius et al., 1999), ozone layer depletion by from herbivores and pathogens (Isman and
methyl bromide (Lee et al., 2001), etc. Akhtar, 2007). Such defensive chemistry is
thought to be extremely widespread among
the plant kingdom.
The body of scientific literature docu-
Biocontrol Agents in menting the bioactivity of plant derivatives
Pest Management to different pests continues to expand; yet
only a handful of botanicals are currently
Considerable attention has also been given used in agriculture in the industrialized
to the potential of biological control of world. In the context of agricultural pest
18 A. Kumar et al.
management, botanical pesticides are well Conclusions

suited for use in industrialized countries
and can play a much greater role in the post- Plants are a virtually untapped reservoir of
harvest protection of food commodities in different valuable chemicals that can be
developing countries (Isman, 2006). used directly or as templates for the formu-
Among the different plant products, the lation of pesticides. Numerous factors
application of essential oils is a very attrac- have increased the interest of the pesticide
tive method for controlling postharvest losses industry and the pesticide market in this
(Table 2.2). Production of essential oils by source of natural products as pesticides.
plants is believed to be predominantly a Pesticides based on plant essential oils or
defence mechanism against pathogens and their constituents have demonstrated their
pests (Oxenham, 2003). Essential oils and their efficacy against a range of fungal pests
components are gaining increasing interest responsible for pre- and postharvest diseases,
because of their relatively safe status, wide as well as mycotoxin production. Encourag-
acceptance by consumers and their exploi- ing results on the use of natural products to
tation for potential multi-purpose use (Sawa- control postharvest fungal spoilage indicate
mura, 2000; Ormancey et al., 2001; Feng and that we should be able to develop natural
Zheng, 2007). The problem of the develop- pesticides that could be as effective as syn-
ment of resistant strains of fungi and other thetic fungicides and presumably safer for
organisms may be solved by the use of man and the environment. Biological com-
essential oils of higher plants as fumigants pounds, because of their natural origin, are
in the management of storage pests because comparatively biodegradable and most of
of synergism between different components them are almost non-residual in nature (Beye,
of the oils (Varma and Dubey, 1999; Dubey 1978).
et al., 2006). During recent years, products of some
The antifungal activity of essential oils pesticidal plants have received global atten-
is well documented and characterized with tion for the protection of several food com-
their bioactivity in vapour phase. The pesti- modities because of their antimicrobial
cidal activities of essential oils are due to properties (Kumar et al., 2007). Such plant
the presence of some aroma compounds. products have been formulated for large-
Fumigation with such aroma compounds scale application as botanical pesticides,
greatly reduces postharvest decay without which are used as alternatives to synthetic
causing any toxicity (Chu et al., 2001; Liu pesticides in crop protection. A consoli-
et al., 2002). Recently, some monoterpenes dated and continuous search of natural
isolated from essential oils exhibited fungi- products may yield safer alternative control
cidal activity and have been shown to measures comparable to azadirachtin and
inhibit fungal rotting of vegetables without pyrethroids, which are being used in differ-
altering taste and quality (Hartmans et al., ent parts of the world as ideal natural fungi-
1995; Oosterhaven, 1995). The fungitoxic cides. The number of options that must be
properties of essential oils from higher considered in the discovery and develop-
plants are well documented but little atten- ment of a natural product as a pesticide is
tion has been paid towards the bioactivity larger than for a synthetic pesticide. How-
of essential oil constituents. The fungitoxic ever, current advances in plant chemistry
activity of some essential oil components and biotechnology, combined with increas-
is listed in Table 2.3 and Fig. 2.1. However, ing need and environmental pressure, are
more work on the bioactivity of plant prod- greatly increasing the interest in plant prod-
ucts including essential oil and constitu- ucts as pesticides. Products from higher
ents in in vitro and in vivo conditions is plants are a safe and economical option in
required. The literature is also silent on the the management of agricultural pests and
mode of action of the essential oils and will be in high demand in the global pesti-
components when used as postharvest cide market.
fungitoxicants.
Table 2.2. Efficacy of some higher plant products in checking fungal growth and mycotoxin production.
Plants Products Fungi/mycotoxins References
Hypericum linarioides EO/PEE/ 6 Fusarium spp. Cakir et al. (2005)

ME/ChlE
Calocedrus macrolepis EO Colletotrichum gloeosporioides, Chang et al. (2008)
Rhizoctonia solani, F. oxysporum
Silene armeria EO/ME/ F. oxysporum, C. capsici, Bajpai et al. (2008)
HexE Botrytis cinerea
Origanum acutidens EO 17 pathogenic fungi Kordali et al. (2008)
Cinnamomum EO Trametes versicolor, Lenzites Cheng et al. (2006)
osmophloeum betulina, Laetiporus sulphureus
Thymus numidicus EO Candida albicans Giordani et al. (2008)
Lantana camara EO Aspergillus niger, Deena and Thoppil (2000)
A. parasiticus
O. glandulosum EO F. oxysporum, Cladosporium Bendahou et al. (2008)
herbarum, A. flavus
Tarchonanthus EO C. albicans Matasyoh et al. (2007)
camphoratus
Syzygium aromaticum AqE/EO A. flavus/aflatoxin B1, Omidbeygi et al. (2007);
A. flavus, Penicillium Aldred et al. (2008);
verrucosum/ochratoxin A Reddy et al. (2008)
Curcuma longa AqE A. flavus/aflatoxin B1 Reddy et al. (2008)
Allium sativum AqE A. flavus/aflatoxin B1 Reddy et al. (2008)
Lippia rugosa EO A. flavus/aflatoxin B1 Tatsadjieu et al. (2009)
Citrus sp. EO A. flavus, P. chrysogenum, Viuda-Martos et al.
P. verrucosum (2008)
Bidens pilosa EO/AqE Corticium rolfsii, F. solani Deba et al. (2008)
Satureja hortensis EO/ME A. flavus, A. parasiticus/aflatoxin Omidbeygi et al. (2007);
Abyaneh et al. (2008);
Dikbas et al. (2008)
T. eriocalyx EO A. niger Rasooli et al. (2006)
T. x-porlock EO A. parasiticus/aflatoxin Rasooli and Abyaneh (2004)
Ocimum basilicum EO A. parasiticus/aflatoxin Atanda et al. (2007)
Pimpinella anisum EO A. niger, P. chrysogenum Matan and Matan (2008)
Salvia officinalis EO C. albicans, Trichophyton Pinto et al. (2007)
rubrum, A. flavus
T. vulgaris EO A. flavus/aflatoxin B1 Kumar et al. (2008)
Cympopogon citratus EO B. cinerea, C. herbarum, Tzortzakis and Economakis
A. niger (2007)
Rosmarinus officinalis EO A. parasiticus/aflatoxin Rasooli et al. (2008)
Trachyspermum copticum EO A. parasiticus/aflatoxin Rasooli et al. (2008)
Cordia curassavica EO/HexE/ R. solani, T. mentagrophytes Hernandez et al. (2007)
ChlE/ME
Sesuvium portulacastrum EO A. niger, A. flavus, P. notatum Magwa et al. (2006)
Calamintha officinalis EO B. cinerea Bouchra et al. (2003)
Olea europaea AE/ME Alternaria alternata, Korukluoglu et al. (2008)
A. flavus, F. oxysporum
Citrus sinensis EO A. niger Sharma and Tripathi (2008)
Azadirachta indica AqE P. citrinum/Citrinin Aparecida et al. (2008)
Agave asperrima ME/AqE A. flavus, A. parasiticus/ Sánchez et al. (2005)
aflatoxin B1
Adenocalymma alliaceum AqE A. flavus/aflatoxin B1 Shukla et al. (2008)
Lupinus albus AqE A. flavus/aflatoxin B1 Mahmoud (1999)
Note: EO, essential oil; ME, methanolic extract; AqE, aqueous extract; PEE, petroleum ether extract; AE, acetone
extract; ChlE, chloroformic extract; HexE, hexane extract.
20 A. Kumar et al.
Table 2.3. Efficacy of some essential oil components in checking fungal growth.
Compounds of
plant origin Fungi References
Ajoene Aspergillus niger, Candida albicans, Yoshida et al. (1987)

Saccharomyces cerevisiae Naganawa et al. (1996)
Allicin C. albicans Ankri and Mirelman (1999)
Myrcene Rhizoctonia solani, Fusarium oxysporum Chang et al. (2008)
Limonene Colletotrichum gloeosporioides, Regnier et al. (2008)
Botryosphaeria parva, F. verticillioides Dambolena et al. (2008)
r-Cymene Fusarium sp. Kordali et al. (2008)
a-Pinene C. albicans, S. cerevisiae, A. niger Yousefzadi et al. (2008)
Sonboli et al. (2006)
Caryophyllene R. solani, F. oxysporum Chang et al. (2008)
Citral A. niger, F. oxysporum, Moleyar and Narasimham
Penicillium digitatum, C. albicans (1986)
Da Silva et al. (2008)
Cinnamaldehyde Lenzites betulina, Laetiporus sulphureus Cheng et al. (2006)
Camphor Fusarium sp., R. solani Pitarokili et al. (2003)
Carvone C. gloeosporioides, B. parva Regnier et al. (2008)
Pulegone C. albicans Duru et al. (2004)
Menthone F. verticillioides Dambolena et al. (2008)
Thujone Phytophthora capsici Shafi et al. (2004)
Linalool C. camelliae Zhang et al. (2006)
Geraniol C. camelliae Zhang et al. (2006)
Citronellol Rhizopus stolonifer Moleyar and Narasimham
(1986)
Terpine-4-ol A. flavus, R. solani, P. commune, F. oxysporum Barra et al. (2007)
Menthol F. verticillioides, R. stolonifer, Dambolena et al. (2008)
Penicillium sp., Monilia sp. Moleyar and Narasimham
(1986)
Serrano et al. (2005)
Thymol C. albicans, Fusarium sp., F. verticillioides Braga et al. (2008)
Kordali et al. (2008)
Dambolena et al. (2008)
Eugenol L. betulina, L. sulphureus, Cheng et al. (2006)
T. mentagrophytes, C. albicans Gayoso et al. (2005)
Fenchone P. capsici Shafi et al. (2004)
1,8 Cineole C. gloeosporioides, B. parva Regnier et al. (2008)
Asarone R. solani, P. infestans, Cladosporium Lee (2007)
cucumerinum, Pythium ultimum Lee et al. (2004)
Zingiberene R. solani Agarwal et al. (2001)
Curcumene R. solani Agarwal et al. (2001)
Verbenone Colletotrichum sp. Meepagala et al. (2003)
Verbenol Colletotrichum sp. Meepagala et al. (2003)
Carvacrol Fusarium sp., Botrytis cinerea Kordali et al. (2008)
Romero et al. (2007)
a-Cadinol R. solani, F. oxysporum Chang et al. (2008)
T-muurolol R. solani, F. oxysporum Chang et al. (2008)
CH2 CH3
CH3
CH3
H3C CH3
CH2
H3C CH3 CH2 H3C CH2 H3C CH3

Myrcene Caryophyllene Limonene P-Cymene
CH3 CH3
OH CH3 CH3
H
CHO
O
CH2 CH2OH H
H3C CH3 H3C CH3 H3C CH3

Linalool Camphor Citronellol Citral
CH3
CH3 CH3
O H
O
H
O O
H3C CH3 H3C CH3 H2C CH3

Pulegone Cinnamaldehyde Menthone Carvone
CH3 CH3
CH3 CH3
O
O
CH3
OH
CH3
H3C CH3 H3C CH3
α-Pinene Terpine-4-ol Fenchone Thujone
CH2
CH3
CH3
CH2OH
H
OH
OCH3
OH H3C CH3 H3C CH3
Eugenol Geraniol Thymol
Fig. 2.1. Chemical structures of some bioactive essential oil constituents.

continued
22 A. Kumar et al.
CH3 H3C CH3 H3C CH3

H H
CH3 CH3
OH
H H
H3C CH3 H3C OH H3C OH
Menthol α-Cadinol T-muurolol
CH2 CH3
S S CH2
H2C S
Ajoene
O
O
S CH2 H3C CH3
H2C S
Allicin 1, 8-Cineole
OCH3
OCH3 CH3 CH3
OH
H3CO
CH3
CH3 H3C CH3 H3C CH3

Asarone Zingiberene Carvacrol
CH3 H3C CH3 H3C CH3
CH3 CH3
CH3
H3C CH3 O OH
Curcumene Verbenone Verbenol
Fig. 2.1. continued.
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3 Deleterious Effects of Fungi on
Postharvest Crops and Their
Management Strategies
A.O. Ogaraku
Plant Science and Biotechnology Unit, Department of Biological Sciences,
Nasarawa State University, Keffi, Nigeria
Abstract
Fungi influence our lives in many ways. The parasitic forms cause serious diseases in crop plants and
pose hazards to the lives of animals and humans whenever they infect consumable crops. Most con-
sumable crops are susceptible to fungal infection. The most prominent types of fungi attacking com-
modities are species of Aspergillus, Penicillium and Rhizopus, etc. Types of crop deterioration caused
by fungi include discoloration, flavours and odour, rotting and caking, destruction of viability and
production of mycotoxins on food before infestation. Conditions that favour the development of fungi
on harvested and stored crops include moisture, preharvest infection and lapses in the processing
method. Method of control involves drying of produce to a safe moisture level, non-mixing of new
produce with old ones, avoidance of pre-storage damage and use of chemicals, fungicides and medici-
nal plants in treating the produce.
Introduction use or reduces the economic value of the

materials (Opadokun et al., 1979).
Fungi are one of the most important groups It is also noteworthy to mention some
of organisms on the planet. They are micro- other factors that have been identified as
scopic, achlorophyllous and non-vascular causing damage to crops, namely:
plants. They cause deterioration of posthar- ● insects and mites
vest crops (Ogundana et al., 1970). ● microorganisms, such as bacteria, actino-
Deterioration means that something is mycetes, yeasts and virus
made to be of less value or worse in quality ● rodents and birds
(Adebayo et al., 1994). It is a common phe- ● physical factors, such as temperature
nomenon in agricultural crops, either on the and relative humidity of the storage
farm, at harvest or during storage. Fungi are environment
known to cause various types of deteriora- ● harvesting, handling and transportation
tion and pose a hazard to humans and ani- (Clarke, 1968).
mals whenever they infect crops. Fungal
deterioration can be defined as any change Before the 17th century, scientists concen-
resulting from the activities of fungi which trated on damage caused by insects on stored
renders a product unsuitable for its intended products. This was because damage by insects
Effects of Fungi on Postharvest Crops 29
was usually conspicuous, easy to quantify in Alabama, while Macrophomina phaseoli

and these insects were visible to the naked causes ‘black mars’ in Gambian groundnuts.
eye. But, awareness of the losses caused by In fruits and seeds, the micropyle is the
fungi, also referred to as ‘moulds’, came with common place for infections to begin, but
the discovery of a toxic metabolite called fungi, bacteria and actinomycetes can develop
aflatoxin in 1968 caused by a fungus called in any other region of the seed or fruit, caus-
Aspergillus flavus, which killed over 100,000 ing abnormal colouring, either localized or
turkeys in Britain when fed with groundnut generalized (Clarke, 1968). However, it is
cakes that were infected by this organism. not all discoloration on produce that is
Studies in Nigeria have revealed the caused by fungi; sometimes, it may be due
presence of aflatoxin in Nigerian ground- to genetic mutations.
nuts and livestock feed maize; hence, there
is a need to take extremely good care of these
products during storage (Akano and Atanda, Flavour and odour
1989). Some crops in which fungal deterio-
ration can take place are as follows: maize,
The flavour and odour of produce caused by
sorghum, millet, cowpea, beans, groundnut,
moulds usually affect the taste of the end
cocoa beans, palm kernels and tubers.
products and are not acceptable to consum-
ers. The change in the flavour and odour is
usually as a result of the biochemical change
Deleterious Effects of Fungi on which takes place in the stored produce.
Postharvest Crops For example, mouldy groundnuts have
a very unpleasant and sour taste when con-
Fungi occur everywhere and have a profound sumed and these are usually spat out from
effect on their environment. Like other micro- the mouth as soon as they are chewed. Unde-
organisms, fungi may be good or harmful, sirable flavour is easily noticed in mouldy
depending on the species involved. The cocoa beans, as it can be detected by tasting
deterioration of postharvest crops by fungi a sample of chocolate which has passed
can be either by destruction of the produce through all the normal manufacturing pro-
itself or by presenting a potential hazard to cesses. Banana and plantain affected by
animals or humans. Some of the deleterious mould also have a detectable flavour and
effects of fungi on postharvest crops are as odour. Mouldy produce can also have an
follows: odour, ranging from the musty odour of
mouldy grains to the foul smell of rotten
grains (Ogundana et al., 1970).
Discoloration
Fungi come in various colours, i.e. green, Biochemical effects

brown, white, grey, black, etc. They impart
these colours on postharvest crops, thereby The development of moulds leads to a great
changing the original appearance. Dis- modification in the chemical composition
coloured produce is often disliked by con- of the infected produce. One such effect is
sumers and manufacturers in that the colours an increase in the free fatty acid (FAA) con-
affect the end products from such produce. tent of the produce. This acid is one of the
Cocoa beans, melon seeds, palm kernels, intermediate products of spoilage in materi-
groundnuts, maize, yam and cassava are als containing fats and oils and its forma-
examples of produce in which deterioration tion results in rancidity. Many of the mould
is accomplished by marked discoloration. species infecting our crops are known to
For instance, Lasiodiplodia theobromae is produce lipases, which can hydrolyse fats
responsible for the disease which discolours into fatty acids by a process called lipolysis,
cocoa, widely known as ‘concealed damage’ thereby increasing the free fatty acid content
30 A.O. Ogaraku
of the produce and resulting in a decrease in absolute weight loss. Scientists have reported
oil content and a low protein content. up to 10% weight loss in rotting yam tubers
Kuku (1972) isolated a number of moulds during storage (Ogundana et al., 1970).
from palm oil and showed that many of these Some of the fungi that can cause weight loss
increased the FAA of palm oil in pure cul- in maize are A. flavus, A. niger, A. candi-
ture studies. Coursey et al. (1963) isolated a dus, Mucor racemosus and P. pallitans.
number of lipolytic fungi from Nigerian
palm kernels. These included A. chevalieri,
A. fumigatus, Paecilomyces variotii and Destruction of viability
P. steckii.
The development of mould on produce
Fungi reduce the viability of seeds by infect-
causes other modifications; generally, an
ing and destroying their embryo. This in
increase in reducing sugars and a loss in
turn affects the germinability of the seeds
protein, which may lead to flours unsuit-
during planting. Broadbent (1967) found
able for bread making. Moreover, mouldy
samples of mouldy maize from government
rice grain breaks easily during polishing. If
farms in southern Nigeria had only 7–14%
we preserve damp grain in an anaerobic
germination, while the mould-free samples
environment, fermentation results in the
had 100% germination.
release of carbon dioxide, alcohol and other
volatile substances. The compounds formed
give a bad taste, which remains even after
drying in the open air. Heating
If crops with high water content are piled

Rotting and caking up together in one place, heat is generated
and decay sets in. Heating or production of
hot spots is one of the characteristics that
Extensive mould activities usually result in
results in rapid fungal development in moist
rotting and caking. Rotting and caking ren-
stored produce, mostly grains and tubers.
ders produce unsightly, decreases milling
Heating in bulk storage is evidence of spoilage
yield and quality. Studies carried out by
in progress or spoilage already completed.
several workers, including Adeniyi (1970)
and Ogundana et al. (1970), revealed that a
number of fungal species, for example A.
niger, Fusarium moniliforme, P. exalicum, Growth abnormalities
etc., caused rotting in Nigerian yams. Oye-
niran (1970) and other workers carried out Groundnut and maize contaminated with
studies that showed that over 30 mould spe- A. flavus produce deformed plants. The
cies could be associated with the deteriora- infected young groundnut or maize plant
tion of maize in Nigeria. will have a greatly decreased growth. The
follicles develop poorly and are elongated
in form. During growth, a large number of
sick plants die. Others are continually
Weight loss
abnormal in appearance, while some evolve
into normal plants.
Fungi growing on plant parts or produce
use them as food substrate. They produce a
variety of enzymes, i.e. amylases, cellulases,
pectinases and lipases, which hydrolyse the Preparation of the Material for
food substances into soluble forms. The Attack by Other Agents
food components easily absorbed and uti-
lized are carbohydrates, proteins, fats and It is sometimes difficult in many crops to
oil. This breakdown invariably leads to separate deterioration or spoilage due to
insects from that caused by fungi, but that since 1960, when it was reported to have
the two are interrelated is in no doubt. What caused the death of about 100,000 turkeys
is in doubt, however, is the exact sequence in Britain when they were fed with ground-
of events and the relative damage caused by nut cakes which was infected with A. flavus.
the two agents. The toxic substance was therefore called
Invasion of stored produce by fungi ‘aflatoxin’. Different mycotoxins affect dif-
prepare such commodities for attack by ferent sites of the body. Aflatoxins produced
other agents of deterioration, especially bac- by A. flavus are the commonest of all the
teria, insects and mites. In fact, some insects toxins and affect the liver, causing aflatoxi-
are known to feed on fungi and in this way cosis or liver poisoning. High levels of afla-
they help to spread the spores. These stor- toxin have also been reported to cause
age insects can live, develop and reproduce infertility (abnormality in the spermatozoa)
entirely on certain fungi and thus undoubt- in samples of semen from men fed on diets
edly play an important part as carriers in contaminated with A. flavus (Ibeh et al.,
the spread of the fungi. An example of such 1994). The production of aflatoxins on maize
an insect is Adhasverus advena. grains and other consumable foods in Nige-
ria has been reported by many researchers,
including Broadbent (1967), Oyeniran (1970),
Opadokun et al. (1979) and Akano and Atanda
Production of Toxic (1989). Other common mycotoxins are:
Metabolites (Mycotoxins)
● Fumonism – this causes oesophageal
Toxic metabolite production is the most cancer in horses and humans. It is pro-
serious effect of microbiological deteriora- duced by F. graminearum on maize.
tion of stored products because of its poi- ● Ochratoxin – produced by A. ochareus,
soning nature. There are two kinds of which causes serious nephropathy in
poisoning by fungi, mycetism and myco- pigs and humans. It is commonly found
toxicosis. In mycetism, the toxic substances in milk and cereals (processed or raw).
are constituents of the fungi, large enough Some examples of mould species and the
to be eaten alone. In mycotoxicosis, the fun- toxins they produce are shown in Table 3.1.
gus is a contaminant of and has produced
toxic product in some food. The effects of
mycetism include diarrhoea and jaundice,
Conditions that Favour Development
while mycotoxicoses were defined by Clarke
(1968) as diseases of animals and humans of Fungi on Harvested and
caused by ingesting poisonous metabolite Stored Crops
fungi that have grown in the food previ-
ously before ingestion. Some notable exam- Fungi, like other living organisms, require
ples of mycotoxicoses are: certain conditions for growth and develop-
ment. These conditions are as follows:
1. Ergotism – diseases of cattle in central
Europe caused by the fungi, Claviceps
purpurea. Moisture
2. Yellow rice disease of humans in Japan
caused by the fungi, P. citrinum.
It is not the moisture content as such that is
3. Alimentary toxic aleukia (ATA) of
the controlling factor in biological deterio-
humans and cattle caused by F. sporotri-
ration; it is the relative humidity of the air
chioides.
in and around the crop. Although relative
4. Importantly, aflatoxicosis of poultry and
humidity is the controlling factor, atten-
livestock caused by A. flavus.
tion is usually focused on the moisture
This last mentioned toxin disease, aflatoxico- content because relative humidity of produce
sis, has been receiving worldwide attention is difficult to measure, while moisture content
32 A.O. Ogaraku
Table 3.1. Examples of mould species and the Oxygen

toxins they produce.
Most fungi are aerobic; they require oxygen
Mould species Toxin produced
to survive, like other living organisms. Any
Aspergillus flavus Aflatoxin device which cuts off oxygen from the stor-
A. ochareus Ochratoxin age environment will reduce, if not totally
A. chevalieri Xanthocillin eliminate, fungi. This is why storage at an
A. nidulans Sterigmatocystin inert temperature has been effective.
A. ruber Rubratoxin
A. niger Oxalic acid
Penicillium islandicum Islanditoxin
P. notatum Xanthocillin
Nutrients
P. rubrum Rubratosin
P. citrinum Citrinin All biological systems, from microorgan-
P. patulinium Patulin isms to humans, share a set of nutritional
Fusarium graminearum Zearalenone requirements with regards to the chemicals
necessary for their growth and normal func-
tioning. The great diversity of nutritional
types required are energy, carbon, nitrogen,
is not. Moisture in stored produce is divisi- sulphur and phosphorus, metallic elements
ble into two main types: chemically bound and vitamins. All these nutritional require-
water, which is the part of the intrinsic com- ments are present in food substances, such
position, and physically bound water, some as carbohydrates, proteins, fats and oil,
of which is held loosely on the commodity. which fungi need in soluble forms for meta-
Moisture in terms of water is necessary bolic processes.
for mould spores to germinate, and it also Fungi produce a variety of enzymes
helps in the process of dissolution of food which break down complex food substances.
materials. The moisture level of a stored Some of these enzymes are as follows:
product therefore determines the develop-
ment rate of the storage fungi. ● Cellulases – break down cellulose in
Mould species vary in their water require- plant materials.
ment; for instance, there are those that thrive ● Amylases – hydrolyse carbohydrates.
at low moisture levels and are said to be xero- ● Lipases – hydrolyse fats to fatty acids
phytic. Examples are A. flavus, A. chevalieri and glycerol.
and A. repens. Others require high levels of ● Proteases – hydrolyse proteins.
moisture before they can survive and are said ● Pectinases – hydrolyse the pectic mate-
to be hydrophilic, e.g. Penicillium species. rials of plant tissues.
Temperature Heating
All living things have a minimum and max- If crops with a high water content are allowed
imum temperature for growth. Fungi are a to overlap or are piled together in one place,
co-exception. Most fungi will grow at tem- yam for example, heat is generated under
peratures between 5°C and 35°C. These are moist conditions and decay will set in.
the mesophilic species. There are those that
thrive at 35°C and above and are said to be
thermophilic. Some thrive at very cold tem- Insufficient drying
peratures and are said to be psychrophilic.
This means that fungi thrive well in a very Some crops grow mouldy if insufficiently
wide temperature range, which gives room dried. Fungi can creep in to destroy the
for existence in postharvest crops. crops.
Preharvest infection Economic loss
Produce destined for storage is sometimes 1. There is a monetary loss because of in-
infected by moulds before harvest. Most fungi accessibility to foreign trade due to the poor
species also invade, especially following nat- quality of the produce. There is also a mon-
ural or artificial wounds. Some examples are etary loss because of the poor health of ani-
attack of cocoa beans by Lasiodiplodia theo- mals fed with inferior feeds.
bromae and other moulds, attack of ground- 2. Some fungi, for example Fusarium spe-
nut by Macrophomina phaseoli and attack cies, can grow on stored animal feeds, gener-
of maize by F. moniliforme and P. citrinum. ating products that are highly toxic to swine
and other animals.
3. Infections leading to disease of crops
are extremely important because of the fam-
Attack during preparation ine, malnutrition and dietary deficiency
they may cause.
During the process of preparation, mould 4. Some plant pathogens cause food intox-
attacks some produce as a result of lapses in ication when eaten by humans or animals;
cultural practices; for example, during for example, the fungus, C. purpurea, which
cocoa fermentation mould could infect and grows on cereal grains and some grasses, re-
penetrate the beans if the fermenting mass places the feed kernels with compact masses
of beans is not stirred or mixed thoroughly of hardened fungus called sclerotia. These
at intervals. In palm produce, mould can contain alkaloids that act on the nervous
attack the fruits and sometimes the kernels system of humans and other animals, caus-
when they are heaped on the ground just ing gangrene, convulsions and death.
before de-husking. In groundnut, the crop
has to be lifted at certain times to avoid
mould contamination. Control of Fungal Deterioration in
Postharvest Crops
If left uncontrolled, these fungi will cause

Types of stores
deterioration of food products and many
other articles of commerce and industry. For
If, for instance, through economy a store is this purpose, a distinction can be made
poorly constructed and the roof is holding between postharvest produce that is stored
water, it is possible to cause leakage and dry, such as grains, cocoa, groundnuts, etc.,
water will drip on to the commodity and and those which are stored with a high water
thereby cause deterioration. content, such as yams and other tubers.
There are other factors which contribute However, some of the measures or sug-
to the development of fungi in crops apart gestions listed below will certainly apply to
from those mentioned above and they are: both types:
1. The degree to which the grain has al- 1. Proper drying of produce to a safe mois-
ready been invaded by storage fungi before ture level, either by retaining maize, millet
it arrives at a given site. or guinea corn on the cob and storing in a
2. The amount of foreign material present condition where gradual drying by heat or
in the grain. aeration takes place, or otherwise by pro-
3. The activity of mites and insects. Bored viding artificial drying.
holes serve as an entry for mould spores. 2. Prevention of damage or wounds on
Some insects, such as A. advena, and mites produce so as to forestall a source of entry
feed on mould spores and therefore help to for moulds.
spread the fungi, as well as increase their 3. Any produce to be stored must be whole-
activities in storage. some and healthy. Bruised yam tubers,
34 A.O. Ogaraku
cassava, oranges and fruits should never be Examples of such chemical preservatives
stored. are propionic acid, ascorbic acid, glycerol,
4. Avoid drying the produce on a bare sulphur dioxide and benzoic acid. Their use
floor because of infestation by soil fungi. in many instances has been limited to live-
5. Hot produce should not be stored. After stock feeds.
drying, allow produce to cool before storage. 16. Other technical methods of control –
6. New produce should not be mixed other methods by which fungal develop-
with an old consignment, to avoid cross- ment in stored products can be controlled
infestation. are refrigeration, irradiation (for yam) and
7. Bagged produce should not be placed storage in airtight containers and inert at-
on the ground but on raised plank platforms. mosphere for grains.
8. Overfermentation should be avoided in
produce like cocoa, cassava, etc. Ogundana et al. (1970) found benomyl and
9. The store or warehouse should be leak- thiabendozole effective in reducing the
proof to prevent moisture reabsorption by activities of fungi in causing yam rot during
the already dried produce. storage, but these chemicals are rather toxic.
10. Prevent pockets of heavy insect activity Research is currently in progress at the
by proper application of insect control Nigerian Stored Products Research Institute
measure to avoid localized moisture in- on the use of safer fungistatic chemicals to
creases and mould growth in the bulk of the preserve yams against microbiological rot
grain. during storage. Adesuyi (1973) stored yams
11. In the case of fruits, harvesting should successfully for up to 6 months by using a
be done promptly as very old fruits are curing method, cutting off sprouts from
highly susceptible to fungi infection. healthy undamaged tubers and using low
12. If possible, dried produce should be temperature and irradiation techniques.
stored in airtight conditions to keep away 17. Precautions in mycotoxicoses – it is
from fluctuating atmospheric relative hu- very important to have a control measure in
midity, which could lead to an increase in harvesting produce in order to eliminate the
moisture content; for example, store in fungi causing mycotoxicoses diseases be-
polythene bags or polythene-lined sacks. cause of their devastating effect on humans
Other methods of controlling deterioration and animals that consume such an infected
of dry produce are: crop. Standard safe limits should be deter-
mined and enforced levels of aflatoxin and
13. Use of fungicides – in the case of other toxins in food and feed. Different
grains not desired for immediate consump- countries have a wide variety of tolerance
tion or use, some fungicides such as cap- level of mycotoxin between 5 and 50 µg/kg
tan, benomyl, thiobendazole, borax, etc., (Hansen, 1993). In the USA, the Food and
have been used to control fungal attack, Drug Association has established an afla-
but their use has been limited because of toxin limit of 20 µg/kg for food and feed
their toxicity. ingredients.
14. Use of plant materials – parts or roots
with medicinal properties can also be used A regular monitoring programme should be
to suppress mould growth in stored crops. arranged for commodities that are suscepti-
Williams and Akano (1985) reported on the ble to aflaxtoxin contamination. Processing,
efficacy of dogonyaro (neem) as a filtrate in packaging, transportation and storage prac-
suppressing rotting fungi growth in stored tices should be well managed to eliminate or
yam tubers. reduce infestation by moulds, especially the
15. Addition of chemical preservative toxigenic strains. Decontamination proce-
agents – the addition of antiseptics to food- dures are to be designed to remove or inacti-
stuffs allows for better preservation under vate the toxins in feed and food. Mycotoxins
certain conditions. The use of these products can be removed from food by detoxification
is subject to regulations in most countries. using chemical agents.
Conclusions is the most important aspect, the precautions

that need to be taken to control or eliminate
The role of fungi in the deterioration of post- the fungi causing mycotoxicoses in humans
harvest crops is enumerated. The contribu- and animals.
tion of some workers in providing an insight It is pertinent to say that knowledge is
into the deleterious effects of fungi on har- far from complete and experts should still
vested and stored crops, economic loss, endeavour to find total solutions to the var-
control of fungal deterioration in posthar- ious aspects of these problems as the strug-
vest crops and precautions in mycotoxico- gle of humans against the menace of fungi
ses diseases is also highlighted. Not forgotten continues.
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Opadokun, J.S., Ikeorah, J.N. and Afolabi, E. (1979) The aflatoxin contents of locally consumed food stuffs.
Nigerian Stored Product Research Institute Technical Report No. 12, 105–108.
Oyeniran, J.O. (1970) Microbiological studies on maize used as poultry and livestock feeds at the research
Farms in Kandan, Western State. Nigerian Stored Product Research Institute Technical Report No. 6,
47–49.
Williams, J.O. and Akano, D.A. (1985) An assessment of wood ash for yam tuber (Dioscorea rotundata) in
storage. Nigerian Stored Product Research Institute Report No. 2, 31–34.
4Exploitation of Botanicals in the
Management of Phytopathogenic and
Storage Fungi
Pramila Tripathi1 and A.K. Shukla2

1Department of Botany, D.A.V.-P.G. College, Kanpur, India; 2Department of Botany,
Rajiv Gandhi University, Rono Hills, Itanagar, India
Abstract
Plants are known to contain a number of secondary substances like phenols, flavonoids, quinines,
essential oils, alkaloids, saponins, steroids, etc. Some of these plant-based metabolites have antimicro-
bial properties and are toxic to phytopathogens. They are also repellant to insects and have fumigant
toxicity against pests. Currently, synthetic pesticides are the primary means of controlling pathogens.
The adverse effects of synthetic pesticides on human health and from the food safety point of view has
enunciated interest in finding an alternative means of controlling phytopathogens and pests. To reduce
dependency on synthetic pesticides, the use of plant-based antimicrobial substances (essential oils,
volatile aromatic compounds, glucosinolates, jasmonates and acetaldehydes) may help in the manage-
ment of phytopathogens and pests as an alternative method for sustainable agriculture. Use of botani-
cals is still on a small scale compared to synthetic chemicals; therefore, it is timely to exploit and
formulate low-cost, effective, free of human hazard and eco-friendly plant-based products for the man-
agement of pests and pathogens.
Introduction 1986). According to WHO estimates, approx-

imately 0.75 million people are becoming
To control fungal diseases, synthetic fungi- ill every year with pesticide poisoning. Fur-
cides are usually applied as effective, depen- ther, the resistance of pathogens to fungicides
dable and economical control measures. has rendered certain fungicides ineffective,
However, the indiscriminate use of chemi- giving rise to a new physiological race of
cal fungicides has resulted in several prob- pathogens. Basic research for over more
lems, such as toxic residues in food, water than 40 years in biology and biochemistry
and soil and disruption of the ecosystem, has made it possible to envisage not only
leading to the fear that their regular use may how new pesticides may be synthesized but
harm the environment further. Hardly also has generated a completely new approach
0.1% of the agrochemicals used in crop pro- to the protection of plants using secondary
tection reach the target pest, leaving the plant products which may be toxic to a spe-
remaining 99.9% to enter the environment cific pest yet harmless to humans. Pesticidal
to cause a hazard to non-target organisms, plants have been in nature and its com-
including humans (Pimentel and Levitan, pounds for millions of years without having

Exploitation of Botanicals 37
any ill or adverse effects on the ecosystem Essential Oils

and, because of their renewability, they have
a distinct advantage in the management of Essential oils from different plant species
disease-causing pests. Plants have a natural are known to exhibit various kinds of bio-
potential to withstand the aggressiveness of logical activities. The volatility, ephemeral
pathogenic species. nature and biodegradability of such volatile
Plants synthesize a dazzling array of components of angiosperms will be espe-
structural variety, which inhibits an almost cially advantageous if they are developed as
equally dazzling array of biological activi- pesticides (French, 1985). Essential oils are
ties. A wide spectrum of secondary sub- a complex natural mixture of volatile sec-
stances is contained in higher plants, ondary metabolites isolated from plants by
namely phenols, flavonoids, quinines, tan- hydro or steam distillation and by expres-
nins, essential oils, alkaloids, saponins and sion. The main constituents of essential oils
steroids. The total number of plant chemi- are mono- and sesquiterpenes, along with
cals may exceed 4000 and of these, 1000 are carbohydrates, alcohols, ethers, aldehydes
secondary metabolites. These secondary and ketones, polyphenolic compounds,
metabolites have a major defensive role for oxides, nitrogen and sulphur compounds
plants (Swain, 1977). The search for botan- and organic acids, etc. The chemical com-
icals from plant species is one of the impor- position of essential oils is extremely com-
tant areas where Indian scientists can take plex and varies with the geographical as
a lead and capture the global market. India well as the environmental conditions where
enjoys the benefits of a varied climate, from the plants are grown (Bhaskara et al., 1998;
an alpine climate in Himalaya to a tropical Vanneste et al., 2002). The essential oils are
one in the south and an arid one in Rajas- extracted from various parts of plants such
than to a highly humid climate in Assam as flowers, fruits, leaves and wood. They are
and Bengal. This is consequently reflected normally formed in special cells or groups
in the rich and diversified flora, which is of cells or as glandular hairs. Oils occur as a
often quite distinct, thanks to the natural globule or globules in the cell and may also
barriers that India has all along its fron- be excreted from cells lining the schizoge-
tiers. It is estimated that India has about nous ducts or canals. They may be present
17,000 species of angiosperms. There is a in glandular regions such as leaves, bark or
need for extensive screening programmes fruit and, when occurring in various organs
at different regional centres of the country in one plant, may possess different individ-
so that knowledge on the various types of ual chemical compounds (Bonner, 1991;
biological properties of angiospermic flora Hili et al., 1997). The general antifungal activ-
may be gathered. This type of scientific ity of essential oils is well documented (Tri-
testing would definitely be helpful in the pathi et al., 2007, 2008). These essential oils
conservation of plant resources and in are thought to play a role in plant defence
proving our sovereign right over our plant mechanisms against phytopathogenic micro-
biodiversity. Under these conditions, in any organisms (Mihaliak et al., 1991). The emerg-
meaningful search for better and cheaper ing picture is that certain specific oils and
substitutes, plant resources for India are a their chemical constituents have tradition-
natural choice. Hopefully, this will lead ally been used to protect stored grains and
to new information on plant application to repel flying insects in the home and have
and a new perspective on the potential use demonstrable contact and fumigant toxicity
of these natural products. This chapter to a number of economically important insects
explores the potential to use a variety of and mite pests, as well as to pathogenic fungi.
botanicals in the form of plant extracts and The essential oils or their major constitu-
essential oils to control various fungal phy- ents could be effective fumigants and also
topathogens and fungi related to the storage could be integrated with other pest manage-
of grains and the postharvest pathogens of ment programmes. Natural pesticides based
perishables.
38 P. Tripathi and A.K. Shukla
on plant essential oils could represent alter- Powdery mildew of Cucurbita maxima is
native crop protectants. The essential oils caused by Sphaerotheca fuliginea. Reynou-
produced by different plant species are, in tria extracts and olive oil were found to be
many cases, biologically active and have effective in controlling the disease (Cheah
antimicrobial, allelopathic, antioxidant and and Cox, 1995). Since olive oil is used in
bioregulatory properties (Caccioni and Guiz- cooking, food additives and medicines, it
zardi, 1994; Vaughan and Spencer, 1994). does not cause any human health or envi-
Sometimes, the chemicals in the oil, as well ronmental problems. Recent studies in
as the oil itself, are registered as pesticide Ghana confirm that Ocimum gratissimum
active ingredients. It is also fairly common and Syzigium aromaticum are very effective
for two or more oils to be used in the same in preventing fungal growth (FAO, 1999).
commercial product. Since the essential oils
as such are a mixture of different major and
minor components which act synergistically
in the biological efficacy of the oil, there Essential Oils Against Fungal
would be less chance of the development of Pathogens of Seeds
physiological races of the target pathogens
if the oils as such were formulated as botan- The fungicidal effect of essential oils against
ical pesticides and fumigants. Essential oils pathogens of cereal grains has been tested
as botanical pesticides may be produced successfully. It is especially significant in
easily, even by small-scale industries, as the case of stored rice, where currently fun-
there is no sophisticated procedure for their gicides are not used to control fungal pests.
distillation and most aromatic plants are Peppermint (Mentha piperata), thyme (Thy-
available locally. They thus constitute a mus copitatus) and caraway (C. carvi) oils
friendly, natural alternative in pest control. have demonstrated effective control against
fungal pathogens like Fusarium sp., Macro-
phomina phaseolina and Colletotrichum
dematium (Abdelmonem et al., 2001). Essen-
Essential Oils Against tial oils from oregano (Origanum vulgare)
Phytopathogenic Fungi and thyme were applied as fumigants against
the mycelia and spores of Aspergillus flavus,
The antifungal activity of essential oils has A. niger and A. ochraceus infesting wheat
been studied by a number of workers grains. Only oregano essential oil exhibited
(Apablaza et al., 2004; Harish et al., 2004; fungicidal activity (Paster et al., 1995). The
Muller-Ribeau et al., 1995). Singh et al. antifungal activity of the essential and fixed
(1980) found that essential oils from Cym- oils of thyme, clove, peppermint, soybean
bopogon spp. and Trachyspermum ammi L. and groundnut were tested against A. fla-
exhibited strong antifungal activity against vus, A. niger, F. oxysporum, F. equiseti and
Bipolaris oryzae. Carvone, a monoterpene Penicillium chrysogenum in vitro on the
isolated from the essential oil of Carum cowpea (Vigna unguiculata) (Kritzinger et al.,
carvi, was found to inhibit the sprouting of 2002). Thyme and clove oils inhibited growth
potatoes during storage. of all the fungi significantly at concentra-
Carvone was also found to have fungi- tions of 500 and 1000 ppm. Peppermint oil
cidal activity that helped to protect potato inhibited growth of the above-mentioned
tubers from fungal rotting without exhibiting fungi successfully at 2000 ppm (Kritzinger
mammalian toxicity (Hartmans et al., 1995). et al., 2002). In blackgram (V. mungo), essen-
It has been introduced in the Netherlands tial oil extracted from wood chips of cedar
under the trade name TALENT. Besides, the (Cedrus deodara) and that from seeds of T.
essential oils of Salvia officinalis have also ammi exhibited antifungal activity, inhibit-
shown practical potency in enhancing the ing the mycelial growth of A. niger and Cur-
storage life of some vegetables by protect- vularia ovoidea, two storage fungi found on
ing them from fungal rotting (Bang, 1997). seeds (Singh and Tripathi, 1999). A. flavus
was also found infesting seeds of guar and F. proliferatum (Marin et al., 2003).
(Cyamopsis tetragonoloba), a native plant of Velutti et al. (2004) reported antimycotoxi-
India which has main commercial value cogenic activity of the essential oils against
due to its seed gum (galactomannan gum). F. graminearum infested seeds. The essen-
In this case, A. flavus was controlled by tial oils of oregano, cinnamon, lemongrass,
cumin (Cuminum cyminum L.) oil extracted clove and palmarosa effect the growth rate
from its seeds (Dwivedi et al., 1991). Chem- of F. graminearum and mycotoxin Zearale-
ical studies indicated that the greater part none (ZEA) and Deoxynivalenol (DON) pro-
of this antimicrobial activity might be duction at two concentrations (500 and
attributed to the cuminaldehyde that is 1000 mg/kg).
present in the dried fruit of this plant (De
et al., 2003). The essential oils of Cassulia
allaris and M. arvens have been reported as
botanical fumigants for management of the Plant Extracts Against
biodeterioration of wheat from A. flavus Phytopathogenic Fungi
(Varma and Dubey, 2001).
The preservative nature of some plant
extracts has been known for centuries and
there has been renewed interest in the anti-
Essential Oils Against microbial properties of extracts from aro-
Aflatoxicogenic and matic plants. The application of the extracts
Mycotoxicogenic Fungi of higher plants to control plant diseases was
first attempted by Democritus as early as
The aflatoxins are well known for their car- 470 BC. Plant extracts have assumed spe-
cinogenic, mutagenic and teratogenic effects cial significance nowadays as an eco-friendly
on humans and domestic animals (Wyllie method for plant disease management. Plants
and Morehouse, 1978). A natural fungicide contain alkaloids, tannins, quinines, cou-
against aflatoxigenic fungi to protect stored marins, phenolic compounds, phytoalex-
rice using the essential oil of lemongrass (C. ins and ipomeamarone in the extract,
citrates) was developed by Paranagama et al. which are known for their antifungal prop-
(2003). Lemongrass oil was tested against erty (Datar, 1999). Use of plant extracts for
A. flavus and the test oil was fungistatic seed treatment is one of the alternative
and fungicidal against the test pathogen at methods of preventing pathogen problems
0.6 and 1.0 mg/ml, respectively. Aflatoxin of agricultural crops. Plant materials as
production was inhibited completely at such can be used as soil amendments that
0.1 mg/ml. Citral has been found as a fungi- can serve as both a nutrient as well as an
cidal compound in lemongrass oil. During antifungal agent. Plant extracts have also
the fumigant toxicity assay of lemongrass been reported to stimulate the growth of
oil, the sporulation and mycelial growth of targeted plant species. This is probably due
the test pathogen were inhibited at a concen- to some hormones and allied substances
tration of 2.80 and 3.46 mg/ml, respectively. like IAA, IBA, etc.
Lemongrass oil could be used to manage afla- However, the active principles of some
toxin production and to inhibit the fungal plants have been isolated phytochemically
growth of A. flavus in stored rice. and have shown a strong inhibitory action
Putative mycotoxicogenic fungi were against a number of fungi. Antifungal activ-
partially or completely sensitive to different ity of plant extracts against a wide range of
essential oils extracted from different medic- fungi has been reported by a number of
inal plants (Soliman and Badeaa, 2002). Seed workers (Grange and Ahmed, 1988; David-
treated with cinnamon, palmarosa and lem- son and Parish, 1989). Bhargava et al. (1981)
ongrass oils at 500 mg/kg showed antimyc- screened extracts of some plant species and
otoxigenic ability against fumonisin B1 found O. canum to be most effective against
accumulation produced by F. vesticillioides A. flavus and A. versiolor. Pandey et al.
(1982) evaluated the seed extract of 30 plants Plant Extracts in the Management
and found soybean, Leonotis nepetaefolis, of Fungal Seed Diseases
Parpalum and Peltophorum to exhibit an
inhibitory effect against the fungi, Alternaria Cereal seeds carry a wide range of fungi that
alternata and A. niger. Ark and Thompson are known to play a significant role in spoil-
(1959) found the leaf extract of Allium sati- age and probably rank second only to insects
vum to be effective against various plant as a cause of deterioration and loss in all
pathogens. Acacia nilotica (leaf and bark) kinds of field and storage crops throughout
and A. farnasiana (bark) of Mimosaceae the world (Christensen and Kaufman, 1974).
showed high activity, while A. catechu of The information on fungal association with
the same family did not show activity either important cereal grains is relevant in assess-
from leaf or from bark (Tripathi, 2005). Four ing the potential risk of mycotoxin contami-
compounds, i.e. iritin A, iritin B, flavonone- nation. In recent years, the use of plant
dehydroulogonin and sesquiterpene pyg- extracts for controlling fungal seed disease
mol, were isolated with dichloro-methane has also been of renewed interest. Carvone
extract of the aerial parts of Chenopodium (monoterpene compound) completely inhib-
procerum. These compounds have been ited F. oxysporum and A. pisi. African yam
found to inhibit the growth of the plant bean, Sphenostylis stenocarpa, is an impor-
pathogenic fungi, Cladosporium cacumeri- tant grain legume in most tropical African
num (Bergeron et al., 1995). Kim et al. countries (Nwachukwu and Umechuruba,
(2004) evaluated Achyranthus japonica and 2001). Major pathogenic fungi associated
Rumex crispus for activity against various with this crop are A. niger, A. flavus, Lasio-
plant pathogenic fungi and control of pow- diplodia theobromae and F. moniliforme.
dery mildew. Methanol extract of the fresh Associated fungi could be controlled by
material of 183 plants was screened in vivo using crude and aqueous extract of basil (O.
for antifungal activity against Magnaporthe basilicum), bitter leaf (Vernonia amyd-
grisea, Corticium sasaki, Botrytis cinerea, alina), neem and pawpaw (Carica papaya).
Phytophthora infestans, Puccinia recondita Parimelazhagan and Francis (1999) reported
and Erisiphe graminis. Among them, 33 plant reduction in the radial growth of Curvularia
extracts showed disease control efficacy. The lunata associated with rice seeds when
methanol extract of Achranthes japonica treated with leaf extract of Clerodendrum
(whole plant) and R. crispus (roots) at a con- viscosum, which also increased seed germi-
centration greater than 11 g fresh weight of nation, root and shoot length of the rice.
plant tissue per litre aqueous Tween 20 The same results were observed by using
solution controlled the development of bar- plant extracts to control B. oryzae on rice
ley powder mildew caused by E. graminis seeds, which have a high natural infection
effectively in an in vivo assay using plant of the fungus (Alice and Rao, 1986). In Ban-
seedlings. Some fungi like F. solani and gladesh, use of the extract of Polygonum
Verticillium alboatrum have been shown to hydropiper, A. cepa, A. sativum and A. jidia
be susceptible to tannins extracted from the demonstrated to be effective against B.
bark of various trees, including chestnut oryzae at higher concentrations. Among
and wattle (Lewis and Papavizas, 1967). them, neem and garlic were the most effec-
The effects of aqueous and methanol, petro- tive at 1:1 dilution and inhibited the occur-
leum ether, chloroform and ethyl acetate rence of the pathogen by 91 and 83%,
extracts of Cyprus rotundus were tested on respectively (Ahmed et al., 2002).
spore germination of F. solani. Ethyl acetate Alternaria padwickii, another impor-
extract exhibited an inhibitory effect on tant seedborne pathogen of rice, was also
spore germination at 1000 µg/ml (Singh and inhibited by aqueous extract of Strychnos
Tripathi, 1999). In the field, reduction of nux-vomica, garlic bulbs, ginger rhizome,
disease incidence has been recorded as a basil leaves and fruits of A. indica (Shetty
result of plant seed treatment with extract, et al., 1989). The ability of natural plant
and an increase in yield was also noted.
extracts to prevent the growth of fungi natu- and the antioxidant (ascorbic acid) contents
rally infesting grains was also studied. Before are maintained at optimum level in botani-
sowing, wheat seeds were soaked in an cally treated seeds (Umarani, 1999). Com-
aqueous plant extract of O. gratissimum and mon botanicals, arrapu (Abizia amaru), neem
disease transmission was evaluated. The (A. indica), notchi (Vitex negundo), Prosopis
rate of infection decreased with the extract sp., pungam (Pongamia glabra), moringa
at concentrations higher than 10% (Rodri- and tamarind, contain an auxin-like sub-
gues et al., 2001). Leaf extracts of Delonix stance which regulates seedling growth
regia, Pongamia glabra and A. nilotica sig- and initial establishment. In botanicals, a
nificantly inhibit spore germination, myce- gibberellin-like substance is also present in
lial growth and spore production of A. addition to saponin and other nutrients,
helianthi, M. phaseolina and F. solani from which interact with amino acids, trypto-
sunflower seeds (Tribuhavanaamala and phane to form the indole acetic acid (IAA),
Narsimhan, 1998). Melon seeds are very which leads to release of plant hormones
important as as condiment and constitute a that are responsible in cell elongation and
very valuable source of oil and protein for vegetative growth. In botanical seed pellet-
many people of West Africa (Oyolu, 1977). ing, the leaf powder acts as a water pad by
After 6 months of incubation, all the melon absorbing/regulating soil moisture availabil-
seeds treated with leaf extract showed no ity, which enhances a better seed–soil rela-
infection except M. phaseolina. Ahmad and tionship (Narasimha, 1994). Seeds are stored
Prasad (1995) evaluated that post-infection by pelleting them with botanical products.
treatment of sponge-gourd fruits with the The aim of botanical pelleting in seed stor-
extracts of Azadirachta indica, Lantana age is to extend storage potential, besides
camara, Murraya exotica, O. sanctum, maintaining its ability to produce normal
Datura fistulosa and Catharanthus roseus seedlings. Jegathambal (1996) found that
almost fully inhibited the spread of disease sorghum seeds hardened and pelleted with
caused by Helminthosporium spiciferum arappu leaf powder could be stored for
and F. scirpi. up to 2 weeks with higher germinability.
Papaya seeds pelleted with botanicals or
presoaked with botanicals gave improved
germination, vigour index and field emer-
Application of Botanicals gence when compared to the control or
in Seed Storage water socking (Ananthakalaiselvi, 1995).
Dry dressing of seeds with botanicals pro-
Quality seed should have higher vigour and longs the storability of the seeds in many
viability and these two characteristics can- crops, especially in pulses, and acts as a
not be maintained in storage because they dual-purpose technologically for seed stor-
deteriorate rapidly under storage conditions age by preventing biotic organisms attack-
and suffer quantitative and qualitative losses ing the seeds during storage. Sabir (1989)
due to pests and diseases. Therefore, treat- reported that soybean seeds treated with
ing seeds with synthetic chemicals is vital sambangi (Polianthes tuberose) seed pow-
for successful storage. However, these chem- der at a ratio of 1:100 maintained a higher
icals are hazardous to humans. Therefore, germination rate (70%), even up to 8 months
use of natural plant products for long-term after storage. Pea seeds dried and mixed
seed storage has multi-purpose benefits as with notchi (V. negundo) powder or sam-
eco-friendly protection against the ageing bangi seed powder at a ratio of 1:100 main-
process, prevention of insects and fungi and tained a higher germination rate after up to
for their cost effectiveness (Vanangamudi 8 months in storage (Paramasivam, 1990).
et al., 2007). During storage, the enzymatic Umarani (1999) reported that dressing
activity (amylase, catalase, peroxidase, dried Casuarina seeds with neem leaf pow-
superoxide dismutase and dehydrogenase) der extended the storability of the seeds for
responsible for maintenance of seed quality up to 9 months.
Biocide Formulation of Essential Oils occurrence as part of the diet, their ephemeral
nature and their biodegradability suggest
The formulation of plant metabolites must low toxic residue problems. Such compounds
be introduced to overcome their degrada- could be extracted and applied to other
tion and to be used practically during han- harvested perishables. Some of the volatile
dling and application as biocides. Such aromatic components, namely acetalde-
formulation could be used easily and hyde, 6-carbon (C6) aldehydes, benzalde-
diluted with water to form the appropriate hyde, hexenel and hexanal, are of significant
concentrations in different applications. importance.
Study should be continued to evaluate the
pesticidal activity of the produced formu-
lated biocides against some plant patho- Aldehydes
genic microorganisms. Narsimhan et al.
(1988) demonstrated that neem oil (A. indica) Vapours of acetaldehyde have been used to
and pungam oil (P. pinnata) emulsifiable control B. cinerea (Prasad and Stadelbacher,
concentrate formulation prevented sheath 1973). Avissar and Pesis (1991) reported
rot (Sarocladium oryzae) of rice. Gascon acetaldehyde to be active against B. cinerea
et al. (1999) showed that the essential oils and Rhizopus stolonifer causing rot to
of rosemary, jarilla, mendocina, tomillo strawberry fruits. Benzaldehyde has been
mendocina, origanum, tarragon, lavandins used in the laboratory to fumigate peaches
and eucalyptus were emulsified with differ- and to protect them against Rhizopus rot. It
ent formulations of water suspensions of inhibits spore germination of B. cinerea
wall support systems using both a hand- totally at 25 µl/l and germination of Monilinia
held propeller blender and a high pressure, fructicola at 125 µl/l (Wilson et al., 1987).
double effect homogenizer. Also, Bowers The aldehydes, benzaldehyde, acetaldehyde
and Locke (2000) report that several com- and cinnamaldehyde, ethanol and benzyl
mercial formulations of botanical extracts alcohol were found to be the strongest
and essential oils have been investigated as growth inhibitors and the most lethal to fun-
possible alternatives for soil fumigation to gal spores and mycelia of fruit and vegetable
control Fusarium wilt disease. Essential oils pathogens like P. digitatum, R. stolonifer and
of fennel, peppermint and caraway have Colletotrichum during in vitro trials.
been formulated in the form of stable emul-
sifiable concentrates.
Hexenal and hexanal
Botanicals in the Management of (E)-2-Hexenal and hexanal are two different

Postharvest Diseases of Perishables volatile flavour compounds. Hexenal vapours
have a number of attributes that may be
Botanicals as antifungal agents important in consumer demand for more
in postharvest disease control of fruits natural measures to combat fruit diseases
with fewer toxic residues. Hexenal vapour
Fruits and vegetables have a number of con- inhibited hyphal growth of apple slices
stituents and inducible volatile aromatic and (Song et al., 1996). Archbold et al. (1999)
flavour compounds (Tripathi, 2007). These showed (E)-2-hexenal to be an efficient fumi-
aromatic and flavour components are gener- gant in controlling mould on ‘Crimson Seed-
ally produced by fruits during ripening less’ table grapes. (E)-2-Hexenal has been
and provide resistance to the fruits at the found to be strongly antifungal in nature
postharvest stage. The flavour compounds and its in vitro and in vivo activity against
are secondary metabolites having unique B. cinerea has been reported by a number of
properties of volatility and low water solu- workers (Hamilton-Kemp et al., 1992; Fallik
bility. As potential fungicides, their natural et al., 1998). The effect of trans-2-hexenal
on the control of blue mould disease (P. given in low doses, jasmonates may provide
expansum) in reducing patulin content and a more environmentally friendly means of
on improving the fruit quality of ‘Confer- reducing the current chemical usage.
ence’ pears has been evaluated and greater
reduction of decay was obtained by treat-
ment at 12.5 µl/l at 20°C for 24 or 48 h after Glucosinolates
inoculation (Neri et al., 2006).
Among natural substances with potential
antimicrobial activity are the glucosinolates,
Acetic acid a large class of approximately 100 com-
pounds produced by members of the family
Acetic acid is a metabolic intermediate that Crucifereae, with well-documented activity
occurs naturally in many fruits (Nursten, (Fenwick et al., 1983). Hydrolysis of glu-
1970). There are several advantages in using cosinolates produces D-glucose, sulphate ion
acetic acid fumigation. It is a natural com- and a series of compounds such as sothio-
pound found throughout the biosphere, cyanate (ITC), thiocyanate and nitril. The
posing little or no residual hazard. Low con- antifungal activity of six glucosinolates has
centrations, i.e. 2.0 or 4.0 mg/l, of acetic acid been tested on several postharvest patho-
in air have been found to be extremely effec- gens, namely B. cinerea, R. stolonifer, M.
tive for controlling B. cinerea conidia on laxa, Mucor piriformis and P. expansum,
apple (‘Red Delicious’) fruit (Sholberg and both in vitro (Mari et al., 1993) and in vivo
Gaunce, 1995). Acetic acid has been shown (Mari et al., 1996). Allyl-isothiocyanate
to be an effective fumigant for commercial (AITC), a naturally occurring flavour com-
use on apricot and plums (Liu et al., 2002), pound in mustard and horseradish, has a
grapes (Sholberg et al., 1996) and sweet cher- well-documented antimicrobial activity.
ries (Sholberg, 1998; Chu et al., 1999, 2001). Exposure of pear fruit to an AITC-enriched
The use of acetic aid and vinegar is the better atmosphere resulted in good control of blue
choice in most cases because it does not have mould, including a TBZ resistant strain on
an objectionable odour and has a long his- pears (Mari et al., 2002). The use of AITC,
tory of use on food (Sholberg et al., 2000). produced from purified sinigrin or from
Brassica juncea, against P. expansum appears
very promising as an economically viable
Jasmonates alternative with moderately low impact on
the environment.
The term ‘jasmonates’ includes jasmonic
acid (JA) and methyl jasmonate (MJ). These
are naturally occurring plant growth regula- Essential oils
tors that are widely distributed in the plant
kingdom and are known to regulate various The antimicrobial effects of essential oils
aspects of plant development and responses (EOs) or their constituents on postharvest
to environmental stresses (Sembdner and pathogens have been studied quite exten-
Parthier, 1993; Creelman and Mullet, 1995, sively (Bishop and Thornton, 1997; Tripathi
1997). Droby et al. (1999) found that posthar- et al., 2007). The advantage of EOs is their
vest application of jasmonates reduced decay bioactivity in the vapour phase, a character-
caused by grey mould, P. digitatum, either istic that makes them attractive as possible
after natural or artificial inoculation of ‘Marsh fumigants for stored product protection.
Seedless’ grapefruit. When applied at low Control of the storage pathogen, B. cinerea,
concentrations, jasmonates are potential post- on Dutch white cabbage (B. oleracea var.
harvest treatments to enhance natural resis- capitata) by the EOs of Melaleuca alternifo-
tance and to reduce decay in fruit. Since they lia in in vitro conditions has been investi-
are naturally occurring compounds and are gated (Bishop and Reagon, 1998). Tripathi
et al. (2008) evaluated some EOs against Treatment of pineapple fruits infested with
moulds of grapes caused by B. cinerea. The C. paradoxa by X. strumarium extract reduced
effect of C. nardus EO on the growth and the severity of the disease (Damayanti et al.,
morphogenesis of A. niger has been tested 1996). The phytochemical investigation of a
(Bellerbeck et al., 2001). The potential of methanolic extract of A. nilotica resulted in
using EOs by spraying or dipping to control isolation of kaempferol. It has shown anti-
postharvest decay has been examined in fungal activity against P. italicum at 500 µg/l
fruits, namely cherries, citrus fruits, apple, (Tripathi et al., 2002). In vitro inhibition of
peaches and cabbage (Tiwari et al., 1988; B. theobromae causing Java black rot in
Smid et al., 1994; Dixit et al., 1995). Thymol sweet potato was induced by phenolic com-
is an EO component from thyme (T. capita- pounds, chlorogenic acid giving the highest
tus). Fumigation of sweet cherries with thy- in vitro inhibition, followed by pyrogallol,
mol was effective in controlling postharvest pyrocatechol, phenol and resorcinol. Low
grey mould rot caused by B. cinerea (Chu concentrations of phenols are required by
et al., 1999) and brown rot caused by M. the fungus during normal metabolism, but
fructicola (Chu et al., 2001). The shelf life higher concentrations are inhibitory to growth
and safety of some perishable foods treated (Mohapotra et al., 2000). The phytochemical
with EOs have been improved remarkably investigations of most plants have resulted
(Ponce et al., 2004; Holley and Patel, 2005). in the isolation of active principles. These
The EO of S. officinalis has also shown compounds when tested against postharvest
practical potency in enhancing the storage fungi have shown pronounced antifungal
life of some vegetables by protecting them activity. A naturally occurring compound
from fungal rot (Bang, 1995). Treatment of isolated from the flavedo tissue of ‘Star Ruby’
oranges by fumigation with the EOs of M. grapefruit (Citrus paradise) identified as
arvensis (100 µl/l), O. canum (200 µl/l) and 7-geranoxy coumarins exhibited antifungal
Zingiber officinale (200 µl/l) has been found activity against P. italicum and P. digitatum
to control blue mould, thereby enhancing during in vitro and in vivo tests (Agnioni et al.,
shelf life (Tripathi et al., 2004). Plaza et al. 1998). Arya (1988) controlled fruit rots by leaf
(2004) evaluated the potential of thyme, extracts of medicinal plants.
oregano, clove and cinnamon EOs against P.
digitatum and P. italicum on citrus fruits.
The postharvest quality of strawberry and
tomato fruit was evaluated after treatment Mode of Action of Essential Oils
with Eucalyptus and cinnamon volatile EO
vapours (Tzortzakis, 2007). The mechanism of action of EOs and other
bioactive phytocompounds against micro-
organisms is a complex process and has
not yet been fully explained. It is generally
Plant extracts recognized that the antimicrobial action of
essential oils depends on their hydrophobic
Some plants extracted in different organic or lipophilic character. Terpenoids may
solvents have shown inhibitory action serve as an example of lipid-soluble agent
against different storage fungi (Singh et al., that affects the activities of membrane catal-
1993; Hiremath et al., 1996; Rana et al., ysed enzymes; for example, their action on
1999; Okigbo and Pandalai, 2005). The respiratory pathways. Compounds of EOs
inhibitory effect of water-soluble extracts of either affect the physiological function of
garlic bulbs, green garlic, green onions, hot microorganisms or cause structural changes
peppers, ginger, Chinese parsley and basil of hyphae and spores (Thompson, 1986;
on the growth of A. niger and A. flavus was Arras et al., 1993; Zambonelli et al., 2004).
examined. Garlic bulbs, green garlic and For instance, the effect of thyme oils and
green onions showed an inhibitory effect thymol on the hyphae cytomorphology of
against these two fungi (Yin and Cheng, 1998). F. solani, R. solani and C. lindemuthianum
increased vacuolization of the cytoplasm itself. Effective antifungal plant compounds

and accumulation of lipid bodies, undula- that can fill the void of phased-out chemi-
tion of the plasmalemma and alteration of cals will require some advances in the study
the mitochondrial and endoplasmic reticu- of regional aromatic plants, their produc-
lum (Zambonelli et al., 2004). However, tion, formulation and possible beneficial
variations in the fungicidal action of the mechanism to prevent or control fungal
compounds seem to depend on solubility, attack, better understanding of how they
as well as on the capacity to interact with will fit into integrated systems and their
cytoplasmic membrane. interaction with the environment and other
IPM components and identification of the
optimum concentration of EOs that can con-
trol seedborne fungal pathogens without
Conclusions affecting seed germination and seedling
growth. The information on the active prin-
Sustainable agriculture in the 21st centaury ciples present in various botanicals on ger-
will rely increasingly on alternative inter- mination and seedling vigour is to be
ventions for pest management that are envi- elucidated. A proper study of the mode of
ronmentally friendly and reduce the amount action and structure activity relationship
of human contact with chemical pesticides. will bring about a new class of interesting
The use of botanicals in crop protection has compounds for future pest control. Despite
now gained popular ground in the world of the common belief that phytocompounds
agriculture as an alternative to the use of are safe, they all have inherent risk, just like
toxic, persistent and synthetic compounds. synthetic compounds. Thus, it is within the
Several factors are now responsible for mak- scope of phytoscientists to elucidate the
ing the use of alternative methods more side effects and appropriate doses and iden-
attractive. A number of studies have been tify bioactive phytocompounds and ways of
conducted on the use of botanicals and sev- extraction and conservation. As a cautionary
eral plants with promising biocidal proper- note, the EOs that are the most efficacious
ties have been identified. Most of these against pests are often the most phytotoxic.
plants have also been used in vitro and in This phytotoxicity requires serious atten-
vivo in the control of various plant diseases. tion when formulating products for agricul-
Certain plant EOs and plant extracts have a tural use. Also, selectivity among invertebrates
broad spectrum of activity against plant is not well documented. Honeybees appear
pathogenic and other fungi. They have con- somewhat susceptible (Lindberg et al., 2000).
siderable potential as crop protectants. Cur- The susceptibility of various natural ene-
rent information indicates that they are safe mies has yet to be reported, although the
to the user and the environment, with few lack of persistence of EOs under field condi-
qualifications. With the modern techniques tions could provide some information on
now available and the attention being given temporal selectivity favouring non-target
to this area, we look forward to intensifying species. Finally, we should maintain our
development of the biological activity of efforts in considering and valorizing our
botanicals so as to exploit them as fungi- natural patrimony, as well as conducting
cides. A consolidated and continuous search more scientific research on aromatic plants
for natural products may yield safer alterna- for chemical analysis and biological, toxico-
tive control measures like azadirachtin and logical and pharmacological investigation
pyrethroids, which are being used. How- of therapeutic aspects. It is important to
ever, in order to consider the use of any remember that just because a pesticide is
plant material seriously, further informa- derived from a plant does not mean that it is
tion is required. The use of locally available safe for humans and other mammals, or that
plants avoids the need to establish complex it cannot kill a wide variety of other life.
mechanisms for pesticide distribution; the Some botanical pesticides can be quite toxic
community can collect or grow the plants to humans and should not be used on plants
for human consumption. For example, methyl collection of this type of information. For
salicylate (oil of wintergreen) is commonly utilization of botanicals on an industrial
used as food flavouring, but it can be quite scale, it may be necessary to obtain such
toxic in large doses (Jonathan and Davis, secondary metabolites from tissue culture-
2007). Few systematic studies have been con- derived materials. There are many advan-
ducted to determine how farmers use plant tages to this method of production, including
protectants, their effectiveness and method immediate response to an increase in demand
of application. The introduction of rapid irrespective of season, freedom from climatic
rural appraisal (RRA) and participatory rural stresses, pests and diseases and product for-
appraisal (PRA) techniques will facilitate the mation in a clear, sterile environment.
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1
2 3
4 5
Plate 1. (a) Perithecia of Gibberella zeae (anamorph Fusarium graminearum) on infected seed of triticale.
(b) Cross-section of a perithecium of G. zeae showing the ostiole and asci bearing ascospores. (Reprinted with
permission from F. Trail and R. Common (2000). Perithecial development by Gibberella zeae: a light microscopy
study. Mycologia 92,130-138. © Mycological Society of America)
Plate 2. Diaporthe phaseolorum (anamorph Phomopsis sojae) causing seed rot on soybean seeds. (Courtesy M.
C. Rollán)
Plate 3. Fusarium sp. Infecting soybean seeds. (Courtesy M. C. Rollán)
Plate 4. Germinating onion seed affected by Botrytis allii. (Courtesy L. du Toit, Diseases in vegetable seed crops:
Identification, biology, and management [Online]. Available at: http://www.seedalliance.org/uploads/pdf/VegSeed-
Diseases.pdf)
Plate 5. Seedborne wilt of spinach by Verticillium dahliae. (Courtesy L. du Toit)
6
7 8
9 10
Plate 6. Spinach seed showing stromatisation due to pseudothecia of Pleospora herbarum (anamorph Stem-
phylium botryosum). (Courtesy L. du Toit)
Plate 7. Rice seed discoloration caused by a fungi complex.
Plate 8. Wheat seed discoloration caused by a fungi complex.
Plate 9. Open pod of soybean showing purple discoloration caused by Cercospora kikuchii. (Courtesy M. C. Rollán).
Plate 10. Conidiophores and conidia of Cladosporium variabile on spinach seed. (Courtesy L. du Toit)
5 Use of Plant Extracts as Natural
Fungicides in the Management of
Seedborne Diseases
Gustavo Dal Bello and Marina Sisterna

Comisión de Investigaciones Científicas de la Provincia de Buenos Aires, Centro
de Investigaciones de Fitopatología (CIDEFI) – Facultad de Ciencias Agrarias y
Forestales, Universidad Nacional de La Plata, La Plata, Argentina
Abstract
Seedborne fungi can cause substantial losses to grains, rendering them unfit for human consumption
and sowing. Several methods have been used for the control of seedborne diseases and among them
chemical control has been the most widely adopted over many decades. The use of most of these fun-
gicides has been restricted because of high and acute toxicity, long degradation periods and bad effects
on human health, plants and animals, which is harmful to our environment. Moreover, recent increases
in the production and sale of organic seed has heightened the scrutiny of organic seed quality and in
particular brought attention to concerns of seedborne disease contamination. In order to meet the
demands of consumers and growers alike, exploration of alternative methods for managing fungal dis-
eases is under way. One such eco-friendly approach of controlling seed fungal diseases is the use of
natural products, specifically plant-derived compounds. They have played a significant role in reduc-
ing the incidence of seedborne pathogens and in the improvement of seed quality and the emergence
of plant seeds in the field. It has long been recognized that several plant compounds, such as essential
oils, have antifungal activity against both pathogens and spoilage fungi. As a rich source of bioactive
chemicals, plants may provide potential alternatives to synthetic fungicides for seed treatment to pro-
tect them against seedborne pathogens. Therefore, this chapter discusses the current status of the use
of plant extracts to control seedborne fungi.
Introduction be carried with, on or in seeds and, in suit-

able environmental conditions, may be
Almost 90% of all the world’s food crops transmitted to cause diseases in developing
are grown from seeds (Schwinn, 1994), which seedlings or plants. With some diseases, the
are widely distributed in national and inter- pathogen attacks the germinating seedling,
national trade. Many plant pathogens can which affects seedling establishment and
be seed transmitted and seed distribution is hence plant populations; with others, dis-
a very efficient means of introducing plant ease symptoms are not seen until a later stage
pathogens into new areas, as well as a means of growth (Rennie and Cockerell, 2006). Fur-
of survival of the pathogen between grow- thermore, seedborne pathogens such as bac-
ing seasons. Disease-causing organisms may teria, fungi, viruses and nematodes have the

52 G. Dal Bello and M. Sisterna
potential to spread disease to the subsequent seed has heightened the scrutiny of organic
crop. Seedborne infection of fungal patho- seed quality, and in particular brought
gens is important not only for its association attention to concerns of seedborne disease
with the seeds but also contamination of the contamination. The number of alternative
soil by permanently establishing its inocula. crop production systems has increased in
Additionally, fungi are significant destroy- the past decade in response to growing
ers of foodstuffs and grains during storage, concerns about agricultural concentration
rendering them unfit for human consump- and interest in a more ecological, farm-
tion by retarding their nutritive value and based agriculture. In these low-input sys-
often by producing mycotoxins (Satish et al., tems, some non-chemical substances, such
2007). as plant extracts, may be used successfully
It is, therefore, necessary to search for as a contact fungicide seed treatment for
control measures that are economical, eco- organic crops. As a rich source of bioactive
logically sound and environmentally safe to chemicals, plants may provide potential
eliminate or reduce the incidence of these alternatives to be used as pathogen-control
important pathogens so as to increase seed agents.
germination and obtain healthy and vigor- Hamburger and Hostettmann (1991)
ous plants with better yield (Hasan et al., report that the total number of plant chemi-
2005). cals may exceed 400,000 and of this, more
Seed treatment is the oldest practice in than 10,000 are secondary metabolites whose
plant protection. Its origin can be traced to major role in plants is defensive in nature.
the 18th century with the use of brine to Thus, plant-based secondary metabolites
control cereal smuts (Neergaard, 1979). The that have a defensive role may be exploited
modern era of seed treatments began with for the management of diseases and pests.
the introduction of organomercury fungi- However, most species of higher plants
cides in 1912, which were widely used for have never been surveyed. Their chemical
several decades. The post-World War II or biologically active constituents that
period saw the development of new fungi- have the potential to be used as new sources
cide chemistry and the first use of seed of commercially valuable pesticides remain
treatment for insect control. Today, the most to be discovered. This is due mainly to the
widely used application of seed treatment is lack of information on the screening/evalu-
the traditional one of protecting the germi- ation of diverse plants for their antifungal
nating seedling against seed- and soilborne potential (Satish et al., 2007). Neverthe-
fungi in the period immediately after plant- less, several higher plants and their con-
ing (McGee, 1995). Chemical fungicides can stituents have shown success in plant
control plant diseases but they have bad disease control and have proved to be harm-
effects on human health, plants and ani- less and non-phytotoxic, unlike chemical
mals, which is harmful to our environment. fungicides.
Besides, using conventional seed treatment
with synthetic fungicides to kill pathogens
is a practice not allowed in organic produc-
tion. Additionally, resistance by pathogens Essential Oils to Reduce
to fungicides has rendered certain fungi- Seedborne Fungi
cides ineffective.
Worldwide ecological awareness Plant extracts have played a significant role
requires more natural foods and products, in reducing the incidence of seedborne
which has influenced the improvement and pathogens and in the improvement of seed
utilization of integrated pest management. quality and the emergence of plant seeds
In this kind of control, alternative methods in the field (Hasan et al., 2005). In recent
are used to protect seeds to decrease the years, much attention has been paid to
use of chemical products. Moreover, recent essential oils, a group of plant-derived com-
increases in the production and sale of organic pounds, for seed treatment to protect them
Use of Plant Extracts as Natural Fungicides 53
against seedborne fungi (Sisterna and Dal mitochondrial structure disorganization (de
Bello, 2007). Billerbeck et al., 2001) and interference
The essential oils arise from a second- with enzymatic reactions of the mitochon-
ary metabolism of the plant, normally formed drial membrane, such as respiratory electron
in special cells or groups of cells as glandu- transport, proton transport and coupled phos-
lar hairs, found on many leaves and stems. phorylation steps (Knobloch et al., 1989).
Oils occur as a globule or globules in the The active components vary between
cell and may also be secreted from cells lin- oils. For example, the main component is
ing the schizogenous ducts or canals. Plant l-carvone in spearmint (Mentha spicata L.),
volatile oils are generally isolated from non- terpinen-4-ol in tea tree (Melaleuca alterni-
woody plant material by several methods, folia (Maiden. & Betche.) Cheel.) oil and
usually distillation, and are a variable mix- α-terpineol in pine (Pinus spp.) (Knobloch
ture of principally terpenoids, specifically et al., 1989). The essential oils of Cinnamo-
monoterpenes [C10] and sesquiterpenes mum zeylanicum Blume (cinnamon) and
[C15], although diterpenes [C20] may also be Syzygium aromaticum (L.) Merr. & Perry
present. A variety of other molecules can (syn. Eugenia cariophyllata Thunb.), con-
also occur, such as aliphatic hydrocarbons, sisting of cinnamaldehyde and eugenol,
acids, alcohols, aldehydes, acyclic esters or respectively, as major components (Parana-
lactones and, exceptionally, nitrogen- and gama, 1991), are known to be potent anti-
sulphur-containing compounds, coumarins fungal materials (Beg and Ahmad, 2002;
and homologues of phenylpropanoids (Dor- Ranasinghe et al., 2002). Citral and geraniol
man and Deans, 2000). Faleiro et al. (2003) are the major components in essential oils
have shown that the antimicrobial action is of Cymbopogon citratus (DC.) Stapf (lemon-
determined by more than one component. grass) and C. martinii (Roxb.) Stapf var.
In such cases, the major component is respon- motia (palmarosa), respectively, which are
sible not only for the antimicrobial activity, antifungal compounds (Paranagama et al.,
but also the synergistic effect that may take 2003; Velluti et al., 2004). Thymol was
place. The mixtures are extremely complex identified as the active ingredient of Oci-
and vary with environmental and genetic fac- mum gratissimum L. (wild basil) and has
tors (Asplund, 1968; Cabo et al., 1986; Arras, been found to suppress fungal growth
1988; Bhaskara et al., 1998; Vanneste et al., (Adekunle and Uma, 2005). Linalool is a
2002). Moreover, the composition of essential major component in the essential oil of Thy-
oils from a particular species of plant can dif- mus mastichina L. subsp. mastichina, with
fer between harvesting seasons and between antimicrobial activity (Faleiro et al., 2003),
geographical sources (Di Pasqua et al., 2005; and both limonene and linalool are the
Di Pasqua, 2006). minor components in the essential oils
Major active compounds from essential derived from different plants. The majority
oils are known for their broad-spectrum of these essential oils and their components
antifungal activity against both human and have proved valuable in protection against
plant pathogens. These constituents can postharvest fungal diseases which cause
either affect the physiological functions of build-up of toxic fungal metabolites in
microorganisms or cause structural changes stored foods (Kishore et al., 2007). There-
of hyphae and spores (Arras et al., 1993; fore, essential oils might substitute agro-
Zambonelli et al., 2004; Kishore et al., 2007), chemicals or contribute to the development
and different fungi appear to react differently of new agents to inhibit both fungal growth
to these components (Szczerbanik et al., 2007). and the production of mycotoxins affecting
The antifungal essential oils reduce hyphal grain and seed crops.
growth and also induce lysis and cytoplas- This chapter discusses the current sta-
mic evacuation in fungi. Growth inhibition tus of plant extracts and the potential use of
by essential oils often involves induction of essential oils as natural antifungal agents to
changes in cell wall composition (Ghfir control the main seedborne pathogens and
et al., 1997), plasma membrane disruption, spoilage fungi.
Symptoms on Seeds B. maydis and B. oryzae in cereals; Col-

Caused by Fungi letotrichum graminicola, Diaporthe phaseo-
lorum (Plate 2) and Fusarium spp. in
Seedborne mycoflora comprise a large soybean (Plate 3); Botrytis allii on onion
number of saprobes and pathogenic fungal (Plate 4); Verticillium dahliae on spinach
species. Pathogenic fungi grown on seeds (Plate 5) and B. cinerea in the seeds of many
can cause heavy damage and reduce yields hosts, including forest trees.
of seed, both quantitatively and qualitatively
(Neergaard, 1979). Other fungi, including Sclerotization and stromatization
saprophytes and very weak parasites (Sis-
Transformation of floral organs or seed into
terna and Lori, 2005), may lower the quality
sclerotia or stromata is an important disease
of seeds by causing discoloration, which
condition in certain categories of fungi and
may seriously depreciate the commercial
host. Ergots produced by Claviceps purpu-
value of seeds, particularly of grain when
rea and other species of Claviceps in cereals
graded for consumption.
and grasses exemplify sclerotia of this type.
Another example is Phomopsis viterbensis
in chestnut, Pleospora herbarum in spinach
Disease and disorder (Plate 6) and Ciboria spp. in the seeds of for-
est trees and grasses.
The following types of disease and disorder
are encountered, often in combination Seed necroses
(Neergaard, 1979):
Many seed-rotting fungi produce superficial
necroses in the seed; other fungi never pen-
Seed abortion
etrate deeply into the tissues, most seed-
The most prominent examples of fungi pro- borne fungi usually not beyond the protective
ducing abortion are the smut fungi, which layers, the seed coat or pericarp. Anthra-
infect cereals and grasses systemically, and cnose fungi, Colletotrichum spp. as well as
the ergot fungi. The floral parts of the hosts Ascochyta spp., often penetrate into the
are replaced by the fructifications of the fleshy cotyledons, producing conspicuous
parasites. Other examples are different spe- necrotic lesions in the seeds of bean, soy-
cies of Fusarium (in wheat, maize and rice); bean, pea, cowpea and other hosts.
Ascochyta rabiei in chickpea may kill the
young seeds; Drechslera verticillata causes Seed discoloration
death of seed primordia in brome grass and
Discoloration of seeds is a very important
in wheat.
degrading factor, both for consumption
(grain) or for industrial purposes (oil seed).
Shrunken seeds, reduced in size It may be a general indication of poor qual-
ity (Plates 7 and 8). Well-known examples
Examples of more or less heavy reduction of are the effects of A. pisi in pea; C. linde-
seed size are: Alternaria brassicicola and muthianum in bean; B. sorokiniana in
Phoma lingam in crucifers, Septoria lini- wheat, B. oryzae in rice, Cercospora kikuchii
cola in flax, D. teres in barley, F. graminearum (Plate 9) in soybean, etc.
and S. nodorum in wheat.
Reduction or elimination of germination
Seed rot capacity, lowered viability
Many seedborne fungi produce seed rot either Obviously, necroses or more deeply pene-
in the crop or during germination. Examples trating rots in seeds reduce the viability of
are F. avenaceum, F. graminearum (Plate 1), the seeds, their longevity in storage and their
F. moniliforme, Bipolaris sorokiniana, emergence in the field.
Physiological alterations or effects in seed bulbs, ginger (Zingiber officinale Roscoe)

rhizomes, basil (O. basilicum L.) leaves, and
Metabolic products of seedborne microor- fruits of Azadirachta indica A. Juss. (neem)
ganisms may affect the seed itself or may (Shetty et al., 1989).
have other, sometimes serious consequences Positive effects have been recorded on
such as toxicity to animals and humans the same fungus with essential oils of C. cit-
(Aspergillus spp., Penicillium spp., Fusar- ratus, O. gratissimum L. and Thymus vul-
ium spp.). garis L. (thyme) (Nguefack et al., 2004). The
Moreover, seed fungi are classified as researchers investigated the ability to con-
field and storage fungi (Christensen and Kauf- trol seedborne infection and seed–seedling
mann, 1965). Genera such as Alternaria, transmission in naturally infected seeds.
Cladosporium (Plate 10), Fusarium and The essential oils increased the germination
Bipolaris invade seeds as they are develop- capacity of the treated seeds.
ing on the plants in the field or after they
have matured, but before they are harvested,
and for this reason, they have been desig-
nated ‘field fungi’. These fungi require mois- Bipolaris
ture content in equilibrium with a relative
humidity of more than 90% to grow and usu- Hasan et al. (2005) demonstrated that plant
ally do not continue to grow in grains after extracts, namely Z. officinale, A. sativum,
harvest, since grains and seeds are stored A. cepa L. (onion), Adhatoda vasica Nees
with moisture contents below those required (vasaka), Achyranthes aspera L. (devil’s
by the field fungi. horsewhip), A. indica, Lawsonia alba Lama-
The storage fungi consist mainly of rck (henna), Cuscuta reflexa Roxb. (giant
several species of Aspergillus. Species of dodder), Vinca rosea L. and Nigella sativa
Penicillium are encountered at times, usu- L. (black cumin), significantly reduced seed
ally in lots of grain stored at low tempera- infection of wheat by B. sorokiniana (Triti-
tures and with moisture contents above cum aestivum L.). Alcoholic extracts of
16%. The storage fungi do not invade grains neem and garlic inhibited the presence of B.
to any appreciable degree or extent before sorokiniana completely, whereas the high-
harvest. est percentage of the fungus was recorded
from untreated seeds (control). Water extract
of all tested plants had the ability to control
Fungicidal Effects of Plant Extracts seedborne fungi of wheat var. Kanchan,
Against Seed Fungi which showed 100% inhibition of B. soroki-
niana with the application of extracts from
Z. officinale, A. sativum, A. cepa, A. indica,
Numerous studies have described the use of
C. reflexa and N. sativa, whereas the highest
botanicals with a view to exploiting their
fungal incidence (11.67%) was observed on
potential as natural fungicides against seed-
untreated seed. After treatment with the
borne fungi. The following section discusses
water extract of L. alba and A. aspera, only
this alternative method, with particular
4.84% and 7.16% incidence of the patho-
emphasis on the main seedborne fungal
gen, respectively, was recorded. Seeds of
pathogens.
wheat treated with A. vasica and V. rosea
gave statistically identical results (5.83%
and 5.90% incidence of B. sorokiniana).
Alternaria Alice and Rao (1986) reported good
results using plant extracts to control B.
A. padwickii, an important seedborne patho- oryzae on rice seeds which have high natu-
gen of rice (Oryza sativa L.), was inhibited by ral infection of the fungus. After soaking in
aqueous extracts of Strychnos nux-vomica L. the filtrates of different extracts, A. sativum
(strychnine tree), garlic (Allium sativum L.) and M. piperita (peppermint) reduced seed
infection by 68%. In Bangladesh, use of fungicidal effect of the oil against numerous
extracts of Polygonum hydropiper L. (water- seedborne fungal pathogens of white jute
pepper), A. cepa, A. sativum and A. indica (Corchorus capsularis L.), one of the most
was demonstrated to be effective against B. important crops from Bangladesh, India and
oryzae at higher concentrations. Among China. The essential oil produced inhibi-
them, neem and garlic were the most effec- tion in both mycelial growth and spore ger-
tive at 1:1 dilution and inhibited the occur- mination of fungi, including C. corchori
rence of the pathogen by 91 and 83%, (Ahmed and Shultana, 1984), which was
respectively (Ahmed et al., 2002). also strongly inhibited in in vitro tests by
Neem and pungam (Pongamia pinnata using crude leaf extracts from Eupatorium
(L.) Pierre) oil-based emulsifiable concen- triplinerve Vehl. (yapana) (Rahman and
trate (EC) formulations were evaluated for Junaid, 2008).
their efficacy to inhibit the mycelial growth Several studies carried out in Burkina
of the fungus Helminthosporium oryzae Faso underlined the antifungal properties of
(syn. B. oryzae) causing grain discoloration extracts from some Cymbopogon spp. against
of rice under in vitro conditions. All three C. graminicola, the causal agent of anthra-
formulations, namely neem oil 60 EC (acetic cnose on sorghum (Sorghum bicolor (L.)
acid), neem oil 60 EC (citric acid) and neem Moench and pearl millet (Pennisetum glau-
oil + pungam oil 60 EC (citric acid), inhib- cum (L.) R. Br.). Somda et al. (2007) demon-
ited mycelial growth of the pathogen; they strated that the essential oil of C. citratus at
were effective even after 9 months of stor- a concentration of 6% was effective in con-
age. These formulations controlled the grain trolling seedborne infection and seed–
discoloration on rice effectively (Rajappan seedling transmission of C. graminicola
et al., 2001). The efficacy of essential oils such without affecting seedling development.
as clove, ginger, lemongrass, basil, pepper- Similarly, the essential oils extracted from
mint, anise (Pimpinella anisum L.) and cin- C. giganteus (Hochst.) Chiov., C. nardus (L.)
namon at different concentrations on growth Rendle and C. schoenanthus Spreng. reduced
inhibition of B. oryzae was examined by sorghum seed infection by the pathogen sig-
Palaoud (2006). Treatments with clove, anise, nificantly. The lowest rates of infected seeds
ginger and cinnamon oils at 500 ppm pro- were recorded on seeds treated with 10 µl
vided the best results in controlling the fun- and 15 µl of C. nardus oil/g seeds. These
gus and, after storage for 4 months, seed doses were more efficient than chemical
viability was as high as 97–98%. Also, the control (Elisabeth et al., 2008).
extracts of C. citratus, O. gratissimum and T.
vulgaris applied to rice seeds infected with
B. oryzae controlled fungal growth and seed-
ling transmission of the pathogen (Nguefack Curvularia
et al., 2004).
In blackgram (Vigna mungo L.), essential
oils extracted from wood chips of cedar
(Cedrus deodara (Roxb. ex Lamb) G. Don) and
Colletotrichum that from seeds of Trachyspermum ammi (L.)
Sprague ex Turrill (ajowan) exhibited abso-
Abdelmonem et al. (2001) screened oils of lute toxicity, inhibiting the mycelial growth
M. piperita, T. capitatus (L.) Hoffmans. and of C. ovoidea, storage fungi found on seeds
Link and Carum carvi L. (caraway) against (Singh and Tripathi, 1999).
various seedborne fungi of soybean (Glycine Parimelazhagan and Francis (1999)
max (L.) Merr.) and lentil (Lens culinaris reported reduction in the radial growth of C.
Medik.) and found all plant extracts to be lunata associated with rice seeds when
highly effective in controlling C. dematium. treated with leaf extracts of Clerodendrum
Among the fibre-producing species, a viscosum Vent. (glory tree), which also
study on garlic bulb extract reported a increased seed germination and root and
shoot lengths of rice. Considerable research tested against F. oxysporum and F. equiseti in
activity has occurred in the Asian-Pacific vitro on cowpea (V. unguiculata (L.) Walp.)
region on the potential for plant extracts to (Kritzinger et al., 2002). Likewise, plant leaf
control seedborne fungi including maize. extracts (crude and aqueous) of basil, bitter
The oils of cassia (C. cassia Blume) and clove leaf (Vernonia amygdalina Del.), neem and
inhibited the growth of established seed- pawpaw (Carica papaya L.) reduced the inci-
borne infections of C. pallescens (Chatterjee, dence of F. moniliforme significantly and
1990). increased seed germination and seedling
emergence of African yam bean (Sphenosty-
lis stenocarpa (Hochst ex. A. Rich) Harms)
when compared with the untreated controls
Fusarium (Nwachukwu and Umechuruba, 2001).
Regarding cereals, several natural plant
The essential oils and their constituents have compounds have been identified as having
been found effective as antifungal agents antifungal activity against seedborne fungi.
against the main species of Fusarium. Among The essential oils of C. citratus, O. gratissi-
several plant extracts, Sitara et al. (2008) mum and T. vulgaris have proved valuable
found that essential oils from seed of neem, in protection against the seedborne fungus,
black cumin and asafoetida (Ferula asafoe- F. moniliforme in rice. This study evaluated
tida L.) showed fungicidal activity of varying the ability to control seedborne infection
degree against F. oxysporum, F. moniliforme and seed–seedling transmission in naturally
(syn. F. verticillioides), F. nivale and F. sem- infected seeds (Nguefack et al., 2004). The
itectum. Of those oils, asafoetida oil at 0.1% extracts applied controlled seed infection
and 0.15% inhibited the growth of all test and seedling transmission of the pathogen
fungi significantly. A variety of wild plants and increased the germination capacity of
from Mexico were evaluated against several the treated seeds. In the field, as a result of
cereal seedborne fungi in in vitro tests extracts seed treatment as compared to the
(Tequida-Meneses et al., 2002). Extracts from non-treated control, reduction of disease
leaves and stems of Larrea tridentata (Sessé incidence and important increases in yield
& Moc. ex DC.), Coville (creosote bush) and were recorded. After rice seeds inoculated
Datura discolor Bernh. (desert thorn apple) with F. moniliforme were soaked in seven
in methanol or ethanol inhibited the radial plant essential oils at ten different concen-
growth of F. poae completely. Next to these trations, anise, ginger, clove and cinnamon
extracts, Proboscidea parviflora (Woot.) oils at 500 ppm provided the best results in
Woot. & Standl. (double claw) also showed controlling the fungus. The percentage of
good fungal inhibition (86.6%), followed by seed germination and the number of normal
Baccharis glutinosa Pers. (saltmarsh bac- seedlings was significantly high when com-
charis) (79.6%), compared to the alcoholic pared with the control. Anise and clove also
controls (0% inhibition). showed the highest seedling dry weight
In legumes, soybean and lentil, carvone (Palaoud, 2006).
(monoterpene compound), among other In another study on wheat, ten plant
tested compounds, manifested the highest extracts were tested for their efficacy in vitro
antimicrobial influence with complete inhi- against seedborne fungi; alcoholic extract
bition to F. oxysporum. It showed broad- of neem and garlic controlled the infection
spectra activity against all the tested isolates of Fusarium sp. completely. Good results of
of fungal strains at low concentrations. Pep- these treatments contributed to increased
permint, T. capitatus and caraway oils also seed germination (Hasan et al., 2005). Fur-
demonstrated a high control effect against thermore, botanicals from male fern (Dry-
Fusarium sp. (Abdelmonem et al., 2001). opteris filix mas (L.) Scott.) suppressed
Also, the antifungal activity of the essential completely the population of F. oxysporum
oils of thyme, clove, peppermint, soybean in the seed mycoflora of wheat (Rake et al.,
and peanut (Arachis hypogaea L.) were 1989). Putative mycotoxicogenic fungi, such
as F. moniliforme, were partially or totally efficacy of indigenous plant extracts against

sensitive to different essential oils extracted seedborne infection of F. moniliforme on
from 12 medicinal plants (Soliman and maize demonstrated that aqueous extracts of
Badeaa, 2002). Results indicated that oils leaves of O. gratissimum, Acalypha ciliata
of thyme, cinnamon and anise (< or = Forssk., V. amygdalina, M. indica L. (mango
500 ppm), marigold (Calendula officinalis tree) and A. indica had significant inhibi-
L.) and caraway (< or = 2000 ppm), spear- tory growth effects on the fungal pathogen.
mint and basil (3000 ppm) inhibited this A. ciliata extract was more effective than
fungus completely. other plant extracts and compared favour-
Bioassays using a poisoning technique ably with benomyl in the control of the
were carried out with C. citratus, O. gratis- pathogen (Owolade et al., 2000). Further-
simum and T. vulgaris for the control of more, to determine whether essential oils
seedborne fungi infecting maize (Zea mays can be used as a contact fungicide seed treat-
L.) seeds. The results disclosed the fungi- ment for organic corn, the essential oils of
cidal properties of theses oils against F. ver- 18 plants were screened for their fungicidal
ticillioides. These natural products control properties. Five oils, cinnamon, clove, O.
the seedborne inoculum of the pathogen by minutiflorum O. Schwarz and P.H. Davis,
90% to 100%. Field trials conducted in the savoury (Satureja montana L.), and thyme,
humid forest and the warm savannah zones controlled Fusarium completely in vitro.
of Cameroon have shown that these prod- The minimum inhibitory concentration
ucts are potential seed treatments that could (MIC) was 800 µl/l and seedlings presented
be used as substitutes for synthetic fungi- no phytotoxicity symptoms in the germina-
cides, which are usually unaffordable to tion test at rates up to 64 µl/kg active ingre-
resource-limited farmers (Tagne et al., 2008). dient (MIC × 20). Field emergence of inbred
Seed treatment with cinnamon, palmarosa and hybrid seeds treated with the essential
and lemongrass oils at 500 mg/kg showed oils were significantly lower than seeds
antimycotoxigenic ability against fumoni- treated with the commercial fungicides,
sin B1 accumulation by isolates of F. verticil- Maxim XL {fludioxonil [4-(2,2-difluoro-1,3-
lioides and F. proliferatum (Marín et al., benzodioxol-4-yl)-1H-pyrrole-3-carbonitrile]
2003). Furthermore, different effects of oreg- 21.4%, mefenoxam [(R)-2-[(2,6-dimethylphe-
ano (Origanum vulgare L.) and herb Louisa nyl) methoxyacetylamino] propionic acid
(Aloysia triphylla (L’Herit) Britton) essen- methyl ester] 8.4%}, which is a conven-
tial oils were observed on F. verticillioides tional fungicide, and Natural 2 (proprietary
M 7075 fumonisin B1 production in corn ingredients), which is an organic fungicide,
grain in Argentina (López et al., 2004). As but were not different from the organic fun-
alternative preharvest natural fungicides, gicide, Yield Shield (Bacillus pumilus GB34
Velutti et al. (2004) showed the antimyco- 0.28%) or an untreated control (Christian
toxigenic activity of the essential oils against and Goggi, 2008).
F. graminearum on corn infested seed. The Bioactivity of different plant extracts on
effect of oregano, cinnamon, lemongrass, F. thapsinum pathogen of sorghum was
clove and palmarosa on growth rate, zearale- evaluated on seeds contaminated with the
none (ZEA) and deoxynivalenol (DON) pro- fungi. Cinnamon, clove, epazote (Teloxys
duction was assessed at two concentrations ambrosioides (L.) Weber), oregano and
(500 and 1000 mg/kg), at different water thyme, singly and in combination, as well as
activity and temperature levels. DON pro- the essential oils of Mentha sp. and rue (Ruta
duction in general was inhibited by all chalepensis L.) and the combination of clove
essential oils at 30°C and, although palma- with cinnamon, had a fungicidal effect. Nev-
rosa and clove were the only essential oils ertheless, only thyme did not affect either
with statistically significant inhibitory effect seed germination or sorghum seedling height.
on ZEA production, an inhibitory trend was The rest of the oils were phytotoxic (Montes-
observed when cinnamon and oregano oils Belmont and Flores Monctezuma, 2001).
were added to maize grain. Studies on the Additionally, plant extracts were also tested
on naturally infected sorghum seeds for studied. The effectiveness of garlic extract
controlling F. moniliforme. More than 50% was comparable to the fungicide, Rovral
of the growth of this fungus was reduced by (Latif et al., 2006).
C. citratus essential oil on seeds, whereas
Eucalyptus camaldulensis Dehnh. (Euca-
lyptus) essential oil was less efficient, even at Macrophomina
high concentrations (Somda et al., 2007).
Elisabeth et al. (2008) investigated the efficacy
Several natural plant compounds have been
of essential oils extracted from C. schoe-
identified as having antifungal activity
nanthus, C. nardus and C. giganteus in con-
against M. phaseolina. The work of Ahmed
trolling Fusarium sp. on seeds of sorghum
and Shultana (1984) reported that garlic oil
and pearl millet. The results indicated that
produced inhibition in both mycelial growth
all the essential oils reduced seed contami-
and spore germination of M. phaseolina, an
nation of both cereals significantly. The low-
important seedborne fungal pathogen of
est rates of infected seeds were recorded on
jute. In sunflower seeds, the leaf extracts of
seeds treated with 10 µl and/or 15 µl of
the flamboyant tree, karanja and gum arabic
essential oil/g seeds. Most of the time, these
tree significantly inhibited the germination
doses were as efficient as the chemical con-
of fungal spores, mycelial growth and spore
trol and oil of C. giganteus used at 15 µl/g
production as well (Thiribhuvanamala and
seeds eliminated pearl millet seed infection
Narasimhan, 1998).
by Fusarium completely.
In vitro experiments conducted by
In another experiment, de Souza et al.
Dwivedi and Singh (1999) confirmed the
(2003) analysed the mycoflora and physio-
fungitoxicity of some higher plant extracts
logical quality of cotton (Gossypium hirsutum
against the mycelial growth of M. phaseo-
L.) seeds treated with chemical fungicides
lina. Among the plant products, the essential
and aroeira (Astronium urundeuva L.) extract.
oils of T. ammi exhibited absolute fungi-
Pure extract did not control the fungal pop-
cidal effect at an MIC of 200 ppm.
ulation but, when mixed with the fungi-
Studies of Abdelmonem et al. (2001)
cides, captan, benomyl and tolylfluanid,
also showed the inhibitory effect of the
they showed reduction in the incidence of
essential oils of M. piperita, T. capitatus
Fusarium sp.
and C. carvi against M. phaseolina associ-
From Leguminosae members, leaf extracts
ated with the seeds of soybean and lentil.
of Delonix regia (Bojer) Raf., flamboyant tree,
Furthermore, when tested in infected cow-
Pongamia glabra Vent. (Karanja) and Acacia
pea seeds, A. indica extract was found to
nilotica (L.) Willd. ex Delile (gum arabic tree)
inhibit the incidence of the pathogen. After
significantly inhibited spore germination,
naturally infected seeds were immersed in a
mycelial growth and spore production of F.
suspension containing neem tree oil at a
solani from sunflower (Helianthus annuus L.)
concentration of 0.5% for 16 h, the infec-
seeds (Thiribhuvanamala and Narasimhan,
tion incidence decreased to 50% in relation
1998). The same pathogen could be controlled
to controls using only water (Mello et al.,
using crude leaf extracts of A. indica and
2005).
O. gratissimum to protect egusi melon (Cuc-
umeropsis mannii Naudin) seed. After 6
months incubation, all the seeds treated
with leaf extracts showed no Fusarium Aspergillus and Penicillium
infection (Adekunle and Uma, 2005).
Efficacy of some plant extracts in con- Putative mycotoxicogenic fungi of wheat
trolling seedborne Fusarium infections of grains were partially or completely sensi-
mustard (B. nigra (L.) W.D.J. Koch) was tive to different essential oils extracted from
evaluated. It was found that garlic and neem 12 medicinal plants (Soliman and Badeaa,
extracts were the most effective in control- 2002). They were tested for inhibitory
ling the pathogen among the plant extracts activity against A. flavus, A. parasiticus and
A. ochraceus. Results indicated that oils of 3.46 mg/ml, respectively. Therefore, lemon-
thyme, cinnamon (< or = 500 ppm), mari- grass oil could be used to manage aflatoxin
gold (< or = 2000 ppm), spearmint and basil formation and fungal growth of A. flavus in
(3000 ppm) inhibited all the tested fungi stored rice. Besides, the essential oil of lem-
completely. Caraway was inhibitory at ongrass inhibited growth of moulds like A.
2000 ppm against A. flavus and A. parasiti- flavus, A. fumigatus and P. chrysogenum of
cus and at 3000 ppm against A. ochraceaus. maize and cowpea grains. Within a storage
Also, the three species were suppressed by period of 10 days, seeds of maize and cow-
anise at < or = 500 ppm. An in vitro initial pea treated with lemongrass powder and
screening of a range of several spice hydro- essential oil showed no physical deteriora-
sols on inhibition of mycelial growth of A. tion. Off-colour, off-odour and mouldiness,
parasiticus revealed that hydrosols of anise, however, characterized untreated control
cumin (Cuminum cyminum L.), fennel seeds (Adegoke and Odelusola, 1996).
(Foeniculum vulgare Mill.), Mentha sp., Another assay on A. flavus determined opti-
oregano, savoury and thyme caused a stron- mal levels of dosages of 11 plant essential
ger inhibitory effect on mycelial growth oils for maize kernel protection, effects of
(Özcan, 2005). combinations and residual effects (Montes-
When essential oil from oregano was Belmont and Carvajal, 1998).
applied as a fumigant against the mycelia Bankole (1997) showed that essential
and spores of A. flavus, A. niger and A. oils from A. indica and Morinda lucida
ochraceus on wheat, the oil vapour exhibited Benth. (brimstone tree) inhibited the growth
a fungicidal effect and a significant reduction of a toxigenic A. flavus and reduced aflatoxin
in the per cent of infested grain was observed B1 synthesis significantly in inoculated
(Paster et al., 1995). Plant extracts of Z. offici- maize grains. Studies in experimental grain
nale, bulbs of A. sativum and A. cepa, leaves bins have demonstrated that soybean oil
of A. vasica, L. alba, A. indica, A. aspera, alone also reduces infection by storage fungi
stem of C. reflexa, root of V. rosea and seeds (White and Toman, 1994). After 12 months,
of N. sativa were tested for their efficacy in kernel infection by Penicillium spp. and
vitro against Aspergillus sp. and Penicillium Aspergillus spp. was 83% and 63.7%, respec-
sp. in wheat. All the plant extracts reduced tively, in untreated corn, compared to 60%
the incidence of seedborne fungi signifi- and 46.2%, in soybean oil-treated corn at
cantly and increased seed germination, the 200 ppm (McGee, 1989). Essential oils from
number of healthy seedlings and the vigour aromatic plants such as cinnamon, clove,
index. Neem and garlic extracts controlled oregano, savoury and thyme inhibited the
the intensity of the fungi completely (Hasan growth of the corn pathogen Penicillium sp.
et al., 2005). completely in vitro. The MIC of the essen-
A natural fungicide against aflatoxi- tial oils in the laboratory was 800 ppm. The
genic fungi to protect stored rice using the growing seedlings were not affected and no
essential oil of C. citratus was developed by phytotoxicity symptoms were seen at rates
Paranagama et al. (2003). Lemongrass oil up to 16,000 ppm concentration of the oils
was tested against A. flavus and the test oil (Goggi et al., 2008). A previous work was
was fungistatic and fungicidal against the undertaken by Chatterjee (1990) to screen
test pathogen at 0.6 and 1.0 mg/ml, respec- some essential oils for their inhibitory activ-
tively. Aflatoxin production was completely ity against fungal infection and mycelial
inhibited at 0.1 mg/ml. The results obtained growth in postharvest maize grains during
from the thin layer chromatographic bioas- storage. It was observed that the oils of Cas-
say and gas chromatography indicated citral sia sp. clove (30 ml/g grain and above), star
a and b as the fungicidal constituents in anise (Illicium verum Hooker fil.) (40 ml/g
lemongrass oil. During the fumigant toxicity grain and above), Geranium sp. (30 ml/g grain
assay of lemongrass oil, the sporulation and and above) and basil (50 ml/g grain) inhibited
the mycelial growth of the test pathogen the in vivo mycelial growth of established
were inhibited at concentrations of 2.80 and seedborne infections of A. flavus, as well as
preventing infection following inoculation native plant of India, whose main commer-
with A. flavus, A. glaucus, A. niger and A. cial value is due to its seed gum (galacto-
sydowi. These oils also preserved the grain mannan gum). In this case, A. flavus was
from natural A. flavus infection during the reduced by cumin oil extracted from seeds
experimental period. Christian and Goggi (Dwivedi et al., 1991). Studies carried out
(2008) studied whether essential oils could have shown that cumin has powerful anti-
be used as a contact fungicide seed treat- microbial properties against diverse species
ment for organic corn. In vitro, the essential of bacteria and fungi. The chemical studies
oils of cinnamon, clove, oregano, savoury indicated that the greater part of this antimi-
and thyme controlled Penicillium com- crobial activity might be attributed to the
pletely. Soybean oil, applied at a rate used cuminaldehyde [p-isopropil benzaldehyde]
to suppress grain dust, reduced storage that is present in the dried fruit of this plant
fungi growth in maize and soybeans in field (De et al., 2003).
storage bins. After 12 months, soybean seed Another study (de Souza et al., 2003)
infection by Penicillium spp. and Aspergil- investigated the mycoflora and physiologi-
lus spp. was 45.7% and 39.2%, respectively, cal quality of cotton seeds treated with
in untreated seeds, 17.7% and 8.2% in soy- chemical fungicides and aroeira extract.
bean oil-treated seeds and 1.7% and 2% in Pure extract did not control the fungal pop-
soybean oil + thiabendazole-treated seeds ulation but, mixed with the fungicides, cap-
(McGee, 1989; White and Toman, 1994). tan, benomyl and tolylfluanid, it showed
Also, soybean oil demonstrated its effec- reduction in the incidence of Aspergillus
tiveness in decreasing by 50% the levels of sp. Garlic extract was also found to be effec-
seed infection and physiological ageing by tive in removal of the seedborne pathogens
the storage fungus, A. ruber, on garden pea of mustard, including species of Aspergillus
seeds (Pisum sativum L.) (Hall and Harman, and Penicillium (Latif et al., 2006).
1991). Peppermint, thyme and clove oils Fungi of the genera Aspergillus and Pen-
were tested in vivo against A. flavus, A. niger icillium are widely distributed storage fungi
and P. chrysogenum on different seed culti- of egusi melon seeds, causing seed discolor-
vars of cowpea. Antifungal activity was ation, decreased nutritive value, increase in
observed for the three oils, depending on free fatty acid and peroxide values, decreased
cultivar and concentrations (Kritzinger et al., seed germination and producing a number
2002). In blackgram, essential oils extracted of toxic metabolites, including aflatoxin. Four
from wood chips of cedar and that from the mould species, A. flavus, A. niger, A. tama-
seeds of ajowan exhibited absolute toxicity, rii and P. citrinum, were inoculated on to
inhibiting the mycelial growth of A. niger on shelled melon seeds. The essential oil of C.
storage seeds (Singh and Tripathi, 1999). citratus at 0.1 and 0.25 ml/100 g seeds
Major seedborne fungi associated with reduced deterioration and aflatoxin produc-
African yam bean like A. niger and A. flavus tion significantly in shelled seeds inocu-
could be controlled by using leaf extracts lated with A. flavus. At higher dosages (0.5
(crude and aqueous) of basil, bitter leaf, and 1.0 ml/100 g seeds), the essential oil
neem and pawpaw. All the plants’ leaf prevented aflatoxin production completely.
extracts reduced significantly the incidence After 6 months in farmers’ stores, unshelled
of fungi tested and increased seed germina- melon seeds treated with 0.5 ml/100 g seeds
tion and seedling emergence when compared of essential oil had a significantly lower
with the untreated controls. The crude proportion of visibly diseased seeds and
extracts were most effective, mainly neem, Aspergillus spp. infestation levels and sig-
which gave complete control of A. niger and nificantly higher seed germination com-
A. flavus. In addition, seed germination was pared to the untreated seeds. The efficacy of
enhanced by this extract and reached nearly the essential oil in preserving the quality of
90% (Nwachukwu and Umechuruba, 2001). melon seeds in stores was statistically on a
A. flavus is also found infesting seeds of par with that of fungicide (iprodione) treat-
guar, Cyamopsis tetragonoloba (L.) Taub., a ment (Bankole et al., 2005).
Conclusions different components of the oils (Varma and

Dubey, 1999; Dubey et al., 2008).
While modern agricultural practices have In recent years, tremendous strides have
resulted in higher and more stable yields, been made in advancing the study of the
they have also weakened the natural bal- natural control of plant pathogens, particu-
ance between pests and their antagonists larly seedborne fungi. As is shown in this
and have reduced soil fertility and health. chapter, plant metabolites and plant-based
Harmful chemicals threaten both the envi- fungicides appear to be one of the better alter-
ronment and human health alike. The ben- natives, as they are known to have minimal
efits of pesticides, in terms of reduced crop environmental impact and danger to consum-
losses, are often overestimated because the ers in contrast to synthetic pesticides. Despite
viability of alternative pest management the potential of these naturally occurring bio-
approaches is not fully understood. Con- chemicals as biorational fungicides, their
versely, the costs of relying predominantly practical development and implementation
on synthetic pesticides in pest control, in will require more detailed studies. Efforts
terms of health, environment, agroecology should be made to search for indigenous
and trade, are also not known completely plants as a source of antifungal compounds
and consequently are often underestimated and to bioprospect the antifungal properties
(SP-IPM, 2008). Integrated pest manage- of these plant products, especially essential
ment (IPM) has emerged as a way towards oils, towards seed fungi. Field trials are
maintaining or increasing agricultural pro- required to assess the practical applicability
ductivity without over-reliance on synthetic of botanical pesticides, together with bulk
chemical pesticides, emphasizes the growth production, extensive usage of active com-
of a healthy crop with the least possible dis- pounds and interaction with other IPM
ruption of agroecosystems and encourages components. Biosafety studies should be
natural pest control mechanisms (FAO, 2002). conducted to ascertain their toxicity to
In this context, development of simple and humans, animals and crop plants. Additional
eco-friendly seedborne disease management screenings might be focused on the quality
methods is necessary to improve the quality assurance of botanicals and its regulation.
of seed in general and farmers’ saved-seed While it is unlikely that biopesticides
in particular (Elisabeth et al., 2008). will replace chemical pesticides completely
Plant-derived compounds as crop pro- in the foreseeable future, we can expect that
tectants represent a vast and rapidly pro- there will be some decline in the use of chem-
gressing resource. Botanical fungicides are icals, particularly in developed countries.
best suited for use in industrialized countries Exploitation of naturally available chemicals
when strict enforcement of pesticide regula- from plants, which retard the reproduction of
tions is impractical, or in the case of organic undesirable microorganisms, would be a
production. However, they can play a much more realistic and ecologically sound method
greater role in protecting crops in developing for plant protection and will have a promi-
countries, where human pesticide poisonings nent role in the development of future
are most prevalent. Among the plant products, commercial pesticides for crop protection
essential oils especially are a very attractive strategies, with special reference to the man-
method of controlling plant diseases. Essential agement of plant diseases (Varma and Dubey,
oils and their components are gaining increas- 1999; Gottlieb et al., 2002). The prospect of
ing interest because of their relatively safe sta- botanical products as fungicides includes
tus, their wide acceptance by consumers and plant compounds with broad-spectrum activ-
their exploitation for potential multi-purpose ity to provide protection against a range of
use. Besides, the problem of developing pathogenic fungi that attack the plant at the
resistant strains of fungi may be solved by same or subsequent growth stages following
the use of essential oils of higher plants as their application. Furthermore, essential oils
fumigants in the management of fungal are made up of many components that may
pathogens because of synergism between have synergistic effects; it may therefore be
expected that blends of essential oils or oil of alternative methods and expect to see
components will be produced to control a synergistic combinations of semi-chemicals
wide range of fungal species (Szczerbanik with other technologies that will enhance
et al., 2007). In the coming years, we envis- the effectiveness and sustainability of inte-
age a broader appreciation of the attributes grated control.
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Part II
Disease Control Through Resistance

6 Resistance to Septoria Leaf
Blotch in Wheat
María R. Simón
Cerealicultura, Facultad de Ciencias Agrarias y Forestales,
Universidad Nacional de La Plata, La Plata, Argentina
Abstract
Mycosphaerella graminicola (Fuckel) Schroeter, in Cohn, is the causal agent of Septoria leaf blotch, an
important disease in many wheat-producing areas of the world which causes significant yield losses.
Breeding for resistance is the most economical approach to control the disease. Advances in the genet-
ics of resistance and genetic variation of the pathogen population, as well as the new tools for a more
efficient incorporation of resistance in breeding programmes, are discussed.
Introduction bacteria, are important production constraints

in almost all wheat-growing environments
Bread wheat (Triticum aestivum L.) is the (Rajaram and van Ginkel, 1996; McIntosh,
most widely grown and consumed food 1998). Globally important fungal diseases of
crop in the world. It is the staple food of wheat caused by obligate parasites include
nearly 35% of the world population and the the three rusts (leaf rust, caused by Puccinia
demand for wheat will grow faster than for triticina Eriks., yellow rust caused by P.
any other major crop (Rajaram, 1999). The striiformis West f. sp. tritici Eriks. and stem
forecast global demand for wheat in the year rust caused by P. graminis Pers. f. sp. tritici
2020 varies between 840 (Rosegrant et al., Eriks & Henn); powdery mildew caused by
1995) to 1050 Mt (Kronstad, 1998). To meet Blumeria graminis tritici (DC) Speer; asexual
this demand, global production will need to form Oidium monilioides (Nees) Link; stink-
increase by 1.6–2.6% annually from the ing smut (Tilletia caries (DC) Tul. and C. Tul.
present production level of 620 Mt. and T. foetida (Wallr. Liro.); loose smut (Usti-
Wheat breeding is focused on develop- lago tritici (Pers.) Rostr.); U. nuda (J.L. Jensen)
ing widely adapted, disease-resistant geno- Kellerm. and Swingle. Those caused by fac-
types with high yields that are stable across ultative parasites include leaf blotch, M.
a wide range of environments. Incorporat- graminicola (Fuckel) J. Schröt., in Cohn,
ing durable resistance is a priority since asexual form S. tritici Rob ex Desm.; glume
breeding for stable yields without adequate blotch (Phaeosphaeria nodorum, asexual
resistance against the major diseases would form Stagonospora nodorum blotch); spot
be impossible (Rajaram, 1999). blotch (Cochliobolus sativus (Ito and Kurib-
Diseases of wheat, mostly caused by ashani) Drechs. ex Dastus, asexual form Bipo-
fungal pathogens and a few by viruses and laris sorokiniana (Sacc.) Shoem.); tan spot,

70 M.R. Simón
Pyrenophora tritici-repentis (Died.) Drechs., may reduce the effect of S. tritici blotch,
asexual form Drechslera tritici-repentis (Died.) genetic resistance is the most cost-effective
Shoemaker; Alternaria spp. (belonging to the and environmentally safe technique to man-
A. infectoria species groups); scab, Fusarium age the disease.
graminearum Schwabe, take all (Gaeu- Monogenic or oligogenic and polygenic
mannomyces graminis (Sacc.) von Arx and resistance coexist in the pathosystem T.
Olivier var. tritici Walker). aestivum/M. graminicola. Monogenic or oli-
Leaf blotch causes important yield gogenic resistance is generally near complete,
losses in many countries. Yield reductions isolate specific, follows the ‘gene-for-gene’
range from 31 to 54% (Eyal et al., 1987), from mode of inheritance and has been found in
10 to 45% (Caldwell and Narvaez, 1960) and several genotypes (Rillo and Caldwell, 1966;
even yield losses higher than 60% have been Rosielle and Brown, 1979; Lee and Gough,
reported (Shipton et al., 1971). Sanderson 1984; Somasco et al., 1996; Arraiano et al.,
(1972) proved the connection between the 2001; Brading et al., 2002; McCartney et al.,
two stages and the sexual (teleomorph) form 2002). Polygenic resistance is generally partial
has been reported in several countries (Hunter and isolate non-specific and is also present in
et al., 1999). The sexual stage in Argentina several genotypes (Jlibene et al., 1994; Simón
was reported by Cordo et al. (1990). and Cordo, 1997, 1998; Brown et al., 2001;
Mycosphaerella graminicola is a hemi- Zhang et al., 2001; Chartrain et al., 2004b).
biotrophic pathogen; early infection is bio- Partial resistance is expressed as a
trophic, followed by a switch to necrophic reduced epidemic development and is sup-
growth just prior to symptom expression. The posed to be durable. Several components
sexual stage is also known to play a role in the contribute to the epidemic-retarding effect.
disease cycle. It causes most of the initial Parlevliet (1979) mentioned four partial
infection of winter wheat crops during autumn resistance components: infection frequency,
in the UK (Shaw and Royle, 1989) and the latent period, spore production and infec-
USA (Schuh, 1990). In Argentina, an increase tion period. The earliest studies on this type
in ascospores at harvest time has been reported, of resistance, previous to the mapping of
suggesting that the sexual stage may be genes and QTLs, investigated the gene effects
important to initiate the infection in the next conditioning these components.
growing season. Following stem elongation, Several of the components of partial
infection of the upper leaves of a crop has been resistance to M. graminicola may be con-
thought to be entirely due to the asexual stage trolled by just a few genes (Jlibene et al.,
of the fungus, in which pycnidia give rise to 1994). Danon and Eyal (1990) determined
splash-dispersed pycnidiospores, which are that additive effects for pycnidial coverage
splash-dispersed from infected basal tissue to were the major variance component, although
the upper leaves by raindrops. However, more dominance effects were also significant.
recent work has shown that upward move- Jlibene et al. (1994) found that general com-
ment of inoculum can occur in the absence of bining ability (GCA) effects accounted for
splashy rainfall, being influenced by the posi- most of the variation of percentage pycnidial
tion of developing leaves in relation to infec- coverage, although specific combining ability
ted leaf layers (Lovell et al., 1997). Another (SCA) effects were detected in some crosses.
possible means of spread within a crop dur- Simón and Cordo (1997, 1998) determined
ing summer is by airborne ascospores, that GCA was preponderant for incubation
which may play a role more important than period, latent period, pycnidial coverage and
previously recognized (Hunter et al., 1999). spore production, although SCA was also
significant. Incubation period was inherited
independently of maturation period and pyc-
Types of Resistance nidial coverage. Those components that are
genetically different and independent could
Although several control methods, including be combined into the same genetic back-
cultural practices and the use of fungicides, ground by crossing (van Ginkel and Rajaram,
Resistance to Septoria Leaf Blotch 71
1999), increasing the level of durable resis- investigated the chromosomal location of
tance. Significant correlations were found resistance using substitution lines. Resis-
between pycnidia/cm2 and spore/ml, indi- tance was found to be located on chromo-
cating the feasibility of selecting for a lower some 7D from a synthetic hexaploid wheat
pycnidial density in order to obtain a reduc- (T. dicoccoides × T. tauschii) in seedling
tion in spore production (Simón and Cordo, and adult stage to some specific isolates
1998). Heritability tends to be only moderate (Simón et al., 2001, 2005b). Also, resistance
(Simón et al., 1998), but progress in breeding was found in chromosomes 1B at the seed-
for resistance may still be possible. Major ling stage and on 5D at the seedling and
genes are interesting because of the high adult stage of the T. aestivum cv. Cheyenne
level of resistance and thus an almost com- (Simón et al., 2001, 2005b); on the 2B, 3A
plete absence of symptoms in the host; par- and 3B of the T. aestivum cv. Cappelle-
tial resistance, however, is very important Desprez at the seedling stage and on 6D and
due to its putative durability and its expres- 7D of T. spelta with some specific isolates
sion under a broad spectrum of isolates of (Simón et al., 2001, 2005b).
the pathogen. A few genes may be enough to During the past decade, several genes
confer resistance that will hold up in farm- (Table 6.1) and QTLs (Table 6.2) have been
ers’ fields (Dubin and Rajaram, 1996). located. Some of them have proved to be
Resistance conditioned by a single domi- effective to isolates from several regions in
nant gene was assigned to some cultivars as the world. Simón et al. (2007) tagged, using
Bulgaria 88 (Rillo and Caldwell, 1966), Oasis isolates from Argentina, a gene in the 7D
(Shaner and Buechley, 1989), Veranopolis chromosome of Aegilops tauschii, which is
(Wilson, 1979) and others. Later, genes were likely Stb5. This would indicate that the
located and it was found for example that Stb1 presence of Stb5 ensures resistance against
conditioned resistance in Bulgaria 88 and some isolates from both Europe (Portugal,
Oasis, Stb2 in Veranopolis, etc. Some other The Netherlands) (Arraiano et al., 2001) and
cultivars showed resistance conditioned by South America (Argentina).
several major genes as Kavkaz 4500 L.6.A.4.
(Jlibene et al., 1992) and the genes were identi-
fied (Stb6, Stb7, Stb10 and Stb12; Chartrain
Breeding for Resistance
et al., 2005a). Also, three major genes were
identified in the Portuguese line TE 9111 (Stb6,
Stb7 and Stb11; Chartrain et al., 2005b). Fur- The incorporation of resistance to the patho-
thermore, commercially grown cultivars range gen has been slow for several reasons, among
from moderately resistant to susceptible, indi- them:
cating the presence of partial resistance. Char- 1. The high variability of the pathogen
train et al. (2004b) found high partial resistance population.
levels in several wheat cultivars from Europe 2. The lack of knowledge of the virulence
and Mexico (Arina, Milan, Senat). Simón et al. spectrum.
(2005a) also found high levels of partial resis- 3. The lack of relationship in the expression
tance in some Argentinian cultivars effective of resistance in seedling and adult stage.
to several isolates (Klein Volcán, Klein 4. The influence of heading date and plant
Dragón) in adult stage. Some germplasm as height on resistance and the difficulty in as-
the Portuguese line TE 9111 (Chartrain et al., sessing real values in breeding programmes.
2005b) also has been proved to carry several
major genes together with partial resistance.
Variability of the pathogen population

Location of the Resistance
The population of the pathogen has been
Studies on the location of resistance began studied and a high variability has been
during the past decade. Some of them found. Variation in virulence patterns within
72 M.R. Simón
Table 6.1. Major genes conditioning resistance to Mycosphaerella graminicola identified in hexaploid
wheat.
Chromosomal
Locus location Linked markers Reference
Stb1 5BL Xbarc74, Xgwm335 Adhikari et al., 2004c

Stb2 3BS Xgwn389, Xgwm533.1, Xbarc133, Xbarc75, Adhikari et al., 2004b
Xgwm493
Stb3 6DS Xgdm132 Adhikari et al., 2004b
Stb4 7DS Xgwm44, RC3, Xgwm11, Xgwm437, Xgwm121 Adhikari et al., 2004a
Stb5 7DS Xgwm44, RC3, Xgwm111, Xgwm437, Xgwm121 Arraiano et al., 2001
Stb6 3AS Xgwm369, Xwmc11 Brading et al., 2002
Stb7 4AL Xgwm160, Xwmc219, Xwmc313 McCartney et al., 2002
Stb8 7BL Xgwm146, Xgwm577, Xgwm611 Adhikari et al., 2003
Stb9 2B Chartrain, 2004
Stb10 1D Xgwm848 Chartrain et al., 2005a
Stb11 1BS Xbarc008, Xbarc137 Chartrain et al., 2005b
Stb12 4AL Xwmc219, Xwmc313 Chartrain et al., 2005a
Stb13 7BL Xwmc396-7B Cowling et al., 2007
Stb14 3BS Xwm632-3B Brule Babel, 2007
Stb15 6AS Xpsr563a, Xpsr904 Arraiano et al., 2007
Table 6.2. Quantitative trait loci (QTLs) conditioning resistance to Mycosphaerella graminicola in
hexaploid wheat.
Locus Chromosomal location Linked marker Reference
QStb.risø-2B 2BL Xwmc1575a-Xwmc175a Eriksen et al., 2003

QStb.risø-3A.1 3AS Xgwm369 Eriksen et al., 2003
QStb.risø-3A.2 3BL Xwmc489-Xwmc505 Eriksen et al., 2003
QStb.risø-3B 3B M62/P38-373 Eriksen et al., 2003
QStb.risø-6B.2 6B Xwmc397-Xwmc341 Eriksen et al., 2003
QtStb.risø-7B 7B M49/P38-229-M49/P11-229 Eriksen et al., 2003
QStb.ipk-1D 1D (seedlings) Xmwg938a Simón et al., 2004a
QStb.ipk-2D 2D (seedlings) Xcdo405a Simón et al., 2004a
QStb.ipk-6B 6B (seedlings) Xksuh4b Simón et al., 2004a
QStb.ipk-3D 3D (adult) Xbcd515 Simón et al., 2004a
QStb.ipk-7B 7B (adult) Xksud2a Simón et al., 2004a
and between populations was shown by high variability within populations has
assessing host response on a selected set of been confirmed (Chen and McDonald, 1996;
cultivars, with little similarity between the Zhan et al., 2001, 2003; Cordo et al., 2007).
results obtained with various sets of differ- The sexual state might have an impact on
entials (Eyal et al., 1995). Evidence for spec- the virulence spectrum in regions where
ificity was also confirmed by several pseudothecia were found and ascospore
researchers (Danon and Eyal, 1990; Kema dispersal coincided with the wheat growing
and van Silfhout, 1997; Simón et al., 2005a). cycle (Shaw and Royle, 1989; Lovell et al.,
Non-specific resistance to a wide set of iso- 1997). No attempts to determine races have
lates was also found (Simón et al., 2005a). been carried out.
During the past decades, the population has Recently, the genome of the pathogen
been studied using molecular markers and a was sequenced completely (Goodwin et al.,
2007). The essentially finished sequence con- that the relationship between those traits
tains 18 chromosomes from telomere to telom- was caused mainly by environmental and
ere, plus five fragments, which presumably epidemiological factors. Associations
make up two additional chromosomes. A between pycnidial coverage percentage and
comparative bioinformatics analysis of M. days to heading were positive or negative,
graminicola with seven other sequenced fun- depending on whether weather conditions
gal genomes revealed that it possessed fewer before the evaluations were more conducive
enzymes than expected for degrading plant to the development of the disease in late or
cell walls. The frequency of transposable ele- early heading cultivars, respectively. Nega-
ments in the genome of the pathogen was tive associations with plant height were
intermediate between those of other sequenced only present in the years where weather
fungi. Availability of the finished genome for conditions were less conducive to the devel-
M. graminicola should aid research on this opment of the disease. Inconducive condi-
organism greatly and will help in the under- tions and longer distances between leaves in
standing of its interaction with wheat. tall cultivars could have reduced the rain-
splash dispersal of pycnidiospores, thus
causing this negative association, mainly
Expression of resistance when the sexual form is not present.
in seedlings and adults In most cases, previous reported asso-
ciations between heading date and resis-
tance could be attributed to the fact that the
Resistance is sometimes expressed in seed-
disease was scored at the same time but not
lings, sometimes at adult stage and some-
at the same growth stage, causing early matur-
times at both stages (Kema and van Silfhout,
ing lines to be exposed to inoculum for a
1997). Some germplasm with resistance at
longer period than later maturing leaves.
both stages have been found (Arama, 1996;
Simón et al. (2009 unpublished) mapped a
Somasco et al., 1996; Simón et al., 2005a).
population derived from T. spelta 7D/Chi-
nese Spring where QTLs conditioning resis-
tance were found, but no genes for heading
Influence of heading date and date were present. Also, some QTLs for resis-
plant height on resistance tance were mapped in a Synthetic 6 × (T.
tauschii × Altar 84) × Opata 85 (Simón et al.,
One complicating factor for the assessment 2004a), which did not coincide with the
of resistance level has been the influence of regions where QTLs for flowering time were
heading date and plant height on the expres- previously mapped. Eriksen et al. (2003)
sion of resistance. Several scientists have located in a double haploid population origi-
reported an increased disease level in ear- nated from the cross of Savanah and Senat, a
lier heading or shorter cultivars (Eyal et al., QTL for increasing plant height linked to a
1987; van Beuningen and Kohli, 1990; Cama- QTL for resistance. Although associations
cho Casas et al., 1995; Chartrain et al., 2004a). could exist in some germplasm, pleiotropic
Baltazar et al. (1990) suggested a genetic effects have not been detected and breeders
association between shortness and suscep- can select for S. tritici blotch resistance within
tibility, while Eyal (1981) and Rosielle and a range of heading dates and plant heights.
Boyd (1985) assumed a genetic association
between earliness and susceptibility. Arama
et al. (1999), Simón et al. (2005a) and Arraiano Resistance and Integrated
et al. (2006) reported no genetic association Management
among those traits. Simón et al. (2004b,
2005a) determined that there was no influ- It is necessary to consider that integrated
ence of heading date when cultivars were management can contribute to the durability
evaluated at the same development stage of resistance. Epidemiological advantages
under similar weather conditions and found can be derived by combining management
74 M.R. Simón
practices and through disease management Conclusions

on a regional scale. Diversifying sources of
partial resistance, on a field or regional Research on Septoria leaf blotch has
basis, might slow pathogen adaptation. Pop- expanded greatly in the past decades. New
ulations of M. graminicola sampled from molecular tools enable the exploration of
mixtures of a susceptible and a partially biological issues associated with the patho-
resistant wheat cultivar were all less fit than gen, the host and the host–pathogen inter-
populations derived from the same cultivars action. Several genes and QTLs have been
grown in pure stand (Mundt et al., 2002). identified and mapped. The major chal-
Cultural practices such as adequate lenge to wheat breeders and plant patholo-
tillage method, planting density and N- gists is the selection and development
fertilization conditions, together with fungi- of cultivars with durable resistance. To
cide applications, are important to the achieve this goal, the incorporation of
appropriated expression of resistance. The marker-assisted selection into breeding
planting of no-till wheat may increase programmes will speed pyramiding several
the level of Septoria leaf blotch because genes or QTLs effective at different stages
increasing levels of crop residue on the soil of plant development into single wheat
surface potentially increase primary inocu- cultivars to develop broad-spectrum and
lum of plant pathogens, mainly under con- durable resistance.
tinuous wheat production or wheat/soybean Management of cultivars should be
sequences in the same year. Since the patho- optimized to minimize the associations
gen can survive in infested wheat residues between heading date, height and resis-
for about 2 years, a rotation where wheat is tance, but selection of early and short lines
planted in only 1 of 3 years is recommended. with high levels of quantitative resistance is
Although there are contrasting results, sev- possible. Progress in the analysis of vari-
eral reports indicate that, under conducive ability and virulence patterns of the patho-
conditions for the development of the dis- gen population is also necessary to test the
ease, an increase in N-fertilization causes a available germplasm with representative
slight increase in severity (Hayden et al., isolates. Durability of the resistance can be
1994; Howard et al., 1994; Leitch and Jen- enhanced by appropriate cultural practices
kins, 1995; Simón et al., 2002, 2003). and diversifying sources of resistance.
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7 Barley and Wheat Resistance
Genes for Fusarium Head Blight
S.A. Stenglein and W.J. Rogers

Laboratorio de Biología Funcional y Biotecnología (BIOLAB)-CEBB, Facultad
de Agronomía, Universidad Nacional del Centro de la Provincia de Buenos
Aires (UNICEN), Buenos Aires, Argentina and Consejo Nacional de Investigaciones
Científicas y Técnicas (CONICET), Argentina
Abstract
The genetic control of resistance to Fusarium head blight (FHB) in barley and wheat is reviewed. This
disease, which can reach epidemic proportions under certain climatic conditions, is caused by various
Fusarium species and affects grain yield and quality detrimentally, resulting in important economic
losses in both crops. Furthermore, FHB infection poses a serious threat to human and animal health,
due to the presence of toxic trichothecenes, of which deoxynivalenol and its derivatives appear to be
the most important. Marker-based mapping studies have identified numerous quantitative trait loci
(QTLs) for FHB resistance, located on all the chromosomes of both species. Only a relatively small
number of these can be detected consistently over a wide range of different environments and genetic
backgrounds. None the less, where genetic effects have been characterized, they have been shown to
be mainly additive in nature, meaning that the accumulation of several QTL factors in a single line
ought to be effective in achieving raised levels of resistance. Indeed, marker-assisted selection has
been directly shown to be feasible for some QTL. A number of QTLs for FHB resistance are associated
with other agronomic characters, such as heading date (HD), flowering time and plant height. In some
cases, QTL alleles favourable for resistance are associated detrimentally with alleles for these charac-
ters, although there appear to be sufficiently large numbers of QTLs for resistance acting indepen-
dently of these characters to imply that reasonable genetic gains for resistance ought to be achievable
in the future. While most studies in barley have addressed Type I resistance (initial infection) and in
wheat Type II (spread between spikelets), or a combination of both Type I and Type II, more recent
studies have addressed other types of resistance, such as Type III (effects on kernel size and character-
istics), Type IV (yield tolerance) and Type V (decomposition or non-accumulation of mycotoxins such
as deoxynivalenol). Besides identifying additional QTLs, these latter studies offer insights into the
mechanisms of the different types of resistance observed, in some cases blurring the distinctions
between them. Other prospects for improvement in FHB resistance, additional to those offered by
marker-assisted selection, are also discussed.
Introduction environments with prolonged wet climatic

conditions from flowering through the soft-
Fusarium head blight (FHB) or scab is a dough stage of kernel development (Parry
destructive disease of wheat and barley in et al., 1995; McMullen et al., 1997). The

Barley and Wheat Resistance Genes 79
disease is of worldwide importance. FHB have been isolated from naturally infected
epidemics have been documented in 26 US wheat or barley spikes and have been asso-
states and five Canadian provinces. Eco- ciated with FHB (Parry et al., 1995; Leonard
nomic losses in wheat since 1990 were esti- and Bushnell, 2003). Fusarium graminearum
mated at US$2.5bn (Windels, 2000). Wheat (teleomorph Gibberella zeae) is the most fre-
yields in 1993 were reduced by about 50% quently encountered pathogen and the most
in north-eastern North Dakota and 40% in virulent species, although F. avenaceum
north-western Minnesota, compared with (teleomorph G. avenacea), F. culmorum and
1992 (National Agricultural Statistics Ser- F. poae are reported to be prevalent in some
vice, 1993–1999). Barley losses have been European and North and South American
equally devastating, with estimated losses countries (Leonard and Bushnell, 2003;
from 1993 to 1999 totalling in excess of Barreto et al., 2004; Bourdages et al., 2006).
US$400m (Windels, 2000). In China, FHB The distribution and predominance of a
has affected more than 7m ha wheat and has Fusarium species in a region is thought to
caused yield losses of more than 1 Mt in be determined by climatic factors, competi-
severe epidemics (Leonard and Bushnell, tion among various Fusarium spp. sharing
2003). In Argentina, during the past 60 years, the same ecological niches, fertilizer use,
several FHB epidemics of varying severity cropping sequence and practices and vege-
have occurred in the central-north area, tation type (Snyder and Nash, 1968; Nelson
where yield losses were estimated to aver- et al., 1981; Doohan et al., 2003).
age between 20 and 50%. FHB reduces kernel set and kernel
FHB is a preharvest disease, but Fusar- weight. Invasion of the kernel by Fusarium
ium species can grow in postharvest phase destroys the starch granules and cell walls
if wet grain is not dried efficiently and and affects endosperm storage proteins,
quickly. More than 17 Fusarium species resulting in a poor quality product (Fig. 7.1).
Fig. 7.1. Shrivelled lightweight seeds of wheat affected by FHB (left) and healthy wheat seeds (right).
80 S.A. Stenglein and W.J. Rogers
Germination rate and seedling vigour are is often associated with more diseases. Gen-
reduced when the seeds are infected. erally, awned genotypes with short pedun-
In addition to causing significant yield cule and a compact spike have faster disease
losses, FHB is of greater significance under spread than genotypes that are awnless,
certain conditions because of the associated have a long peduncule and a lax spike (Rudd
mycotoxin accumulation which can occur et al., 2001). In addition, short saturated
in infected grain. Fusarium graminearum, genotypes with a long grain-filling duration
F. avenaceum, F. culmorum and F. poae can generally get more disease than tall geno-
produce a range of mycotoxins and contam- types that have rapid grain fill (Mesterhazy,
inated grain is unsuitable for animal and 1995). These morphological characteristics
human consumption because of the adverse contribute to resistance, but are often consid-
effects of such toxins on health (Placinta ered nuisance factors in screening nurseries,
et al., 1999; Gutleb et al., 2002). Within and it is generally agreed that they are of
Fusarium mycotoxins, some of the most minor significance compared with physio-
important from the point of view of animal logical resistance (Rudd et al., 2001). How-
health and productivity, are the trichoth- ever, morphological traits have also been
ecenes, zearalenone and the fumonisins associated with FHB resistance in barley.
(D’Mello et al., 1999). Type A and B tricho- Two-rowed barley is more resistant to FHB
thecenes represent the most important than six-rowed barley and, in crosses between
members of these mycotoxins. Type A tri- six-rowed and two-rowed genotypes, two-
chothecenes include T-2 toxin, HT-2 toxin, rowed progenies are most resistant, followed
neosolaniol (NEO) and diacetoxyscirpenol by genotypes heterozygous for spike type.
(DAS), while type B trichothecenes include Six-rowed types are most susceptible (Takeda
deoxynivalenol (DON, also known as vomi- and Heta, 1989; Xihang et al., 1991).
toxin) and its 3-acetyl and 15-acetyl deri- Mesterhazy (1995) described five types
vates (3-DON and 15-DON, respectively), of physiological resistance, expanding the
nivalenol (NIV) and fusarenon-X (FUS-X). two types described by Schroeder and Chris-
A common feature of many Fusarium spe- tensen (1963). These include Type I resis-
cies is their ability to synthesize zearale- tance to initial infection. It may be passive,
none (ZEN or F-2 toxin) and its co-occurrence involving morphological characteristics of
with certain trichothecenes raises important wheat head. Alternatively, Type I resistance
issues regarding additivity and/or syner- may be active and include defence reactions
gism in the aetiology of mycotoxicosis in such as the activation of enzymes degrading
animals (Placinta et al., 1999). Fumonisins the fungal cell wall or pathogenesis-related
are an increasingly important group of tox- (PR) proteins (Nicholson et al., 2005). This
ins as they have been postulated as the type of resistance is estimated by spraying a
causative agent for several endemic dis- spore suspension over flowering spikes and
eases, both in humans and animals (Syden- counting diseased spikelets. Type II refers
ham et al., 1990; Chu and Li, 1994). to the resistance of movement of the patho-
Host resistance has long been consid- gen from one infected spikelet to another via
ered the most practical and effective means the rachis. The mechanisms involved in
of disease handling, but breeding for FHB Type II resistance are thought to be active,
resistance has been hindered by a lack of but again may be due to morphological char-
effective resistance genes and by the com- acteristics. This type of resistance is estimated
plexity of the resistance in identified sources by delivering conidia into a single floret of a
(Mesterhazy, 1997). No source of complete spike and counting the blighted spikelets after
resistance is known and current sources a period of time. The other types of resistance
provide only partial resistance. include: kernel size and number retention
Resistance types are generally classified (Type III), yield tolerance (Type IV) and
as either morphological or physiological. decomposition or non-accumulation of myco-
Head anatomy or positioning that contribu- toxins (Type V). Type III resistance is
tes to higher humidity around the spikelets measured by threshing infected spikes and
observing the damage to the kernels. Kernel was identified as one of the most resistant
number reduction, kernel weight, test weight, two-rowed barley accessions and also accu-
or visual estimates of Fusarium-damaged mulated low concentrations of DON (Urrea
kernels (tombstones) are common measure- et al., 2005).
ments used to assess this resistance. Type Six-rowed types are preferred for malt-
IV resistance, or yield tolerance, can be ing, but they are generally more susceptible
assessed by measuring grain yield of natu- to FHB than two-rowed barley. Chevron, an
rally or artificially inoculated spikes or plots old cultivar from Switzerland, is a six-rowed
and comparing the data with spikes or plots malting barley and a popular parent in bar-
that do not show disease symptoms (Rudd ley breeding programmes. It has high resis-
et al., 2001). Finally, Type V resistance is tance to kernel discoloration, which is a
identified by measuring DON concentration disease complex caused by several different
at a given level of FHB (Rudd et al., 2001). fungal pathogens, including Fusarium.
This resistance is important from a grain In China and Japan, over 10,000 barley
utilization perspective, for example for accessions from different countries have
malting barley, because even trace levels of been screened for FHB resistance, but only
DON may reduce beer quality significantly. several dozen accessions have a low level of
Considerable progress in the search for FHB (Xihang et al., 1991; Zhou et al., 1991).
host resistance has been made. Improve- To date, no wild species of Hordeum have
ment of cultivar resistance has become a shown greater resistance than that of two-
major breeding objective worldwide. Recent rowed barley. DON content in even the best
developments in genomic research and bio- sources of resistance are still well above the
technology hold promise for understanding specification for the brewing industry
the genetic mechanisms of FHB resistance (< 0.5 mg/kg), but much lower than that of
and allow more effective utilization of FHB current commercial malting barley cultivars
resistance genes to develop new resistant (Leonard and Bushnell, 2003).
wheat and barley cultivars. Investigation of the genetics of resis-
tance to FHB in barley has not been very
extensive and published reports on the iden-
tification of loci controlling FHB resistance
Genetics of FHB Resistance in Barley and DON accumulation are limited (Rudd
et al., 2001). Barley producers currently
Few sources of FHB resistance have been attempt to manage the disease through crop
found in barley and the level of their resis- rotation and fungicide application. How-
tance is modest. Although FHB in barley ever, these measures alone are not sufficient
usually does not spread from spikelet to to reduce the risk of the disease. Resistant
spikelet within a spike (up and down the barley cultivars are the most cost-effective
spike), barley seems to be very susceptible measures for controlling the disease, but
to initial infection. Severe disease usually breeding for FHB resistance has been diffi-
results from multiple initial infections in cult for several reasons. One, genetic resis-
the spike. tance is complex. There seem to be many
Of primary importance to barley breed- QTLs that have relatively small effects and
ers are data on FHB severity and DON con- are subject to genotype × environment inter-
centration, since these are traits that affect actions. Two, FHB screening experiments
the marketing of grain in malting most are labour-intensive and expensive. Three,
severely. The first sources of resistance used assessing FHB severity in both the field and
were the breeding lines Gobernadora from the greenhouse is difficult. Disease severity is
ICARDA/CIMMYT in Mexico and Zhedar 1 correlated strongly with HD and other agro-
and Zhedar 2 from China. All three lines had nomic and spike morphology traits. Since
the two-rowed spike morphology. Other two- infection can occur only after the spike
rowed barley with low DON content were emerges from the boot, differences in HD
CI 4196, Svanhals and Imperial. CI 4196 make it difficult to distinguish ‘true’ disease
resistance from ‘apparent’ resistance that is and was found on chromosome 2(2H). The
due to host escape from the pathogen. Both QTL on chromosome 4(4H) explains 4–12%
of these problems necessitate the identifica- of the phenotypic variation for FHB resis-
tion of molecular markers linked to QTLs for tance. This QTL was also associated signifi-
FHB resistance that can be used in marker- cantly with morphological traits including
assisted breeding. In addition, since disease plant height, seeds per inflorescence, inflores-
expression is influenced strongly by the cence density and lateral floret size. In each of
environment, comparisons among barley the previous mapping studies, QTLs for accu-
genotypes that differ in HD are themselves mulation of DON in harvested grain were also
confounded by the effect of the environment detected. These QTLs were also distributed
on disease development. However, because throughout the genome and were, in some
of the complex nature of genetic resistance to cases, coincident with FHB QTL. Taken
FHB, QTL identification is not always very together, these studies indicate resistance is
robust. Therefore, validation of these QTLs is conditioned by many loci and that there is a
important before implementing marker- strong association between certain morpho-
assisted selection in a breeding programme. logical traits and FHB resistance.
To gain a genetic understanding of FHB Two major traits associated with FHB
resistance in barley, multiple sources of severity are spike type and HD. The Vrs1
resistance including Chevron (de la Pena and Int-c loci control lateral floret fertility
et al., 1999; Ma et al., 2000), Gobernadora and hence determine whether a spike is two-
(Zhu et al., 1999), Fredrickson (Mesfin et al., rowed (Vrs1; int-c/int-c) (Lundqvist and
2003; Smith et al., 2004), Zhedar 2 (Dahleen Franckowiak, 1997) or six-rowed (vrs1/vrs1;
et al., 2003) and CI 4196 (Horsley et al., 2006) Int-c/Int-c) (Hockett and Nilan, 1985). In sev-
have been used in QTL mapping studies. eral studies, the two-rowed spike type has
QTLs providing resistance to FHB and been associated with FHB resistance (Chen
DON accumulation in barley have been et al., 1991; Xihang et al., 1991; Steffenson
identified on all seven chromosomes. QTLs et al., 1996; de la Pena et al., 1999). In a genetic
for FHB resistance were identified on chro- study, Takeda (1990) demonstrated an asso-
mosomes 1(7H), 2(2H), 3(3H), 4(4H), 5(1H) ciation between the Vrs1 locus and FHB
and 7(5H) in the Chevron (resistant)/M69 resistance. In two-rowed barley (Vrs1) with
(susceptible) population (de la Pena et al., the Int-c/Int-c genotype, the laterals can be
1999). A major QTL on chromosome 2(2H) inflated and lateral floret size has been asso-
explains 13.5% of the phenotypic variation ciated with FHB severity (Zhu et al., 1999).
for FHB resistance. However, this QTL is also The FHB mapping studies published to date
associated with HD and the resistant allele is have used populations derived from either
linked to late heading. Ma et al. (2000) used a six-rowed × six-rowed or two-rowed × two-
population derived from the cross Chevron/ rowed crosses (de la Pena et al., 1999; Zhu
Stander and reported nine QTLs for FHB et al., 1999; Ma et al., 2000). Therefore, the
resistance located on chromosomes 1(7H), Vrs1 locus was not segregating in these pop-
2(2H), 3(3H), 6(6H) and 7(5H). A QTL on ulations. HD may also strongly influence the
chromosome 2(2H) was detected consistently severity of FHB on barley and QTLs for HD
in five environments and explained 11.8– and FHB resistance are coincident (de la
20.7% of the phenotypic variation for FHB Pena et al., 1999; Ma et al., 2000). Generally,
resistance. This QTL, in addition to the QTL late heading plants tend to have lower sever-
on chromosome 2(2H) discovered by de la ity, while early heading plants have higher
Pena et al. (1999), is also associated with days severity, indicating that the late heading
to heading. Using a population derived from plants are exposed to the inoculum for a
the two-rowed parents, Gobernadora and shorter period of time (Leonard and Bush-
CMB 643, Zhu et al. (1999) found QTLs for nell, 2003).
FHB resistance on all barley chromosomes In all of these studies except the one
except chromosome 7(5H). The largest QTL using Gobernadora, the bin 8 region of the
explained 33% of the phenotypic variation long arm of chromosome 2H designated by
Horsley et al. (2006) as Qrgz-2H-8 was asso- F. poae are also the cause of the disease in
ciated consistently with FHB severity, HD some environments. Epidemics may cause
and DON concentration. The approximate major losses when climatic conditions are
size for the overlapping QTL region ranged favourable after flowering (Paillard et al.,
from 22cM in the Fredrickson/Stander pop- 2004). As in barley, agricultural management
ulation (Mesfin et al., 2003) to 45cM in and fungicide treatments, while reducing the
Chevron/M69 (de la Pena et al., 1999) and damage (Gervais et al., 2003), are not wholly
CI 4196/Foster (Horsley et al., 2006) popu- effective (Stack, 1989; Bai and Shaner, 1994;
lations. Depending on the population and Parry et al., 1995). Unfortunately, complete
the environment, Qrgz-2H-8 explained 7–60% FHB resistance is unknown, although long-
of the variation in FHB resistance, 12–30% term control of the disease is probably most
of the variation in HD and 10–30% of the likely to be achieved through genetic resis-
variation in DON concentration. In all of the tance research, involving QTL mapping and
studies, FHB severity and DON concentra- other procedures (see below), and its conse-
tion were correlated negatively with HD. In quent application in the breeding of resis-
a validation study of this QTL, the Chevron tant cultivars. This appears to be the case, in
introgression at the Qrgz-2H-8 region reduced spite of the complexity of the genetic control
FHB by 42% and increased HD by 3.8 days involved, the presence of confounding envi-
(Canci et al., 2004). ronmental effects, the influence of geno-
The association between lower FHB type × environment interaction and the fact
severity and late heading may be due to that laborious inoculation and evaluation
shorter inoculum exposure (pleiotropy) or procedures in mature host plants are required
tight linkage of separate genes for flowering in order to identify useful marker associa-
time and disease resistance (Leonard and tions (Snidjers, 1990; van Ginkel et al., 1996;
Bushnell, 2003). To determine if the associa- del Blanco et al., 2003). A further complica-
tion between late HD and FHB resistance is tion is that associations between FHB resis-
due to linkage or pleiotropy, Nduulu et al. tance with HD, flowering time (FT) and
(2007) constructed a fine map for the chromo- plant height (PH) have also been observed
some 2(2H) QTL region using recombinant (Mesterhazy, 1997; Hilton et al., 1999; Buer-
near isogenic lines (rNILs) derived from a stmayr et al., 2000).
cross between a BC5 line carrying the Chev- For breeding purposes, three broad ori-
ron alleles for markers at the Qrgz-2H-8 region gins of resistant germplasm have been rec-
and the recurrent parent M69, and concluded ognized (Gilbert and Tekauz, 2000; Paillard
that the relationship between FHB and HD at et al., 2004): (i) spring wheat from Asia (e.g.
the Qrgz-2H-8 region was likely due to tight cv. Ning 7840 [China], cv. Sumai 3 [China],
linkage rather then pleiotropy. cv. Nobeokabozu [Japan]); (ii) spring wheat
from South America (e.g. cv. Frontana [Bra-
zil]); and (iii) winter wheat from Europe
(e.g. Arina, Praag-8, Novokrumka). Further
Genetics of FHB Resistance in Wheat examples of individual resistant cultivars
are given in the studies described below,
Besides similar considerations as for bar- which are all concerned with bread wheat,
ley regarding the detrimental effects of FHB unless specified otherwise.
on grain yield and quality in general, and In contrast to barley, FHB generally
the effects of mycotoxins on human and live- spreads between spikelets (although it is
stock health, the fact that the disease results currently unclear whether this is so for F.
in the degradation of the endosperm storage poae) and most genetic research has there-
proteins means specifically that the quality fore concentrated on Type II resistance (most
of bread, biscuit, pasta and other industrial frequently evaluated after single-spikelet
products can be seriously prejudiced. World- inoculation with F. graminearum), although
wide, the species F. graminearum predomi- combined evaluation of Type I and Type II
nates, but F. avenaceum, F. culmorum and resistance through spray inoculation has
also been widely carried out. However, explaining the differences in means between
there are an increasing number of studies parental, F1, F2 and backcross generations
that address other types of resistance, such (Mather and Jinks, 1982), that most of the
as the ability to detoxify DON (Type V) and observed genetic variation could be explained
the ability to maintain grain yield in spite of by additive effects, where dominant and
disease symptoms (Type IV). epistatic effects accounted for only a small
The first QTL mapping studies were proportion of the genetic effects present in
carried out in the mid-1990s (Bai, 1995; the crosses analysed. The authors pointed
Moreno-Sevilla et al., 1997), involving the out that this implied that it should be possi-
use of RFLP and RAPD markers to map Type ble to accumulate different genes to improve
II resistance. However, the marker associa- resistance to FHB. The mainly additive
tions identified individually accounted for nature of genetic effects was also observed in
only a small proportion of the variation, per- the soft red winter wheat, Ernie (Liu et al.,
haps due to the relatively low level of poly- 2005).
morphism observed for the markers employed In a subsequent study involving Type II
(Bai et al., 1999). Subsequently (Bai et al., resistance after inoculation with F. grami-
1999), AFLP markers were applied to a map- nearum and F. culmorum (applied separately)
ping population involving the relatively of a mapping population derived from the
resistant cv. Ning 7840 (Type II resistant bread wheat cross cv. CM-82036 (resistant,
cultivar), where the main specific character a line derived from Sumai 3) × cv. Remus
measured was the area under disease prog- (susceptible) and using RFLP, AFLP, SSR
ress curve (AUDPC) after F. graminearum and endosperm storage protein markers
single-spikelet inoculation. One major QTL (Buertsmayr et al., 2002), the large effect of
was identified accounting for up to 60% of Qfhs.ndsu-3BS (up to 60% of variation
the observed variation, which, although orig- accounted for) was again confirmed and two
inally thought to be located on 7B, was iden- further QTLs were located to 5A and 1B.
tified subsequently as being equivalent to the The 3BS and 5A QTLs were flanked with
QTL identified on chromosome arm 3BS SSR markers and the 1B QTL associated
(designated Qfhs.ndsu-3BS) (Waldron et al., with the Glu-B1 locus encoding high molec-
1999) and present in one of the ancestral cul- ular weight glutenin subunits. In a second
tivars of cv. Ning 7840, namely cv. Sumai 3. part of this study (Buertsmayr et al., 2003),
Two years later (Anderson et al., 2001), the the authors extended the analysis to include
same group verified the presence of this QTL combined Type I and Type II resistance; they
(up to 41.6% of the variation accounted for) in found that, under spray inoculation, Qfhs.
Sumai 3 and located two further QTLs from ndsu-3BS had a much larger effect than the
Sumai 3 on 6AS (up to 11.6%) and 6BS (up to 5A QTL, which they named Qfhs.ifa-5A,
9.2%). The susceptible parent, cv. Stoa, was whereas after single-spikelet inoculation, the
also shown to carry two QTLs for resistance, two loci showed effects of similar magni-
on 2AL (up to 14.3%) and 4BS (up to 7.2%). tude. Qfhs.ndsu-3BS appeared to be associ-
A further QTL from a third line, ND2603 ated mainly with resistance to fungal spread
(partially resistant), was located on 3AL (up (Type II), whereas Qfhs.ifa-5A appeared to
to 9.1%), in this case in a cross with the sus- be associated principally with fungal pene-
ceptible cv. Butte 86. These studies referred tration, and might contribute primarily
to Type II resistance (0–100% FHB severity towards Type I resistance and, to a lesser
scale after F. graminearum single-spikelet extent, towards Type II. In both these stud-
inoculation). ies, no isolate × wheat genotype interaction
During this period, in crosses between was observed, consistent with the previ-
six resistant Chinese bread wheat cultivars ously observed non-specific or horizontal
with two susceptible cultivars (Bai et al., nature of resistance (Mesterhazy, 1995; van
2001), where AUDPC was evaluated after F. Eeuwijk et al., 1995), which was particu-
graminearum single-spikelet inoculation, it larly interesting in this case since the two
was shown, from joint scaling tests aimed at isolates used belonged to different species
(F. graminearum and F. culmorum). The detected were located on 2AL, 3AL, 3BL,
authors concluded that FHB resistance 3DS and 5AL. The authors concluded that
depended on a few (2–3) major QTLs, operat- FHB resistance was polygenic, rather than
ing together with unknown numbers of minor the bimodal distribution observed in some
genes. They pointed out that marker-assisted previous studies (Bai et al., 1999; Waldron
selection (MAS) for the major QTLs ought to et al., 1999; Buertsmayr et al., 2002). The
be a feasible method of accelerating the 2AL QTL was located at the same map posi-
development (through breeding that included tion as one originating from cv. Stoa (Wal-
use of backcrosses) of resistant cultivars that dron et al., 1999; Anderson et al., 2001) and
combined Type I and Type II resistance. the 5AL QTL in the same position as one
They felt that marker-mediated transfer of identified previously (Gervais et al., 2003).
the QTL to durum wheat also ought to be In contrast, the 3DS QTL was located differ-
feasible, given that no D genome chromo- ently compared to one identified previously
somes were involved in the QTL identified. on this arm (Shen et al., 2003a). The major
The effect of Qfhs.ndsu-3BS was also 6D and 5B QTL overlapped completely with
observed in several other studies (Kolb a QTL for HD and the 6D QTL overlapped
et al., 2001; Zhou et al., 2002; Bourdoncle partially with a QTL for PH. However, QTLs
and Ohm, 2003; del Blanco et al., 2003; for PH were identified that were not associ-
Shen et al., 2003a; Xie et al., 2007). Effects ated with FHB resistance. The data could
on 2A and 2B have also been observed in not distinguish pleiotropic effects from
analyses involving Sumai 3 (Zhou et al., linkage. A further study involving cv. Arina
2002). In one study (Yu et al., 2006), it was (crossed to cv. NK93604) failed to detect the
suggested that the 3BS, 5AS and 6BS resis- same QTL (Semagn et al., 2007); instead,
tance QTLs of Sumai 3 were derived from QTLs on 1BL and 6BS from Arina and on
the Chinese landrace, Taiwan Xiaomai. 1AL and 7AL from NK93604 were detected.
QTLs on chromosomes 2A, 3A, 3B and A study of Arina crossed to the susceptible
5A, which had been observed previously in UK cultivar, Riband, identified at least 10
Asian wheats, were also observed in RILs QTLs, very few of which were coincident
derived from a cross between the European with the other Arina studies; the most con-
winter wheat cultivars, Renan (resistant) and sistent was a major QTL on 4DS (Draeger
Récital (susceptible), using spray inoculation et al., 2007), detected in four of the five
of F. culmorum (Gervais et al., 2003). In the environments evaluated.
same study, new QTLs were identified on In the winter wheat cross cv. Patter-
2BS and 5AL. Although co-localization of son × cv. Fundulea F201R (resistant cultivar
QTLs for resistance with awnedness (5A), from Rumania), QTLs for Type II resistance
PH (5A) and FT (2B) was observed, the were found on 1B, 3A, 3D and 5A, with the
authors considered that it should be possi- 1B and 3A consistent over experiments
ble to produce resistant lines independent (Shen et al., 2003b).
of these characters. It appears that, whereas Sumai 3 and its
In RILs obtained from the Swiss winter derivatives have major QTLs on 3B and 5A,
wheat cross cv. Arina (resistant) × cv. Forno the three winter wheat populations so far
(susceptible) characterized with microsatel- characterized seem to depend more on the
lite and RFLP markers and subjected to spray accumulation of moderate and minor QTLs.
inoculation with F. graminearum (combined The 3BL QTL located in the Renan/Récital
Type I and II resistance), eight QTLs were population may be the same as that observed
identified that together explained 47% of in the Arina/Forno population.
the variation (Paillard et al., 2004). Three of In a cross of the resistant Brazilian cv.
these were considered of major effect: 6DL Frontana with the susceptible cv. Remus
(22%), 5BL (14%, contributed by the sus- (Steiner et al., 2004) inoculated with F.
ceptible parent) and 4AL (10%). The authors graminearum and F. culmorum, a major
considered that these were different from QTL accounting for 16% of the variation in
QTLs previously reported. The other QTLs FHB severity and incidence was located on
3A and a QTL accounting for 9% of the vari- fungal DNA content (FDNA), relative spike-
ation in FHB severity was located on 5A. let weight (RSW) and per cent of Fusarium-
Smaller effects for severity were located on damaged kernels (FDK); although this may
1B, 2A, 2B, 4B, 5A and 6B. The resistance of be due to linked genes, the authors consid-
Frontana was found to be due principally to ered it more likely to represent one resis-
the inhibition of fungal penetration (Type tance gene (which appeared to be linked to
I), but with a minor effect on fungal spread the Rht-D1 locus, an association that may
(Type II). PH, FT and spike morphology prejudice attempts to improve resistance in
influenced FHB reaction, but co-localization germplasm containing the Rht-D1b (Rht)
of QTLs was observed only for minor QTL, semi-dwarfing allele). In this study, further
and sufficient QTLs for FHB resistance act- QTLs were observed as follows, whose
ing independently of these characters were detected presence varied over environments:
observed in order to allow selection of resis- AUDPC: 1BL, 2B, 6BL, 7AL, 7BL, 7DL; DON
tant lines with any height, flowering date content: 6BL, 7DL; FDNA: 3DL, 6BL, 7BL;
and spike morphology. RSW: 1BL, 2AS, 6BL, 7DL; FDK (Type III):
Seven QTLs for Type I and II resistance 5AS, 7AL; yield loss (Type IV): 7AL.
were found on 1BS, 1DS, 3B, 3DL, 5BL, 7BS In a study involving lines derived from
and 7AL in a cross between cv. Cansas the cross CM-82036 × Remus (Lemmens
(moderately resistant) and cv. Ritmo (sus- et al., 2005), the QTL on 3BS derived from
ceptible). The 1DS QTL seemed primarily to Sumai 3, closely associated with resistance
involve resistance to fungal penetration, to spread of the disease (Type II), appears to
while the other QTLs were concerned mainly convert DON to DON-3-O-glucoside. The
with resistance to fungal spread (Klahr authors hypothesized that the 3BS QTL
et al., 2007). Significant correlations with encoded a DON-glucosyl-transferase or reg-
PH and HD were observed. ulated the expression of this.
The Qfhs.ndsu-3BS region of Sumai 3 In a cross involving cv. CJ 9306 (Jiang
has been fine mapped and named Fhb1 et al., 2007), two QTLs were found for resis-
(Cuthbert et al., 2006), as well as being vali- tance to DON accumulation, QFhs.nau-2DL,
dated by near-isogenic line studies (Cuth- explaining up to 20% of the observed varia-
bert et al., 2007). A second region on 6BS tion, and QFhs.nau-1AS, explaining 4–6%.
has also been fine mapped and named Fhb2 The QTLs, QFhs.ndsu-3BS (up to 23% of
(Pumphrey et al., 2007). the variation) and QFhs.nau-5AS (4–6%)
Over recent years, attention has turned were also validated. QTL × environment
towards other types of resistance. For exam- interaction was found for QFhs.nau-2DL
ple, in the partially resistant cultivars, only. The authors suggested that marker-as-
Wuhan-1 and Maringa, QTLs for the accu- sisted selection would be effective and made
mulation of DON (Type V) were located on suggestions for the particular markers to be
2DS and 5AS (as well as QTLs for FHB resis- used, either singly or in combination. They
tance on 2DL, 3BS and 4B) (Somers et al., also validated QFhs.ndsu-3BS for resistance
2003). QTLs were located on 5A (12.4%), to grain yield loss (Type IV). No QTL inde-
2A (8.5%) and 3B (6.2%) for low DON con- pendent of Type II resistance was found.
tent in the Chinese landrace, Wangshuibai In many of the above studies, markers
(as well as QTLs for Type II resistance on 3B closely linked to the FHB resistance QTL
and 2A (Ma et al., 2006)). In the previously were identified, enabling MAS to be con-
cited study on Arina × NK93604 (Semagn templated. For example, SSR markers for
et al., 2007), the QTLs located on 1AL and the 3A and 5A QTL in Frontana have been
2AS were associated with DON content, identified, allowing these to be combined
although only 1AL was associated with FHB through MAS with the QTL in Sumai 3 and
resistance. In the additional Arina study its derivatives. The feasibility of MAS has
cited, involving Arina × Riband (Draeger been directly demonstrated (Wilde et al.,
et al., 2007), the major 4DS QTL identified 2007), involving the 3B and 5A resistance of
was found to affect AUDPC, DON content, Sumai 3 and the 3A resistance of Frontana;
MAS for the two Sumai 3 QTLs gave signifi- of wheat, whose detected presence and mag-
cant reductions in FHB severity and DON nitude of effects depend greatly on environ-
content, although MAS for the Frontana mental factors and the particular genetic
QTL had no effect. Additional phenotypic background in which they are evaluated. In
selection acting on other unmarked QTLs this sense, the genetic control of resistance
should give additional gain. Some markers appears to be complex, even though genetic
have been used extensively in breeding pro- effects appear to be mainly additive in
grammes (Guo et al., 2006). nature. The situation may be set to become
Some of the above reports are particu- more complicated still: although, as men-
larly illuminating, since they appear to be tioned previously, FHB resistance is thought
showing that the various types of resistance to be non-specific or horizontal, recent stud-
are not necessarily truly distinct categories. ies indicate that interactions may be more
For example, the Sumai 3 3BS resistance complex (Xihang et al., 1991).
generally has been regarded as being of
Type II. However, this locus may in fact be
involved in detoxifying DON (Type V resis-
tance). That is, it may be that at least a part Conclusions
of the mechanistic basis of the Type II resis-
tance associated with this locus is its Type Although handling of FHB requires the
V nature. application of several different disease man-
The map-based cloning of QTL ought to agement strategies, substantial progress has
contribute to understanding resistance mech- been made in understanding the genetic
anisms further (Liu and Anderson, 2003; basis of resistance to FHB in wheat and bar-
Shen et al., 2006). An expressed sequences ley. Quantitative resistance usually is caused
tag (EST) rich in leucine and with low simi- by the simultaneous segregation of several to
larity to a protein kinase domain of the Rpg1 many genes and diverse non-genetic factors.
gene in barley was identified on 3BS and Of the several types of resistance that have
might represent a portion of a gene for FHB been hypothesized or reported, Type II
resistance (Shen et al., 2006). This EST resistance is the most stable and well stud-
could be used in MAS and for map-based ied. The Chinese wheat cultivar, Sumai 3,
cloning. Resistance gene analogues (RGA) and its derivates are one of the best sources
associated with 1AL have been identified of resistance to FHB and may provide the
(Guo et al., 2006); all RGA markers studied maximum degree of Type II resistance. The
contained a heat shock factor that initiated major QTL on chromosome 3BS is found in
the production of heat shock proteins. Other most of the resistant cultivars from China.
promising areas for improvements in FHB However, QTLs located on all the other
are: (i) the introduction of genes from related chromosomes have also been reported but,
species (QTLs for FHB resistance have been for many of them, their expression is not
identified on 3A in Triticum dicoccoides stable over different environments or in all
(Otto et al., 2002) and on 4A in T. macha genetic backgrounds.
(Steed et al., 2005)); and (ii) the genetic Only a few barley cultivars have a rela-
engineering of FHB resistance by, for exam- tively higher level of FHB resistance. Most
ple, the expression in wheat of Arabidopsis of these resistant cultivars are two-rowed
NPR1 (Makandar et al., 2006). barley. Within six-rowed barley, which is
The above studies (and others not preferred for malting, the cultivar, Chevron,
included here due to space confines, some has the best degree of resistance, but its
of which are cited in the ‘Catalogue of gene DON level is still too high and far from
symbols for wheat’ [Mclntosh et al., 2003] meeting the safety requirements of the brew-
and subsequent annual supplements pub- ing industry. In contrast to wheat, Type I
lished in the Annual Wheat Newsletter) resistance is the major resistance type in
demonstrate that QTLs for FHB resistance barley. Molecular mapping indicates that
have been identified on all the chromosomes many QTLs, spread over many chromosomes
and with minor effects, control this resis- and the application of high-throughput mark-
tance. Correlation between FHB severity ers for FHB-resistant QTLs may improve
and other spike-related traits has presented selection efficiency significantly. Moreover,
a major barrier to breeding for FHB resis- recent developments in genomics and bio-
tance in barley. Using MAS for the Chev- technology hold promise for understanding
ron allele at the Qrgz-2H-8 locus should the genetic mechanism of FHB resistance
help breeders surpass this barrier (Nduulu and for more effective development of resis-
et al., 2007). tant wheat and barley cultivars. Functional
Marked-assisted selection may provide genomics tools such as microarray analysis
such a technique for dissecting and stacking and ESTs open a new way for genome-wide
different resistant QTLs for FHB resistance gene expression profiling.
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8 Sustainable Management of Rice Blast
(Magnaporthe grisea (Hebert) Barr):
50 Years of Research Progress in
Molecular Biology
S. Nandy,1 N. Mandal,2 P.K. Bhowmik,1 M.A. Khan3 and S.K. Basu4

1Bioproducts and Bioprocesses, Lethbridge Research Center, Agriculture and
Agri-Food Canada, Lethbridge, Canada; 2Bidhan Chandra Krishi Vishavidalay,
Nadia, India; 3Department of Weed Science, NWFP Agricultural University,
Peshawar, Pakistan; 4Department of Biological Sciences,
University of Lethbridge, Lethbridge, Canada
Abstract
Rice blast fungus (Magnaporthe grisea (Hebert) Barr) as a species has a very broad host range, infecting
more than 40 Graminaceous hosts and some other non-grass hosts. The seedling stage, the rapid tiller-
ing stage after transplanting and the flower emergence stage have been identified as the most suscep-
tible to rice blast. In developing countries, poor farmers cannot afford to control blast disease by the
application of expensive fungicides. Therefore, sustainable rice blast disease management is more
important for environmental concern, as well as for better financial returns to farmers in Third World
countries. During the past few decades, a substantial amount of research has been conducted all over
the globe to cope with blast fungus. In this chapter, we emphasize specifically the molecular biological
aspect of the study on rice blast fungus over the past 50 years.
Abbreviations used: BRV: blast-resistant varieties; HR: hypersensitive response; RBD: rice blast
disease; RBF: rice blast fungus; RGAs: resistance gene analogues; ROI: reactive oxygen intermediates;
PCR: polymerase chain reaction; RAPD: random amplification of polymorphic DNA; RFLP: restriction
fragment length polymorphism.
Introduction threat to the supply of this staple food for

nearly one-half of the world’s population
Many rice researchers consider blast to be (Zhu et al., 2000; Talbot, 2003). The rice
the most important disease of rice worldwide blast fungus (RBF), scientifically known as
(Valent and Chumley, 1994). This is because M. grisea (Hebert) Barr (anamorph: Pyricu-
the disease is widely distributed (85 coun- laria grisea Sacc.), is a filamentous Ascomy-
tries) and can be very destructive when cetous fungus that parasitizes over 40
environmental conditions are favourable. grasses, including economically important
Rice blast causes between 10–30% yield crops like wheat, rice, barley and millet
losses worldwide in rice, posing a constant (Ou, 1985), but the pathogen is best known

Sustainable Management of Rice Blast 93
as the casual agent of the rice blast disease occurred within 6–10 h at 20–30°C in the
(RBD). RBD is one of the most serious dis- presence of water on the surface of the leaf
eases in all rice-growing regions of the world. (Asuyama, 1965; Ou, 1985). The formation
Under heavy dew, all aerial parts of the plant of dew or a little rainfall or the occurrence
can be affected; leaf surfaces become speck- of fog provided the necessary water required
led with oval to globular lesions and severely for the germination of spores. Analysis of
infected plants are liable to lodging if stems the intensity of infection recorded in differ-
are infected. The infected panicle results in ent long-term experiments of several years
severe yield loss (Ou, 1985). The fungus has revealed that blast infection had occurred
the capacity to overcome resistance in a under natural conditions when the mini-
short period of time, soon after the release mum temperature during the night was
of a resistant cultivar, and thus has made 26°C and below, with the concomitant
breeding for resistance a constant and diffi- occurrence of relative humidity of 90% and
cult challenge to address for rice breeders higher (CRRI Annual Report, 2001–2002).
and pathologists (Shao et al., 2008). Analy-
sis of the existing genetic variation in plant
pathogen populations is an important pre-
requisite for understanding the mechanism Grouping of Blast Fungal Isolates
of co-evolution in the plant pathological sys-
tem (McDonald et al., 1989). Several popula- M. grisea as a species has a very broad host
tions of rice blast pathogen all over the globe range, infecting more than 40 Graminaceous
have been studied for their characteristic hosts and some other non-grass hosts (Asuy-
phenotypic and genotypic variations (Levy ama, 1965; Ou, 1985). Ou (1980) studied
et al., 1991, 1993; Shull and Hamer, 1994; variability in the pathogen and the host resis-
Chen et al., 1995; Kumar et al., 1999). Blast tance of M. grisea. Monoconidial cultures
disease was first reported in China (1637) showed continued segregation for virulence
and then in Japan (1704), Italy (1828) and in pattern and generated diverse lesion types
the USA (1996) (Asuyama, 1965; Ou, 1985; on individual leaves. Conidial and mycelial
CRRI Annual Report, 2001–2002). In this cells of M. grisea were reported to contain
chapter, we discuss the 50 years of research nuclei with a different number of chromo-
on M. grisea and the available sustainable somes. These observations offered the best
disease resistance management in rice. genetic explanation for the variation. Latter-
ell and Rosi (1986) studied the longevity and
pathogenic stability of M. grisea for 30 years.
They suggested that the species comprised a
Epidemiology of Blast Disease wide range of pathotypes (races), each char-
acterized by its capacity to attack certain
Seedling stage, rapid tillering stage after cultivars of rice, and that these races were
transplanting and flower emergence stage basically stable and mutations (or parasexual
were identified as the most susceptible to recombination) were the exception rather
rice blast. The fact that the age of the leaves than the rule, resulting in broader host range
influences the susceptibility to blast was also or increased sporulating capacity. The detec-
brought out. The older the leaves on the plant, tion of parasexual DNA exchanges in wild-
the more they are resistant to blast (Ou, 1985; type strains and the existence of merodiploids
CRRI Annual Report, 2001–2002). Excessive in nature suggest that parasexual recombina-
exposure to nitrogen and cold night tempera- tion occurs in field populations of M. grisea
tures predisposed susceptible varieties, but (Zeigler et al., 1997).
did not show any effect on highly resistant Three DNA probes were developed by
varieties. The critical range of temperature Hamer et al. (1989), which reliably and spe-
for penetration and establishment of infec- cifically identified the genetic backgrounds
tion was around 25–26°C, whereas germina- of the full spectrum of the rice blast fungal
tion of spores and appressoria formation pathotypes. One of these probes consists of
94 S. Nandy et al.
cloned fragments of repeated DNA obtained weeds of rice, cutgrass and torpedo grass.
from the RBF genome and which are called Levy et al. (1993) studied the genetic diver-
MGR586 (M. grisea repeat elements, pre- sity of RBF in a disease nursery in Colom-
viously referred to as PCB586). The probe bia. DNA fingerprints using MGR586, 115
hybridizes with approximately 50 EcoRI frag- haplotypes from 151 fungal isolates were
ments, ranging in size from 1.5–20.0 kb in the identified and partitioned into six discretely
genome of all M. grisea isolates pathogenic to distinct genetic lineages. Xia et al. (1993) con-
rice. Worldwide conservation of MGR586 ducted a DNA fingerprinting study to exam-
sequences in RBF suggests that they descend ine microgeographic variations in the M.
from a common ancestral source, genetically grisea population in two different rice fields
isolated from other host-limited forms of M. in Arakans in South-east Asia. The DNA fin-
grisea. The use of MGR shows that sequences gerprints of 113 isolates were grouped based
are dispersed randomly on all chromosomes on restriction fragment length polymorphism
of the pathogens and segregate as genetic loci (RFLP) similarity. Seven distinct fingerprint
(Zeigler et al., 1997; Suzuki et al., 2007). groups were identified and four fingerprint
Borromeo (1990) studied the Philippine groups were common in both fields.
isolates of RBF with MGR586 and MGR613. A study examining the relationship
Valent and Chumley (1994) discussed the between phylogeny and pathotypes for iso-
recent application of tools for molecular lates of the RBF in the Philippines revealed
genetic analysis of M. grisea and past and cur- that the distribution of virulence was non-
rent research in the problem areas. Iwano random with respect to lineage for the culti-
(1990) and Chen (1993) reported that the vars under study (Zeigler et al., 1995). Sivaraj
racial composition in a field in Yongnan (1995) reported six different lineages (L, A, B,
province, China, and the Philippines showed E, F and H) from Karnataka in southern India,
wide yearly fluctuations. Iwano (1990) using the MGR DNA fingerprinting approach.
claimed that isolates from the same lesion The repetitive DNA element, MGR586, has
changed their reaction on a set of several cul- been widely used for fingerprinting and phy-
tivars annually. Silue et al. (1992) studied the logenetic analyses of M. grisea. George et al.
patterns of inheritance of avirulence in M. gri- (1998) developed a polymerase chain reac-
sea in seven different rice cultivars. Aviru- tion (PCR)-based marker to DNA fingerprint
lence to four cultivars has been reported as the Magnaporthe species coming from dif-
being controlled by one gene, whereas for ferent biogeographic zones. Roumen et al.
the other three cultivars, it was controlled (1997) studied the genetic variability among
by two genes. 41 isolates of the blast pathogen from five
In another study using DNA polymor- rice-growing countries from the European
phism, common ancestral patterns were Union, including Spain, France, Hungary,
found among Magnaporthe infecting rice Italy and Portugal. DNA fingerprinting grou-
isolates and their associated weed hosts (Bor- ped the isolates into five discrete lineages,
romeo et al., 1993) However, the pathogenic which typically showed less than 65% band
populations infecting the weed hosts do not similarity. Srinivasachary et al. (1998) clas-
supply pathogenic inoculums for the rice. sified 27 single spore isolates of M. grisea
Weeds can act as alternative hosts for the from Karnataka in southern India over three
disease in greenhouse tests; but their role in different locations using random amplified
the field is not yet quite clear (Kato, 2001). polymorphic DNA (RAPD) primers. They
Rice, as a widely and intensively cultivated found three clear groups at 70% similarity
crop, could be a potential target for parasitic level. But Srinivasachary et al. (2002a,b) used
‘host shifts’ and a potential agent for ‘shifts’ 27 isolates from Ponnampet, Mandya and
to accompanying weeds (Couch et al., 2005). Bangalore for genetic analysis using 30
The authors also reported the single origin RAPD primers. Three distinct lineages
of rice-infecting M. oryzae after a ‘host shift’ were reported by the authors. Chadha and
from a Setaria-millet and that it was proba- Gopalakrishna (2005) also used 20 isolates
bly closely followed by additional ‘shifts’ to from seven different locations in India using
123 RAPD primers for cluster analysis. Sci- takes place in response to infection deter-
entists have sequenced the M. grisea genome mines the tissue resistance to the pathogen;
and it is now available online at http:// (iii) the presence of two toxic cinnamate
www-genome.wi.mit.edu/annotation/fungi/ derivatives (ferulate and coumarate) in the
magnaporthe/. It is, however, important to cell walls forming toxic oxidized products/
note that for the first time in the USA, the polymers like lignin and melanin-like com-
genomic structure of a significant plant pounds on oxidation forming a mechanical
pathogen has been made publicly available. barrier for the fungus and thereby arresting
the spread of the pathogen to adjacent cells,
thus restricting disease lesions; and (iv) the
synthesis and accumulation of antimicrobial
Physiology of Disease Resistance compound(s) (diterpenoid in nature) known
as ‘phytoalexins’ in response to infection
Plants develop defence mechanisms to rec- toxic to the growth of the pathogen. However,
ognize pathogens and protect them from none of these mechanisms seemed to be uni-
attack. These defence reactions are triggered versal in nature and the defence mechanism
by the recognition of pathogens by plant was dependent on the varieties tested (CRRI
disease resistance (R) genes. After the recogni- Annual Report, 2001–2002).
tion of pathogens, a signalling pathway is acti-
vated, resulting in resistance to pathogens
(Hammond-Kosack and Jones, 1997). Dur-
ing the early steps in R gene-mediated dis- Finding the Right Gene
ease resistance, reactive oxygen intermediates
(ROI) such as O2– and H2O2 are generated The generation of cultivars that possess
rapidly after infection; and, subsequently, non-specific resistance to M. grisea would
hypersensitive response (HR) leading to cell provide an economically effective and envi-
death has been observed. An understanding ronmentally sound approach to rice blast
of how pathogens induce disease, how the control. One promising approach to the
plants become diseased and how they defend achievement of non-specific resistance to M.
themselves against the pathogens would grisea is to incorporate genes that elicit gen-
help us to understand the functions of the eral defence responses in rice (Dang and
genes governing resistance, which remains Jones, 2001; Stuiver and Custers, 2001).
unknown, and eventually to develop novel Much effort has been devoted to understand-
methods for controlling RBD. The nature of ing the genetic and molecular basis of resis-
resistance to blast disease operating at both tance in RBF and several genes have been
the pre- and post-penetrative stages of the cloned (Parson et al., 1987; Leung et al.,
disease was investigated using several mod- 1990; Khang et al., 2008; Shao et al., 2008).
els involving cultivars differing in their Although earlier studies focused on
reaction to the disease, nitrogen fertilization pathotypic variability (Ou, 1985), later stud-
and temperature-induced tissue suscepti- ies focused extensively on molecular markers
bility and resistance induced by certain to characterize population diversity (Nandy
chemicals (CRRI Annual Report, 2001–2002). et al., 2004). Extensive use of the MGR586
Four different mechanisms govern blast resis- heterodispersed element (Roumen et al.,
tance in rice: (i) the epicuticular wax present 1997; Kumar et al., 1999; Correll et al., 2000;
on the surface of the leaves influences the Viji et al., 2000; Srinivasachary et al., 2002a,b;
infection by suppressing the appressorium Chadha and Gopalakrishna, 2005) to delin-
formation by the pathogen, thus offering a eate DNA fingerprint lineages has helped to
partial resistance resulting in a reduced identify and classify the genetic structure of
number of lesions being formed; (ii) free phe- this important pathogen. PCR-based molec-
nolic compounds and their oxidases toxify ular markers are useful tools for detecting
the tissue in the infected region: the speed genetic variation within populations of
and magnitude at which the toxification important plant pathogens (Vakalounakis and
96 S. Nandy et al.
Fragkiadakis, 1999; Kolmer and Liu, 2000; on the perfect state of M. grisea in India
Srinivasachary et al., 2002a,b; Chadha and (Dayakar et al., 2000; Mandal et al., 2004).
Gopalakrishna, 2005). RAPD (Welsh and The sexual cycle does not seem to be a
McClelland, 1990; Williams et al., 1990) source of variation for the rice blast patho-
and markers have been widely used for esti- gen in India (Kumar et al., 1999). Similar
mating genetic diversity in wild populations results have also been reported from other
(Annamalai et al., 1995), mainly because the corners of the globe (Valent et al., 1986).
technique does not need previous molecular The wide range of diversity among collected
genetic information and increases marker isolates of M. grisea from different locations
density for evaluating genetic kinship. The in West Bengal can be explained mainly by
RAPD technique has also been used to study evolution resulting from natural and stress-
genetic diversity among RBF from different induced transposition (Ikeda et al., 2001).
geographical locations in the world (Lima, Other mechanisms like horizontal gene
1999; Suzuki et al., 2007). transfer between RBF and its host (Kim et al.,
The dynamic virulence of the rice blast 2001) may also be of importance because
pathogen could be the main cause for the varieties deployed within a region are based
breakdown of resistance in several rice vari- on crop seasons, along with several other
eties. The diversity and variability of the biotic and geographic factors (Babujee and
pathogen population may originate from the Gnanamanickam, 2000).
clonal mode of reproduction, coupled with
mutation, migration, selection or random
drift, heteroploidy and parasexuality of the
fungus (Gesnovesi and Magill, 1976; Daya- Using Genetic Diversity
kar et al., 2000; Noguchi et al., 2007). A of Disease Resistance
repeat sequence termed MGR586 was iden-
tified in the genome of rice-infecting strains Genetic studies of qualitative resistance
of M. grisea (Shull and Hamer, 1994). This were started when Goto (1970) established
sequence has been widely used for DNA fin- the differential system for races of P. grisea
gerprinting of M. grisea to investigate the or M. grisea in Japan. Thirteen major genes
epidemiology of the RBD (Roumen et al., for qualitative resistance have been reported
1997; Kumar et al., 1999; Correll et al., 2000; by several researchers (Kiyosawa et al.,
Viji et al., 2000; Chadha and Gopalakrishna, 1981). Several rice cultivars with durable
2005). Molecular analysis of isolates of M. blast resistance have been identified and
grisea from different regions within a state ‘Moroberekan’ have been cultivated in the
(West Bengal, India) revealed the occur- world for many years without high losses
rence of a high level of polymorphism, indi- from blast (Notteghem, 1985). These plants
cating a wide and diverse genetic base have been used as resistance donors in breed-
(Mandal et al., 2004). Overall, a high genetic ing programmes. Major resistance genes have
diversity was also obtained in Indian RBF been used successfully for developing blast
(Roumen et al., 1997; Kumar et al., 1999; resistance cultivars (Khush, 2004) and sev-
Correll et al., 2000; Mandal et al., 2004, eral dominant resistance genes have been
Chadha and Gopalakrishna, 2005). identified which confer complete blast resis-
Genetic mechanisms, namely simple tance (Kiyosawa et al., 1981). Atkins and
mutations, meiotic recombination and para- Johnson (1965) identified two independent
sexual recombination, could explain such genes designated Pi-1 and Pi-6. Hsieh et al.
genetic diversity (Yamasaki and Niizeki, 1965; (1967) in China found four dominant genes
Zeigler, 1998; Zeigler et al., 2000, Khang, for pathogen resistance in japonica cultivars,
2001). Some indirect evidence suggests that named as Pi-4, Pi-13, Pi-22 and Pi-25 using
M. grisea has the potential for sexual repro- RFLP techniques. Yu et al. (1991) mapped
duction in specific geographic zones and three major resistance genes, namely Pi-1,
localities (Viji et al., 2000, Adreit et al., Pi-2 and Pi-4 in the Philippines. Several genes
2007). There have been few investigations from tropical cultivars like ‘Tetep’, ‘Pai-kan
tao’, ‘5173’, ‘LAC23’, Moroberekan and of natural screening, which is quite cumber-
‘Apura’ were identified and mapped using some, time-consuming and season specific.
RFLPs (Yu et al., 1991; Miyamoto et al., There has been considerable achievement
1996; Rybka et al., 1997) (Table 8.1). Recent in the development of blast-resistant varieties
reports identified at least four clusters, with (BRV), particularly using vertical-resistant
five to eight loci each, located on chromo- genes (Nandy et al., 2004). Nevertheless,
somes 4, 6, 11 and 12 (Roumen et al., 1997; durable resistance alone can protect irri-
Rybka et al., 1997, Tabien et al., 2000; Gao gated rice crops in the tropics adequately.
et al., 2002). Exploitation of durable resistance has been
Many pathogenic races have been iden- proposed for less blast-conducive envi-
tified in M. grisea and pathogenic variabil- ronments (Buddenhagen, 1983; Notteghem,
ity has been cited as the principal cause for 1985; Parlevliet, 1988; Bonman et al., 1992).
the breakdown of resistance in rice varieties Artificial inoculation in Karnataka, south-
(Baker et al., 1997). Therefore, an artificial ern India, was also carried out by Srinivasa-
inoculation study can be practised in place chary et al. (2002a) to study involving the
Table 8.1. List of blast disease-resistance genes with chromosome numbers, donor varieties and linked
markers of rice.
Gene Chromosome
symbol number Donor variety Linked marker Reference(s)
Pi-1(t) 11 LAC23, C101LAC Npb181, RZ536 Atkins and Johnson (1965);

Yu et al. (1991); Leung et al.
(1998)
Pi-2(t) 6 BL245, C101A51, 5173 RG64 Yu et al. (1991);
Sridhar et al. (1999)
Pi-4(t) 12 Tetep, Pai-kan-tao, RG869, RZ397 Yu et al. (1991); Hittalmani
BL245, C101PKT et al. (1995); Tabien et al.
(2000)
Pi-5(t) 4 RIL 45, RIL 249, RG498 Wang et al. (1994);
Moroberekan Sridhar et al. (1999)
Pi-6 12 – RG869 Causse et al. (1994);
Atkins and Johnson (1965)
Pi-7(t) 11 Moroberekan, RIL 29 RG103 Wang et al. (1994)
Pi-9 6 O. minuta derivative RG16 Leung et al. (1998);
WHD-IS-75-1-127 Khush et al. (1999)
Pi-10 5 Moroberekan RRF6, RRH18, Naqvi et al. (1995),
OPF6(2700) Tabien et al. (2000)
Pi-11 8 Oryzica Llanos 5 BP127, RZ617 Zhu et al. (1992); Roca et al.
(1996); Khush et al. (1999)
Pi-12 12 Moroberekan, RIL 10 RG869 Khush et al. (1999)
Pi-b 2 F-145-2 RZ123 Miyamoto et al. (1996);
Khush et al. (1999)
Pi-z5 6 C101A51 RG64, RG612 Fukuoka and Okuno (1997);
Leung et al. (1998);
Sridhar et al. (1999)
Pi-k 11 F-129-1 – Chao et al. (1999); Bryan
et al. (2000)
Pi-ta and 12 Taducan, C101PKT, RZ397, RG241 Shigemura and Kitamura
Pi-ta2 IR64, F-124-1, (1954); Rybka et al. (1997);
F128-1 Leung et al. (1998); Bryan
et al. (2000)
98 S. Nandy et al.
reaction of representative single-spore cul- strategy is modified as a phylogenetic patho-

ture PPT-4 to rice varieties Moroberekan, type exclusion. Lineage exclusion presumes
isolines of Co39, namely Pi-1, Pi-2, Pi-4, that lineage-specific avirulences represent
Pi-2 + Pi-1, Pi-1 + Pi-4, along with IRAT177, an evolutionary genetic barrier to pathotype
Apura and Doddi showed resistant reaction. diversification within the lineage. IRAT212/
Of these, Pi-1, Pi-2, Pi-4, Pi-2 + Pi-1 and N22, RR18-3/Bala, Bala/Tetep, Azucena/Gau-
Pi-1 + Pi-4 are known to contain major rav and several lines from the natural cross of
genes conferring resistance to blast disease. CR314-5-10 were resistant to leaf blast disease
Yamada et al. (1976) and Kiyosawa et al. (CRRI, Annual Report 2000–2001). A combi-
(1981) selected 12 differential varieties for nation of genes is also considered useful to
resistance genes Pi-ks, Pi-a, Pi-k, Pi-km, Pi-z, confer resistance to the pathogen lineages
Pi-ta (Pi-4), Pi-ta2, Pi-zt, Pi-kp, Pi-b and Pi-t. prevalent in China, the USA and Latin Amer-
These differential varieties were used in ica (Babujee and Gnanamanickam, 2000).
Japan especially, but were not readily avail-
able in other countries. Monogenic lines
including only a single gene in each genetic
background and targeting for 24 different Molecular Genetic Analysis
resistance genes – Pi-a, Pi-b, Pi-i, Pi-ks, Pi-k, of the Pathogen
Pi-k-h, Pi-km, Pi-kp, Pi-sh, Pi-t, Pi-ta (Pi-4),
Pi-ta2, Pi-z, Pi-z5 (Pi-2), Pi-zt, Pi-1, Pi-3, Plant disease resistance (R) genes confer
Pi-5(t), Pi-7(t), Pi-9, Pi-11(t), Pi-12(t), Pi-19 resistance to a wide range of pathogens (fungi,
and Pi-20 – were developed by Tsunematsu viruses, bacteria and nematodes); they share
et al. (2000) as the first international stan- various conserved motifs, suggesting the
dard differential variety set. The polymor- existence of a common defence signal trans-
phic RG-64 marker was used by Hittalmani duction pathway in different plant–microbe
et al. (2001) to identify rice plants carrying interaction systems (Dang and Jones, 2001;
Pi-2(t) from an F2 population derived from Martin et al., 2003). In general, the R genes
the cross between Co39 and C101A51. More fall into six distinct classes, the most preva-
than 30 blast-resistant genes (Babujee and lent of which is the nucleotide-binding site
Gnanamanickam, 2000) and QTLs have been plus leucine-rich repeat (NBS–LRR) genes
identified in rice by conventional genetic (Martin et al., 2003; Qu et al., 2006). The
studies based on linkage analyses and recom- LRR domains are generally thought to be
bination frequencies (Kinoshita, 1991; Mack- involved in the interaction with avirulence
ill et al., 1993). Some major genes for blast (AVR) proteins and to be the major deter-
resistance have been identified in recombi- minant of resistance specificity (Hulbert
nant inbred lines (RILs) (Wang et al., 1994). et al., 2001). The AVR-Pita avirulence gene
Zeigler et al. (1995) proposed that orga- family has been cloned recently at Kansas
nization of the blast fungus population into State University, USA, by Khang et al. (2008).
well-defined lineages and their distribution They have studied isolates of the M. grisea
in specific geographic locations have led to species complex from diverse hosts and have
the employment of resistance genes targeted found that AVR-Pita is a member of a gene
against pathogen populations prevalent in family, which led them to rename it AVR-
that region. This has been known as the ‘lin- Pita1. Using the dominant DNA markers
eage exclusion’ hypothesis. Sivaraj et al. derived from portions of the Pi-ta gene, 141
(1996) proposed a model to support gene rice germplasm accessions were rapidly
pyramiding based on lineage exclusion. They determined and the results were confirmed
consider traditional plant breeding as a strat- by inoculating rice germplasm with an M.
egy of pathotype exclusion, which leads to grisea strain containing AVR-Pita (Wang
frequent resistance breakdown when appro- et al., 2007). The Pi-ta gene was found in
priate pathotypes appear within 1 or 2 years accessions from major rice-producing coun-
after such resistance is deployed in large tries, including China, Japan, Vietnam, the
areas. In lineage exclusion, the conventional Philippines, Iran and the USA.
In another recent study, Shao et al. avoiding damp or most soil with high mois-
(2008) have reported that the expression of ture content for seed sowing, etc. However,
a hairpin-encoding gene (hrf1), derived chemical control is the most commonly
from Xanthomonas oryzae pv. oryzae, con- used approach in most parts of the globe for
fers non-specific resistance in rice to the effective disease control. Several fungicides
blast fungus, M. grisea. Transgenic plants are used against blast disease, including
and their T1–T7 progenies were highly resist- benomyl, fthalide, edifenphos, iprobenfos,
ant to all major M. grisea races in rice-growing tricyclazole, isoprothiolane, probenazole,
areas along the Yangtze River, China. The pyroquilon, felimzone (= meferimzone),
expression of defence-related genes was acti- diclocymet, carpropamid, fenoxanil and
vated in resistant transgenic plants and the metominostrobin, and antibiotics such as
formation of melanized appressoria, which blasticidin and kasugamycin (Kato, 2001).
is essential for foliar infection, was inhib- The composition, quantity, time and appli-
ited on plant leaves. These results suggest cation method of fungicides applied in field
that hairpins may offer new opportunities trials are dependent on the disease forecast
for generating broad-spectrum disease resist- for a particular region or zone, or on the
ance in other crops. However, occurrence of local disease prevalence rate (Kato, 2001).
clustered multigene families is a major Carbendazim, chlorobenthiozone, cora-
obstacle in the cloning of R genes (Dixon top, fungorene, hinosan and kitazin fungi-
et al., 1996; Ori et al., 1997), which makes it cides and antibiotic kasumin were effective
even more difficult to determine the func- against foliar and neck blast in India (CRRI
tional copy of these genes. Therefore, fine Annual Report, 2001–2002). Rice seed treat-
mapping of R-gene analogues on different ment with Carbendazim + TMTD 25 was
chromosomes would be helpful in the iden- effective in controlling seedborne blast (CRRI
tification of multigene families in rice, which Annual Report, 2001–2002). The control of
in turn will lead to the establishment of cor- rice blast relies on the use of resistant culti-
relation between the chromosomal position of vars and the application of fungicides, but
known R genes and their analogues. Recently, neither approach is particularly effective in
Kumar et al. (2007) cloned and also carried different geographic locations (Shao et al.,
out in silico mapping of resistance gene ana- 2008) because management of rice blast via
logues (RGAs) isolated from rice lines con- breeding BRV has had only short-term suc-
taining known genes for blast resistance. They cess due to the frequent breakdown of resist-
have amplified RGAs from the genomic DNA ance under field conditions (Valent and
of 10 rice lines having varying degrees of Chumley, 1994). The frequent appearance of
resistance to M. grisea by using degenerate new races (or pathotypes) of the fungus that
primers. Twenty RGAs were mapped near are capable of infecting previously resistant
to the chromosomal regions containing varieties has been proposed as the principal
known genes for rice blast, bacterial leaf cause for the loss of resistance (Ou, 1980).
blight and sheath blight resistance. Thirty- Host resistance in rice to M. grisea func-
nine RGA sequences also contained an open tions via a classical gene-for-gene interaction
reading frame representing the signature of in which a single dominant resistance gene
potential disease-resistance genes. corresponds with a dominant avirulence
gene in the pathogen (Hammond-Kosack and
Jones, 1997; Talbot, 2003). Because of the
apparent instability in the genome of M.
Control Measures grisea, new pathogenic races evolve rapidly
and thus host resistance typically lasts for a
Kato (2001) suggests burning and composting few years only (Zhu et al., 2000; Talbot,
of infected plant parts; use of non-infected or 2003). Few fungicides are available for the
certified healthy seeds and disease-resistant effective control of rice blast, but rapid muta-
cultivars; appropriate regulation of fertilizer tion in the pathogen leads to the emergence
application; proper cultural control and of fungicide-resistant variants (Takagaki
100 S. Nandy et al.
et al., 2004); thus, higher-dose applications have been used for rice blast management
of fungicides pose risks both to humans and for the past 50 years.
the environment.
Conclusions
Sustainable Rice Blast
Disease Management We have reviewed here the past 50 years of
research progress in the genetics and molec-
In developing countries, poor farmers cannot ular biology of rice blast disease, but differ-
afford to control blast disease by the applica- ent approaches can be taken for sustainable
tion of fungicides. Chemical control of plant disease control with recent advances in
pathogens is most effective and yet the use of genomics, proteomics and diverse genetic
chemicals is not generally desired due to the resistance mechanisms. Liu et al. (2002)
serious environmental threat it poses. Envi- recently reported the application of candi-
ronmental effects and resistance are not date defence genes to develop blast-resistant
considered a major concern in developing breeding lines with resistance to diverse
countries. Farmers are more interested in pathogen populations. Several biocontrol
short-term strategy for disease control. How- agents for blast have been deployed suc-
ever, the continuous use of fungicides leads cessfully to combat the disease in the labo-
to the resurgence of resistant races of the ratory, greenhouse and field tests (Chatterjee
pathogen under selection pressure. Therefore, et al., 1996; Krishnamurthy et al., 1998;
sustainable rice blast disease management is Gnanamanickam et al., 1999). The feasibil-
more important for environmental concern. ity of such strategies on a commercial scale
Figure 8.1 shows the basic components that still remains to be tested. Hence, use of
Using
the genetic
Finding
diversity of
the right gene
disease
resistance
Sustainable rice
blast disease
management
Understanding Molecular
the physiology genetic
of disease analysis of
resistance the pathogen
Fig. 8.1. The four basic components of sustainable rice blast management.
resistant cultivars is the best available alter- complementation that result in durable
native to overcome severe yield losses. resistance. Gene pyramiding is one of the
The objective of the green revolution strategies recommended to increase the
has not changed; there is the added impetus durability of blast disease resistance (Robin-
that crop protection should be conducted in son, 1973; Nelson, 1978; Buddenhagen, 1983;
the context of improving the livelihood of Pedersen and Leath, 1988).
rural people and preserving limited natural Pyramided resistance will be durable in
resources (Leung et al., 2003). However, the places where compatibility to the compo-
gene revolution has opened up newer and nent resistance genes is distributed among
better possible ways of preventing yield loss the prevalent lineages. Agricultural practices
from pathogen attack, conservation and uti- such as soil preparation, low nitrogen fertil-
lization of wild species for resistance genes. ization, low sowing density, optimized use
The variability of the pathogen and the his- of water and seed selection contribute to
tory of resistance breakdown have led to the reduce the virulence of M. grisea popula-
development of a number of different plant tions. Optimized integration of genetic resis-
breeding and molecular approaches to tance in agricultural management is the
achieve durable blast resistance. Combina- preferred strategy to protect cultivated rice
tions of resistance genes are thought to pro- from RBD in a way that is affordable, feasi-
vide broader spectra of resistance through ble, durable and, overall, compatible with
both ordinary gene action and quantitative environmental protection.
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Part III
Biological Control Mechanisms

9 Postharvest Technology – Yeast
as Biocontrol Agents: Progress,
Problems and Prospects
Neeta Sharma and Pallavi Awasthi

Mycology and Plant Pathology Division, Department of Botany,
University of Lucknow, Lucknow, India
Abstract
Storage losses of fruits in India are high owing to temperature and humidity conditions. Losses in
fruits are estimated to vary between 20 and 30%, valued at nearly 8000 crores annually, depending on
the fruit variety and the postharvest handling system. The application of fungicides to fruits after har-
vest to reduce decay has been increasingly curtailed due to the development of resistance in pathogens
to many key fungicides, lack of replacement with better fungicides, negative public perception regarding
the safety of pesticides and consequent restrictions on fungicide use. Biological control of postharvest
diseases has emerged as an effective alternative and several products are available in the market. One of
the major limitations with biological disease control is inconsistency in the efficacy of the product. The
limitations of biocontrol products can be addressed by enhancing biocontrol through genetic and envi-
ronmental manipulations and integration with other alternative methods that, alone, do not provide
adequate protection but, in combination with biocontrol, provide additive or synergistic effects.
Introduction vegetables were 16 per cent and 21 per cent,

respectively; many more “qualitative” refer-
Approximately half of the population in the ences, not included here, indicate estimates
Third World does not have access to ade- of 40–50 per cent and above.’
quate food supplies. There are many reasons The application of effective fungicides
for this, one of which is food losses occurring just prior to or shortly after harvest gener-
in the postharvest and marketing system. A ally controls postharvest decay (Eckert
study on ‘Postharvest Food Losses in Devel- and Ogawa, 1988). About 23m kg of fungi-
oping Countries’ conducted by a committee cides is applied to fruits and vegetables
of the US National Research Council con- annually and it is generally accepted that
cludes that, ‘postharvest losses are “enor- production and marketing would not be
mous”’. The committee extrapolated from possible without their use (Ragsdale and
apparent loss patterns and expected produc- Sisler, 1994). However, use of fungicides
tion trends and projected postharvest food has been restricted due to their carcinoge-
losses to be, at a minimum, 47,000,000 Mt nicity, teratogenicity, residual toxicity and
of durable crops and 60,000,000 Mt of per- long degradation period causing environ-
ishable crops. ‘The average minimum losses mental pollution (Unnikrishnan and Nath,
reported for roots and tubers and fruits and 2002).
110 N. Sharma and P. Awasthi
The Food Quality Protection Act (FQPA) M. fructicola, Penicillium digitatum, P. ital-
in the USA, the Food and Environment Pro- icum, P. expansum and Rhizopus stolonifer
tection Act (FEPA) 1985 and Control of (Droby et al., 1989; Wisniewski et al., 1991;
Substances Hazardous to Health (COSHH) Sharma, 1992, 1993, 2000; Mehrotra et al.,
Regulations 1988, made under the Health 1996, 1998; Sharma et al., 1997; Spadaro
and Safety at Work Act, 1974, in the UK are et al., 2002) . In the past 25 years, research
the guiding forces in the regulation of pesti- on biological control of postharvest diseases
cide use in their respective countries. Sev- has moved from laboratory to practical
eral countries have implemented their own applications (Wisniewski and Wilson, 1992;
specific policies to reduce pesticide use Wilson and Wisniewski, 1994; Mari and
(Matteson, 1995). Similarly, the fruit indus- Guizzardi, 1998; Droby et al., 2001; Jan-
try worldwide has accepted the concept of isiewiez and Korsten, 2002; Korsten, 2006).
integrated fruit production (IFP). IFP aims to By early 2000, there were three post-
produce high-quality fruit in harmony with harvest biological products available in the
the consumer and the environment. This market: Aspire™, a product developed from
implies minimum usage of chemicals, espe- C. oleophila (limited to the USA and Israel);
cially after harvest. Globally, greater restric- BioSave™, developed from P. syringae to
tions on pesticide use in the developed control decay caused by P. italicum and P.
nations have resulted in increasing trends for digitatum (limited to the USA); and Yield-
natural, non-chemical or organic approaches Plus™ (limited to South Africa). Avogreen™,
to disease control. Understandably, alterna- a commercial product of B. subtilis, was
tives to chemical pesticides or products that developed to control diseases caused by Cer-
allow reduced usage in terms of fewer or cospora spot and anthracnose of avocado.
reduced rates of application are beginning to
appear on the market in the form of biologi-
cal control agents (BCA). The present chap-
ter reviews the status of yeast as a biocontrol Isolation of Antagonist
agent and the problems associated with its
commercialization and registration. Often, carposphere, phylloplane, flowers and,
Cook and Baker (1983), in their book on in a few cases, other matrixes have provided
biological control, cited only one example the major source for antagonists (Filonow
of the biocontrol of postharvest disease of et al., 1996; Sharma, 2003; Belve et al., 2006).
strawberry fruit rot using Trichoderma sp. Various strategies have been employed to
Subsequently, Wilson and Pusey (1985) isolate antagonists and these include isola-
presented their initial research on Bacillus tion from natural cracks on the fruit surface;
subtilis to control brown rot on peaches, agar plates containing apple juice that were
caused by Monilinia fructicola, and the seeded with a rot pathogen (Wilson et al.,
organism was patented. A number of micro- 1993); freshly made wounds on apples in
organisms (bacteria, yeasts and fungi), which the orchard that were exposed to coloniza-
effectively control postharvest pathogens, tion by fruit-associated microbiota from 1 to
have been identified for the control of post- 4 weeks before harvest (Janisiewiez, 1996);
harvest diseases and some of these have been and from an apple juice culture resulting
patented and registered (El-Ghaouth and from seeding diluted apple juice with the
Wilson, 1997, 2002). In several studies, yeast orchard-colonized wounds and repeated
strains (Aureobasidium pullulans, Candida reinoculation to fresh apple juice. Isolation
oleophila, C. guilliermondii, C. sake, Crypto- of the antagonists can be improved by using
coccus laurentii, Debaryomyces hansenii, fruit from unmanaged orchards (Falconi and
Metschnikowia pulcherrima, Pichia gullier- Mendgen, 1994) where natural populations
mondii, Sporobolomyces roseus) are reported have not been disturbed by chemical usage
for biocontrol of postharvest fungal decays and the pool of potential antagonists is
of fruits caused by Alternaria alternata, greater than in a chemically managed orchard
Botrytis cinerea, Geotrichum candidum, (Smolka, 1992).
Postharvest Technology 111
Natural microflora maintains a balance ● does not produce metabolites that are
among the microbes normally present and deleterious to human health
inhibits the growth of newer arrivals. Sharma ● resistant to pesticides
(2005) reported that undiluted fruit wash- ● compatible with commercial process-
ings when plated on agar plates exhibited a ing procedures
dense population of yeast and bacteria and, ● does not grow at 37°C and is not associ-
on dilution, filamentous fungi of the patho- ated with infections in humans
genic type were isolated. This suggests that ● non-pathogenic to host commodity.
bacteria and yeast, naturally present on the
surface, may inhibit the growth of other
microorganisms, including plant pathogenic
fungi. Later, it was observed that the citrus Biocontrol Activity
fruits, when washed and stored, rotted faster
than the unwashed fruits, suggesting that Most antagonistic yeasts are efficient colo-
these bacteria and yeast provide protection nizers, even under adverse environmental
to fruits against postharvest pathogens. conditions, as they utilize nutrients rapidly,
Rather than in vitro screening of organ- produce extracellular materials that enhance
isms in Petri plates, which favoured the their survival on fruit surfaces and restrict
identification of antibiotic-producing organ- both colonization sites and flow of germina-
isms, a selection strategy was developed to tion caused to fungal propagules (Dugan
identify suitable yeast antagonists (Wilson and Roberts, 1995). In order to optimize dis-
et al., 1993). The method involved placing ease control, it is important to understand
washing fluids obtained from the surface of the mode of action of the antagonists so that
the fruit into fruit wounds that subsequently these attributes can be utilized to improve
were inoculated with a rot pathogen. Organ- performance. The antagonist activity can be
isms were then isolated from the surface of expressed in a number of ways. The most
wounds that did not develop infections. common is antibiosis (production of metabo-
These were plated out and isolated. lites such as pyrrolnitrin or iturins), attributed
Pure cultures of potential antagonists mainly to bacterial antagonists (Smilanick
were produced and then each organism was and Dennis-Arrue, 1992). The antibiotic
screened individually to assess its potential pyrrolnitrin, produced by Pseudomonas
as a biocontrol agent. This method identi- cepacia LT-4-12W (Janisiewiez and Roit-
fied a number of antagonists that were stud- man, 1988), reduced in vitro growth and
ied more intensely and measured against conidia germination and controlled the
the criteria set for suitability for commercial pome fruit pathogens, P. expansum and B.
production, as outlined by Wilson and Wis- cinerea, and citrus fruit pathogen, P. itali-
niewski (1989) and Hofstein et al. (1994): cum. However, the significance of the anti-
biotics in these biocontrol situations was
● genetically stable not clear, since strain LT-4-12W still pro-
● effective at low concentrations vided substantial control of blue mould
● not fastidious in its nutrient require- decay on oranges inoculated with laboratory-
ments derived mutants of P. italicum resistant to
● ability to survive adverse environmen- pyrrolnitrin. Spadaro et al. (2002), in stud-
tal conditions (including low tempera- ies on M. pulcherrima, found that in the
ture and controlled atmosphere storage) in vitro antagonism studies on different sub-
● effective against a wide range of patho- strates, the yeast could produce some metab-
gens on a variety of fruits and vegetables olites toxic to the pathogen, as distinct from
● amenable to production on an inexpen- the application of culture filtrates in vivo. In
sive growth medium recent years, the use of antibiotic-producing
● amenable to a formulation with a long bacteria has been abandoned in order to pre-
shelf life vent the appearance of resistance in patho-
● easy to dispense gen strains for humans or animals.
Competition for nutrients and/or space and C. albidus exhibited tenacious attach-
is the major mechanism involved for P. guil- ment with pathogen hyphae, along with
liermondii, C. laurentii, C. utilis, C. oleo- secretion of extracellular lytic enzymes
phila, D. hansenii and several other yeasts (Chan and Tian, 2005). Ultrastructural and
employed as bioagents (Chalutz and Wil- cytochemical studies on yeast, C. saitoana,
son, 1990; Arras, 1996; Arras et al., 1997; Spa- when co-cultivated with B. cinerea, showed
daro et al., 2002; He et al., 2003; Chan and cytological damage as papillae and protu-
Tian, 2005; Zhang et al., 2005). Janisiewiez berances in the cell wall and degeneration
et al. (2000) developed a non-destructive of the cytoplasm. It was also found to stim-
method using tissue culture plates having a ulate structural defence response in the
defusing membrane at the lower end of host. Host cell walls were well preserved and
cylindrical inserts for in vitro study of com- displayed an intense and regular cellulose-
petition for nutrients separated from the labelling pattern, as seen in transmission
competition for space. Living cells of the electron microscopy (El Ghaouth et al.,
antagonist are necessary to guarantee fungal 1998).
control. The ability to prevent infection by Yeast cells are able to produce hydro-
pathogen was lost when the antagonist cells lytic enzymes capable of attacking the cell
were killed. It was also observed that com- walls of pathogens and extracellular poly-
petition for nutrients was not visible when a mers that appear to have antifungal activity.
surplus of nutrients was available. There- Yeast, P. anomala strain K, effective in the
fore, the nutritional environment available control of grey mould of apple, increased
at the wound site may create a favourable production of exo-b-1,3-glucanase threefold
microenvironment for antagonists to colonize, in the presence of cell wall preparations of
multiply and compete effectively (Zheng B. cinerea in apple wounds. Higher b-1,3-
et al., 2004). The activity of an antagonist is glucanase and chitinase activity was also
dependent on the concentration of the detected in apple wounds treated with
antagonist: the higher the concentration, the strains of another antagonist, A. pullulans,
more effective the control. The antagonist effective in controlling various decays on
cell concentration of 106 – 108 CFU/ml or apple, table grape and other fruits (Ippolito
more of Candida spp., D. hansenii and Pan- et al., 2000; Castoria et al., 2001). Yeast, P.
toea agglomerans provided satisfactory lev- membranefaciens and C. albidus, show
els of control (Droby et al., 1989; McLaughlin b-1,3-glucanase and exo-chitinase activity
et al., 1990). However, different isolates of in the presence of cell wall preparations of
M. pulcherrima at 106 CFU/ml were not R. stolonifer, M. fructicola and P. expansum
found to provide satisfactory levels of con- (Chan and Tian, 2005).
trol against B. cinerea and P. expansum Yeasts like C. famata are reported to
(Spadaro et al., 2002). control green mould due to induction of
While early studies indicated that nutri- phytoalexins, scoparone and scopolectin
ent competition and the fast growth rate of (Arras, 1996). However, the role of enzymes
antagonists played a major role in biocon- and phytoalexins in biocontrol activity war-
trol activity, subsequent studies indicated a rants further investigation. Fajardo et al.
much more complex interaction, such as (1998) reported differential induction of
direct interaction with the pathogen (Wis- proteins in orange flavedo by biologically
niewski et al., 1991; Spadaro et al., 2002), based elicitors. More recently, molecular
induced resistance in host tissue (Wilson approaches to examine the mode of action
et al., 1994; Droby et al., 2002) or a gamut of have been studied on the biocontrol agent. A
interactions between the antagonist, patho- transformation system for C. oleophila yeast
gen and commodity. Pichia guilliermondii produced yeast lines with either higher or
US-7 (Droby et al., 1989) and M. pulcherrima lower levels of a b-1,3-glucanase gene/enzyme
(Spadaro et al., 2002) exhibited nutrient com- expression compared to the wild type. Bio-
petition along with direct parasitism against control activity did not differ between the
B. cinerea in apples. Pichia membranefaciens different yeast lines, but the results did not
rule out a role for this gene in biocontrol as osmotolerance, temperature, oxygen requi-
activity. It was also demonstrated that over- rements, optimum pH and optimum growth
expression of a lytic peptide belonging to the rate. Growth rate of yeast is very high, but
defensin family of antimicrobial peptides in lower than that of bacteria; longer fermen-
yeast could enhance biocontrol activity tation durations pose the risk of yeast cul-
(Segal et al., 2002; Yehuda et al., 2003). tures becoming contaminated. Yeast is also
sensitive to low pH (below three), which is
used generally as a measure to check bacte-
Constraints in Product rial contamination because pH above five
Development and Registration is favourable for bacteria that may contam-
inate yeast culture. Aeration of fermen-
In the early years, several yeast antagonists tors, to fulfil the oxygen requirement for
that had commercial potential were mis- maximum output, can also be a source of
identified, such as strain US-7 of C. guillier- contamination during the early phases of
mondi, which was misidentified originally production and, to prevent such contamina-
as D. hansenii. This caused some confusion tion, other technologies must be used. The
in the patenting process and emphasized contaminants should be identified at each
the need to have at least two confirming stage of production and quantified in the
identifications by reputable yeast taxonomic end product.
services. It also emphasized the weakness of Yeast fermentation is an exothermic
using physiological tests as the basis for mak- process; therefore, the fermentation temper-
ing taxonomic determinations (McLaughlin ature can never be below ambient and, since
et al., 1990). Also, few isolates of C. guilllier- yeasts appear sensitive to high temperatures
mondii were abandoned because they were (above 28°C), a cooling system more effi-
found to be pathogenic to humans. cient than the evaporative system routinely
Potential biocontrol agents often have used has to be employed. This, however,
some significant limitations: sensitivity to adds to the cost of production.
adverse environmental conditions such as A major obstacle to the commercializa-
extreme dryness, heat and cold, limited tion of biocontrol products is the develop-
shelf life, limited biocontrol efficacy in situ- ment of a shelf-stable product that retains
ations where several pathogens are involved bioactivity similar to that of fresh cells. For-
in decay development and an inability to mulations can influence the survival and
control latent infections. For commercial- activity of biocontrol agents. An accurate
ization, several semi-commercial and com- formulation has a profound effect on the
mercial trials have to be conducted, for which efficacy of a biocontrol agent, including its
large volumes of antagonist are required. The shelf life, ability to grow and survive after
mass production of the bioagent by rapid, application, effectiveness in disease con-
efficient and inexpensive fermentation of the trol, ease of operation and application and
antagonist is a key issue. Therefore, it is fun- the cost (Fravel et al., 1998). A biofungicide
damental to find carbon and nitrogen sources should be effective for at least 6 months,
that provide maximum biomass production and preferably for 2 years (Pusey, 1994).
at minimum cost, while maintaining biocon- This can be achieved by supplementing the
trol efficacy. Cheap industrial waste materi- yeast with protectants, carriers or additives.
als such as cottonseed meal, corn steep liquor, Alternatively, yeast can be conditioned dur-
partially digested peptone, yeast extract, dry ing fermentation by using an emulsifier.
brewer’s yeast, sucrose and molasses have Drying the product and maintenance in
been used as growth media for the multiplica- a dry environment or suspension in oil are
tion of cells (Hofstein et al., 1994; Costa common approaches. Products are available
et al., 2001). as wettable powder, as frozen cell concen-
Large-scale production of any yeast trated pellets or as liquid formulations. It
depends on the amount of technical infor- was found that freeze-dried cells were sig-
mation available on that specific strain, such nificantly less effective than fresh cells.
Certain freeze-drying protective agents and use throughout the period when active con-
rehydration media enhanced the viability of trol is required, which may be several months
the antagonist, P. agglomerans strain CPA-2, for some pathogens. During this time, it
effective against blue mould and grey mould must survive fluctuations in the physical
of pome fruits (Costa et al., 2000). Survival environment and the action of the indige-
of cells of the antagonistic yeast, C. sake, nous and competitive microbiota. The use
was improved from 0.2% to 30–40%, by of appropriate inoculum production, for-
using freeze-drying protective media con- mulation and application technologies,
sisting of skim milk and other protectants, together with quality control checks, should
such as 10% lactose or glucose and 10% also help in this process. Nevertheless, even
fructose or sucrose. The presence of treha- if reliable BCAs can be produced, they must
lose in liquid formulations appeared to help still be easy to use and cost-effective or they
preserve the viability of C. sake during stor- will either never reach the marketplace or
age. It is known that intracellular trehalose not be used by growers.
exerts a protective effect on yeast under By early 2000, there were two yeast-
extreme environmental conditions such as based postharvest biological products avail-
desiccation, freezing, osmotic stress and able on the market: Aspire™ (C. oleophila
heat shock, and it also provides thermal sta- I-182) and YieldPlus™ (El-Ghaouth and
bility to the cells (Abadias et al., 2001). Wilson, 1997, 2002; Wilson and El-Ghaouth,
The application of adjuvant can protect 2002). The commercial development of Aspire
and stimulate the establishing of the antago- by Ecogen-Israel Partnership Ltd, focused on
nist on the host surface. The addition of the biocontrol of postharvest decays of citrus,
xanthan gum to A. pullulans L47, applied to mainly blue mould and green mould caused
strawberries in the field from bloom to fruit by P. italicum and P. digitatum, respectively,
at the green stage, improved survival of the which invade through wounds after har-
antagonist and increased biocontrol of stor- vest. Throughout the course of developing
age rot caused by B. cinerea (Ippolito et al., Aspire™, considerable research went into
1998). Formulations may include wetters finding methods to enhance the reliability
(humectants) to facilitate reabsorption of and efficacy of the product and other
moisture from air. Wetters not only make selected antagonists as well.
water spray stay on plants but, like oil carri- As a result, second generation biocon-
ers, they also enable organisms to reach other- trol products were developed using a com-
wise inaccessible places such as depressions, bination of natural products along with a
stomata and lenticels, thereby improving the yeast antagonist to address the poor ability.
chances of establishing antagonists for dis- Research efforts led to the development of
ease control. Oil carriers are expensive, but two new products whose main components
formulations containing oils can enhance consisted of the yeast antagonist, C. saitoana,
the reliability of biological control agents and either a derivative of chitosan (Biocoat)
(Jones and Burges, 1998). Research is needed or lysozyme (Biocure) (El Ghaouth et al.,
to determine the value of each additive alone 2000a). Both compounds have been tested
and also in the presence of other ingredi- worldwide and have shown strong eradicant
ents, as well as to ensure the requirements activity. Both products contain additional
for ecological safety. additives, such as sodium bicarbonate, to
One of the major limitations with bio- enhance efficacy and perform as well as the
logical disease control is the inconsistency postharvest fungicides currently available.
in efficacy that is often observed when use- Another constraint concerns registra-
ful antagonists reach the stage of large-scale tion. Currently, there are no fungal biocon-
testing, and which can arise from a variety trol products registered and sold worldwide.
of causes reflecting the biological nature of Some products are available in several coun-
the control microorganism. Essentially, the tries, while others are sold in their respective
organism must first survive application and countries. This reflects the problems asso-
then retain activity in the environment of ciated with registration requirements in
different countries and includes concerns 1996) when a mixture of antagonists was
about releasing non-indigenous microorgan- applied. The mixtures are either paired at
isms. The legislation drafted essentially for random or after screening, for minimum
chemical pesticides is not always applicable mutual niche overlap. To determine further
to biological pesticides and the require- compatibility of the strains selected, it is
ments for the registration of biological pesti- important to conduct coexistence studies
cides are currently under discussion for using De Wit displacement series in fruit
appropriate review. wounds (Wilson and Lindow, 1994). The
The position of the biocontrol product benefits of this approach are clear, but its
in the market governs its future. For exam- implementation requires approval from the
ple, if the product enters the agrochemical industry. It also entails doubling of the cost.
market, it competes against synthetic fungi- However, this can be overcome by using in
cides that can kill pathogenic organisms, the mixture at least one antagonist which
while yeast only-based products cannot do has been commercialized.
so and neither do they have systemic action. Some exogenous substances, such as
They act mainly as protectants that may also chitosan, amino acids, antibiotics, calcium
induce resistance in the hosts. The other salts and carbohydrates, have been studied
option is to position the product in the ‘all to enhance the biocontrol capability of
green’ category in markets such as those of antagonists against fungal pathogens. Cal-
perishables, where no other option is avail- cium chloride improved biological control
able, thus eliminating any competition and of the yeast, P. guilliermondii (Droby et al.,
fulfilling the principal objective of con- 1997). Combining 0.2% glycolchitosan with
sumer and environmental safety. the antagonist, C. saitoana, was more effec-
tive in controlling green mould of oranges
and lemons, caused by P. digitatum, and
grey and blue moulds of apples than either
Integrated Control treatment alone (El- Ghaouth et al., 2000a,b).
In a recent study by the authors, a combina-
Since, biological agents alone are not capa- tion of chitosan and the yeast, C. utilis, was
ble of providing commercially acceptable found effective in controlling postharvest
levels of control, their integration with other pathogens on tomato (Sharma et al., 2006).
control measures is expected to provide The studies also showed that several yeast
greater stability and effectiveness. It is also genera were compatible with low concen-
desirable that the use of antagonists must be trations of chitosan and the protection
compatible with current handling and stor- afforded by this combination was superior
age practices which could otherwise cause a to the stand-alone treatments.
reduction in the effectiveness of antagonist GRAS (generally recognized as safe) sub-
strains. For biological control to be effective, stances such as sodium carbonate, sodium
use of antagonists must be compatible with bicarbonate and ethanol reduced conidial
other control measures. An effective biocon- germination of P. digitatum, the causal
trol based on a mixture of several comple- agent of green mould of citrus. Ethanol at
mentary and non-competitive antagonists 10%, in combination with ethanol-resistant
has several advantages: apart from a wider S. cerevisiae strains 1440 and 1749, reduced
spectrum of activity, they increase efficacy, the incidence of grey mould decay on apples
are more reliable and allow reduction in from more than 90% to close to 0%, respec-
application times and treatment costs. They tively, whereas either treatment alone did
also permit the combination of different not reduce decay. The same concentration
genetic characteristics, minimizing the need of ethanol reduced green mould of lemons to
for genetic engineering. In a study on apples, less than 5% (Smilanick et al., 1995, 1999).
a broader spectrum of pathogens was con- A. pullulans, in combination with cal-
trolled and less total biomass of the antago- cium chloride or sodium bicarbonate, was
nist was needed to control decay (Janisiewiez, found effective in controlling postharvest
pathogens on sweet cherries (Ippolito et al., to exploit the nitrogen compounds present
1998). or with a higher transport or metabolism
Pre-storage hot air treatment of apples rate of the limiting factor can be developed,
reduced or eliminated blue mould decay because nitrogen is often a limiting sub-
caused by P. expansum and grey mould stance when the biocontrol mechanism of
decay (Fallik et al., 1995). Heat also action is competition for nutrients; and use
improved biocontrol with heat-tolerant of mutants that use new substrates, not
yeasts when applied to apples up to 24 h metabolized by the pathogen, to provide a
after inoculation with the pathogen. The nutritional advantage or attempt to obtain
heat treatment alone provided little resid- strains resistant to phenolic compounds
ual protection, but the residual protection (Bizeau et al., 1989).
provided by Ca and the antagonist in combi- Early experiments in transformation
nation enhanced the control by heat. When for marker genes have been successful.
antagonists were applied to apple wounds Metschnikowia pulcherrima was trans-
before heat treatment, the heat reduced formed with the green fluorescent protein
populations of P. syringae and increased gene (Nigro et al., 1999) and histidine
populations of the two heat-tolerant yeasts auxotrophs of C. oleophila were trans-
more than tenfold. formed with HIS3, HIS4 and HIS5 genes
(Chand-Goyal et al., 1999). In all cases, the
transformed antagonists maintained their
biocontrol capability and there were no
Conclusions detectable differences between the wild type
and the transformants. All these studies were
Future lines of research should be directed accomplished only to obtain variants of the
to find methods of enhancing the reliability antagonistic strains with a genetically stable
and efficacy of selected antagonists, and the marker. Jones and Prusky (2002) investi-
field is gaining momentum. It should aim at gated the possibility of expressing a DNA
finding additives or physical control meth- sequence in S. cerevisiae to allow the pro-
ods that will act synergistically with the duction of a cecropin A-based antifungal
antagonist. This involves combining the peptide. Yeast transformants inhibited the
product with a low-level of postharvest fun- growth of germinated Colletotrichum coc-
gicide or GRAS substances. It has been coides spores and inhibited decay develop-
reported that physical treatments such as hot ments caused by the pathogen in tomato
air, curing, hot water brushing and combina- fruit. The lack of activity toward non-target
tions of the above with pressure infiltration organisms by the peptide and the use of S.
of calcium could also increase the efficacy of cerevisiae as a delivery system suggest that
antagonists. Using mixtures of antagonists, this method could provide a safe alterna-
or combining antagonists with specific nutritive for postharvest disease control. How-
ents or sugar analogues, is also suggested as ever, attempts to overexpress genes involved
an approach to increase efficacy. in biocontrol, for example, lytic enzymes,
Genetic manipulation of antagonists is or engineering strains with desired biocon-
a field in its infancy. Current efforts are trol traits may soon yield positive results.
focused on developing efficient transforma- Biological control of plant diseases in gen-
tion procedures for yeast antagonists and eral and on fruit after harvest in particular
inserting genes for tracking the antagonist is a niche market, with a relatively small
in the environment rather than enhancing profit potential. However, it is clear that the
biocontrol (Yehuda et al., 2001). stage is set for biological control agents to
Other approaches could be: the inser- play a greater part in agriculture and horti-
tion of the gene for amylase under the con- culture. This approach undoubtedly would
stitutive promoter in some BCAs to allow encourage environmentally desirable prod-
effective use of the fruit carposphere starch; ucts that are desired by the public to reach
biocontrol strains with a higher capability the marketplace rapidly.
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10 Biological Control of Plant Diseases:
An Overview and the Trichoderma
System as Biocontrol Agents
Abhishek Tripathi,1 Neeta Sharma2 and Nidhi Tripathi1

1Department of Bioscience and Biotechnology, Banasthali University,
Banasthali Vidyapith, India; 2Mycology and Plant Pathology Division,
Department of Botany, University of Lucknow, Lucknow, India
Abstract
Biocontrol is the reduction of inoculum density or disease-producing activities of a pathogen in its
active or dormant state by one or more microorganisms, accomplished naturally. Research on biologi-
cal control agents has utilized naturally occurring saprophytic soil fungi to compete with and/or
destroy soilborne pathogens. Biological control has attracted attention from researchers for over 30 years,
primarily because of the interest in developing more ‘environmentally friendly’ means of disease manage-
ment in the absence of agricultural pesticides. Despite considerable effort in the area of biological control,
few practical applications have become established in agriculture for the control of plant diseases. Com-
mon biocontrol agents include Trichoderma, Gliocladium, Aspergillus, Penicillium, Chaetomium, Dac-
tylella, Glomus, etc. Biological control is achieved by competition, hyperparasitism, induced resisitance,
hypovirulence, etc. Mycoparasitism and production of volatile and non-volatile antibiotics are important
mechanisms operating in the case of Trichoderma, besides commercial uses and mass multiplication of
the novel biocontrol agent. The future of biocontrol lies perhaps with the development of better applica-
tion methods and the use of genetic engineering to increase the efficacy of various wild strains.
Introduction against certain classes of fungicides has fur-

ther reduced the number of disease control
Empirical approaches to chemical disease measures available. In recent years, it has
control have been practised since ancient become evident, as a result of public opin-
times, when concoctions consisting of salt ion and environmental laws, that new and
brine, sulphur, lime, ashes and salts of cop- safer alternatives to traditional synthetic
per, mercury and arsenic were used to com- pesticides are both desirable and mandated.
bat disease. Reports of pesticide residues in Research emphasis has therefore been on
food, soil, river and groundwater undermine the development of alternative approaches
consumers’ trust. Thus, the increasing to control the pathogens and pests of orna-
concern, particularly in developed nations, mental crops using biocontrol agents.
is that modern methods of crop protection There are considerably more success sto-
have an overall negative impact on the envi- ries involving the control of insect pests. Gar-
ronment and on society. Pathogen resistance rett’s (1965) definition of biological control

122 A. Tripathi et al.
of plant disease was, ‘any condition under agents are Trichoderma, Gliocladium, Asper-
which or practice whereby survival or activ- gillus, Penicillium, Neurospora, Chaetomium,
ity of a pathogen is reduced through the Dactylella, Arthrobotrys and Glomus, etc.
agency of another living organism (except According to Baker (1987), biological
man himself), with the result that there is a control is the decrease of pathogen activity
reduction in the incidence of the disease accomplished by one or more organisms
caused by the pathogen’. Although biologi- including the host plant but excluding
cal control consists of diverse methods and humans. Harman (2000) defined biological
approaches to suppress plant disease, in most control as a critically needed component of
cases antagonists to pathogens are added to plant disease management. Biocontrol agents
the agroecosystem. Various approaches of are known as antagonists. The most impor-
biocontrol are directed at suppressing ini- tant, well-studied antagonists against several
tial disease induced by a soilborne pathogen plant pathogens are fungi like Ampelomy-
or the application of an avirulent isolate of ces sp., Aspergillus spp. (particularly A.
the pathogen that ‘competes’ with the viru- niger and A. terreus), Chaetomium globo-
lent pathogen on or in the host. Biological sum, Coniothyrium minitans, Fusarium sp.,
control employs living agents (usually antag- Gliocladium virens, Penicillium citrinum,
onists or competitors of the causal agent) to Peniophora gigantea, Trichoderma spp.
control plant diseases. Effective biological (particularly T. harzianum and T. viride)
controls take advantage of the natural compe- and Sporodesmium sp.; and bacteria like
tition of living organisms for limited resources Agrobacterium radiobacter strain K84, spe-
or ecological niches. Thus, two organisms cies of Bacillus, Enterobacter, Micromono-
cannot occupy the same space at the same spora, Pseudomonas and Streptomyces.
time, they cannot consume the same resource
(e.g. food source) at the same time and, in
some cases, one organism produces com-
Mechanisms of Biological
pounds that are inhibitory to the growth
and development of the other organism. Control of Plant Diseases
Certain microorganisms that normally com-
pete for and live off debris and dead animal Competition
and plant cells in the soil environment have
developed, through mutation, the ability to Competition occurs between microorgan-
invade a host plant and escape the effects of isms when space or nutrients (i.e. carbon,
antagonists. These invading organisms are nitrogen and iron) are limiting and its role
referred to as pathogens. The lack of sur- in the biocontrol of plant pathogens has
vival of the pathogen and the superior com- been studied for many years, with special
petitiveness of the antagonists relative to emphasis on bacterial biocontrol agents. An
pathogens brings promise to the theory of important attribute of a successful rhizo-
using antagonists to control pathogens. sphere biocontrol agent would be the ability
Tubeuf (1914) coined the term ‘biologi- to remain at a high population density on
cal control’ in relation to plant pathogens, the root surface, providing protection of the
while Hartley (1921) first attempted to con- whole root for the duration of its life. Myc-
trol the root diseases of plants with intro- orrhizal fungi can also be considered to act
duced microorganisms. Cook and Baker as a sophisticated form of competition or
(1983) defined biological control as, ‘the cross-protection, decreasing the incidence
reductions of the amount of inoculum or of root disease. Fomes (Heterobasidion)
disease-producing activity of a pathogen annosum colonizes stumps of freshly cut
accomplished by one or more organisms pine and other conifers and spreads via root
other than man’. Microorganisms, which are grafts to other healthy trees, where it causes
used in the management of plant diseases, root rot (refer to Chapter 26). Spraying
are referred to as ‘biocontrol agents’. The freshly cut stumps with spore suspensions
important genera of fungi used as biocontrol of Phlebia (Peniophora) gigantea will prevent
Biological Control of Plant Diseases 123
H. annosum from getting a foothold, and Hypovirulence

this is standard practice in the UK.
Hypovirulence is a term used to describe
reduced virulence found in some strains of
Antibiosis pathogens. This phenomenon was first
observed in Cryphonectria (Endothia) para-
Antibiosis is the inhibition of an organism sitica (chestnut blight fungus) on European
by a metabolic product (such as an antibiotic) Castanea sativa in Italy, where naturally
from another organism. Many organisms, occurring hypovirulent strains were able to
especially soil fungi and Actinomycetes, reduce the effect of virulent ones. These
produce antibiotic substances. The produc- slower-growing hypovirulent strains contain
tion of antibiotics by Actinomycetes, bacte- a single cytoplasmic element of double-
ria and fungi is demonstrated very simply stranded RNA (dsRNA) similar to that found
in vivo. Numerous agar plate tests have been in mycoviruses, which is transmitted by anas-
developed to detect volatile and non-vola- tomosis in compatible strains through natural
tile antibiotic products by putative biocon- virulent populations of C. parasitica. Hypo-
trol agents and to quantify their effects on virulence has also been reported in many
pathogens. In general, however, the role of other pathogens, including R. solani, Gaeu-
antibiotic production in biological control mannomyces graminis var. tritici and Oph-
in vitro remains unproved. Three diseases iostoma ulmi, but the transmissible elements
can be controlled by antibiosis: Armillaria responsible for hypovirulence or reduced
root rot by T. viride, Pythium and Rhizocto- vigour of the fungi are subject to debate and
nia damping off and stem and root rot dis- may be due to dsRNAs, plasmids or viruses.
eases by P. fluorescens and crown gall by A.
radiobacter. The most widely accepted
commercial example is the control of crown Induced Resistance and
gall using strain 84. Cross-Protection
Induced resistance is a plant response to

Hyperparasitism and mycoparasitism challenge by microorganisms or abiotic
agents such that, following the inducing
Biological control can occur through direct challenge, de novo resistance to pathogens
parasitism. Parasitism involves the direct uti- is shown in normally susceptible plants.
lization of food of one organism by another Both localized and systemic-induced resis-
organism. Hyperparasites are organisms par- tance are non-specific and can act against a
asitic on other parasites. Some have referred whole range of pathogens, but whereas
to this as ‘natural biocontrol’. A few exam- localized resistance occurs in many plant
ples of hyperparasitism include: Darluca species, systemic resistance is limited to
(Sphaerellopsis) filum parasitizes rust fungi some plants. Cross-protection differs from
and species of Ampelomyces parasitize pow- induced resistance in that, following inocu-
dery mildews; Tuberculina maxima parasit- lation with avirulent strains of pathogens or
izes the aecial stage of Cronartium ribicola, other microorganisms, both inducing micro-
cause of white pine blister rust; T. viride, organisms and challenge pathogens occur
and a number of other species, are known to on or within the protected tissue. The most
parasitize hyphae of R. solani. The most commonly reported examples of cross-
common example of mycoparasitism is that protection involving fungi are probably
of Trichoderma sp., which attack a great those used against vascular wilts. Inocula-
variety of phytopathogenic fungi responsi- tion with non-pathogenic formae speciales
ble for the most important diseases suffered of Fusarium and Verticillium species, or
by crops of major economic importance with other fungi or bacteria, has shown dif-
worldwide. ferent levels of cross-protection.
Predation that AM colonization was reduced at higher

phosphorus level.
Predation has also been examined as a poten-
tial form of biocontrol. Nematode-trapping
fungi and predaceous nematodes have been Biocontrol of Airborne Diseases
studied in detail as potential biological con-
trol agents, but ultimately have had little Many naturally occurring microorganisms
effect on the numbers of plant parasitic have been used to control diseases on the aer-
nematodes in the soil. ial surfaces of plants. The most common
bacterial species that have been used for the
control of diseases in the phylloshpere include
Mycorrhizae P. syringae, P. fluorescens, P. cepacia, Erwinia
herbicola and B. subtilis. Fungal genera that
have been used for the control of airborne
Mycorrhizae are symbiotic (mutualistic)
diseases include T. ampelomyces and the
associations between fungi and plant roots.
yeasts, Tilletiopsis and Sporobolomyces.
The increased surface area provided by
Biocontrol agents normally must achieve
mycorrhizal fungi allows for increased
a high population in the phyllosphere to
nutrient uptake, which indirectly benefits
control other strains, but colonization by the
disease management derived by healthier,
agent may be reduced by competition with
more vigorous roots. Because of the gener-
the indigenous microflora. Integration of
ally beneficial effect of mycorrhizae on plant
chemical pesticides and biocontrol agents
growth and their common occurrence, many
has been reported with Trichoderma spp.
investigations have looked into the poten-
and P. syringae. Biocontrol agents tolerant
tial of root–fungus associations as potential
to specific pesticides could be constructed
biological control agents.
using molecular techniques. Resistance to
Vesicular arbuscular mycorrhizal (VAM)
the fungicide benomyl is conferred by a sin-
fungi were recognized and described in the
gle amino acid substitution in one of the
last few decades of the 19th century. The
b-tubulins of T. viride, the corresponding
term ‘VAM’ was changed to ‘AM’ by Draft and
gene thereby producing a biological control
Nicolson (1974) because some species did not
agent that could be applied simultaneously
form vesicles. AM fungi occur throughout
or in alternation with the fungicide.
the terrestrial ecosystem in almost all the
herbaceous and woody plants, forming a
symbiotic relationship with the roots (Ger-
demann, 1968; Trappe and Fogel, 1977). This Biocontrol of Soilborne Diseases
symbiotic association has been reported to
play an important role in plant mineral nutri- Chemical control of soilborne plant diseases
tion (Gianinazzi and Gianinazzi-Pearson, is frequently ineffective because of the
1986). It has been observed by several work- physical and chemical heterogeneity of the
ers that these fungi facilitate the uptake of soil, which may prevent effective concen-
many nutrients (phosphorus, zinc, copper, tration of the chemicals from reaching the
sulphur, potassium, iron, calcium, etc.), pathogen. Biological control agents colonize
resulting in increased biomass (Wani and the rhizosphere, the site requiring protec-
Lee, 1992). Nutrient content of N, P and K, tion and leave no toxic residues, as opposed
and also Fe, Mn and Cu, increased due to to chemicals. Microorganisms have been
AM inoculation in papaya. Among all the used extensively for the biological control of
AM species, G. mosseae was recorded as the soilborne plant diseases, as well as for pro-
most efficient for nutrient uptake. Rajesh- moting plant growth. Fluorescent Pseudo-
wari et al. (2001) reported that G. fascicula- monads are the most frequently used bacteria
tum at low phosphorus level increased the for biological control and plant growth
root and shoot biomass. They also recorded promotion, but Bacillus and Streptomyces
species have also been commonly used. expressed in Pseudomonas spp. and the
Trichoderma, Gliocadium and Coniothyrium plant symbiont, Rhizobium meliloti. The
are the most commonly used fungal biocon- modified Pseudomonas strain controlled
trol agents. Perhaps the most successful the pathogens, F. oxysporum f. sp. rodelens
biocontrol agent of a soilborne pathogen is and G. graminis var. tritici.
A. radiobactor strain K84, used against crown
gall disease caused by A. tumefaciens.
Molecular techniques have also facili- Commercial Biocontrol Agents
tated the introduction of beneficial traits
into rhizosphere competent organisms to
The following is a list of commercially avail-
produce potential biocontrol agents. Chitin
able products formulated for the biocontrol
and b-(1,3)-glucan are the two major struc-
of plant pathogens and/or plant growth pro-
tural components of many plant pathogenic
motion involving the induction of plant host
fungi, except Oomycetes, which contain cel-
defence. The list originated in 2000 through
lulose in their cell wall and no appreciable
the efforts of Dr Deborah Fravel, USDA-ARS,
levels of chitin. Biological control of some
and is now being updated by the APS Bio-
soilborne fungal diseases has been correlated
logical Control Committee (Table 10.1).
with chitinase production. Bacteria produc-
ing chitinases or glucanases exhibit antag-
onism in vitro against fungi. A recombinant
Escherichia coli expressing the chiA gene The Trichoderma System as
from S. marcescens was effective in reduc- Biocontrol Agents
ing disease incidence caused by Screrotium
rolfsii and R. solani. In other studies, chi- Trichoderma spp. are free-living, saprophytic
tinase genes from S. marcescens have been fungi that exhibit a high rate of interactions
Table 10.1. Fungi, bacteria, activators and their available commercial products.
Commercial products
Fungi
Ampelomyces quisqualis AQ10
Candida oleophila Aspire
Coniothyrium minitans Contans, Intercept WG, KONI
Fusarium oxysporum Biofox C, Fusaclean
Gliocladium sp. Primastorp, SoilGard
Myrothecium verrucaria DiTera
Paecilomyces lilacinus Paecil
Phlebia gigantea Rotstop
Pythium oligandrum Polyversum
Trichoderma sp. Bio Fungus, Binab T, Root Pro, RootShield/PlantShield, T-22G,
T-22 Planter Box, Trichodex, Trichopel, Trieco
Bacteria
Agrobacterium radiobacter Galltrol, Nogall
Bacillus sp. BioYield, Companion, EcoGuard, HiStick N/T, Kodiak, Rhizo Plus,
Serenade, Subtilex, YieldShield
Burkholderia cepacia Deny, Intercept
Pseudomonas sp. BioJect Spot-Less, Bio-save, BlightBan, Cedomon
Streptomyces sp. Actinovate, Mycostop
Activators of host defence
Bacteria Actinovate, BioYield, YieldShield
Bacterial protein Messenger
Synthetic chemical Actigard
with root, soil and foliar environments. The described the mode of action of Tricho-
antagonistic nature of fungi from the genus derma sp. against plant pathogens. Recently,
Trichoderma was demonstrated more than Herrera-Estrella and Chet (2004) discussed
70 years ago. Furthermore, excellent progress the role of Trichoderma spp. as a biological
has been made towards the improvement of control agent; the expression of mycopara-
Trichoderma sp. as a biological control sitism related genes (MRGs); antibiosis; the
agent in the past few years. Many Tricho- role of MRGs in biological control and strain
derma isolates have been used as biocontrol improvement; competition; induced resis-
agents against soilborne pathogens (Wein- tance; plant growth promotion; and Tricho-
dling, 1934). Trichoderma is a ubiquitous derma spp. as a source of genes for crop
genus present in almost all types of habitat improvement.
fungal antagonists. It comprises 3% of the The biocontrol action is due largely to
total fungal population in forests and 1.5% the inherent nature of inhibition or degrada-
of the total fungal population in other soils. tion of pectinases and other enzymes, which
It also exhibits the property of competition are deemed essential for phytopathogenic
with fellow plant pathogenic fungi for key fungi in order to cause pathogenesis in
exudates from seeds that stimulate the germi- plants. These direct effects on other fungi
nation of propagules of plant pathogenic fungi are remarkable yet complex and, until now,
in soil, and also with soil microorganisms for were attributed to being the basis for the
nutrients and space. Trichoderma spp. act action exerted by Trichoderma sp. on plant
against a range of economically important growth and development.
aerial and soilborne plant pathogens. They
have been used in the field and greenhouse
against silver leaf on plum, peach and nec-
tarine; Dutch elm disease on elms, honey Mechanism of Action of Trichoderma
fungus (A. mellea) on a range of tree species
and against rots on a wide range of crops, Several modes of action have been proposed
caused by Fusarium, Rhizoctonia, Pythium to explain the suppression of plant patho-
and Sclerotium (Table 10.2). Lacicowa and gens by Trichoderma spp. These include
Pieta (1994) reported that Trichoderma spp. mycoparasitism, antibiosis, competition,
and Gliocladium sp. gave significant control siderophore production, induction of sys-
against soilborne pathogenic fungi of pea, temic resistance, growth promotion, etc.
which was better than that obtained with (Dennis and Webster, 1971; Upadhyay and
the use of chemicals. Spiers et al. (2004) Mukhopadhyay, 1986; Chet, 1987).
Table 10.2. Trichoderma as biocontrol agents and their target pathogens which cause diseases in
various host plants.
Biocontrol agent Pathogens Host crop
Trichoderma spp. Pythium sp. Bean, pea, cucumber

T. harzianum Fusarium oxysporum Cucumber, cotton, wheat, muskmelon, tomato, ginger
Fusarium sp. Lentil, cotton
Pythium sp. Pea, radish, cucumber, tomato
Rhizoctonia solani Pea, radish, snapbean
Sclerotinia sclerotiorum Cucumber, Mentha sp.
Sclerotium rolfsii Sugarbeet, groundnut, chickpea, Mentha sp.
Gaeumannomyces sp. Wheat
T. viride Pythium sp. White mustard
R. solani Potato
Direct action of biocontrol the antagonism of Trichoderma as a biocon-

agent Trichoderma trol agent. The process apparently includes:
1. Chemotropic growth of Trichoderma;
Mycoparasitism 2. Recognition of the host by the myco-
Mycoparasitism is the phenomenon in which parasite;
fungal parasites attack other fungi. It is 3. Secretion of extracellular enzymes;
divided into necrotrophic (destructive) and 4. Penetration of the hyphae; and
biotropic (balanced) parasitism (Barnett and 5. Lysis of the host.
Binder, 1973). Trichoderma spp. are grouped
in necrotrophic mycoparsites. Velikanov Antibiosis
et al. (1994) noticed hyperparasitism with
The high percentage of effectiveness of the
different strains of T. viride, T. harzianum
biocontrol ability of Trichoderma is conferred
and G. virens, which were tested against five
most likely by more than one exclusive
phytopathogenic fungi, namely F. oxyspo-
mechanism. Another known mechanism of
rum, F. solani, Pythium sp., R. solani and
biocontrol is antibiosis, which is the release
S. sclerotiorum causing root rot of pea.
of antibiotics and other metabolites that are
Trichoderma recognizes signals from the
harmful to the pathogen and inhibit their
host fungus, triggering coiling and host pen-
growth. Many such substances have been
etration. Remote sensing is due at least par-
isolated from Trichoderma sp., namely glio-
tially to the sequential expression of cell wall
toxin and glyoviridin from T. viride (Sharma
degrading enzymes. Different strains can fol-
and Dohroo, 1991), viridin, alkyl pyrones,
low different patterns of induction, but the
isonitriles, polyketides, diketopiperazines
fungi apparently always produce low levels
and some steroids (Upadhyay and Mukho-
of an extracellular exochitinase. The possible
padhyay, 1986). Many Trichoderma spp.
role of agglutinins in the recognition process
are reported to produce volatile and non-
determining fungal specificity has been
volatile antibiotics, chloroform soluble anti-
examined recently. Barak et al. (1985) pro-
biotics, including trichodermin, and peptide
posed that lectins of plant pathogenic fungi
antibiotics active against a range of plant
might play a role in recognition. Inbar and
pathogenic fungi (Dennis and Webster,
Chet (1992) proved the role of lectins in rec-
1971). Indeed some isolates of Trichoderma
ognition during mycoparasitism using a
excrete growth-inhibitory substances. In
biometric system. Secretion of lytic enzymes,
fact, it seems advantageous for a biocontrol
including b-1,3-glucanase(s), proteinase(s),
agent to suppress a plant pathogen using
chitinases and lipases, enables Trichoderma
multiple mechanisms.
spp. to degrade the host cell wall, thereby
reducing the incidence of disease (Harman, Competition
2001). Ordentlinch et al. (1990) reported that
there was no correlation between in vivo and This mode of action implies the competi-
separated in vitro dual culture or enzyme tion among microorganisms for space and
assays. Involvement of chitinase and b-1,3- nutrients when these factors are limiting in
glucanase in Trichoderma-mediated biologi- nature. It is considered a ‘classical’ mecha-
cal control was also reported by Harman nism of biocontrol. The mechanism is con-
(2001). Involvement of b-1,6-glucanases and sidered involved when no evidence of either
b-1,4-glucanases may also play an important mycoparasitism or antibiosis is found in a
role in mycoparasitism (Thrane et al., 1997). particular interaction. Since Trichoderma is
T. harzianum-mediated mycoparasitism may an omnipresent fungus and is found in agri-
involve 20 separate genes and gene products; cultural and natural soils throughout the
most of these gene products are synergistic world, it is enough proof of it being an
with one another (Lorito, 1998). excellent competitor for space and nutri-
It is considered that mycoparasitism is tional resources. Excellent competitiveness
one of the main mechanisms involved in for space and nutrition is supposed to be
useful for biological control in the absence root area. Similarly, an increase in P and Fe
of mycoparasitism or antibiosis (Cook and concentration was observed in Trichoderma
Baker, 1983). Elad (2000) reported that when inoculated plants.
conidia of T-39 were sprayed on leaves, ger- In recent times, there has been tremen-
mination of conidia of B. cinerea was slowed dous progress related to pathways of resis-
down, because the pathogenic conidia tance and much has been done to elucidate
required external nutrients for germination them. In many instances, salicylic acid or jas-
and infection. monic acid, together with ethylene or nitrous
oxide, induce a cascade of events that lead to
the production of a variety of metabolites
Indirect action of biocontrol agents and proteins with diverse functions. Differ-
ent pathways are induced by different chal-
lenges, although there seems to be crosstalk
In addition to the ability of Trichoderma
or competition between pathways.
spp. to attack or inhibit the growth of plant
There has been a great leap in explain-
pathogens directly, recent discoveries indi-
ing the ISR pathway activated by rhizobac-
cate that they can also induce systemic and
teria; the best part is that it is the closest
localized resistance to a variety of plant
analogue of induced resistance activated
pathogens.
by Trichoderma. The rhizobacteria-induced
systemic resistance (RISR) pathway pheno-
typically resembles systemic acquired resis-
Biochemical elicitors of disease tance (SAR) systems in plants. Heil (2001)
resistance and induced defined ISR as the set of changes by which
systemic resistance plants respond to an initial infection or elic-
itor treatment in becoming systemically
Induced systemic resistance (ISR) is another resistant against pathogen attack. Several
phenomenon of biocontrol exhibited by the workers demonstrated that Trichoderma
plant to combat the harmful effects of the spp. could also affect the host plant, which
pathogen. It implies the elicitation of resis- shows an induced resistance-type response.
tance or plant response against the microor- Chang et al. (1986) reported hastened
ganism or abiotic agent, such that following flowering, increased number of blooms in
the inducing challenge posed to the plant, Chrysanthemum and an increase in the height
de novo resistance to pathogens is shown in and weights of other plants as a result of T.
normally susceptible plants. Localized and harzianum inoculation in steamed soil. Tri-
systemic induced resistance occurs in all or choderma viride-coated seeds of broad bean
most plants in response to attack by patho- resulted in increased fresh and dry weight of
genic microorganisms, physical damage due shoots, roots and nodules (Yehia et al., 1985).
to insects or other factors, treatment with Pea seeds treated with apple pomace-based
various chemical inducers and the presence Trichoderma inoculant extracts resulted in
of non-pathogenic rhizobacteria. Specific increased emergence, rapid plant growth,
strains of fungi in the genus Trichoderma increased seedling vigour and phenolics
colonize and penetrate plant root tissues content. The increase in overall phenolic
and initiate a series of morphological and content may contribute to improved lignifi-
biochemical changes in the plant, which are cation and antioxidant response (Zheng and
considered to be part of the plant defence Shetty, 2000). Altomore et al. (1999) reported
response. Finally, it leads to ISR in the for the first time the ability of a Trichoderma
entire plant. The capability of T. harzianum strain (T-22) to solubilize insoluble or spar-
to promote increased growth response was ingly soluble minerals by three possible
verified both in greenhouse experiments mechanisms, namely acidification, produc-
and in the hydroponic system. A 30% increase tion of chelating agents and redox activity.
in seedling emergence was observed and Further, they reported the solubilization of
these plants exhibited a 95% increase in Fe2O3, MnO2, Zn and rock phosphate by the
cell-free culture filtrate of T-22. Tricho- Oligosaccharides and low molecular

derma strains are also supposed to induce weight compounds
the production of hormone-like metabolites
on release of nutrients from soil or organic Another finding in this sphere has been the
matter (Kleifeld and Chet, 1992). transformation of Trichoderma mutants
with reporter based on green fluorescent
protein or specific enzymatic activities (glu-
Chemicals Produced by Trichoderma cose oxidase) under the control of biocontrol-
related promoters. One of the advantages of
this discovery has been the possibility that
What has been stated above is the induced
biomolecules released by the action of
resistance exhibited by some plants that is a
Trichoderma secreted cell wall degrading
result of some microorganism, in this case,
enzymes on the cell walls of fungal pathogens
Trichoderma. In this context, it has been
and plants can be isolated. These molecules
found that Trichoderma produces three
function as inducers of the antagonistic
classes of compounds to exert its effect and
gene-expression cascade in Trichoderma
induce resistance in plants. These include:
and some also function as elicitors of plant
defence mechanisms.
Proteins with enzymatic or

other functions
Plant Growth Promotion
With regards to the first class of biochemical
Fungal as well as bacterial biocontrol agents
elicitors of Trichoderma, it is stated that
are reported and known to induce growth
much before the discovery of the induction
of various crops and also increase crop
of resistance by Trichoderma, a small 22-kDa
yield. Trichoderma spp., and other benefi-
xylanase protein was shown to induce ethyl-
cial root-colonizing microorganisms, also
ene production and plant defence. Working
enhance plant growth and productivity.
in the direction of Trichoderma, it has been
Mukhopadhyay (1996) has reported increa-
found very recently that a series of proteins
sed growth of several crop plants following
and peptides that are active in inducing ter-
seed treatment with T. harzianum and T.
penoid phytoalexin biosynthesis and per-
virens. The reason attributed to this effect
oxidase activity in cotton are produced by
of Trichoderma and other microbes on
strains of T. virens.
plants has been explained based on the fol-
lowing arguments.
1. Suppression of harmful root microflora,
Avr homologues
including those not a direct causal organism
of disease.
Another class is the protein product of Avr 2. Production or activation of growth-
genes, which have been identified in a vari- stimulating factors.
ety of fungal and bacterial plant pathogens. 3. Increased nutrient uptake through solu-
These are usually seen functioning as race-
bilization and sequestering of nutrients.
or pathovar-specific elicitors, possessing the
capability of inducing hypersensitive res- It is a well-established fact that microorgan-
ponses and other defence-related reactions isms closely associated with the roots of a
in plant cultivars that contain the corre- plant can influence plant growth and devel-
sponding resistance gene. Proteome analysis opment directly. Although the ability of
of T-22 identified proteins that are homo- species of Trichoderma spp. to promote or
logues of Avr4 and Avr9 from Cladosporium inhibit plant growth directly has been noted
fulvum; T. atroviride strain P1 also produces for many years (Ozbay and Newman, 2004),
similar proteins. efforts to define and exploit these influences
have met with limited success. Many work- have been selected or modified to be resis-
ers have reported plant growth promotion by tant to specific agricultural chemicals.
different strains of Trichoderma spp. Chang
et al. (1986) observed plant growth promo-
tion resulting in enhanced germination, more
rapid flowering, increased flowering and Mass Multiplication of Trichoderma
increased height and fresh weight in pep-
per, periwinkle, Chrysanthemum and sev- The most critical obstacles to the application
eral others after treatment of the soil with of biological control fungi as an effective
peat/bran inoculum or conidial suspension means of disease management are the lack
of T. harzianum. of knowledge of methods for mass culturing
and a proper delivery system, which is needed
to augment the soil directly with fungal
Solubilization and Sequestration of antagonists (Papavizas, 1985; Singh et al.,
Inorganic Plant Nutrients 2002, 2004; Dissevelt and Ravensberg, 2004).
Solid media for the experimental produc-
tion of Trichoderma sp. and Gliocladium
It is a common natural occurrence that plant
sp., two of the most common fungal antago-
nutrients undergo a complex, intricately
nists, have been used frequently in laboratory
woven conversion from soluble to insoluble
and greenhouse studies (Bateman, 2004).
forms when in the soil; this is a precursor to
Some workers have tried composted
the ease of access and absorption by roots. It
hardwood bark as a substrate for the large-
is here that microorganisms may influence
scale production of biocontrol fungi (Nelson
these transitions (Altomare et al., 1999). The
and Hoitink, 1983). Sundheim (1977) used
most commonly and extensively studied
bark pellets as a medium for mass produc-
nutrients are iron and manganese. Tricho-
tion of Trichoderma and Gliocladium sp. to
derma sp. has been reported to produce some
control Phomopsis sclerotioides in cucum-
compounds called siderophores (Sen, 2000).
ber. A variety of media have been used by
Iron chelated with these siderophores is in
various researchers for the production of
the unavailable and bound form for plant
Trichoderma sp. in stationary flasks, shak-
pathogens and so they do not have access to
ers (Jin et al., 1991) and liquid fermenters
iron. On the contrary, plant roots are capa-
(Jin et al., 1996).
ble of absorbing iron in this form, so these
Backman and Rodriguez-Kabana (1975)
are accessible to the plant. This is one of the
used diatomaceous earth granules along
mechanisms that operate for the growth of
with molasses for developing a formulation
plants and the supply of nutrients to them.
of biocontrol agents for application in soil.
Trichoderma sp. increases the uptake and
Hadar et al. (1979) used wheat bran formu-
concentration of a variety of nutrients
lations for mass-multiplying biocontrol agents
(copper, phosphorus, iron, manganese and
for field application. Papavizas et al. (1984)
sodium) in roots of hydroponic culture,
developed a liquid fermentation technology
even under axenic conditions. This increased
for mass production of fungal antagonists by
uptake indicates an improvement in plant
employing a combination of molasses and
active uptake mechanisms.
brewer’s yeast. Sivan et al. (1984) developed
a formulation of T. harzianum on wheat bran
and peat. Mukhopadhyay et al. (1986) used
Pesticide Susceptibility sorghum grains to prepare the powdered for-
mulations of fungal antagonists.
Another aspect and quality of Trichoderma Tapioca rind, cow dung, biogas slurry,
sp. lies in the fact that it possesses innate farmyard manure, paddy chaff, rice bran,
and natural resistance against most agricul- groundnut shell, sugarcane bagasse, sheep
tural chemicals, including fungicides. The manure, chickpea husk, maize cob, etc.,
capability differs with strain. Some lines are some of the substrates used for mass
multiplication of T. harzianum and T. viride would be beneficial to a larger degree than

(Kousalya and Jeyarajan, 1990). Conway individual components.
et al. (1996) used oat seeds for mass cultur- A primary obstacle in the commercial
ing of T. harzianum isolate OK-86. Alginate use of Trichoderma spp. for both disease con-
pellets were used for formulating a biomass trol and growth enhancement is the mass
of G. virens and T. hamatum and various production and delivery methods of its for-
food bases like wheat bran, maize cobs, mation to the plants (Papavizas, 1985; Muk-
groundnut hulls, soy fibre, castor pomace, hopadhyay, 1996). The problem lies in the
cocoa hulls and chitin were used. They fact that biocontrol products represent living
found that the pellets with G. virens and all systems. A large number of growth media are
the food bases with bran, soy fibres, castor, reported to be suitable for the genus Tricho-
pomace or chitin resulted in stands similar derma, but most of these are either food
to those of the control, except cocoa hull grains or are expensive. For solid-state fer-
meal significantly reduced damping-off of mentation substrates like sorghum grain,
Zinnia caused by R. solani and P. ultimum. wheat grains, wheat bran, tea leaf waste, cof-
Kumar and Marimuthu (1997) tested fee husk, sawdust, etc., have been used (Gogoi
the effect of decomposed coconut coir pith and Roy, 1996; Mishra, 1998). A liquid fer-
(DCCP) added to normal nursery media on mentation method consisting of molasses,
the survival of T. viride. The pure DCCP wheat bran and yeast is proposed for large-
gave efficient sporulation of T. viride popu- scale production of Trichoderma (Montea-
lation. Lewis et al. (1998) used commercially legre et al., 1993). Bioefficacy of T. harzianum
manufactured cellulose granules (Biodac) in produced by solid fermentation, which con-
a mixture with a sticker and fermenter- tains only conidia, was found more effective
produced biomass of Trichoderma sp. and than when produced by liquid fermentation,
G. virens to produce a formulation in which where a mixture of chlamydospores, hyphal
chlamydospores in the biomass were acti- fragments and conidia were present.
vated with dilute acid. Tiwari et al. (2004) Conidia of Trichoderma in pyrophyl-
suggested that among the eight substrates, lite survived better than alone at between –5
namely grains of Sorghum vulgare [S. bicolor], and 30°C. A temperature range from –5 to
wheat, Pennisetum typhoides [P. glaucum], 5°C was found most suitable for an impro-
S. vulgare cv. M.P. Chari and Sorghum sp., ved shelf life (Mukherjee, 1991). Mukherjee
a locally available millet; wheat bran; rice reported that shelf life of T. virens was almost
bran; and sugarcane bagasse were evaluated constant on coated chickpea seeds at 5°C
for the mass propagation of T. viride. Sor- and, at room temperature, it was decreased
ghum sp., a locally available millet, resulted by 12%. Chlamydospore-based formulations
in the greatest spore concentration, spore exhibited longer shelf life than conidia-
viability and total biomass of the fungal based formulations (Mishra et al., 2001).
antagonist. The greatest spore concentration
(8 × 109) was observed after 15 days of incu-
bation at 27 ± 1°C. The spores of T. viride Basic Components of
remained viable for 6 months at 5°C. Biocontrol Systems
There are three basic components of bio-

control systems. These are as follows:
Commercial Use of Trichoderma
Commercialized systems for the biological Biocontrol strain

control of plant diseases are few. It has been
stressed that microbes cannot be used in The first step towards successful biocontrol
isolation and exceptional results expected. is to obtain or produce a highly effective bio-
On the contrary, a biocontrol system or control strain or other material (Table 10.3).
consortia needs to be developed, which For instance, the development of the T-22
Table 10.3. Inexpensive production and formulation of the biocontrol agent using various base
materials.
Base material Biocontrol agent Formulation References
Blackgram shell, shelled maize cob, Trichoderma viride, Powder Kumar and
coir pith, peat, gypsum, barley grains T. harzianum Marimuthu, 1997
Coffee fruit skin + biogas slurry T. harzianum Pellets Sawant and
Sawant, 1996
Coffee husk T. harzianum, Pellets Bhai et al., 1994
T. viride, T. virens
Coffee berry husk T. harzianum, Pellets Sawant and
T. viride, T. virens Sawant, 1989
Fruit skin and berry mucilage T. harzianum, Pellets Sawant and
T. viride, T. virens Sawant, 1989
Groundnut shell T. viride Powder
Mustard oil cake T. viride Pellets
Soil T. harzianum, T. viride Powder Singh, 2002
Sorghum grain T. harzianum, Powder Upadhyay and Muk-
T. virens hopadhyay, 1986;
Mishra, 1998
Sugarcane straw T. harzianum, T. viride, Pellets Singh et al., 2004
T. reesei, T. koningii
Wheat bran T. virens Powder Singh et al., 2002
Rice husk, maize cob powder, spent tea T. harzianum Powder Tripathi, 1998
leaves, wheat bran, citrus fruit pulp (MTCC 3843)
strain of T. harzianum by Harman and fel- Compatibility Testing of Trichoderma

low researchers was the result of a decade
and more of hard work. Still, its commercial The success of a biocontrol agent depends on
product, Root Shield, picked up pace in the its compatibility with other disease manage-
late 20th century (Harman, 2000). Besides ment systems. This requires holistic testing
the usual properties of a biocontrol agent, of biocontrol agents (BCA) in combination
the strain must also possess the following: (i) with other disease management practices in
to be able to compete and persist in the envi- a system approach. Once the BCA is found
ronment in which it must operate and (ii) ide- to be compatible, it can be integrated suc-
ally, to be able to colonize and proliferate on cessfully with the disease management
existing and newly formed plant parts well modules for each cropping system. Csinos
after application. Sundaram (1996) developed et al. (1983) evaluated the compatibility of
fusants of two isolates of T. harzianum (Th-1 Trichoderma spp. with fungicides for the
and Th-2), among them some showed mor- management of S. rolfsii in groundnut. T. har-
phological characters immediately between zianum, Rhizobium and carbendazim were
Th-1 and Th-3. When T. harzianum (Th-3) integrated successfully for the management
was fused with T. virens, many fusants were of stem rot of groundnuts caused by S. rolf-
developed and few exhibited improved bio- sii. A combination of either Trichoderma or
control activity (Ghosh, 1996) (Table 10.4). Gliocladium with fungicides like carboxin
or metalaxyl protected crop plants against
soilborne pathogens and was emphasized
Ease of delivery and application by several workers (Sawant and Mukhopad-
hyay, 1990; Mukhopadhyay et al., 1992).
Some delivery methods for Trichoderma The alternation of BCA with fungicides was
are listed in Table 10.5. found to be more effective than mixtures.
Table 10.4. Commercial products of Trichoderma currently in the open market or under registration.
Product Biocontrol agent Effective against Manufacturer/distributor
Antifungus Trichoderma sp. Various fungi Grondortsmettigen De

Cuester n.v., Belgium
Bas-derma T. viride Basarass Various fungi Biocontrol Res. Lab., India
Binab T T. harzianum Control of wound decay and Bio-innovation AB, UK
(ATCC 20476) and wood rot
T. polysporum
(ATCC 20475)
Bioderma T. harzianum/T. viride Various fungi Biotech International Ltd.,
India
Biofungus Trichoderma sp. Sclerotinia, Phytophthora, Grondortsmettigen De
Rhizoctonia solani, Pythium Cuester n.v., Belgium
spp., Fusarium, Verticillium
Bio-trek 22G T. harzianum Various fungi Bioworks, Inc. of Geneva,
NY
Ecofit T. viride Various fungi Hoechst Schering Agro
Evo Ltd., India
Root pro, Root T. harzianum R. solani, Pythium spp., Efal Agr, Israel
Protato Fusarium spp. and
Sclerotium rolfsii
Root shield, Plant T. harzianum Pythium spp., R. solani, Bioworks Inc., USA
shield, T-22 Rifai strain Fusarium spp.
Planter Box KRL-AG(T-22)
RUTOPIA Trichoderma sp. Organic Soil Amendment NaEx Corp/Poulenger
Turfgrass Biostimulant USA, Inc
SoilGard Trichoderma sp. Damping-off diseases USA
(formerly caused by Pythium and
GlioGard) Rhizoctonia spp.
Supresivit T. harzianum Various fungi Borregaard and Reitzel,
Czech Republic
T-22 G, T. harzianum strain Various fungi THT Inc., USA
T-22 HB KRL-AG2
Trichoderma Trichoderma spp. R. solani, S. rolfsii, Myocontrol Ltd., Israel
2000 Pythium spp., Fusarium spp.
Trichodex, T. harzianum Botrytis of vegetables and Makhteshim Chemical
Trichophel grapevines Works Ltd., USA
Trichophel, T. harzianum and Armillaria, Botryosphaeria, Agrimm Technologies Ltd.,
Trichoject, T. viride Chondrosternum, Fusarium, New Zealand
Trichodowels, Nectria, Phytophthora,
Trichoseal Pythium, Rhizoctonia
Tri-control Trichoderma sp. Various fungi Jeypee Biotechs, India
Trieco T. viride Rhizoctonia spp., Pythium spp., Ecosense Labs Pvt. Ltd.,
Fusarium spp., root rot, Mumbai, India
seedling rot, collar rot, red rot,
damping-off Fusarium wilt
TY Trichoderma spp. Various fungi Myocontrol, Israel
Tusal Trichoderma spp. Damping-off diseases caused Spain
by Pythium, Phoma and
Rhizoctonia species,
rhizomania disease of
sugarbeet and drop of lettuce
Table 10.5. Mass production and delivery methods of Trichoderma.
Biocontrol agent Mass production Delivery method
Trichoderma viride Commercially produced pellets Applied directly to the soil along
(BINAB T SEPPIC). Also produced with food base
on wheat bran: sawdust and tap water
(3:14). Have been produced on a variety
of growth media (autoclaved rye, barley
and sunflower seeds)
T. harzianum As in T. viride; also produced on Backman and Rodriguez-Kabana
molasses and enriched clay applied it @ 140 kg/ha after 70
granules as food base days of planting
Integration of T. harzianum with a sublethal fungicides applied at reasonable rates can-

dose of methyl bromide (300 kg/ha) and not do. It can also be used in conjugation
soil solarization yielded maximum control of with other microbes, which thereby increa-
Fusarium crown and root rot of tomato caused ses its efficiency. The two-pronged advan-
by F. oxysporum f. sp. radicis-lycopersici tage would be a reduction in the use of
(Sivan and Chet, 1993). pesticides and limiting root-attacking dis-
In order to get the maximum efficiency eases, plus protection of transplants in the
from Trichoderma, it is important that it field by virtue of its ability to colonize
should be applied properly. It is effective as roots. Besides this, powdered formulations
a seed treatment with or without fungi- can be made and applied to the seed
cides. The basic reason why this is used is directly, and then the seeds are sown. This
its multifaceted nature and broad range. It would reduce the amount of biocontrol
colonizes roots, increases root mass and agent used, as well as protect the plants
improves plant health, and consequently from pathogen attack. Further, plant growth
provides yield increases, which chemical would also improve.
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11 Physiological Specialization of
Ustilaginales (Smut) of Genera Bromus,
Zea and Triticum in Argentina
Marta M. Astiz Gassó1 and María del C. Molina1,2

1Instituto Fitotécnico de Santa Catalina (IFSC) and 2Consejo Nacional de
Investigaciones Científicas y Tecnológicas (CONICET), Facultad de
Ciencias Agrarias y Forestales, Universidad Nacional de La Plata,
Llavallol, Buenos Aires, Argentina
Abstract
The objective of this project was to determine the existence of physiological forms of Ustilaginales in
Bromus, Zea and Triticum types in Argentina. Studies were carried out on the physiological special-
ization of Ustilago bullata Berk on Bromus spp., Zea seedlings’ reaction to inoculation with U. maydis
(D.C.) Corda and physiological specialization of Tilletia laevis Wallr. (common bunt) on Triticum spp.
The smut was collected in different agricultural and cattle-raising regions in the country, using Usti-
laginales taxonomic keys for smut identification and classification. The experiments were carried out
in greenhouses and in fields at the Instituto Fitotecnico de Santa Catalina (FCAyF-UNLP). For U. bul-
lata and T. laevis, the techniques used were as follows: inoculation by sprinkling of teliospores on host
seeds and inoculation by hypodermic syringe with suspension of U. maydis sporidia on plantlets of
Z. mays and related wild species. As a result of said studies, it was determined that: (i) different
physiological forms exist in each of the kinds of smut analysed; (ii) genetic variability exists in the
hosts which have genes that express different degrees of resistance to the disease; and (iii) genetic
improvement is the most efficient and least environmentally harmful method.
Introduction als, where the pathogens produce important

economic losses (Fischer and Holton, 1957;
Smuts are pathogens of plants that belong to Hirschhorn, 1986; Snetselaar and Mims,
Phylum Basidiomycota, Class Ustilaginomy- 1992). Until the 20th century, they were con-
cetes, Order Ustilaginales. Smut has the sidered, worldwide, one of the most serious
characteristic of forming greyish-black pow- causes of loss of grain and/or seeds, similar
dery masses of teliospores (basidiospores) to the effects produced by rust.
on different organs such as the seeds, stems, In Argentina, between 1934 and 1995,
leaves, flowers and fruit of the hosts. Approx- Hirschhorn and collaborators carried out sev-
imately 1400 species of smut are known, eral studies on Ustilaginales covering the
which attack around 75 families of Angio- taxonomic classification of the species, geo-
spermae; the most familiar diseases are those graphical distribution, germination types and
affecting Monocotyledoneae, especially cere- histopathology and cytology of the different
Physiological Specialization of Ustilaginales (Smut) 139
species of smut (Hirschhorn, 1986). Currently, in samples of B. catharticus, B. mollis, Hor-

control of these diseases is by means of agro- deum jubatum and H. compresum. The author
chemicals and, on a smaller scale, by obtain- also determined that Bromus head smut in
ing species with resistant genes through Argentina was represented by U. bullata, U.
improvement programmes and studies on bullata cv. macrospora (Hirschhorn, 1977,
variability of these pathogens. 1986). Astiz Gassó (1983, 1985, 1994) reported
The objective of this project was to the presence of genes for resistance to the
determine the existence of physiological pathogen and in vitro U. bullata teliospore
forms of Ustilaginales in Bromus, Zea and formation, a phenomenon unrecorded for
Triticum in Argentina: this pathogen and uncommon in other smuts.
The objective of this work was to determine
1. Physiological specialization of U. bul-
the existence of physiological forms in U. bul-
lata Berk on Bromus spp.
lata populations on several Bromus species.
2. Zea seedlings’ reaction to inoculation
In this experiment, we used seeds of B.
with U. maydis (D.C.) Corda.
catharticus. B. parodii, B. brevis, B. auleti-
3. Physiological specialization of T. laevis
cus and B. inermis cv. gombaszpuzta were
Wall. (common bunt) on Triticum spp.
provided by the Instituto Fitotécnico de
Santa Catalina, FCAyF and Department of
Genetics, and the Experimental Estación
Physiological Specialization of of Pergamino (INTA). The seeds were de-
Ustilago bullata Berk on Bromus spp. infested with a 2% formaldehyde solution
for 20 min and then washed in sterile water
Head smut (U. bullata Berk) is a pathogen three times. For identification of the patho-
which affects the growth of various grass gen, spores from each isolate harvested from
species, especially within the genus Bro- plants naturally infected in the field were
mus. The disease is initiated when fungal examined microscopically (Table 11.1).
hyphae penetrate seedlings; the attack devel- Viability of teliospores was tested by plat-
ops from the inflorescences at the expense ing them in PDA medium 2% (Fischer and
of the ovaries, forming a typical sorus. Severe Holton, 1957). The seeds were infested with
infection affects limbs and glumes, reduc- teliospores (1.8 × 10 3g teliospore/g seed),
ing seed and forage production. In the USA, placed in sulphite paper envelopes and
Fischer and Holton (1957) and Hirschhorn shaken well, so that spores would stick to
(1977, 1986) verified experimentally the the seed. Precautions were taken to avoid
existence of genes for resistance, physiolog- contamination with the different isolates of
ical forms and the ability to cross-breed U. the pathogen. Thirty live seeds per isolate
bullata and U. striiformis. Kreizinger et al. in three replications were inoculated during
(1947) recorded the different reactions of U. 4 consecutive years. An uninoculated sam-
bullata on Bromus which grew in the moun- ple was also included during the study.
tains and Bromus which grew on the plains; Inoculated samples were sown in experi-
these experiences indicated that resistant mental plots 1.5 m × 0.40 m in three rows
Bromus varieties and lines could be obtained
by artificial infection under controlled con- Table 11.1. Ustilago bullata isolates collected in
ditions and in the field. Also, 13 physiologi- different localities in Argentina.
cal forms of the pathogen could be studied
(Meinrs and Fischer, 1953). In New Zealand, Locality Province
Falloon (1976, 1979a,b) carried out studies
on the effect of U. bullata infection on B. Pergamino Buenos Aires
catharticus. Also, Falloon and Hume (1988) Tres Arroyos Buenos Aires
reported the effects of the pathogen on B. Llavallol Buenos Aires
Gowland Buenos Aires
willdenowii productivity and endurance in
General Roca Río Negro
the field. In Argentina, Hirschhorn (1977)
Check Mixture
studied teliospore morphological variations
140 M.M. Astiz Gassó and M. del C. Molina
with a distance of 0.20 m between them. The but the levels of infection were lower than
experimental design used was a randomized B. catharticus; B. brevis gave a resistant
complete block. Evaluations in the field reaction to isolate Gowland, a moderately
were conducted by head countings, record- resistant reaction to Pergamino, Llavallol
ing the percentage of infection based on the and the mixture, a moderately susceptible
number of infected and healthy heads. Then, reaction to isolate Tres Arroyos and a sus-
the average infection for the 4 years was cal- ceptible reaction to General Roca. Similar
culated. The level of resistance/susceptibil- results were reported previously by Astiz
ity was determined using a disease rating Gassó (1983). B. auleticus and B. inermis cv.
scale (Table 11.2). gombaszpuzta were resistant to all isolates
Isolates showed an 80–90% teliospore and the uninoculated check did not show
germination, approximately 20–25 h after any infection.
they were cultivated on PDA. The teliospore Four physiological forms in the popula-
germination rate increased with tempera- tions of U. bullata are shown in Fig. 11.1: (i)
ture from 20 to 25°C, with significant among- Tres Arroyos; (ii) Pergamino and Llavallol;
population differences. Boguena et al. (2007) (iii) Gowland; and (iv) General Roca. The spe-
also obtained similar results when they exam- cies B. brevis would be the differential host.
ined the effect of temperature from telio-
spore germination. Table 11.3 shows the
reaction of the Bromus species tested with
the different U. bullata isolates. Bromus Reaction to Inoculation with Ustilago
catharticus was susceptible to all isolates maydis (D.C.) Corda on Zea seedlings
including the mixture and similar results
were reported for Astiz Gassó and Aulicino Ustilago maydis is a smut that promotes the
(1999); B. parodii showed similar reactions, development of galls in Zea, the relation
with the host being necessary to fulfil its life
cycle. Damage produced in plants by the
Table 11.2. Disease rating scale for Ustilago presence of corn stunt is: chlorosis, seedling
bullata. death and tumours in leaves, stems, ears
and tassels. At first, it was considered that
Reaction Infection (%) U. maydis attacked Z. mays and Z. mexi-
cana, but it was later verified that it also
Resistant (R) 0–5
attacked Z. perennis, Z. diploperennis, Z.
Moderately resistant (MR) 6–10
parviglumis, Z. luxurians and their hybrids
Moderately susceptible (MS) 11–30
Susceptible (S) 31–100
with the grown species (Hirschhorn, 1986;
Duran, 1987).
Table 11.3. Reaction of Bromus species to different Ustilago bullata collected in different localities in
Argentina.
Ustilago bullata isolates
HOSTS Pergamino Tres Arroyos Gowland Llavallol General Roca Mixture
Bromus catharticus S S S S S S
B. parodii S S S S S S
B. brevis MR MS R MR S MR
B. auleticus R R R R R R
B. inermis cv. R R R R R R
gombaszpuzta
Nor-inoculated check 0 0 0 0 0 0
100
90
80
70
Infection (%)
60
50
40
30 Bromus catharticus
Bromus parodii
20 Bromus brevis
Bromus auleticus
10 Bromus inermis cv
Non-inoculated check
0
PERGAMINO TRES GOWLAND LLAVALLOL GENERAL MIXTURE
ARROYOS ROCA
Isolates
Fig. 11.1. Reaction of Bromus spp. to U. bullata isolates.
Until 1964, corn stunt did not any have U. maydis are presented. This was done
incidence at the Instituto Fitotécnico de with the purpose of determining resistance
Santa Catalina, but in that year, a Z. peren- of the species and/or inbreds to U. maydis.
nis from Jalisco (México) was introduced The host materials used were the popula-
and later on Z. mexicana, Z. parviglumis, Z. tion ‘Colorado Klein’, the inbreds SC66,
luxurians and Z. diploperennis were also B73, E624A688 of Z. mays, as well as clones
grown and hybridized to Z. mays. As the of Z. perennis and Z. diploperennis. Over a
hybrids are grown in the field as well as in time period of 2 years, 1296 plants were
the greenhouse, vegetative plants are avail- inoculated with different strains of U. may-
able throughout the year (Astiz Gassó and dis isolated from the province of Buenos
Molina, 1996). Aires (Santa Catalina, Balcarce, Necochea
The pathogen multiplies on these plants and 25 de Mayo), the province of Entre Ríos
with the corresponding increase in the (Paraná) and the province of Córdoba (Río
number of spores disseminated by air and Cuarto). These strains were cultivated in a
in the soil. Losses from corn smut range liquid medium of PDB 2% on a shaker for
from 1% to up to 10% of all Zea species and 18–24 h running at 25°C ± 2. The pathogen
hybrids are also attacked, depending on the was inoculated by puncturing the base of
environmental conditions favouring patho- the seedling with a hypodermic syringe and
gen development; sweet corn may show the sporidial suspension with concentra-
losses approaching 100% from corn smut in tions 105–106 sporidia/ml was then forced
localized areas (Callow and Ling, 1973; up into the leaf whorl (Callow and Ling,
Hirschhorn, 1986; Banuett, 1995; Astiz Gassó 1973; Snetselaar and Mims, 1992, 1993;
and Molina, 1999). Banuett, 1995; Edmunds, 1998; du Toit and
In this chapter, the results from analysing Pataky, 1999). In many previous works, this
the response of Z. mays, Z. perennis and method was very successful in producing
Z. diploperennis seedlings when they disease galls in seedlings (Astiz Gassó and
are inoculated with six populations of Molina, 1999).
The trial involved three replications Río Cuarto (4.55%); Z. perennis: Santa Cat-
and a tester (non-treated plants). The plants alina (1.67%) and Z. diploperennis: 25 de
were evaluated using a reaction scale to Mayo (13.89%), Paraná (2.78) and Santa
determine the mean percentage of infection Catalina (1.67%).
with U. maydis (Table 11.4). The first symp-
toms in seedlings were observed 4–6 days Table 11.4. Reaction scale in hosts.
after inoculation and gall development occu-
rred 7–8 days after the treatment (Fig. 11.2). Behaviour Host reaction
The behaviour of the host when inocu-
lated with six populations of U. maydis was 0 = Immune No reaction
analysed in Fig. 11.3. The hosts that reacted 1 = Resistant Partial chlorosis
forming galls (grade 4) were cv. Colorado 2 = Medium Accent chlorosis and/or
Klein: Necochea (8.34%) and Balcarce resistant presence of stripe or
(2.78%); B73: Río Cuarto (14.15%), 25 de anthocyanin stain
Mayo (11.11%), Santa Catalina (5.84%) and 3 = Medium Necrosis and reduction
susceptibility of growth in plant
Balcarce (1.04%); E642A688: 25 de Mayo
4 = Susceptibility Formation of tumours
(8.33%) and Santa Catalina (3.34%); SC66:
(a) (b) (c)
(d) (e) (f)
Fig. 11.2. Reaction of hosts after inoculations with U. maydis. (a) No reaction, immune; (b) partial
chlorosis; (c–d) accent chlorosis and/or presence of stripe or anthocyanin stain; (e) necrosis and
reduction of growth in plant; (f) formation of tumours (galls).
60.00
50.00
40.00 40.00
% reaction
% reaction
30.00
20.00 20.00
10.00
0.00 0.00
Pobl. Pobl. Pobl.25 Pobl. Pobl.Rio Pobl.Sta. Pobl. Pobl. Pobl.25 Pobl. Pobl.Rio Pobl.Sta.
Necochea Balcarce de Mayo Parana Cuarto Catalina Necochea Balcarce de Mayo Parana Cuarto Catalina
(a) Isolates (b) Isolates
50.00
60.00
40.00
% reaction
% reaction
40.00 30.00
20.00
20.00
10.00
0.00 0.00
(c) Isolates (d) Isolates
100.00
80.00
80.00
% reaction
% reaction
60.00 60.00
40.00 40.00
20.00 20.00
0.00 0.00
(e) Isolates (f) Isolates
Grade 0 Grade 1 Grade 2 Grade 3 Grade 4
Fig. 11.3. Reaction of Zea mays (lines and populations), Zea perennis and Zea diploperennis to six
strains of U. maydis isolates: (a) Colorado Klein (Z. mays); (b) Lines E642A688 (Z. mays); (c) Line SC66
(Z. mays); (d) Line B73; (e) Z. perennis; and (f) Z. diploperennis.
Physiological Specialization of establishing the variability or physiological

Tilletia laevis Wallr. (Common Bunt) specialization of Tilletia species. Histori-
on Triticum spp. in Argentina cally, pathogenic races that are virulent to
resistant cultivars have appeared, so new
germplasm is screened continually for resis-
Common bunt of wheat is caused by T. trit-
tance. Investigations to determine disease
ici and T. laevis; infection takes place in the
resistance were incorporated into breeding
coleoptile when teliospores are found on
programmes (Meinrs and Fischer, 1953;
the coleoptile surface and/or the ground
Kendirck, 1961; Metzger and Hoffmann,
(Fischer and Holton, 1957; Hirschhorn, 1986;
1978; Gaudet, 1990; Johnsson, 1991; Gaudet
Wilcoxson and Saari, 1996). Chemical con-
et al., 1994; Wilcoxson and Saari, 1996).
trol is achieved through seed treatments;
In Argentina, Hirschhorn and collabo-
however, the disease is aggravated due to
rators studied the morphology, taxonomy,
inefficiency in the method of fungicide
symptomatology, spore germination, basidial
application and the widespread use of sus-
cytology and geographical distribution of
ceptible wheat cultivars. In common bunt,
pathogens, T. tritici and T. laevis, to common
the spores survive in the soil for long peri-
bunt of wheat (Hirschhorn, 1986; Astiz Gassó,
ods and can cause infection of seedlings.
1992; Astiz Gassó and Hirschhorn, 1994).
The most effective control method is by
The presence of 12 T. foetida (= T. laevis)
genetic resistance to the pathogen and by
physiological forms and cultivar wheat dif- based on the number of infected and healthy
ferentials for identification of T. foetida were heads. Results were transformed through
reported by Astiz Gassó (1992, 1997a,b) and the Arcosen and the average for 6 years of
Astiz Gassó and Hirschhorn (1994). The testing was calculated. Data were subjected
objective of this work was to establish the to ANOVA (Statistix, 2008). Where signifi-
physiological forms of T. laevis and to study cant differences were detected, treatment
the reaction of commercial wheat cultivars means were separated using HSD Tukey
to the pathogen in Argentina. test (P < 0.05). Our field research to date
In this experiment, we used ten hexa- indicates that T. laevis shows several physi-
ploid bread wheat cultivars with different ological forms: Tandil, Rio Cuarto, Villa
levels of resistance and two tetraploid culti- María, Cabildo, Castelar and Casilda. The
vars considered resistant. Seeds were de- rest of the 19 populations of common bunt
infested with a formaldehyde solution (3:1) showed homogeneous behaviour, so it
and washed in sterile water. Pathogens from could be considered as one physiological
25 localities in the Argentine wheat belt form (Table 11.5).
were tried. Wheat cultivars were inoculated Tetraploid cultivar, Buck Cristal, proved
with 0.5 g of teliospore/100 g of seed. Exper- the presence of resistant genes. The hexa-
imental field plots consisted of three rows ploid wheat cultivars, Buck Ñapuca and
2 m long per cultivar/pathogen isolate. Field Buck Yapeyú, were moderately resistant to
evaluations were carried out by head count- pathogen incompatibility to different iso-
ings and the percentage of infection was lates (Table 11.6). The rest of the hexaploid
Table 11.5. Means of infections of 25 T. laevis populations.
Tilletia laevis populations Province Mean
1. Tandil Buenos Aires 21.50 a

2. Río Cuarto Córdoba 19.83 ab
3. Bordenave Col.1 Buenos Aires 17.12 abc
4. Sta Rosa La Pampa 16.60 abcd
5. Venado Tuerto Santa Fé 16.45 abcd
6. Tres Arroyos Col.1 Buenos Aires 15.70 abcde
7. Lincoln Buenos Aires 15.58 abcde
8. Tres Arroyos Col.2 Buenos Aires 15.38 abcde
9. Laboulaye Córdoba 14.48 abcde
10. Rafaela Santa Fé 14.10 abcde
11. Pergamino Buenos Aires 14.01 abcde
12. San Francisco Córdoba 13.70 abcde
13. Villa María Córdoba 12.92 bcde
14. Salliquelo Buenos Aires 12.61 bcde
15. Marcos Juarez Córdoba 11.89 bcde
16. Necochea Buenos Aires 11.87 bcde
17. Cabildo Buenos Aires 10.91 cde
18. Bordenave Col.2 Buenos Aires 10.73 cde
19. Cañada de Gomez Santa Fé 10.30 cde
20. Bragado Buenos Aires 19.49 cde
21. Río Tercero Córdoba 19.07 cde
22. Paraná Entre Ríos 18.32 cde
23. Castelar Buenos Aires 18.28 de
24. Casilda Santa Fé 17.29 e
25. Bolivar Buenos Aires 17.17 e
Note: Means followed by different letters within column indicate significant

differences according to Tukey’s test (P < 0.05).
Table 11.6. Means of infection of common bunt L1avallol, Gowland and General Roca. Bro-
in wheat cultivars. mus brevis is the differential host for the
fungus populations and shows genetic resis-
Hosts Mean tance to the Gowland isolate. Bromus aule-
ticus and B. inermis cv. gombaszpuzta were
Buck Charrua 19.97 a
resistant to all the fungus isolates. This was
Buck Ombú 18.78 a
Buck Catriel 17.07 ab the first report in Argentina determining the
Buck Bagual 16.58 ab physiological forms of smut U. bullata of
Buck Fogón 13.01 bcd Bromus spp. It can be concluded that the
Buck Guaraní 11.21 bcd wild species and the grown species of the
Buck Ñapuca 18.88 cd genus Zea reacted in different ways (toler-
Buck Yapeyu 18.72 e ant and/or resistant to moderately suscepti-
Buck Cristal 12.85 f ble), depending on the geographic origin of
U. maydis populations. These results might
Note: Means followed by the same letter with a column
indicate cultivars that are homogenous according to
be considered when selecting germplasm to
Tukey’s test (P < 0.05). obtain new forage plants from interespecific
hybrids of the genus Zea. The wheat culti-
vars evaluated would also be used as differ-
wheat was moderately susceptible. Also, the
entials for identification of T. laevis races.
interaction among wheat cultivar populations
Six physiological forms were detected
of T. laevis was significantly high and the
among the used populations of T. laevis.
interaction among pathogen population
This is the first report in Argentina deter-
replications was significantly high accord-
mining the physiological forms of smut T.
ing to Tukey’s test (P < 0.05).
laevis of Triticum spp. The most effective
methods to control the disease are genetic
resistance and establishing the variability of
Conclusions the smut populations. Determination of the
physiological forms of U. bullata, U. maydis
From this analysis, four physiological forms and T. laevis and genetic improvement is
of U. bullata were found in the isolates the most efficient and least environmentally
studied: Tres Arroyos, Pergamino and harmful method.
References
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Argentinas de Micología. Resúmenes, 40 pp.
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shop on the Smut Fungi. CIMMYT, El Batán D.F., México, pp. 90–97.
Astiz Gassó, M.M. and Molina, M.C. (1996) Estudios preliminares para determinar el grado de resistencia
a Ustilago maydis DC. Corda en especies cultivadas y silvestres del Gro Zea. Proceedings Xth Bien-
nial Workshop on the Smut Fungi. University of Calgary, Calgary, Alberta, Canada, pp. 57–62.
Astiz Gassó, M.M. and Molina, M.C. (1999) Zea seedling reaction to inoculation with Ustilago maydis (DC)
Corda. Maize Genetics Cooperation Newsleter 73, 58–60.
Banuett, F. (1995) Genetics of Ustilago maydis, a fungal pathogen that induces tumors in maize. Annual
Review of Genetics 29, 179–208.
Boguena, T., Meyer, S.E. and Nelson, D. (2007) Low temperature during infection limits Ustilago bullata
(Ustilaginaceae, Ustilaginales) disease incidence on Bromus tectorum (Poaceae, Cyperales). Biocon-
trol Science and Technology 17, 33–52.
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the infection of sporidia of Ustilago maydis (DC) Corda. Physiological Plant Pathology 3, 489–494.
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Ustilago bullata. Phytopathogy 43, 200–203.
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Part IV
Endophytes in Plant Disease Control

12 Status and Progress of
Research in Endophytes from
Agricultural Crops in Argentina
Silvina Larrán and Cecilia Mónaco

Centro de Investigaciones de Fitopatología (CIDEFI), Facultad de Ciencias
Agrarias y Forestales, Universidad Nacional de La Plata, La Plata, Argentina
Abstract
Plants harbour a heterogeneous population of endogenous microorganisms, comprising both patho-
gens and non-pathogens, including fungi, bacteria, actinomycetes, etc. Their association has substan-
tial impact on plant health and fitness. Endophytes reside inside healthy plant tissues without
producing any disease symptoms. They are helpful in modifying biochemicals produced by plants and
may add to their protection from insect herbivores, fungal pathogens and even grazing by animals.
However, the ecological role of these endophytes is not yet fully understood. This chapter reports on
endophytic fungi present in beet and tomato leaves. Isolation and analysis of endophytic microorgan-
isms of soybean and wheat are also described. It is advocated that endophytes may have a definite role
in the biological control of Drechslera tritici-repentis, responsible for tan spot disease in wheat.
Introduction the term endophyte has been used lately in

a broad sense to include any fungi isolated
Before beginning, the term ‘endophyte’ from symptomless plant tissues, but the con-
must be defined. Literally, an endophyte is cepts of endophytic colonization and latent
an organism which lives inside a plant, infection by fungi are clearly different. Endo-
‘endo’ meaning within and ‘phyte’ is derived phytic colonization or infection cannot be
from the Greek word ‘phyton’, meaning plant. considered as causing disease, since a plant
There are several definitions of endophytes, disease is an interaction between the host,
such as ‘endophyte’ is an all-encompassing parasite, vector and the environment over
topographical term that includes all organ- time, which results in the production of dis-
isms that are living in plant tissues during a ease signs and/or symptoms. Endophytic
more or less long period of their life, colo- fungi may be described as mutualistic (Clay,
nizing symptomlessly the living internal 1991). Latent infecting fungi are parasitic
tissues of their hosts (Petrini, 1991). Such but cannot be considered mutualistic. Latent
infections are termed ‘endophytic’, particu- infection is the state in which a host is infected
larly when the association is believed to be with a pathogen, but does not show symp-
mutualistic or at least non-pathogenic, or toms and persists until signs or symptoms
‘latent infections’, where a latent pathogen are prompted to appear by environmental or
is involved (Cabral et al., 1993). Therefore, nutritional conditions or by the state of

150 S. Larrán and C. Mónaco
maturity of the host or pathogen (Sinclair N. lolii (Latch, Samuels & Christensen) Glenn,
and Cerkauskas, 1996). Bacon & Hanlin. In tall fescue, N. coenophi-
Petrini’s definition of endophytes (1991) alum causes enhanced tillering and root
encompasses not only mutualistic and neu- growth, increases drought tolerance (Arecha-
tral symbionts, but also those pathogens valeta et al., 1989) and protects against cer-
known to live latently within their hosts. tain nematodes (Kimmons et al., 1990),
Therefore, Wilson (1995) has expanded the fungal pathogens (Gwinn and Gavin, 1992)
‘endophyte’ definition to include internal and insect herbivores (Rowan and Latch,
bacteria that live inside plant tissues with- 1994). The protective nature of endophytes
out causing disease. A wide range of bacte- is due to the presence of alkaloids, whereas
rial genera has been isolated from healthy these alkaloids are responsible for poisoning
plant species of agricultural and horticul- domestic animals. Ergovaline is associated
tural crops (Chanway, 1996, 1998; Sturz with various maladies often observed among
et al., 1998). Endophyte associations may cattle that graze N. coenophialum-infected
range from intimate contact where the fun- tall fescue and collectively called ‘tall fes-
gus inhabits the intercellular spaces and cue toxicosis’. Likewise, lolitrem B is asso-
xylem vessels in the plant, to more or less ciated with the malady ‘ryegrass staggers’,
superficial colonization of peripheral, often most commonly observed in sheep grazing
dying or dead tissues (Petrini, 1996). They perennial rye grass in New Zealand (Schardl
may colonize single cells (Stone et al., 1994) and Phillips, 1997).
or tissues (Schulz et al., 1999). On the other hand, symptomless endo-
The endophytes of aerial parts of plants phytes of plants other than grasses have
could be assembled in two different groups: been known for more than 80 years (Lewis,
the fungal endophytes of grasses and non- 1924; Carroll and Carroll, 1978; Fisher et al.,
grass endophytes (fungi and bacteria). Grass 1992; Menendez et al., 1995; Faeth and
endophytes are a particular type of systemic Hammon, 1997; Gasoni and Stegman, 1997;
symbiosis, these are fungi of the family Clavi- Fröhlich et al., 2000; Larran et al., 2007).
cipitaceae, which grow between host cells in Endophytes are found in all plants and are
vegetative tissues, ovules and seeds and are extremely abundant and very diverse. Endo-
seed transmitted vertically (Stone and phytes of a non-grass host represent a broad
Petrini, 1997). Most studies of endophytes range of genera. Taxonomically, the endo-
have dealt with grasses due to their economic phytic fungi recovered from plants belong
importance to livestock (Clay, 1988, 1991). mainly to the phylum Ascomycota and
The close association of an endophyte (Neo- Basidiomycota (fungi) and some Oomycetes
typhodium coenophialum (Morgan-Jones & (phylum Oomycota, Chromista) have been
Gams) Glenn, Bacon & Hanlin = Acremo- isolated as endophytes (Sinclair and Cerkaus-
nium coenophialum) and tall fescue (Festuca kas, 1996), along with members of phylum
arundinacea L.) has been widely studied. As Ascomycota and their conidial form or ana-
a result of the association between host and morphic form lacking a sexual state.
fungus, alkaloids are produced. These are The strategy of endophytes is commonly
responsible for fescue toxicosis in livestock characterized by early occupation of living
(Bacon et al., 1977). host tissue, ensuring possession of the nutri-
Since the initial work of Bacon et al. tional resource (Dingle and McGee, 2003).
(1977), numerous researchers have come to Host colonization by these fungi is frequently
understand further the relationship between localized in foliage, roots, stems and bark
fungal endophytes of grasses and animal and they are transmitted horizontally via
toxicosis. On the other hand, it has been spores. Frequently, colonization is more
well documented that grass endophytes often non-systemic. These endophytic infec-
provide their host with a number of benefits tions are often presumed to form mutualistic
that increase host fitness. The most intensely association with their hosts in a manner sim-
studied symbioses are tall fescue with N. ilar to the endophytes in grasses (Stone and
coenophialum and perennial ryegrass with Petrini, 1997). The plant tissues act as host
Research in Endophytes from Agricultural Crops in Argentina 151
for complex fungal communities. In the past distribution, biodiversity and biochemical
few years, several works have provided evi- characteristics could be important in improv-
dence for the development of a highly spe- ing plant fitness. Moreover, they could play
cific endophytic assemblage for a given host an important role in the interactions present
(Bertoni and Cabral, 1988; Petrini and Fisher, in an ecological agriculture.
1988; Sieber et al., 1988, 1991; McInroy and In the past few years, research on endo-
Kloepper, 1991; Pereira et al., 1999; Larran phytes has been carried out at the CIDEFI
et al., 2000, 2001, 2002a,b). Organ specific- Research Centre in the city of La Plata, Bue-
ity, probably the result of adaptation by some nos Aires, Argentina. It is thought that endo-
endophytes to the particular microecological phytes could be used as biocontrol agents. In
and physiological conditions present in a Argentina nowadays, biological control is
given organ, has been demonstrated in sev- an attractive option for the management of
eral studies (Fisher et al., 1991; Petrini et al., some plant diseases. A considerable amount
1992). Whereas a large number of species of knowledge on endophytes has been accu-
can be isolated from a given host, in general, mulated. Preliminary studies have focused
only a few species are present in significant mainly on determining the biodiversity of
amounts (Petrini et al., 1992). The ecological endophytes on economically important
roles of endophytes are not yet clarified in all plants. Likewise, species composition from
associations. Only the interaction of Neoty- different organs has been investigated.
phodium/grass has been studied in depth, Finally, research will be undertaken to test
but less is known about other endophytic the antagonistic interactions between endo-
associations (Clay, 1990). phytes and plant pathogens. Significant
The endophytes may provide a rapidly research is summarized in this chapter.
evolving defence mechanism against her-
bivory (Carroll, 1988, 1991; Findlay et al.,
1995) and many are potential producers of
secondary metabolites and enzymes that Endophytic Fungi in Beet
will probably find diverse applications in (Beta vulgaris var.
the most diverse fields of biology (Petrini esculenta L.) Leaves
et al., 1992; Schulz et al., 1995; Istifadah and
McGee, 2006; Istifadah et al., 2006). Several The aim of this investigation was in order to
studies have demonstrated auxin and cyto- document the species composition of endo-
kinin production (Pugh, 1972; Bacon and phytic fungi of healthy cultivated beet leaves;
De Battista, 1991) and antibiotic compounds to determine their infection frequencies and
(Clark et al., 1989; Brunner and Petrini, 1992). to verify possible qualitative and quantita-
Competition for infection site, their capac- tive changes of species isolated during the
ity to produce secondary metabolites and growing season (Larran et al., 2000). Sam-
their potential to stimulate defence reac- ples were collected from healthy beet leaves
tions may contribute to antagonism by the of plants cultivated in the experimental
endophytes against pathogens living in the field of the Facultad de Ciencias Agrarias y
same tissues (Dingle and McGee, 2003; Isti- Forestales, Universidad Nacional de La
fadah and McGee, 2006). Plata (UNLP), Buenos Aires, Argentina. The
Also, several authors have proposed that plants were sampled three times during the
endophytes could be used as vectors of genes growing season. Leaves were cut, surface-
to be introduced artificially in the popula- sterilized and then leaf disks were incubated
tion of the host, due to natural genomes on 2% potato dextrose agar (PDA) for 8 days.
showing useful characteristics and attributes Nested ANOVA and Tukey tests were applied
that could be selected. For example, endo- to evaluate the differences in infection fre-
phytes used as vectors of genetic information quencies for different fungi. Data were trans-
could also be of particular interest for the formed according to y = arcsin R2 (P/100).
development of mycoherbicides (Petrini Microscopic examinations were made from
et al., 1992). The knowledge of endophyte leaf disks previously surface-sterilized and
then incubated in a humid chamber for 48 h. Endophytic Fungi in the Leaves of

The disks were cleared and stained. Hyphae Lycopersicon esculentum Mill.
were the principal fungal structures observed
(Fig. 12.1). They could be observed emerg- We have selected tomato plants for this
ing through the stomata or growing intercel- investigation because both greenhouse and
lularly under the cuticle and could be field production in La Plata horticultural
followed between the layers of cells. No vis- area are economically important (Larran
ible disruption or impairment of the plant et al., 2001).
cells by the fungi was noted. The endo- Tomato production is used mainly for
phytes isolated from healthy beet leaves are fresh consumption, as well as being a source
shown in Table 12.1. of many value-added products. The investi-
Fungi colonized 100% of the leaves gation reports the endophyte frequencies
sampled. Twelve taxa of endophytic fungi from healthy tomato leaves (cultivar Tommy)
were isolated and identified. Yeast, Alter- cultivated in the field of the Facultad de
naria alternata, Pleospora herbarum, Stem- Ciencias Agrarias y Forestales, UNLP, Bue-
phylium sp. and Epicoccum nigrum were nos Aires, Argentina. Samples were collected
the most frequently isolated fungi. The fre- for 2 years to determine possible qualitative
quency of A. alternata and P. herbarum and quantitative changes of species. Data
increased significantly in time, whereas were analysed by ANOVA for factorial exper-
yeast decreased along the growth stages. iments. Differences between means were
There were no relevant quantitative changes separated with Tukey’s test (P ≤ 0.05). Like-
in the frequency of colonization by other wise, different surface-sterilized techniques
species. The diversity of isolated fungi were evaluated previously and the technique
species decreased from the first to the last selected was used. The diversity of isolated
sampling. endophytes is shown in Table 12.2.
Fig. 12.1. Hyphae emerging from stomata.

Table 12.1. Mean density of colonization (%) of endophytic fungi from beet leaves at three different time
intervals during the growing season.
Sampling dates
Endophytes 1 2 3
Alternaria alternata (Fr.) Keissler 12.5a** 23.0** 31.0**

Chaetomium sp. 1.0 0 0
Cladosporium spp. 1.0 1.0 1.0
Colletotrichum dematium (Pers.) Grove 1.0 0 0
Epicoccum nigrum Link. 5.3 3.0 3.0
Glomerella cingulata (Stonem.) Spaulding & Schrenk 2.0 1.0 0
Penicillium spp. 1.0 4.0 1.0
Phoma betae Fr. 0 1.0 0
Phomopsis sp. 1.0 0 0
Pleospora herbarum (Pers. ex Fr.) Rabenh. 7.3 9.0 11.0
Stemphylium sp. 6.1 9.0 8.0
Yeast 18.0 10.0 7.0
Sterile mycelia 0 1 0
Total number of endophytes 11 10 7
Total segments sampled: 300
Note: aMean of ten replications. Numbers followed by ** differ statistically according Tukey’s test (P ≤ 0.05).
Table 12.2. Mean frequencies (%) of endophytic fungi isolated from tomato
leaves in 1998 and 1999.
Mean frequencies (%)
Endophytes 1998 1999
Alternaria alternata (Fr.) Keissler 8.75 25.8*

Arthrinium sp. 3.78 0
Bipolaris cynodontis (Marig.) Shoem. 0 1.44*
Chaetomiun globosum Kunze ex Fries 2.50 0
Cladosporium sp. 3.75 5.48
Colletotrichum coccodes (Wallr.) Hughes 2.50 0
C. gloeosporioides (Penz.) Sacc. 13.75* 0
Epicoccum nigrum Link. 0 1.59*
Cryptococcus sp. 0 1.87*
Nigrospora sphaerica (Sacc.) Mason 2.50 0
Penicillium spp. 2.50 2.55
Phomopsis sp. 3.75 0
Ulocladium alternariae (Cooke) Simmons 2.50 0
Stemphylium botryosum (Pers.ex Fr.) Rabenh. 1.25 0
Rhodotorula sp. 0 2.25*
Note: Means followed by * differ significantly according to Turkey’s test (P ≤ 0.05).

Total segments sampled at each growth stage: 75.
Different endophytic species were iso- registered, as several authors observed that
lated in 1998 and 1999, although some of various climatic conditions – site moisture,
them were isolated in both years. This could rainfall and wind exposure – yielded
be due to the different climatic conditions distinct endophyte assemblages (Chapela,
1989; Petrini et al., 1992). Alternaria alter- bean leaves and their infection frequency
nata was the fungus isolated most frequently and to verify possible qualitative and quanti-
from tomato leaves in 1999, but it was the tative changes of species isolated at two
second most common species in 1998. In growth stages: R2–R3 and R4–R5 (according to
contrast, C. gloeosporioides was the fungus Fehr et al., 1971). Fifty asymptomatic plants
isolated most frequently in 1998, but it was were randomly sampled at each growth stage
not found in 1999. Species of other genera, from a segregating population (F3 generation)
such as Cladosporium and Penicillium, were cultivated at the experimental field of the
isolated in both years. These two genera have Facultad de Ciencias Agrarias y Forestales,
been described as endophytes from other UNLP, Buenos Aires, Argentina. Samples
plants as well (Fisher et al., 1992; Cabral were surface-sterilized and incubated over 9
et al., 1993). days. The student t-test and percentage dif-
ferences test were used to evaluate differ-
ences in infection frequencies for various
Endophytic Fungi in fungi. The results are shown in Table 12.3.
Healthy Soybean Leaves Twelve genera of endophytic fungi were iso-
lated and identified from healthy soybean
Soybean (Glycine max (L.) Merr.) in Argen- leaves. In general, in both growth stages, the
tina is one of the most important crops, not same species were isolated and most of them
only by its production but also because of the did not show significant differences in their
volume exported, and it is planted on about infection frequencies, except for Phomopsis
16.5 m ha. A study (Larran et al., 2002b) was sp., P. longicolla and Cladosporium sp.
undertaken to document the diversity of The endophytic fungi isolated more fre-
endophytic fungi of healthy cultivated soy- quently from healthy leaves of soybean were
Table 12.3. Mean percentage frequencies of endophytic fungi and their variations from soybean leaves
at R2–R3 and R4–R5 stages (total segments sampled: 591).
Frequencies (%)
Endophytes R2–R3 stagea R4–R5 stage Variation (%)
Alternaria alternata (Fr.) Keissler 78.48b 68.79 –12.34 NS

A. tenuissima (Kunze ex Pers.) Wiltshire 0 1.60 – NS
Bipolaris sorokiniana (Sacc.) Shoem. 0.94 0 –100.00 NS
Cladosporium sp. 0 2.06 – *
Colletotrichum sp. 1.28 0 –100.00 NS
Curvularia lunata (Wakker) Boedijni 0 0.40 – NS
Epicoccum nigrum Link. 1.23 1.93 +56.90 NS
Glomerella cingulata (Stoneman) 17.20 14.04 –18.40 NS
Spauld. & Schrenk
G. glycines Lehm. & Wolf 0.51 0.40 –21.60 NS
Nigrospora sphaerica (Sacc.) Mason 1.33 2.00 +50.40 NS
Penicillium sp. 0 1.86 – NS
Phomopsis longicolla Hobbs 1.99 0 –100.00 *
P. sojae Lehman 3.48 3.86 +10.90 NS
Phomopsis sp. 2.89 5.50 +90.30 **
Pleospora herbarum (Pers. ex Fr.) Rabenh. 0.66 0.99 +50.00 NS
Stemphylium sp. 3.32 2.99 –9.90 NS
Note: aBased on the plant stages designated by Fehr et al. (1971). bThe infection frequency was calculated as the
number of subsamples infected by a given fungus divided by the total number of subsamples incubated. *Significant
difference (P < 0.05); **highly significant difference (P < 0.01); NS, no significant difference.
A. alternata and G. cingulata. Most of the microorganisms × cultivars and the triple
fungi isolated in this work are cited as soy- interaction were not significant. The fre-
bean pathogens in different parts of the world quency of the microorganisms isolated
(Farr et al., 1989). Because it is known that increased with crop age, but it was statisti-
most fungal pathogens of soybean have an cally similar for the three wheat cultivars
asymptomatic or latent period after infection tested. Rhodotorula rubra, A. alternata, C.
or colonization, these fungi could be either herbarum and E. nigrum were isolated in
avirulent or hypovirulent, or virulent but in the highest frequency. The other microor-
a latent phase. Pathogenicity tests would be ganisms were present at intermediate or low
needed to investigate this hypothesis. Soy- values. Most fungal endophyte isolates from
bean leaves are hosts to an abundance of wheat leaves have been described as endo-
endophytic fungi, but only A. alternata is the phytes of wheat and others plants (Sieber
dominant species. Further studies will be et al., 1988; Petrini et al., 1992; Gindrat and
carried out to evaluate the potential use of Pezet, 1994).
endophytes from soybean leaves in biologi- A variation in the number of taxa iso-
cal control. lated was recorded along the growing season
of wheat. A change in species composition
from the three growth stages was observed;
Isolation and Analysis of Endophytic however, no differences were noted between
Microorganisms in Wheat Leaves cultivars. Further studies were needed to
analyse endophyte composition and varia-
The presence of endophytic fungi in healthy tion from other organs and cultivars. There-
wheat crops has been demonstrated previ- fore, the following study was undertaken.
ously in other countries of the world. The
present investigation was undertaken in order
to document the spectrum of endophytes of Endophytic Fungi from Wheat
healthy leaves from three wheat cultivars (Triticum aestivum L.)
and to determine their infection frequencies
at three growth stages in Argentina (Larran In this work, five wheat cultivars (Buck Pon-
et al., 2002a). Wheat cultivars, Buck Ombú, cho, B. pronto, Klein Cobre, K. Dragón and
Klein Centauro and Klein Dragón, were Pro INTA Federal) were grown in the exper-
grown in the experimental field of the Facul- imental field of the Facultad de Ciencias
tad de Ciencias Agrarias y Forestales, UNLP, Agrarias y Forestales, UNLP, Buenos Aires,
Buenos Aires, Argentina. Ten asymptomatic Argentina. The purpose of this investigation
plants of each cultivar were randomly sam- was to document the diversity of endophytes
pled at three defined growth stages: second from different cultivars and to determine
node detectable, medium milk and soft dough their infection frequencies from different
stages (32, 75 and 85, according to Zadoks plant organ (leaves, stems, glumes and grains)
et al., 1974). Samples were surface-sterilized (Larran et al., 2007). Samples were collected
and incubated on 2% PDA and, after 9 days, at five growth stages from crop emergence
identifications were made. Data were analy- to harvest (GS 2, GS 8, GS 10.5, GS 11.1 and
sed by ANOVA for factorial experiments. GS 11.4) (Large, 1954), with the aim of veri-
Differences between means were separated fying possible qualitative and quantitative
by LSD (P ≤ 0.05). changes of the species isolated. Pieces of
From the 450 wheat leaf segments incu- tissues were surface-sterilized and incu-
bated, 3 bacterial isolates and 130 fungal bated on 2% PDA over 9 days. An ANOVA
isolates were obtained (Table 12.4). From including organs, microorganisms, cultivars
all the isolates, 19 fungal species were iden- and growth stages as a source of variation was
tified. There were significant differences carried out but, due to differences between
between microorganisms, stages of growth organs, an ANOVA was performed consid-
and stages × microorganism interactions. Diff- ering each organ separately. Differences
erences between cultivars, stages × cultivars, between means were separated by LSD
Table 12.4. Frequencies of endophytes isolated from wheat leaves of three cultivars at three
growth stages.
Samplings
Endophytes Gs. 35* Gs. 75 Gs. 85 Average
Alternaria alternata (Fr.) Keissler 0a 0.67 ab 14.0 d 4.89 de

Alternaria sp. I 0a 0a 0.67 ab 0.22 a
Alternaria sp. II 0a 0a 2.0 abc 0.67 a
Arthrinium sp. 1.33 a 0a 0a 0.44 a
Aspergillus sp. 0a 0a 0.67 ab 0.22 a
Bipolaris sp. 0a 2.67 abc 2.0 abcd 1.56 ab
B. cynodontis (Marig.) Shoem. 0a 3.33 bc 2.0 abcd 1.78 abc
B. sorokiniana (Sacc.) Shoem. 0a 0.67 ab 0.67 ab 0.44 a
Chaetomium globosum Kunze ex Fries 0a 0a 1.33 abc 0.44 a
Cladosporium herbarum (Pers.: Fr.) Link. 0a 3.33 bc 7.33 f 3.56 cd
Cryptococcus sp. 0a 4.0 c 1.33 abc 1.78 abc
Epicoccum nigrum Link. 0a 5.33 c 4.67 de 3.33 bcd
Fusarium sp. 0a 2 abc 0a 0.67 a
Penicillium sp. 0a 0a 0.67 ab 0.22 a
Phoma sp. 0.67 a 0a 0a 1.33 a
Phomopsis sp. 0.67 a 0a 0a 0.22 a
Pleospora herbarum (Fr.) Raben. 0a 0a 4 cde 0.22 a
Rhodotorula rubra Harrison 0a 9.33 d 6.67 ef 5.33 e
Stemphylium sp. 0a 0.67 ab 3.33 bcd 1.33 a
SM I 0a 0.67 ab 0a 0.44 a
SM II 0a 0a 1.33 abc 0.22 a
Bacillus sp. 0a 0.67 ab 1.33 abc 0.67 a
Average of growth stages 0.12 aa 1.52 b 2.45 c
Gs. 85: soft dough stage. *Growth stages according to Zadoks et al. (1974). Data are the mean of 150 leaf pieces
(5 pieces × 10 replications × 3 cultivars)/growth stage. Means followed by same letter in the same column are not
statistically different according to LSD (P ≤ 0.05). aFor the average of growth stages means followed by the same letter
in the same row are not statistically different (P ≤ 0.05). Gs.35: second node detectable. Gs.75: medium milk.
(P ≤ 0.05). A total of 1750 plant segments of taxa isolated was greater in the leaves
were processed from wheat tissues and 33 than in the other organs analysed. Respec-
microbes were recovered. Three bacteria, 27 tively, 25, 17, 12 and 15 were the number of
fungal taxa and 3 non-sporulating mycelia, taxa recovered from leaves, stems, glumes
assigned as ‘sterile mycelia’, were registered and grains. Few species were dominant
(Tables 12.5 and 12.6). A. alternata, C. her- from grains, whereas they had the highest
barum, E. nigrum, Cryptococcus sp., R. percentages of isolates from the total sam-
rubra, Penicillium sp. and Fusarium ples analysed.
graminearum were the fungi that showed Likewise, a variation occurs in the spe-
the highest colonization frequency in all the cies composition of endophytes isolated
tissues and organs analysed. As is shown, from different organs and growth stages.
the bacterial isolates (Serratia sp., Bacillus No significant differences between cultivars
sp. and unidentified yellow bacteria) were were obtained, except when the glumes were
registered with high frequencies. The results analysed. Whereas Bacillus sp. was isolated
of this statistical analysis showed that from stems and grains, Serratia sp. and yel-
organs, microorganisms and interaction of low bacteria were recovered from all organs
organs × microorganisms were significant. analysed.
On the other hand, as results of ANOVA Although most of the microorganisms
from each organ, we obtained that the number followed a similar pattern in the four organs,
Table 12.5. Frequency (means) of microorganisms isolated from leaves, stems,

glumes and grains on five wheat cultivars.
Endophytes Means (all organs) and growth stages
Alternaria alternata (Fr.) Keissler 8.48 e*

A. infectoria species group 0.56 a
Arthrinium sp. 0.58 ab
Bacillus sp. 1.26 ab
Bipolaris sorokiniana (Sacc.) Shoem. 0.73 ab
B. spicifera (Bainier) Subramanian 0.00 a
Bipolaris sp. 0.26 a
Candida albicans (C.P. Robin) Berkhout 0.04 a
Cephalosporium sp. 0.06 a
Chaetomium globosum Kunze ex Fries 0.19 a
Cladosporium herbarum (Pers.:Fr.) Link. 6.55 d
Cryptococcus sp. 2.14 b
Cochliobolus spicifer Nelson 0.14 a
Curvularia lunata (Wakker) Boedijni 0.01 a
Epicoccum nigrum Link. 4.38 c
Fusarium oxysporum Schlechtend.: Fr. 0.53 a
F. graminearum Schwabe 1.01 ab
Helicocephalum sp. 0.00 a
Nigrospora sp. 0.04 a
Penicillium sp. 1.16 ab
Phoma sp. 0.00 a
Pleospora herbarum (Fr.) Raben. 0.00 a
Rhodotorula rubra Harrison 1.27 ab
Septoria tritici Roberge in Desmaz. 0.00 a
Serratia sp. 8.95 e
Stachybotrys sp. 0.00 a
Stemphylium botryosum Wallr. 0.09 a
Trichoderma hamatum (Bonord.) Bainier 0.17 a
Ulocladium sp. 0.04 a
SM 1 0.00 a
SM 2 0.00 a
SM 3 0.38 a
Yellow bacteria 4.33 c
Organs
Leaves 0.94 a
Stems 1.27 a
Glumes 0.98 a
Grains 2.03 b
Cultivars
Klein Dragon 1.54 a
Buck Pronto 1.37 a
Klein Cobre 1.33 a
Buck Poncho 1.39 a
Pro INTA Federal 0.91 a
Note: *Means followed by the same letter in the same column within the same treatment are not
statistically different according LSD (P ≤ 0.05). SM, sterile mycelia.
Table 12.6. Means of the frequencies of microorganisms, cultivars and growth stages for each organ
(leaves, stems, glumes and grains) of five wheat cultivars.
Endophytes Leaves Stems Glumes Grains
Alternaria alternata (Fr.) Keissler 4.8 e* 2.4 efg 9.33 e 17.6 d

A. infectoria species-group 1.4 abc 0.0 a 0.26 ab 0.8 a
Arthrinium sp. 1.2 abc 0.16 ab 0.00 a 1.2 a
Bacillus sp. 0.0 a 3.68 gh 0.00 a 1.6 a
Bipolaris sorokiniana (Sacc.) Shoem. 1.0 abc 0.0 a 0.53 abc 1.6 a
B. spicifera (Bainier) Subramanian 0.2 a 0.0 a 0.00 a 0.0 a
Bipolaris sp. 0.4 a 0.48 abc 0.00 a 0.4 a
Candida albicans (C.P. Robin) Berkhout 0.0 a 0.0 a 0.00 a 0.4 a
Cephalosporium sp. 0.0 a 0.48 abc 0.00 a 0.0 a
Chaetomium globosum Kunze ex Fries 0.2 a 0.0 a 0.00 a 0.8 a
Cladosporium herbarum (Pers.: Fr.) Link. 1.4 abc 1.28 abcde 2.13 cd 21.6 e
Cryptococcus sp. 2.4 cd 4.8 h 1.60 abc 0.0 a
Cochliobolus spicifer Nelson 0.0 a 0.0 a 0.00 a 0.4 a
Curvularia lunata (Wakker) Boed. Boedijni 0.4 a 0.0 a 0.26 ab 0.0 a
Epicoccum nigrum Link. 3.0 d 2.88 fg 1.86 bc 10.8 c
Fusarium oxysporum Schlechtend.: Fr. 2.2 cd 0.16 ab 0.00 a 0.0 a
F. graminearum Schwabe 1.0 abc 2.88 fg 0.00 a 0.8 a
Helicocephalum sp. 0.0 a 0.16 ab 0.00 a 0.0 a
Nigrospora sp. 0.4 a 0.0 a 0.00 a 0.0 a
Penicillium sp. 2.0 bcd 2.08 def 0.80 abc 0.0 a
Phoma sp. 0.2 a 0.0 a 0.00 a 0.0 a
Pleospora herbarum (Fr.) Raben. 0.2 a 0.0 a 0.00 a 0.0 a
Rhodotorula rubra Harrison 2.4 cd 3.04 fg 0.26 ab 0.0 a
Septoria tritici Roberge in Desmaz. 0.2 a 0.0 a 0.00 a 0.0 a
Serratia sp. 3.3 d 13.6 i 12.53 f 6.8 b
Stachybotrys sp. 0.2 a 0.0 a 0.00 a 0.0 a
Stemphylium botryosum Wallr. 0.2 a 0.0 a 0.00 a 0.4 a
Trichoderma hamatum (Bonord.) Bainier 0.6 ab 0.64 abcd 0.26 ab 0.0 a
Ulocladium sp. 0.0 a 0.0 a 0.00 a 0.4 a
SM 1 0.2 a 0.0 a 0.00 a 0.0 a
SM 2 0.0 a 0.0 a 0.00 a 0.0 a
SM 3 0.0 a 1.76 cdef 0.00 a 0.0 a
Yellow bacteria 3.0 d 1.6 bcdef 3.73 d 9.6 bc
Cultivars
Klein Dragon 0.90 a 1.21 a 1.53 c 2.73 a
Buck Pronto 1.09 a 1.01 a 0.89 ab 2.73 a
Klein Cobre 0.83 a 1.40 a 1.31 bc 2.18 a
Buck Poncho 1.12 a 1.26 a 1.05 abc 2.36 a
Pro INTA Federal 0.97 a 1.48 a 0.48 a 1.39 a
Growth stages
2 0.73 a 0.85 a
8 0.65 a 2.16 b
10.5 0.80 a 1.02 a 0.24 a
11.1 1.76 b 1.26 a 1.87 c 1.72 a
11.4 1.09 a 0.94 b 2.83 b
Note: *Means followed by the same letter in the same column within the same treatment are not statistically different
according to LSD (P ≤ 0.05). SM, sterile mycelia.
there were some, A. alternata for example, that endophytes may have a role as biocon-
with higher values in grains and glumes trol agents against D. tritici-repentis.
than in leaves and stems. The spectrum of
species isolated ranges from potential sap-
robes over taxa that probably are present Conclusions
as natural symbionts to known pathogens
(Fisher et al., 1992). Whereas A. alternata, The study of endophytes began with the
C. herbarum and E. nigrum are species com- aim of studying their biodiversity and dis-
monly abundant in the phylloplane and are tribution from different hosts. We confirmed
considered primary saprobes and minor that endophytes were present in all the
pathogens, others like B. sorokiniana, C. hosts evaluated. Then, we found that endo-
lunata and F. graminearum are economically phytes colonized distinct ecological niches
important pathogens of wheat (Zillinsky, and could suggest their organ specificity
1984). according to several authors (Sieber, 1988;
Due to the fact that some of these endo- Fisher et al., 1991). On the other hand, in our
phytes adapted to a given organ may benefit studies, we have isolated a large number of
the host against pathogens, further studies species from healthy tissues of beet, tomato,
were undertaken. soybean and wheat but only few species
were dominant, in agreement with Petrini
et al. (1992). Distinct endophyte assemblages
were obtained from healthy tomato leaves
A Biological Control Approach in 1998 and 1999, which could be explained
to Infection of Drechslera because of the different climatic conditions
tritici-repentis in Wheat prevailing in both years.
Endophytes could be adapted to their
The investigation was carried out to study hosts and be antagonists for their pathogens
the interactions between some endophytes and, depending on their antagonistic capac-
isolated from healthy wheat plants and ity, they would be able to displace, reduce,
Drechslera tritici-repentis and to determine suppress or induce resistance against them.
its possible significance in the biological Nowadays, in accordance with the sta-
control of tan spot (Larran et al., unpub- tus of our investigation, we consider that
lished). Endophytes isolated previously from further studies are needed to evaluate the
wheat cultivars in Buenos Aires Province, possible use of endophytes as biocontrol
Argentina, were selected for the assay. They agents against pathogens of agricultural crops.
were: A. alternata, Bacillus sp., C. globosum, Intensive work is needed to understand the
C. herbarum, E. nigrum, Penicillium sp., R. role of endophytes and, mainly, their pos-
rubra, Trichoderma hamatum and P. lilaci- sible use as agents of biocontrol. Likewise,
nus. Mycelial and conidial morphological it is very important to study the nature of
alterations and inhibition of colony growth plant–endophyte–pathogen interactions and
of D. tritici-repentis were registered under the mechanism of antagonism (antibiosis,
in vitro conditions. Likewise, greenhouse hyperparasitism, competition) with the aim
experiments were also carried out. The results of improving the efficiency of the biological
obtained from all tests have demonstrated control of pathogens.
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13 Effect of Tillage Systems on the
Arbuscular Mycorrhizal Fungi
Propagule Bank in Soils
Santiago Schalamuk1,2 and Marta N. Cabello1,3

1Institutode Botánica ‘Spegazzini’; 2CONICET (Consejo Nacional de
Investigaciones Cientificas y Technicas), Universidad Nacional de La Plata,
La Plata, Argentina; 3CICBA (Comision de Investigaciones Cientificas
de la Provincia de Buenos Aires), Argentina
Abstract
In this chapter we discuss the effects of tillage and no-tillage systems on the characteristics of the
arbuscular mycorrhizal fungi (AMF) propagule bank in soils. These fungi, which belong to the phylum
Glomeromycota, are of great interest in agriculture. AMF are often assumed to be solely beneficial;
however, in certain environmental conditions, growth depressions related to AMF have been observed.
In soils under no-tillage, an intact hyphal network is present, whereas under conventional tillage, this
network can be damaged and AMF spores may remain as propagule sources. Some direct effects of
tillage on AMF propagules are: (i) disruption of the hyphal network; (ii) dilution of the propagule-rich
topsoil; and (iii) accelerated root decomposition. Spore counts in soils should be considered as useful
indicators for AMF activity in situ; however, the presence of spores does not always imply recent
activity of AMF and mechanical disturbance may change their spatial distribution in the soil profile.
Therefore, the information about spore numbers in agricultural systems needs to be analysed cautiously.
The different environmental conditions and direct effects related with tillage and no-tillage on AMF
communities generate shifts not only in the composition of the AMF soil propagule bank, but also in its
diversity. If the differential use of the various types of propagules by the Glomeromycota families, as
many authors suggest, is confirmed, the lack of disruption of the hyphal network in no-tillage can help
to explain the differences in Glomeromycota diversity that are found in field experiments.
Importance of AMF in Agriculture their cosmopolitan distribution (Harley and

Smith, 1983). They have been found from
Arbuscular mycorrhizae (AM) show symbi- the Antartic Peninsula to the tropics (Huante
oses between plant roots and fungi belong- et al., 1993; Cabello et al., 1994). The wide
ing to the phylum Glomeromycota (Schübler host range of these fungi and their ability to
et al., 2001). These fungi are obligate biotro- grow in different environments are the reason
phs and form associations with most plant why arbuscular mycorrhizal fungi (AMF)
species (Trappe, 1987). AM associations are are usually considered ‘generalists’ with
the most frequent symbioses in nature because low host specificity (Smith and Read, 1997).
of their broad association with plants and Studies have confirmed that mycorrhizal

Effect of Tillage Systems 163
fungi colonize most agricultural plants and fungal interactions, such as competition,
that they can have a substantial impact on antagonism and dominance (Allen et al.,
crop productivity (Johnson, 1993). 2003). Because of the importance of AMF in
The interaction between the fungus and agrosystems, their study is relevant both for
its host plant consists mainly in nutrient the manipulation of indigenous AMF in the
transfer: the plant provides the fungus with field through appropriate agricultural prac-
carbon compounds, while the fungus deliv- tices and for the development of a success-
ers nutrients to the plant. The increased ful inoculation.
nutrient uptake from the soil, particularly of
phosphorus and nitrogen, is the main benefit
attributed to mycorrhizal symbioses (Smith
and Read, 1997; Govindarajulu et al., 2005). Agricultural Practices
Other benefits may include enhancement of and Mycorrhizae
resistance to root parasites (Borowicz, 2001),
improvement of drought tolerance (Augé, Agricultural practices for annual crops,
2001) and reduction of the impact of envi- such as crop rotations, tillage, sowing, fer-
ronmental stresses such as salinity (Ruiz- tilization, pest, weed and disease control,
Lozano et al., 1996). AMF also have an and harvest, generate changes that affect the
important role in the improvement of soil microbial communities in the rhizosphere.
stability, which can possibly diminish ero- Conventional tillage is characterized by the
sion (Rillig et al., 2002). use of disc or mouldboard ploughs, fol-
AM fungi are often assumed to be solely lowed by harrowing for seedbed prepara-
beneficial, since they are widely thought to tion. In no-tillage, seeds are drilled directly
function as mutualists. However, their effects into the soil with an appropriate planting
on host growth often depend on environ- machine (Crovetto, 1992). No-tillage sys-
mental conditions such as nutrient avail- tems are characterized by the accumulation
ability and soil moisture (Peng et al., 1993; of crop residues on the soil surface, leading
Al-Karaki et al., 1998; Graham and Abbott, to greater carbon, nitrogen and surface water,
2000; Valentine et al., 2001). As AMF draws compared to conventional tillage (Doran and
C from the host, the overall effect on host Linn, 1994). Several changes in soil proper-
growth depends on the cost–benefit rela- ties have been reported with no-tillage
tionship of the symbiosis (Johnson et al., management systems: improved aggregate
1997; Grimoldi et al., 2005). Consequently, stability, moisture availability with residue
in fertile soils, growth patterns of mycor- retention, changes in the distribution of
rhizal plants often do not differ significantly organic matter residues down the soil pro-
from those of non-mycorrhizal ones (News- file, for example, a more even distribution
ham et al., 1995) and even growth depres- of organic matter in cultivated soil as com-
sions related to AMF have been observed in pared to that in non-tilled soil, where resi-
many plant species (Johnson et al., 1997; dues are concentrated on the surface
Allen et al., 2003). In such plant–AMF inter- (Alvarez et al., 1998). One of the problems
actions, only the fungal symbiont has a net that may occur in no-tillage is the nutri-
benefit, and this has sometimes been inter- tional deficiency because of the reduced
preted as parasitism (Johnson et al., 1997). mineralization of the soil organic matter
AM fungi are grouped into genera that (Fox and Bandel, 1986).
encompass more than 150 species described In the case of AMF, the lack of soil
to date and the effects that they have on their physical disturbance in no-tillage might
host plants, or ‘effectivity’, differ greatly wrongly suggest that soils with annual crops
between fungal strains or species (Miller et al., under this system may be similar to those of
1985; Modjo and Hendrix, 1986). Since a sin- natural grasslands. However, agroecosys-
gle root can be colonized simultaneously by tems have particular characteristics which
various Glomeromycota species, AMF root influence AMF activity. Natural ecosystems
colonization is mediated by interspecific present various plant species hosting AMF,
164 S. Schalamuk and M.N. Cabello
at different phenological stages. Annual crops, network and consequently lowers mycor-
however, inherently represent a change for rhizal colonization (McGonigle and Miller,
AMF, because of the reduction in host 1996a). At the final crop stages, the AMF
biodiversity. In addition, cropped systems colonization levels in no-tillage and con-
show two clearly different periods: a period ventional tillage often do not differ signifi-
with high density of host plants of the same cantly; however, at the early stages, crop
species growing simultaneously and, after plants under no-tillage often show higher
harvesting, the fallow period with no host mycorrhizal colonization (Schalamuk et al.,
or, in some cases, scarce presence of sponta- 2004). As already mentioned, in no-tillage
neous vegetation (i.e. weeds). As obligate systems, the reduced mineralization of the
symbionts, Glomeromycota relies on the soil organic matter often generates plant
plant host for the supply of C assimilates nutritional deficiencies. Nevertheless, a
required for its growth, maintenance and higher nutrient concentration related to a
functioning. Therefore, dynamics and bio- rapid AMF colonization has been observed
diversity are clearly affected by agricultural under no-tillage systems (McGonigle and
practices (Kurle and Pfleger, 1994). Miller, 1996a; Mozafar et al., 2000; Schala-
muk et al., 2004). By using the method of
Plenchette et al. (1989), we have previously
found higher levels of mycorrhizal soil
Significance of the AMF Propagule infectivity in no-tillage systems (Schalamuk
Bank on Root Colonization et al., 2004). As already pointed out, coloni-
zation of roots by AM fungi can arise from
Effect of tillage different sources of inoculum. Colonized
root fragments (Rives et al., 1980), spores
Colonization of roots by AM fungi can arise (Gould and Liberta, 1981; Jasper et al., 1987,
from three sources of inoculum: spores, col- 1988) and hyphae (Jasper et al., 1989) lose
onized root fragments and hyphae. The their ability to initiate colonization with
propagules in soils therefore may be called soil disturbance, which can be related to
a ‘propagule bank’ that is ‘waiting’ for suit- physical damage to the propagules by till-
able conditions to germinate, grow and age and/or unfavourable conditions for ger-
eventually colonize new plant roots (Öpik, mination or colonization after disturbance
2004; Schalamuk, 2005). Most of the host (Stahl et al., 1988; Bellgard, 1993).
plant benefits obtained by AM symbiosis, Mycorrhizal soil infectivity (MSI)
mainly phosphorus acquisition, depend on (Plenchette et al., 1989) compares the abil-
the early colonization of roots. The rapid ity of different soils to induce colonization
colonization is related to AMF propagule in plants and depends on the activity of all
density and composition, i.e. the so-called the propagule types in soil. It is difficult to
propagule bank. A graph of the percentage distinguish the relative contributions of the
of the root length colonized against time has different types of propagules to the coloni-
a sigmoid form showing three phases: lag zation of root systems (Smith and Read,
phase, linear phase and a plateau (Sieverd- 1997), and mycorrhizal infectivity does not
ing, 1991). A higher AMF propagule density provide information about the relevance of
often reduces the length of the lag phase and each propagule type in any particular field
thereby accelerates the process of mycor- situation. Although a number of different
rhizal colonization (Smith and Read, 1997). propagule types exist in the soil, they may
Numerous studies have shown that not be equally effective at producing new
mycorrhizal colonization is affected nega- infection units (Klironomos and Hart, 2002).
tively by tillage (Douds et al., 1995; McGoni- In many habitats, the hyphal network in the
gle and Miller; 1996a; Kabir et al., 1998; soil, together with root fragments, is proba-
Mozafar et al., 2000). Soil disturbance bly the main means by which plants become
reduces AMF propagule density since till- colonized, even when significant spore
age of soil breaks up the AM fungi hyphal populations are also present (Hepper, 1981;
Tommerup and Abbott, 1981; Birch, 1986; increases during the growing cycle (Cabello,
Jasper et al., 1992). Studies have shown that 1987) and sporulation is frequently linked
AMF extraradical hyphae are affected severely to host phenology in the field (e.g. maxi-
by soil disturbance at tillage (Fairchild and mum spore production occurs near the mid-
Miller, 1990; McGonigle and Miller, 1996b; dle or the end of a growing season) (Morton
Kabir et al., 1997; Wright and Upadhyaya, et al., 2004). At the early stages of the crop,
1998). Jasper et al. (1989) have stated that higher spore densities are usually found in
due to the importance of the AMF hyphal untilled soils, in comparison with conven-
network as inoculum in undisturbed soil, a tional systems, whereas at the more advanced
lower infectivity of soil propagules after the phenological stages, differences between
disturbance usually can be determined by tillage systems are reduced (Schalamuk et al.,
the damage on the network, rather than on 2003).
spores and colonized root fragments. Another It is well known that spores can survive
effect of tillage on the AMF propagule bank, in soils for several years (Sieverding, 1991).
which occurs simultaneously with the dis- Thus, spore counts reflect both the sporula-
ruption of the hyphal network, is the dilution and the action of many factors that
tion of the topsoil rich in propagules, with affect their survival and accumulation in
the poorest part in the subsurface (Sieverd- the soil. Consequently, spore density is a
ing, 1991). Clearly, mechanical soil mixing result of a complex balance and, while spo-
affects all types of AMF propagules. rulation is probably related to the recent
As a conclusion, it is suggested that till- activity of the AMF, spore counts in the soil
age affects all types of AMF propagules include structures formed at different times.
directly, to a greater or lesser extent, through Spore production depends on carbon
different mechanisms acting together: (i) supply from the host to the fungus (Furlan
disruption of the hyphal network; (ii) dilu- and Fortin, 1977; Daft and El Giahmi, 1978).
tion of the propagule-rich topsoil; and (iii) Douds et al. (1993) have indicated that the
accelerated root decomposition. Through production of fungal AM spores can decrease
all these direct effects, tillage may reduce when soils are tilled. Increases in spore num-
soil mycorrhizal infectivity and thereby AM bers have been associated with root growth
root colonization at the early stages of crop (Hayman, 1970) and/or with host maturity or
growth. senescence (Hayman, 1970; Koske and Hal-
vorson, 1981; Giovannetti, 1985; Gemma
et al., 1989; Troeh and Loynachan, 2003).
Agricultural practices generate disturbances
Effects of Tillage and Cropping on that affect AMF colonization and, in turn,
AMF Spore Densities in Soils spore formation in soils (Kurle and Pfleger,
1994). Therefore, tillage, either through
AMF spores are formed by differentiation of changes in mycorrhizal colonization or
vegetative hyphae in soil or roots and appear through indirect effects, such as changes in
to be long-term survival structures. In agri- the soil environment and plant growth, largely
cultural systems with annual crops, other affect AMF spore production in soils.
propagule types (i.e. hyphae inside and out- The survival of a spore depends on its
side the roots) seem to be more important to morphological traits, determined mainly by
start colonization in particular conditions. the species of Glomeromycota to which it
Nevertheless, spore counts in soils should belongs, as well as on the characteristics of
be considered as useful indicators for the soil environment. Spore survival is an
AMF activity in situ. Several studies have important factor determining the variations
found higher spore numbers in no-tillage in AMF spore counts in soils; however,
than in conventional tillage (Crovetto, 1985; information about spore survival is scarce
Kabir et al., 1998; Jansa et al., 2002; Schala- as compared to that about sporulation (Lee
muk et al., 2003). In agroecosystems with and Koske, 1994a). In natural ecosystems,
annual crops, the number of spores generally decreases in spore numbers have been
attributed mainly to their germination, the these variations can be associated with the
activity of macro and micro fauna and their utilization of different propagule types by
destruction by other soil fungi and parasites AMF families (i.e. Acaulosporaceae, Gigaspo-
(Gerdemann and Trappe, 1974; McIlveen raceae and Glomeraceae) (Tommerup and
and Cole, 1976; Ross and Ruttencutter, 1977; Abbott, 1981; Biermann and Linderman,
Ross and Daniels, 1982; Rabatin and Stin- 1983; INVAM, 1993; Braunberger et al., 1996;
ner, 1985, 1988). AMF spores are commonly Brundrett et al., 1999; Klironomos and Hart,
infected either by other fungi (Daniels and 2002; Hart and Reader, 2002, 2004). Jansa
Menge, 1980; Lee and Koske, 1994a; Rous- et al. (2002), in an intensively used agricul-
seau et al., 1996) or by actinomycetes (Lee tural soil under long-term reduced tillage
and Koske, 1994b), and environmental con- management, found that the presence of
ditions have a strong influence on these certain AMF species, especially those that
processes (Janos, 1980; Koske, 1988). In did not belong to Glomus spp., had a ten-
agricultural systems, another effect that dency to increase. However, we have found
directly reduces spore counts is the dilu- that the contribution of species belonging to
tion of the topsoil rich in spores with the the Glomeraceae family increases in no-
part in the subsurface poorer in propagules tillage plots, to the detriment of Acaulospo-
(Crovetto, 1985; Sieverding, 1991). For all raceae and Gigasporaceae (Schalamuk et al.,
these reasons, spore survival and accumula- 2006). In that experiment, the greatest contri-
tion may have a great influence on spore bution of Glomeraceae species in no-tillage
counts, and the largest spore numbers in no- indicated a lower equitability in the distribu-
tillage at the early stages may be the result tion among the families of Glomeromycota,
of either higher or faster sporulation and/or and thereby a lower diversity, in compari-
the presence of residual spores produced son with conventional tillage. These find-
during the fallow or the previous crop. As ings differ from those of Jansa et al. (2002).
the presence of spores does not always Nevertheless, it is important to point out
imply recent activity of AMF, and mechani- that mycorrhizal communities are site-
cal disturbance may change their spatial specific and that each AMF species can be
distribution in the soil profile, the informa- affected in several ways by different agricul-
tion about spore numbers in agricultural tural management practices; therefore, gen-
systems is useful, but needs to be analysed eralization is difficult.
cautiously. De Souza (2005), based on life history
strategy studies, suggested that members
of the Gigasporaceae family were ‘K’ strat-
egists in contrast to single spore-producing
AMF Propagule Bank and ‘Glomus’ species. Hart and Reader (2004)
Biodiversity found that the Gigasporaceae family was
less sensitive to soil disturbance than the
As already pointed out, tillage may alter the Glomeraceae. The basis for this difference
AMF propagule bank in several ways and between both families is due probably to
the lack of disturbance in continuous no- differences in their colonization strate-
tillage systems can generate accumulative gies. AM fungi in the Gigasporaceae colo-
effects. Therefore, in soils under no-tillage, nize primarily from spores, whereas those
an intact hyphal network can be present, belonging to the Glomeraceae can colo-
whereas under conventional tillage, this net- nize from hyphae (Tommerup and Abbot,
work can be damaged and AMF spores may 1981; Biermann and Lindermann, 1983).
remain as propagule sources. Little informa- Hyphae are more sensitive to soil distur-
tion exists on the effect of tillage systems on bance than spores and thus subsequent
Glomeromycota diversity (Jansa et al., 2002; colonization of additional roots is affected
Schalamuk et al., 2006). Several studies more.
have shown that Glomeromycota taxa may Tillage or the lack of disturbance in
vary in their colonization strategies and that continuous no-tillage determine different
environmental conditions and direct effects Conclusions

on AMF communities, and thereby shifts in
the composition of the AMF soil propagule Tillage and continuous no-tillage systems
bank. Consequently, if the differential use change the composition of the AMF propagule
of the various types of propagules by the banks in the soil, whereas mechanical soil
Glomeromycota families, as many authors mixing affects all types of AMF propagules.
suggest, is confirmed, the lack of disruption Continuous no-tillage systems favour the
of the hyphal network in no-tillage for a presence of an intact hyphal network in
period of several years can help to explain soils. Possible differences in colonization
the higher proportions of Glomeraceae that strategies among Glomeromycota taxa might
have been found previously in the system have a great influence on the impacts of till-
(Schalamuk et al., 2006). age on AMF diversity.
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14 Mechanism of Action in Arbuscular
Mycorrhizal Symbionts to Control
Fungal Diseases
Arun Arya, Chitra Arya and Renu Misra

Department of Botany, Faculty of Science, The Maharaja Sayajirao University
of Baroda, Vadodara, India
Abstract
Currently, the world over, especially in developing countries, maintenance of soil fertility and control
of plant diseases have become crucial issues in meeting the biomass needs for food, fodder and fuel,
as well as preserving a clean environment. An ideal fertile soil is characterized not only by optimum
physical properties and chemical constituents conducive for plant growth, but also by microbiological
processes that are maintained in equilibrium. More than 90% of land plants are estimated to form
arbuscular mycorrhizal (AM) associations with soilborne fungi in the phylum Glomeromycota. They
have a wide host range, yet certain host and fungal combinations are more effective from either the
perspective of the fungus, i.e. greater spore/hyphae production, or from that of the host, i.e. enhanced
growth, nutrient acquisition or pathogen resistance. Besides improving uptake of phosphorus, AM
fungi improve plant health through improved resistance to various biotic and abiotic stresses. Of par-
ticular importance is the bioprotection conferred to plants against many soilborne pathogens, such as
species of Aphanomyces, Cylindrocladium, Fusarium, Macrophomina, Phytophthora, Pythium,
Rhizoctonia, Sclerotium, Thielaviopsis and Verticillium, as well as various nematodes by AM fungal
colonization of the plant roots.
Achieving the effective and sustainable control of plant diseases remains a formidable challenge
for all agricultural systems. Despite the continued release of resistant cultivars and pesticides, patho-
gens still cause crop damages and losses that exceed 12% worldwide. Studies have shown that root rot
in wheat caused by S. rolfsii was prevented by the inoculation of Glomus fasciculatum. Reduced quan-
tum of lesioned roots was found in take-all diseases caused by Gaeumannomyces graminis tritici due
to G. deserticola in wheat. The association of G. radiatum with apple has been studied in the USA. It
was found that soilborne fungi, Cylindrocarpon, Pythium and the parasitic nematode, Pratylenchus
spp., were common with replant diseases of apple. In this disease, young trees are stunted and develop
fewer branches than healthy trees.
The exact mechanisms by which AM fungal colonization confers the protective effect are not
completely understood, but a greater understanding of these beneficial interactions is necessary for the
exploitation of AM fungi in organic and/or sustainable farming systems. The mechanisms employed
by AM fungi indirectly to suppress plant pathogens include enhanced nutrition to plants; morpho-
logical changes in the root; increased lignification; changes in the chemical composition of the plant
tissues like antifungal chitinases, isoflavonoids, etc.; alleviation of abiotic stress and changes in the
microbial composition in the mycorrhizosphere. Bioprotection within AM fungal-colonized plants is
the outcome of complex interactions between plants, pathogens and AM fungi. In this chapter, the
different diseases of cereals, pulses, fruits and vegetables and the potential mechanisms by which AM
fungi contribute to bioprotection against plant soilborne pathogens are discussed.

172 A. Arya et al.
Introduction Phytophthora root rot in soybean. The

known interaction may include a number
Arbuscular mycorrhizal (AM) symbiosis is of mechanisms such as exclusion of patho-
the most commonly occurring underground gens, lignification of plant cell wall and
symbiosis in plants. It can be found in a change in phosphorus nutrition, leading to
large majority of terrestrial plants (Newman exudation by roots and the formation of
and Reddell, 1987) and in almost a quarter inhibitory low molecular weight com-
of a million plant species. It is as normal for pounds. The mycorrhizal fungi can pro-
the roots of plants to be mycorrhizal as it is duce certain compounds that inhibit or kill
for the leaves to photosynthesize (Mosse, the pathogenic fungi.
1986). The AM fungi are included in the phy-
lum Zygomycota, order Glomales (Redecker
Interaction of AM Fungi
et al., 2000), but recently they have been
classified into the phylum Glomeromycota
with Fungal Pathogens
(Schussler et al., 2001). The phylum is
divided into 4 orders, 8 families, 10 genera Cereal crops
and 150 species; the common genera are
Aculospora, Gigaspora, Glomus and Scutel- Achieving the effective and sustainable con-
lospora (Schussler, 2005). They are charac- trol of plant disease remains a formidable
terized by the presence of extra-radical challenge for all agricultural systems. Despite
mycelium, branched haustoria-like struc- the continued release of resistant cultivars
tures within the cortical cells, termed arbus- and pesticides, pathogens still cause crop
cules. These are the main sites of nutrient damages and losses that exceed 12% world-
transfer between the two symbiotic partners wide (Johar, 2005). Root rot in wheat caused
(Hock and Verma, 1995; Smith and Read, by S. rolfsii was prevented by inoculation of
1997). G. fasciculatum (Harlapur et al., 1990). Gra-
AM fungi colonize plant roots and pen- ham and Menge (1982) reported reduced
etrate the surrounding soil, extending the quantum of lesioned roots in take-all dis-
root depletion zone and the root system. ease caused by G. graminis tritici due to
They supply water and mineral nutrients G. deserticola in wheat.
from the soil to the plant, while AM benefit It was found that root dry weight of
from carbon compounds provided by the paddy was not affected by R. solani in
host plant (Smith and Read, 1997). AM mycorrhizal plants, but the pathogen
fungi are associated with improved growth caused 29% loss in root dry weight in non-
of host plant species due to increased nutri- mycorrhizal plants (Khadge et al., 1990).
ent uptake, production of growth-promoting Also, the pathogen multiplied less in mycor-
substances, tolerance to drought, salinity rhizal plants. Cochliobolus sativus negated
and synergistic interactions with other the effect of VAM inoculation in locally
beneficial microorganisms (Sreenivasa and adapted WI 2291 cultivar of barley, whereas
Bagyaraj, 1989). The beneficial role of AM in the absence of the pathogen, AM inocula-
fungi in plant biomass production is associ- tion increased grain yield from 31.9 g to
ated with their capacity to reduce or prevent 46.6 g in phosphorus fertilized plants but
the development of plant disease (Mano- did not have fertilized plants (Grey et al.,
harachary, 2004). The protective ability of 1989). Contrary results were obtained by
mycorrhizae is generally observed against Schonbeck and Dehne (1979), who observed
soilborne diseases and is often related to the increase in disease due to Erysiphe graminis
nature of the host plant, mycorrhizal symbi- and Helminthosporium sativum in barley.
onts, plant pathogens and the condition of The severity of common root caused by
the soil (Tello et al., 1987). AM fungi are Bipolaris sorokiniana in barley was reduced
helpful in controlling disease; however, Ross by three species of Glomus (Boyethko and
(1972) reported increased development of Tewari, 1990).
Mechanism of Action to Control Fungal Diseases 173
Pulses and oil crops earlier in mycorrhizal plants. However,

2 months later, disease severity was reduced
Gigaspora calospora exerted an inhibitory significantly in these plants. Between the
effect on the development of pigeon pea two species tested, G. etunicatum was more
blight caused by P. drechsleri f. sp. cajani effective than G. mosseae (Sharma and Johri,
(Bisht et al., 1985). Similarly, in Tamil Nadu 2002). Brassica oleracea infected with AM
Agricultural University, India, studies showed fungus had lower infection by R. solani;
that another AM fungus, G. etunicatum, higher moisture content (25%) enhanced
induced tolerance to cowpea (Vigna unguic- disease incidence (Iqbal et al., 1988). Stud-
ulata) against Macrophomina root rot. Dis- ies conducted at the University of Jordan,
ease incidence was 16% in inoculated plants Jordan, showed that the mycorrhizal plants
as against 33% in uninoculated plants (Ram- of tomato inoculated with F. oxysporum
raj et al., 1988). Rosendahl (1985) observed a had significantly higher root and shoot
decrease in disease incidence in peas due weights and plant heights than plants inoc-
to Aphanomyces euteiches. Similar results ulated with F. oxysporum only (Al-Momany
were observed for soybean (Zambolin and and Al-Radded, 1988). Only the presence
Schenck, 1983) and groundnut (Abdalla and of G. intraradices resulted in a significant
Abdel-Fattah, 2000) due to F. solani. Krishna decrease in the population of F. oxysporum
and Bagyaraj (1983) observed a reduction in and root necrosis (Caron et al., 1986).
disease due to M. phaseolina in soybean. Early infestation of G. fasciculatum enhanced
Studies conducted at the University of Bay- tomato plant growth and reduced Fusarium
reuth, Germany, showed that in leachates of wilt (Manian et al., 2006). They also observed
AM rhizospheric soil of Zea mays and Tri- that the percentage disease index was less
folium subterraneum, fewer sporangia and in mycorrhizal than in non-mycorrhizal
zoospores were produced by P. cinnamomi tomato plants when inoculated with Alter-
as compared to non-AM plants, suggesting naria solani.
that sporangium-induced microorganisms The presence of G. mosseae decreased
declined or sporangium inhibitors increased both weight reduction and root necrosis in
(Meyer and Linderman, 1983). tomato caused by P. nicotianae var. para-
Pandey and Upadhyay (2000) studied sitica (Trotta et al., 1996). In vitro experi-
the effect of microbial populations on the ments in which Ri T-DNA transformed
development of pigeon pea in Pusa, Bihar, roots of alfalfa were inoculated with AM
India. Screening for resident antagonists fungi showed normal mycorrhizal formation
was carried out and the mode of mycopara- by G. intraradices and hypersensitivity-like
sitism was studied. Dual inoculation with response to G. margarita. Colonized cells
AM endophyte (G. mosseae) and M. phaseo- became necrotic and HPLC studies indicated
lina restricted the progression of the patho- concentration of phenolics and isofla-
gen significantly in the roots of mungbean vonoids in these roots. The data strongly
(V. radiata). Disease incidence was reduced support the existence of a degree of specific-
from 77.9% in pathogen inoculated to 13.3% ity between AM fungi and the host (Douds
in AM + pathogen inoculated plants (Jalali et al., 1998).
et al., 1990). G. fasciculatum reduced the Onion pink rot caused by Pyrenochaeta
number of sclerotia produced by S. rolfsii in terrestris and tomato root rot caused by T.
groundnuts (Arachis hypogaea) (Krishna basicola are controlled by mycorrhizal
and Bagyaraj, 1983). fungi (Vidhyasekaran, 2004). Inoculation of
G. mosseae in tomato and eggplant seed-
lings controlled the incidence of Verticil-
lium wilt caused by V. dahliae in Greece
Horticultural crops (Karagiannidis et al., 2002). Trotta et al.
(1996) studied the interaction between the
The early wilt symptoms caused by F. soilborne root pathogen P. nicotinae var.
oxysporum on tomato appeared 8–10 days parasitica and the arbuscular mycorrhizal
174 A. Arya et al.
fungus G. mosseae in tomato plants. Treat- and G. mosseae, exhibited a medium level
ment with Phytophthora resulted in a visible of resistance to the disesases. Rhizome rot
reduction in plant weight and in a wide- of ginger caused by P. aphanidermatum was
spread root necrosis in plants without mycor- controlled by G. mosseae and G. fascicula-
rhiza. The presence of AM fungus decreased tum (Sivaprasad et al., 2006).
both weight reduction and root necrosis. Field application of a commercially
The percentage reduction of root necrosis available formulation of AM marketed as Josh
ranged between 63 and 89%. by Cadila Pharmaceuticals, Agro Division,
Utkhede et al. (1992) studied the effect was tried for the management of charcoal
of G. mosseae on replant disease of apple. It stump rot disease caused by Ustulina zonata
was found by Graham and Egel (1988) in (Chakraborty et al., 2005). Commercial pro-
Florida, USA, that G. intraradices did not duction of the medicinal plants in arid and
increase the resistance or tolerance of sweet semi-arid areas of the Thar Desert is affected
orange seedlings to Phytophthora root rot mostly by the soilborne plant pathogens
unless mycorrhizae conferred a phospho- ready to attack any seedlings transplanted
rus nutritional advantage over the non- into the field. Mycorrhizal symbiosis resulted
mycorrhizal plants. Citrus root rot caused in significant disease severity in Chlorophytum
by P. parasitica and T. basicola can be con- borivillianum, Convolvulus microphyllous
trolled by AM fungi (Vidhyasekaran, 2004). and Withania somnifera (Vyas, 2005).
Prior root colonization by mycorrhizal fungi,
G. margarita or G. macrocaropum, reduced
the damage caused by P. parasitica in two Role of AM fungi in forestry
citrus root stocks, Carrigo citrage and Sour
orange (Schenck et al., 1977). To ensure Studies conducted at the Northern Forest
good mycorrhizal establishment in citrus Research Centre, Canada, showed that Fusar-
roots, plants were exposed for 110 days to ium wilt disease severity in Albizia procera
mycorrhizal fungi before challenging them and Dalbergia sissoo was reduced signifi-
with the pathogen. In phalsa (Grewia subin- cantly when inoculated with mycorrhizal
aequalis), better root growth and feeding fungi (Chakravarty and Mishra, 1986). The
sites of nematodes during the rainy season effect of AM fungi, Pseudomonas and Rhizo-
promoted better colonization of AM fungi bium, was observed on the rate of photosyn-
(Hasan and Khan, 2006). thesis and colonization in D. sissoo (Bisht
et al., 2006). The rate of photosynthesis was
significantly higher in plants inoculated
with AM consortium. Arya and Chaterjee
Cash crops (1995–1996) found better plant biomass and
good growth of neem seedlings after inocula-
Studies conducted at the Rajasthan Agricul- tion of G. fasciculatum. Arya (2006) recorded
ture University, India, showed that Cuminum a change in soil mycoflora after inoculation
cyminum in association with G. calospora, G. of AM fungus in neem seedlings. Fungi
fasciculatum, G. mosseae and Acaulospora like Aspergillus fumigatus, A. nidulans, A.
laevis enhanced nutrient uptake and reduced ochraecous and F. pallidoroseum were not
wilt severity due to F. oxysporum f. sp. recorded after 3 months.
cumini (Champawat, 1991). In Germany, G. A significant increase in dry weight of
etunicatum reduced leaf blight in rubber Santalum (Krishnamurthy et al., 1998) and
plants caused by Microcycles ulei (Feld- Tamarindus (Bagyaraj and Reena, 1990) seed-
mann et al., 1990). G. monosporum inocu- lings has been observed after inoculation of
lated tobacco plants showed better tolerance AM fungi. In ectomycorrhizae, the presence
against T. basicola (Giovannetti et al., 1991). of a mantle around the root prevents the entry
Sivaprasad et al. (2006) controlled foot rot of pathogens, while in endomycorrhizae, the
of black pepper by inoculation of G. mono- better nutrient uptake makes the plant more
sporum. Two other species, G. etunicatum resistant to various pathogens.
Fungi are harmful agents to humans but with plants reduce the damage caused by
mycorrhizal fungi are indispensable for lux- plant pathogens (Harrier and Watson, 2004).
uriant growth of forest trees. Contrary to These interactions have been documented
popular belief, the luxuriance of rainforest for many plant species. With the increasing
is not because the rainforest soil is more fer- cost of inorganic fertilizers and the environ-
tile (as torrential rains over millennia leach mental and public health hazards associ-
out soluble minerals), but because the roots ated with pesticides and pathogens resistant
associate with fungi, whose spreading hyphae to chemical pesticides, AM fungi may pro-
increase the area of absorption of scarce nutri- vide a more suitable and environmentally
ents and transport this to the plant in return acceptable alternative for sustainable agri-
for photosynthetically fixed carbon (Mahesh- culture (Table 14.1).
wari, 2005). In Ghana and the Mopri Forest
Reserve of Cote d’Ivoire, Terminalia ivoren-
sis plantations are susceptible to dieback,
the cause of which is unknown; poor myc-
Mechanism of Disease Control
orrhizal infection may be a contributory
factor (Wilson et al., 1994). Any one or more mechanisms may be oper-
ative in plants, imparting them with resis-
tance against pathogens.
Signalling Pathway in Mycorrhiza 1. Physical alteration in plant body.
2. Physiological changes.
The signalling pathway to activate the 3. Biochemical mechanisms
mycorrhiza-specific phosphate transporter
has its origin in the PL (phospholipid) PC
(phosphatidylcholine), imager component Physical alteration in plant body
of membranes of plants and probably, also
of the AM fungus. However, PC is not active
According to some scientists, AM affects
in itself. It gains activity only after treatment
soilborne plant pathogens on the basis of
with PLA2 and PC from plants, fungus or
physical alterations. Lignification of cell wall
both remains to be explored further. Several
and production of other polysaccharides has
PLA2s have been identified in plants and all
been reported, which prevents penetration
are secretory proteins. Their regulation and
of mycorrhizal plants by F. oxysporum
substrate specificity are unknown. This
(Dehne and Schonbeck, 1979) and Phoma
might hint at extracellular production of the
terrestris (Becker, 1976). Mycorrhizal inoc-
LPC (lyso-phosphatidylcholine) signal might
ulation improves plant growth. Arya (2006)
be generated more specifically in the arbus-
found better growth of neem seedlings after
cules containing cells. LPCs are highly mobile
inoculation with three isolates of G. fascicu-
within the intact cells and LPC is therefore
latum. It has also been suggested that a
a good candidate for a cytoplasmic messen-
stronger vascular system of the mycorrhizal
ger that transduces signals to activate down-
plants is likely to increase the flow of nutri-
stream processes and gene expression in the
ents, impart greater mechanical strength and
nucleus (Drissner et al., 2007).
diminish the effect of vascular pathogens
(Schonbeck, 1979). A few electron opaque
structures resembling the deposits were
Bioprotectant Nature of AM Fungi found in some cells and intercellular spaces
of non-infected mycorrhizal carrot roots, but
Plant diseases can be controlled by manip- were absent in infected, non-mycorrhizal
ulation of indigenous microbes or by carrot roots. Restriction of pathogen growth,
introducing antagonists to reduce the disease- together with an increase in hyphal altera-
producing propagules (Linderman, 1992). tion and accumulation of new plant prod-
AM fungi and their associated interactions ucts in mycorrhizal roots, but absent in
176 A. Arya et al.
Table 14.1. Effects of AM fungi on fungal diseases of certain crops.
Crop AM fungi Pathogen Reference
Tomato Glomus intraradices Fusarium oxysporum f. Caron et al., 1986; Akkopru

sp. lycopersici and Demir, 2005
G. mosseae F. oxysporum Al-Momany and Al-Raddad,
1988
G. etunicatum F. oxysporum f. sp. lycopersici Bhagawati et al., 2000
G. mosseae Phytophthora parasitica Pozo et al., 2002
G. intraradices Rhizoctonia solani Berta et al., 2005
Banana Glomus sp. Cylindrocladium Declerck et al., 2002
G. proliferum Spathiphylli Declerck et al., 2002
G. etunicatum R. solani Yao et al., 2002
Cucumber G. etunicatum Pythium ultimum Rosendahl and Rosendahl,
Glomus sp. 1990
G. etunicatum F. oxysporum f. sp. cucumerinum Hao et al., 2005
Pepper G. mosseae F. oxysporum Al-Momany and Al-Raddad,
1988
P. capsici Ozgonen and Erkilic, 2007
Onion Glomus sp. Sclerotium cepivorum Torres-Barragan et al., 1996
Pea G. fasciculatum Aphanomyces euteiches Rosendahl, 1985
Cowpea G. fasciculatum Macrophomina phaseolina Devi and Goswami, 1992
G. fasciculatum F. oxysporum Sundaresan et al., 1993
Chickpea G. fasciculatum F. oxysporum f. sp. ciceris Siddiqui and Singh, 2004
G. intraradices M. phaseolina Akhtar and Siddiqui, 2006
G. fasciculatum M. phaseolina Akhtar and Siddiqui, 2007
Cotton G. mossae V. dahliae Liu, 1995
G. vesiformae
non-mycorrhizal roots, shows that mycor- phosphorus, AM fungi are known to enhance
rhizal infection is responsible at least in uptake of Ca, Cu, S and Zn (Gerdemann,
part for the plant defence system which pro- 1968; Sharma, 1990). Glomus monosporum
vides protection against pathogen attack was found effective against P. capsici in
(Benhamon et al., 1994). black pepper (Sivaprasad et al., 2006). The
authors found resistance due to improved
nutrient uptake. Host susceptibility to infec-
Physiological changes tion by the pathogen and tolerance to dis-
ease is influenced by the nutritional status
AM fungi can interact directly with the of the host and the fertility status of the soil
pathogens through phenomen like antago- (Wallace, 1973). For example, nematode-
nism, antibiosis or predation. The studies damaged plants frequently show deficien-
conducted so far suggest that they affect cies of B, N, Fe, Mg and Zn (Good, 1968).
the host–pathogen relationship indirectly High levels of P fertilization in the absence
through physiological alteration or by com- of AM fungi can interact with minor ele-
peting for space or host resources. Through ments, creating a deficiency situation which
increased P nutrition, AM fungi enhance predisposes plants to root knot nematodes
root growth, expand the absorptive capacity (Smith et al., 1986). AM fungi may, therefore,
of the root system for nutrients and water also increase host tolerance to pathogens
and affect cellular processes in roots (Hussey by increasing uptake of essential nutrients
and Roncadori, 1982; Reid et al., 1984; other than P which are otherwise deficient
Smith and Gianinazzi, 1988). In addition to in non-mycorrhizal plants. Production of
siderophore can suppress root pathogens resistant to pathogenic attack. Since the first
(Sharma and Johri, 2002). Higher levels of report of mycorrhiza-related chitinase in
amino acids, especially arginine, in combi- tobacco (Dumas-Gaudot et al., 1992), addi-
nation with root exudates of the mycor- tional ones have been demonstrated in vari-
rhizal plant have been reported to reduce ous plant species. Lambias and Mehdy (1996)
chlamydospore production of T. basicola evaluated the expression of mycorrhiza-
(Baltruschat and Schoenbeck, 1975). Increa- specific chitinases and ß-1,3-glucanases in
sed levels of phenylalanine and serine have soybean root infected with G. intraradices.
been observed in tomato roots inoculated The efficacy of six AM species, A. mor-
with G. fasciculatum. High concentrations rawae, G. margarita, G. fasciculatum, G.
of orthodihydroxy (O-D) phenols in mycor- macrocarpum, S. calospora and Sclerocys-
rhizal roots suppressed the growth of S. tis rubiformis obtained from rhizosphere of
rolfsii (Goodman et al., 1967; Krishna and C. microphyllus was evaluated for enhance-
Bagyaraj, 1983). The presence of HCN pre- ment of PRO (peroxidase), PPO (polyphenol
cursors has been observed in rubber plant oxidase) effect, with S. calospora being the
infected with G. etunicatum (Lieberei and most promising of all the fungi. Good results
Feldmann, 1990). were observed with G. fasciculatum in
W. somnifera.
AM fungi ensure protection against cer-
Biochemical mechanisms tain soilborne pathogens (Diop, 1996). An
AM fungus influences microbial populations
The production of phytoalexins in AM- and improves soil texture by the secretion of
containing plants has been demonstrated mucilaginous compounds (Strullu et al.,
conclusively. Enhanced accumulation of 1991). Vesicles are lipid-filled and are initi-
glyceollin I, a highly antifungal phytoalexin, ated after the formation of arbuscules, but
has been reported in the roots of mycorrhizal live longer after the senescence of arbuscules
soybeans (Morandi et al., 1984). According (Diouf et al., 2003). In Medicago truncatula,
to Sharma and Johri (2002), it is not clearly at an early stage of arbuscule development
understood how AM fungi induce the pro- by G. versiforme, bright diffuse florescence
duction of phytoalexins and elicitors. It may is seen around the arbuscular branches fol-
be possible that mycorrhizal fungi perturb lowing antitubulin labelling. At later stages
root tissues so that the plant elicitors are lib- of development, short microtubules are
erated. Cell damage, which is closely asso- closely associated with plasma membrane
ciated with the production of isoflavanoids surrounding the labyrinthine surface of the
in legumes (Bailey, 1982), has been observed arbuscule. γ Tubulin has been shown to be
rarely in mycorrhizal soybean roots. The associated with the nuclear envelope and
concentration of coumestrol increased in perifungal membrane in tobacco arbuscular
mycorrhizal roots (25 µg/g) and was much mycorrhizas (Genre and Bonfante, 1999).
greater than that of glyceollin I (Morandi Mycorrhizosphere changes in populations
et al., 1984); coumestrol inhibits the growth of antagonists to specific pathogens depend
of bacteria and nematodes. on having those antagonists present in back-
According to Chakraborty et al. (2005), ground soil. If antagonists are absent and
induction of disease resistance in pea deleterious microbes are present in signifi-
plants against charcoal stump rot was asso- cant numbers and enhanced by AM, the
ciated with the accumulation of defence incidence of disease can be increased
enzymes, followed by stimulation of anti- (Sharma et al., 2002).
fungal phenolics.
Roots colonized by an AM fungi exhibit
high chitinolytic activities. These enzymes Conclusions
can be effective against other fungal patho-
gens under the direct influence of mycor- The use of AM fungi as a biofertilizer is the
rhizal fungi and root tissues become more only alternative for successful farming.
178 A. Arya et al.
With this approach, we can obtain maxi- production of phytoalexins (glyceolin I,

mum return, not only for a season or a daidzein and coumestrol), chitinase, ß-1,3-
year, but also over centuries. Mycorrhizal– glucanases and b1 (PR) protein are important
disease interaction has been studied in dif- components of AM-induced changes leading
ferent plants by various workers. In many to the resistance of the host to other patho-
cases, a better growth of plant is reported gens. The systemic effect of AM fungi to Phy-
after inoculations of an exotic AM fungi. A tophthora infection in tomato has been
low level of fungal aggressiveness and a demonstrated by Pozo et al. (2002). Spores of
weak plant reaction are no doubt two key AM fungi are reported from different soils in
factors that help in the establishment of a the country. These symbionts improve plant
successful symbiotic relationship between growth. Their utilization as biocontrol agents
the two organisms. is gaining importance after many successful
Increase in peroxide activity which is trials. Multiplication and inoculum produc-
localized in plant vacuoles and the cell tion of indigenous efficient AM fungi should
wall (Schloss et al., 1987) in AM-infected be undertaken. Efforts are needed to commer-
root is one example of a mechanism of cialize these novel microbes to bring about a
plant resistance to microorganisms. Likewise, second green revolution in the country.
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15 Role of Fungal Endophytes
in Plant Protection
S.K. Gond, V.C. Verma, A. Mishra, A. Kumar and R.N. Kharwar

Mycopathology and Microbial Technology Laboratory, Centre of Advanced Study
in Botany, Banaras Hindu University, Varanasi, India
Abstract
Endophytes are the microorganisms that reside inside healthy plant tissues without causing any
detectable disease symptoms to the host. Often, each and every plant harbours either one or a battery
of endophytic microorganisms. The study of endophytes is now on a voyage of interest, not only
because of their role in filling the divide between discovered and undiscovered microbial diversity,
but also due to their harbouring a great potential to produce novel natural products. Other than soil,
higher plants also act as an alternative resource to isolate potential microorganisms. Natural com-
pounds ranging from crop protection to human welfare have been isolated from this alternative source
of endophytes. Several anticancer, antibiotic, antimycotic, antiviral, antioxidant, nematicide, insecti-
cide and immunosuppressive compounds have been reported from endophytes, such as cytochala-
sines, ambuic acid, oocydin, jesterone, cryptocandin, lolitrem B, and 3-hydroxypropionic acid and
taxol, etc. Many of them produce some toxic alkaloids and protect their hosts from herbivores. They
also improve the growth and yield of crops under various stressed conditions. Endophytic fungi have
been emerging as a new tool in genetic engineering, the pharmaceutical industry and in crop protec-
tion as well. In this chapter, the ability and role of endophytic fungi to ward off pests and environmen-
tal stresses on plants is discussed.
Introduction was phased out in 2005. The utilization of

biological materials is an alternative and
The use of agrochemicals as a single control safe way to protect plants from phytopatho-
measure in the field to protect crops from gens. The control of plant pathogens by
their pests has been generating resistance in phylogenetically diverse microorganisms
these pests, and also represents a high risk acting as natural antagonists has been dem-
to field workers and consumers. Most of onstrated repeatedly over the past 100 years.
these chemicals are non-biodegradable and The antifungal ability of Trichoderma sp.
are responsible for polluting the environ- has been well known since the 1930s and
ment. The control of phytopathogens has extensive efforts have been made since then
relied mostly on chemical control agents to use them seriously for plant disease con-
such as methyl bromide (Jarvis, 1993) but trol (Harman, 1996).
after the Montreal Protocol (1991), the man- Although the term ‘endophyte’ was used
ufacturing of and trade in methyl bromide much earlier in 1866 by German scientist,

184 S.K. Gond et al.
Heinrich Anton De Bary, the presence of (Backman and Sikora, 2008). Endophytes are
endophytes in plant was only recorded in recorded from lower plant to higher plant
1904 when Freeman (1904) described an hosts (Stone et al., 2000). Each and every
entire plant’s endophyte life history in the plant is a reservoir of one or a suite of endo-
seeds of darnel (Lolium perenne sub sp. phytes. In angiosperms, Poaceae members
temulentum = L. temulentum). This is a are studied more for their endophytes.
topographical term and includes bacteria, Endophytic fungi are now attracting
fungi, actinomycetes and algae which spend great interest from researchers as an alterna-
their whole life, or a period of their life tive source in controlling plant and human
cycle, inside healthy plant tissue without pathogens. Some of the earlier workers before
causing any disease symptoms. Among all the 1970s documented the endophytic fungi
endophytes, after bacteria, fungi are domi- residing inside the plant, exploring the bio-
nant in higher plants (Figs. 15.1 and 15.2). It diversity of hidden fungi. The period of 1981
seems that other microbial forms almost to 1985 can be considered a historical one in
certainly exist in plants as endophytes such the study of endophytes, as plant protection
as mycoplasmas (pleuro pneumonia-like against herbivore insects was demonstrated
organisms – PPLO), rickettsia and archae- by endophytic microorganisms. Webber
bacteria; however, no evidence of them has (1981) demonstrated for the first time the
yet been observed. On the basis of their nature, role of endophytic Phomopsis oblonga in the
endophytes may be categorized in three protection of elm trees against the beetle
groups: (i) pathogens of another host that are Physocnemum brevilineum. This report gen-
non-pathogenic in their endophytic rela- erated interest in the role of endophytes in
tionship; (ii) non-pathogenic microbes; and plant protection. Now, their beneficial role
(iii) pathogens that have been rendered non- to plants as well as to humans is being con-
pathogenic but still capable of colonization sidered. In this regard, a large number of
by selection methods or genetic alteration antimicrobial compounds have been isolated
Fig. 15.1. Endophytic fungal mycelia and spores within plant tissue stained with aniline-blue.
Role of Fungal Endophytes 185
Fig. 15.2. Leaf pieces in Petri plate (21 days old) showing emergence of endophytic fungal mycelia.
from these endophytic microorganisms (Stro- Trichoderma sp. and Fusarium sp. (Huang
bel, 2002, 2003; Zhang et al., 2006; Kharwar et al., 2001). In a similar study, fermentation
et al., 2009). Endophytic fungi are now rec- broths of 9 (4.8%) out of 187 endophytic
ognized as a new tool in the production of fungi isolated from mainly woody plants
antimicrobials and pharmaceutical com- were highly active against Phytophthora
pounds. In the search for bioactive com- infestans in tomato plants (Park et al., 2005).
pounds, several endophytic fungi have been Induced resistance against Fusarium wilt by
reported from the medicinal plants of North endophytic F. oxysporum was generated in
India (Gond et al., 2007; Verma et al., 2007; tomato plants (Duijff et al., 1998). Sclerotinia
Kharwar et al., 2008). sclerotiorum is a common root, crown and
stem rot causing pathogen to several hosts
such as cabbage, common bean, citrus, celery,
coriander, melon, squash, soybean, tomato,
Antimicrobials and their Activities lettuce, cucumber, etc. Cyclosporine is char-
Produced from Endophytes acterized as a major antifungal substance
against S. sclerotiorum from the fermentation
Antifungal activity of endophytes broth of endophytic F. oxysporum (Rodri-
guez et al., 2006). Out of 510 isolates of
Fungi are major causal organisms of various endophytic fungi, 64 isolates gave antifun-
diseases in plants. Many synthetic fungi- gal activities against Candida albicans, C.
cides are available on the market, but they glabrata, C. krusei, Cryptococcus neoformans,
are giving resistance to pathogens and are Aspergillus fumigatus, A. flavus, Rhizopus
also assisting in increasing the hazards to oryzae, Trichophyton rubrum and Microspo-
human health. Data show that 52.3% of rum canis (Anke et al., 2003).
endophytic fungal fermentation broths Narisawa et al. (2000) found that the
display growth inhibition to at least one root endophytic hyphomycete, Heteroconium
pathogenic fungus, such as Neurospora sp., chaetospira, suppressed Verticillium sp. in
Chinese cabbage in the field. Verticillium diacetamide. Consequently, a tetramic acid,

wilt is one of the most destructive diseases cryptocin, has also been isolated from the
of aubergine. Eleven out of 123 isolates of cultures of C. quercina, which exhibits strong
endophytic fungi, especially H. chaetospira, antifungal activity against Pyricularia oryzae,
Phialocephala fortinii, Fusarium, Penicil- the causal agent of blast of rice, as well as
lium, Trichoderma and Mycelium radicis some other plant pathogenic fungi (Li et al.,
atrovirens ( MRA), after being inoculated on 2000).
to axenically reared aubergine seedlings, Colletotrichum gloeosporioides was iso-
almost completely suppressed the patho- lated from A. mongolica, which produced
genic effects of a post-inoculated, virulent antifungal metabolite colletotric acid, against
strain of V. dahliae (Narisawa et al., 2002). the fungus Helminthosporium sativum (Zou
Out of 39 endophytes of Artemisia annua, et al., 2000). Another Colletotrichum sp.,
21 showed in vitro antifungal activity isolated from A. annua, produced bioactive
against a number of fungal pathogens (Liu metabolites that were fungistatic to several
et al., 2001). The extracts of endophytic plant-pathogenic fungi (Lu et al., 2000).
Alternaria sp., isolated from medicinal Pestalotiopsis microspora is a commonly
plants of the Western Ghats of India, inhib- isolated and well-identified fungus from
ited the growth of C. albicans (Raviraja every rainforest plant and, as a single endo-
et al., 2006). Colletotrichum gloeosporioides phytic species, it contributes a high percent-
was isolated as an endophyte from healthy age to the total mass of fungal endophytes in
leaves of Cryptocarya mandioccana, giving any host. Pestalotiopsis is observed to pro-
antifungal activity against phytopathogenic duce many antimicrobial secondary metab-
fungi Cladosporium cladosporioides and C. olites. One such secondary metabolite is
sphaerospermum (Inacio et al., 2006). Fun- ambuic acid, an antifungal agent which has
gal endophytes Chaetomium and Phoma sp., been described from several isolates of P.
isolated from asymptomatic leaf of wheat, microspora (Li et al., 2001). P. jesteri, iso-
reduced the number and the area of pustules lated from the Sepik River area of Papua New
of Puccinia recondita f. sp. tritici. A study Guinea, produced jesterone and hydroxy-
showed 40%, 65% and 27% antagonistic jesterone which exhibited antifungal activity
interaction by endophytic morphospecies against a variety of plant-pathogenic fungi
in vitro against cacao pathogens, Monilio- (Li and Strobel, 2001). Two new metabolites,
phthora roreri, P. palmivora and Crinipellis ethyl 2,4-dihydroxy-5,6-dimethylbenzoate
perniciosa, respectively, while in the field and phomopsilactone, have been isolated
the endophytic C. gloeosporioides produced from P. cassiae, an endophytic fungus in
a significant decrease in pod loss (Mejia Cassia spectabilis, with strong antifungal
et al., 2008). activity against the phytopatogenic fungi, C.
Cryptosporiopsis quercina is an endo- cladosporioides and C. sphaerospermum
phytic fungus of a medicinal plant, Trip- (Silva et al., 2005).
terigium wilfordii. It was observed that C. An aquatic plant, Rhyncholacis penicil-
quercina produced an antimycotic com- lata, is known worldwide to harbour a
pound, cryptocandin, which was active potent antifungal microbe, Serratia marce-
against a number of human and plant patho- scens, which produces an antioomycetous
genic fungi, including C. albicans, S. sclero- compound named oocydin A (Strobel et al.,
tiorum and Botrytis cinerea (Strobel et al., 1999b). Oocydin A provides the plants with
1999a). A number of antifungal compounds a strong protection against several water
have been identified by Yue et al. (2000) moulds.
from the cultures of Epichloe and Neoty-
phodium species which showed activity
against chestnut blight fungus, Cryphonec- Antibacterial activity of endophytes
tria parasitica. These compounds were
indole derivatives, indole-3-acetic acid and The antimicrobial activity of endophytic fungi
indole-3-ethanol, a sesquiterpene and a has been observed in a range of bacteria
representing pathogens to plants and humans. and P. fluorescens (Shu et al., 2004). Peri-
The broths of 16 endophytic fungi isolated conicins A and B were isolated from
from the medicinal herb, Cynodon dactylon endophytic fungus Periconia sp. of Taxus
(Poaceae), were identified as having potent cuspidata and exhibited antibacterial activ-
anti-Helicobacter pylori activity. The most ity against many pathogenic bacteria. The
active endophyte, identified as Aspergillus minimum inhibitory concentration (MIC) of
sp. (strain number: CY725), produced four periconicin A was even less (3.12 µg/ml)
active fractions and was identified as: (i) than that of gentamicin (12.5 µg/ml) against
helvolic acid; (ii) monomethylsulochrin; Klebsiella pneumoniae (Kim et al., 2004).
(iii) ergosterol; and (iv) 3β-hydroxy-5α, The endophytic fungus, Xylaria sp., isolated
8α-epidioxy-ergosta-6, 22-diene with corre- from Ginkgo biloba, showed strong antibac-
sponding MICs of 8.0, 10.0, 20.0 and 30.0 µg/ terial activity in vitro against S. aureus
ml against H. pylori, respectively (Li et al., (MIC 16 µg/ml), E. coli (MIC 10 µg/ml), S.
2005). Bioactivity of endophytic fungi of Cof- typhae (MIC 20 µg/ml) and S. typhimurium
fea arabica and C. robusta was screened (Liu et al., 2008). Recently, some bioactive
against Salmonella choleraesuis, Staphylo- nitronaphthalenes have been isolated from
coccus aureus, Pseudomonas aeruginosa and endophytic fungus, Coniothyrium sp. (Krohn
four different Escherichia spp. Out of these et al., 2008). Javanicin, an antibacterial
endophytic fungi, T. harzianum, Guignardia naphthaquinone, has been isolated from
sp. and Phomopsis sp. have inhibited four to neem endophyte, Chloridium sp., which
five bacterial species successfully (Sette et al., was significantly active against Pseudomo-
2006). Out of 377 isolates of endophytic fungi nas spp. (Kharwar et al., 2009).
from Garcinia plants, 18.6% isolates displa-
yed antimicrobial activity against at least one
pathogenic microorganism, such as S. aureus, Antiviral activity of endophytes
a clinical isolate of methicillin-resistant
S. aureus, C. albicans and C. neoformans Viruses are an important causal agent of var-
(Phongpaichit et al., 2006). ious diseases in plants and animals. Endo-
Epicoccum purpurascens and Trunca- phytes can induce plant resistance against
tella hartigii were found to have significant viral diseases, but there is a contradiction
action against human pathogenic bacteria. and Guy (1992) found no correlation between
E. purpurascens expressed a good antibac- virus infection and the incidence of endo-
terial effect on S. aureus and P. aeruginosa phyte in perennial ryegrass (L. perenne),
and a very good antibacterial effect on E. whereas other correlative studies have
coli, while T. hartigii exhibited a significant revealed that some endophyte-infected tall
antibacterial effect on Enterococcus faecalis fescue (Festuca arundinaceum) seem to be
(Janes et al., 2007). Fusarium was the most more resistant to barley yellow dwarf virus
frequently isolated endophyte from the Chi- (BYDV) than the others (Mahmood et al.,
nese traditional medicinal plant, Dioscorea 1993; Guy and Davis, 2002). Lehtonen et al.
zingiberensis, and F. redolens showed the (2006), when releasing the viruliferous
most potent antibacterial activities against aphid vectors to endophyte-infected and
B. subtilis, S. haemolyticus, E. coli and X. endophyte-free L. pretense plants in a com-
vesicatoria (Xu et al., 2008). mon garden, found the number of aphids
Two antibacterial cerebrosides, one and the percentage of BYDV infections were
new and another known, were isolated from lower in endophyte-infected plants com-
Fusarium sp., an endophytic fungus found pared to endophyte-free plants. Human
in Quercus variabilis. The new cerebroside cytomegalovirus (hCMV) is a ubiquitous
was named fusaruside with structure (2S,2′R, opportunistic pathogen. Two novel human
3R,3′E,4E,8E,10E)-1-O-b-d-glucopyranosyl- cytomegalovirus protease inhibitors, cytonic
2-N-(2′-hydroxy-3′-octadecenoyl)-3-hydroxy- acids A and B, have been isolated from the
9-methyl-4,8,10-sphingatrienine. Both of solid-state fermentation of the endophytic
them were active against B. subtilis, E. coli fungus, Cytonaema sp. (Guo et al., 2000).
Nematicidal activity of endophytes Insecticidal activity of endophytes
Endophytic fungi are known to produce Fungi are known to produce a large number
some compounds which are toxic to nema- of insecticidal metabolites such as destrux-
todes. The first report on antagonistic activ- ins, ibotenic acid, pantherine, tricholomic
ity of endophytic fungi against plant acid, etc. Endophytic fungi are also known
parasitic nematodes was observed in tall to deter insect pests (Clay, 1989; Carroll,
fescue (F. arundinacea) infected by Praty- 1991, 1995; Azevedo et al., 2000). Several
lenchus scribneri. The nematode popula- toxins are produced by endophytic fungi
tion was found to be comparatively less in and these substances confer host protection
the soil surrounding endophyte-infected against different herbivores. The endophytic
plants. Since the root of tall fescue (F. arun- fungus, P. oblonga, was responsible for
dinacea) was infected by Acremonium reducing the spread of Dutch elm disease
coenophialium, it was considered that the causal agent, Ceratocystis ulmi, by controlling
presence of A. coenophialium deterred the its vector beetle (P. brevilineum) (Webber,
nematode population. The colonization of 1981). In 1985, Claydon and his co-workers
fungal endophyte, F. oxysporum, in the confirmed that endophytic fungi belonging
roots of tomato plant reduced 60% infection to the Xylariaceae family synthesized sec-
of Meloidogyne incognita successfully. ondary metabolites in host Fagus sp. and
Endophyte-free perennial ryegrass plants are that these substances affected the beetle lar-
shown to have a larger number of M. incog- vae. Susceptible and resistant cultivars of
nita population in roots than endophyte- perennial rye grass (L. perenne L.) against
containing plants (Ball et al., 1997). sod webworms (Crambus spp.) were analy-
Pregaliellalactone and structurally related sed for the presence of an endophytic fun-
lactones were isolated with nematicidal gus. All resistant cultivars were found to
activity from non-graminaceous endophytes have a high infection of endophytic fungi.
and related saprophytic ascomycetes (Kop- Several highly infected ryegrass species
cke et al., 2002a,b). Another endophytic with endophytic fungi consequently have
microbe, Burkholderia ambifaria, isolated shown less attack frequency of Argentine
from corn root, produced some toxic metab- stem weevils (Listronotus bonariensis)
olites which inhibited egg hatching and (Gaynor and Hunt, 1983). Barker et al. (1984)
mobility of second-stage juveniles of M. and Prestidge et al. (1984) also observed
incognita (Li et al., 2002). that the same grass infected with endo-
Diedhiou et al. (2003) demonstrated the phytic Acremonium sp. was more resistant
successful nematicidal activity of an arbus- to stem weevils in New Zealand. In the
cular mycorrhiza, Glomus coronatum, and an white spruce, Picea glauca, the death rate of
endophytic fungus, F. oxysporum, against the Homoptera, Adelges abietis, was con-
the M. incognita in tomato plant. Several siderably higher when galls were infected
endophytic fungi isolated from above-ground with the endophytic fungus, C. sphaerospe-
plant organs produced 3-hydroxypropionic rum (Lasota et al., 1983). In L. perenne and
acid (HPA) by bioactivity-guided fraction- a few members of genus Cyperus, insect-
ation of extracts and showed selective nem- pest Spodoptera frugiperda was affected
aticidal activity against the plant-parasitic adversely by endophytic fungus like Balan-
nematode, M. incognita, with LD50 values of sia cyperi (Clay et al., 1985a,b). Ahmad
12.5–15 µg/ml (Schwarz et al., 2004). Rado- et al. (1985) showed that endophytic Acre-
pholus similis is an important parasitic monium sp. deterred the grasshopper,
nematode on banana and other plants. It is Acheta domesticus. Patterson et al. (1992)
suggested that the dual inoculations of observed the production of alkaloids by
endophytic fungal isolates reduce a large endophytic Acremonium in plants Lolium
number of the R. similis population (Felde and Festuca that reduced the attack of the
et al., 2006). Japanese beetle, Popilla japonica. Muscodor
vitigenus, an endophytic fungus of Paullinia Plant Protection in Abiotic Stresses

paullinioides, from the Peruvian Amazon,
is known to produce naphthalene, which Endophytes are also involved in the protec-
effectively repels the adult stage of the wheat tion of plants in various abiotic stresses like
stem sawfly, Cephus cinctus (Daisy et al., drought, temperature, pH, heavy metals, etc.
2002). Endophyte-mediated resistance was (Rodriguez et al., 2004). Water stress toler-
reported in strong creeping and chewings ance was observed in epacrids and their
fescue species against red thread (Bonos endophytic partners in south-west Australia
et al., 2005). Beauveria bassiana is a highly (Hutton et al., 1996). In drought conditions,
effective entomopathogen of a wide range of water content of some endophyte-associated,
insects. Grass varieties infected by Neoty- field-grown tall fescues may be maintained
phodium endophyte have affected the feed- at higher levels than those of endophyte-free
ing performance and preference of newly plants (Elbersen and West, 1996; Buck et al.,
hatched nymphs of the hairy chinch bug, 1997). This phenomenon may be explained
Blissus leucopterus hirtus, a common turf- by enhanced accumulation of solutes in tis-
grass pest in north-eastern USA (Steeve et al., sues of endophyte-infected plants as com-
2007). Akello et al. (2007) incorpotated B. pared to non-infected plants, or by reduced
bassiana as an artificial endophyte in banana leaf conductance and a slowdown of the
plants to combat the banana weevil, Cos- transpiration stream, or due to thicker cuticle
mopolites sordidus. The endophytic fungi, formation (Malinowski and Belesky, 2000).
B. bassiana and Clonostachys rosea, isolated The endophytic mutants and wild-type
from coffee plant, showed strong antagonis- C. magna confer drought tolerance that
tic activity against coffee berry borers (Vega allows symbiotic tomato and pepper plants
et al., 2008). to survive desiccation for 24 and 48 h lon-
Two new insecticidal compounds, ger than non-symbiotic plants, respectively
5-hydroxy-2-(1′-oxo-5′-methyl-4′-hexenyl) (Redman et al., 2001). Endophytic coloniza-
benzofuran and 5-hydroxy-2-(1′-hydroxy- tion was observed to increase the minimum
5′-methyl-4′-hexenyl) benzofuran were iso- leaf conductance in Theobroma cacao, a
lated via bioassay-directed fractionation of measure of leaf water loss after maximal sto-
culture extracts of an unidentified endophytic matal closure under drought stress (Arnold
fungus obtained from wintergreen, Gaultheria and Engelbrecht, 2007). However, no evi-
procumbens (Findlay et al., 1997). These dence for endophyte-mediated drought tol-
compounds exhibited toxicity to spruce erance was observed in Acremonium-infected
budworm (Choristoneura fumiferana Clem.) tall fescue (White et al., 1992). It is sug-
cells. Peramine and lolines, potent insecti- gested that endophyte-mediated drought
cides, are produced in endophyte-infected resistance may be due to alterations in
perennial ryegrass and protect them from the drought avoidance.
Argentine stem weevil, Listronotus bonar- Malinowski and Belesky (1999) observed
iensis (Rowan and Latch, 1994; Tanaka et al., that the pH of a limed, acidic soil increased
2005). Nodulisporic acids, novel indole faster as a result of the root activity of
diterpenes, have potent insecticidal proper- endophyte-infected tall fescue compared
ties against the larvae of the blowfly by with non-infected plants under phosphate-
activating insect glutamate-gated chloride deficient conditions. Liu et al. (1996) observed
channels. Nodulisporium, an endophytic that aluminium tolerance in endophyte-
species from the plant, Bontia daphnoides, infected fine fescues (Festuca spp.) was
produces such nodulisporic compounds greater as compared to non-infected plants.
(Demain, 2000). A strain of endophytic Pen- In an experiment, endophyte-infected clone
icillium sp., isolated from the fresh roots of grew significantly better in high alumin-
Derris elliptica, produces some insecticidal ium soils relative to the endophyte-free
compound analogues to rotenone against clone (Zaurov et al., 2001). L. perenne, sym-
the adult turnip aphid, Lipaphis erysimi biotic with N. lolii, showed higher values of
(Hu et al., 2005). total dry weight and tiller number compared
to non-symbiotic plants in Zn stress (Monnet Indian Contributions to

et al., 2001). In low NaCl salt stress condition, Fungal Endophyte Research
the endophyte Piriformospora indica-infected
barley plants showed higher biomass than The past history of endophytic research in
non-infected plants (Waller et al., 2005). The India, especially with fungi, is not so encour-
mechanism of endophyte-conferred salt tol- aging. It seems that workers who started this
erance has not been investigated so far. research in India are still actively involved
In the USA, a plant species, Dichanthe- in advancing their research manifesto with
lium lanuginosum, has been found growing this ‘under-studied’ group of microbial
in the geothermal soils of Yellowstone population and have not advanced to the
National Park (YNP) and Lassen Volcanic fields and forests of the countryside looking
National Parks (LVNP) at temperatures as for novel microbe/plant associations. Prof
high as 57°C (Stout and Al-Niemi, 2002). Suryanarayanan and his group (Chennai)
Redman et al. (2002) observed that those have initiated biodiversity and distribution
plants colonized by an endophytic fungus, patterns of fungal endophytes with some
Curvularia protuberata, were able to toler- medicinal plants in India and have pub-
ate the higher temperature and, thus, we lished several papers along this line. He has
might conclude that endophytes supported also isolated some bioactive compounds
the plant to withstand heat or drought and melanin from endophytic fungi (Sury-
stresses. However, an in-depth investiga- anarayanan et al., 2004). Several research
tion by Marquez and his colleagues (2007) groups have started paying more attention
showed that this was not only because of to various aspects of endophytic fungi.
plant–fungus symbiosis but it also included No more than a dozen research groups at
a virus as a third partner, which parasitized various locations in India are vigorously
on C. protuberata. Thus, it is a complex tri- involved in either biodiversity or natural
partite symbiosis and the heat tolerance product discovery from this untapped and
ability of the fungus is, in fact, related to the alternative resource (Table 15.1).
virus. That mycovirus is called a Curvularia It has become obvious to many workers
thermal tolerance virus (CThTV). throughout the world that endophytic
Two mechanisms are involved in the microbes have enormous potential to solve
endophyte-conferred biotic and abiotic many of mankind’s problems. Thus, with
stress tolerance: (i) rapid activation of host the discovery of new compounds, we can
stress response systems in exposure to stress protect our agriculture and medicine indus-
(Redman et al., 1999); and (ii) synthesis of tries, as well as plant health. After more than
anti-stress biochemicals in the host, either 20 years of effort, the total number of publi-
by endophytes or through endophyte induc- cations from Indian researchers, including
tion (Bacon and Hill, 1996). Endophyte- some fairly recent ones (Shankar et al.,
produced anti-stress biochemicals are 2003; Seena and Sridhar, 2004; Amna et al.,
mostly alkaloids. In addition to anti-stress 2006; Tejesvi et al., 2007; Gangadevi and
biochemicals, plant and fungal mutualism Muthumarry, 2008), is relatively small.
has been maintained over an evolutionary Due to the great variation in plant bio-
time by the ability of fungi to control the diversity and seasonal changes in India, we
activation of host stress response systems may have a better opportunity to collect/
and, in core, act as ‘biological triggers’ isolate various types of promising endo-
(Rodriguez et al., 2004). When a plant inter- phytic fungi, especially from rainforests and
acts with environmental biotic and abiotic mangrove swamps, which may be able to
stresses, it produces several damaging reac- produce an enormous variety of potential
tive oxygen species (ROS). Therefore, it is bioactive natural compounds. An increasing
hypothesized that endophytes inside plants population of AIDS and immunocompro-
scavenge these ROS rapidly and protect mised patients in India compels us to bear
their host (Rodriguez and Redman, 2005; them in mind when searching for safe drugs.
Tanaka et al., 2006).
Table 15.1. List of Indian workers involved in endophytic fungal research.
Sr. No. Name of group leader Place of work Work specialization E-mail addresses
1. Dr T.S. Suryanarayanan Department of Botany, Vivekanand College, Endophytic fungal diversity and tssury@md3.vsnl.net.in
Chennai natural product discovery ts_sury2002@yahoo.com
2. Dr K.R. Sridhar Department of Biosciences, Mangalore University, Endophytic fungal diversity and sirikr@yahoo.com
Role of Fungal Endophytes

Mangalagangotri, Mangalore bioactive molecules
3. Dr H.S. Prakash Department of Botany, University of Mysore, Endophytic fungal diversity and hasriprakash@gmail.com
Manasagangotri bioactive molecules
4. Dr D.J. Bhat Department of Botany, Goa University, Panji, Goa Endophytic fungal diversity bhatdj@rediffmail.com
5. Dr J. Muthumarry Department of Botany, University of Madras, Endophytic fungal diversity and mm_j@rediffmail.com
Chennai bioactive fungal compounds
6. Dr Arun Arya Department of Botany, MSU, Baroda Endophytic fungal diversity aryaarunarya@rediffmail.com
7. Dr R. Uma Shaankar University of Agriculture, Karnataka Endophytic fungal diversity and umashaanker@gmail.com
bioactive molecules
8. Dr Absar Ahmad National Chemical Laboratory, Pune Synthesis of silver and gold particles aahmad@dalton.ncl.res.in
from endophytes
9. Dr S.K. Singh Agharkar Research Institute, Pune Endophyte diversity singhsksingh@rediffmail.com
10. Dr S.C. Puri/ Regional Research Laboratory, Kanal Road, Natural product development khajuriark@yahoo.com
R.K. Khajuria Jammu Tawi
11. Dr R.N. Kharwar Department of Botany, B.H.U., Varanasi-221005 Endophytic fungal diversity and rnkharwar@yahoo.com
natural product discovery kharwar1@rediffmail.com
191
India really needs a variety of novel antimi- The role of endospheric or so-called endo-
crobial compounds of biological origin, so phytic fungi in plant protection is quite clear
that we can solve the problems of eco- in the above-mentioned examples. Besides
friendly farmers and the weaker sections of protecting plants from biotic and abiotic
society in which the above-mentioned dis- stresses, endophytes also improve the health
eases are prevalent. The fungi, as a group, and yield of plants by producing some
hold enormous potential as sources of anti- growth-regulating phytohormones. Although
microbials. Observations prove that this endophytes are still poorly investigated
group of organisms resides inside healthy microorganisms, they have shown that they
plant tissues as endophytes without causing are going to play a prominent part in the dis-
any detectable symptoms. Therefore, we covery of many bioactive natural compounds.
feel strongly that India needs to gear up and Bioactive natural products of endophytic ori-
exact its research to exploit the maximum gin can change the scenario of existing agrope-
potential of the promising endophytes for sticides because of their easy and sustainable
natural product discovery, which could at production. Many scientists throughout the
least facilitate some of the existing problems world are engaged in the search for bioac-
of its huge population. tive compounds from endophytes. There is
a gap in the knowledge on the genetic and
biochemical communications between the
Conclusions plant and endophytic symbionts. We have
to minimize this gap for better utilization of
endophytic microorganisms.
In the study of mycodiversity, we often for-
get the endospheric fungi as researchers
focus their attention on the phyllospheric
and rhizospheric fungi. The endosphere is a Acknowledgements
special niche where endophytic microor-
ganisms reside and, in response, produce a The authors are thankful to the Head of the
variety of metabolites, which are mostly Department of Botany, BHU, Varanasi, for
toxic to plant and human pathogens. In this providing the necessary facilities. They also
aspect, plant pathogens interact with the extend their thanks to the CSIR, New Delhi,
plant itself, as well as the plant’s endophytes. for financial support.
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Part V
Managing Fungal Pathogens

Causing Leaf Damage
16 The Rust Fungi: Systematics,
Diseases and Their Management
M.S. Patil1 and Anjali Patil2

1Department of Botany, Shivaji University, Kolhapur, India; 2Department of Botany,
Rajaram College, Kolhapur, India
Abstract
The rust fungi (Uredinales) consist of 7000 species belonging to 163 genera in 14 families and com-
prise about 10% of all described species in the Kingdom Fungi. All the rust fungi are ecologically
obligate parasites on ferns, gymnosperms and angiosperms. There are six vital processes in plants and,
correspondingly, six ways in which rusts affect their hosts adversely. Rusts as pathogens damage foli-
age, the main organ of photosynthesis, destroy seedlings, impair growth and interfere in the metabo-
lism of the hosts. Management of any disease begins with correct identification of the pathogen; hence,
some important concepts in rust systematics are discussed, along with detailed information about rust
diseases of some economically important crops. Of course, discussion on plant diseases would not be
complete without recent management strategies. The discussion includes the following;
1. Rust systematics, including characteristic features of rust fungi, their occurrence and geographi-
cal distribution, vegetative and reproductive propagules, pleomorphism, autoecious and heteroecious
nature and host range, etc.
2. Rust diseases of crops, including field crops – medicinal, ornamental, cereals, pulses, millets,
oilseeds, fruit and plantation crops, etc. – nature of disease, epiphytotics, disease development index;
X = XoeRT, assessment of crop losses.
3. Management strategies citing food crisis, need for another green revolution, crop losses, famines,
social impact of rust diseases, e.g. change in coffee-drinking habit due to coffee rust, management
methods – Sharvelle’s strategy (1961): (i) protective, (ii) preventive; and (iii) corrective (physiological dis-
orders), cultural, chemical, biological, breeding, biotechnology – transgenic plants are described in detail.
Introduction: Rust Systematics recently, a rust, Uredo vetus Henne, has

been reported for the first time on Selag-
The rust fungi (Uredinales) consist of 7000 inella sp. The parasitism of rust fungi to the
species belonging to 163 genera in 14 fami- host plant is highly specific; however, this
lies and comprise c.10% of all described specialization varies with species, for exam-
species in the Kingdom Fungi (Kirk et al., ple, two well-known rusts of soybean, namely
2001; Ono, 2002). The Uredinales are Phakopsora pachyrhizi Syd. and Syd. and
believed to be monophyletic taxa and recent P. meibomiae Arthur (American rust) (Ono
molecular–phylogenetic analysis (Swann and et al., 1992), occur on a large number of spe-
Taylor, 2001) supports this perspective. Very cies of family Leguminosae. The rust fungi

202 M.S. Patil and A. Patil
are unique in having complex life cycle pat- many economically important pathogens
terns with elaborate spore forms. Accord- of vascular plants. Dietel (1900) divided
ingly, many types of life cycles are known the order Uredinales into four families based
(Laundon, 1973) with modified spore types on sessile or pedicillate teleutospores as
with immense functional diversities; for follows:
example, aecioid teleutospores in endoform
rusts like Endophyllum, Monosporidium Pedicillate teleutospores -------Pucciniaceae
and Kulkerniella or uredinoid teleutospores Sessile teleutospores
in Hemileia vastatrix Berk. & Br., which In a single layer --------Melampsoraceae
Rajendren (1967) described as ‘Kamat Phe- In 1–2 layers forming waxy crust --------
nomenon’. The spermogonial–aecial host/s ---------------------------Coleosporiaceae
and uredinial–telial host/s are closely asso- In chains ---------------------Cronartiaceae
ciated eco-geographically. Heteroecious life
cycle is widespread in the uredinales. If all
spore forms are produced in unidirectional Families Genera
order, the life cycle is said to be macrocyclic
(Puccinia graminis, P. helianthi, the het- Pucciniastraceae (Arthur) 06
eroecious and autoecious rusts of wheat and Gaumann
sunflower, respectively). There is a tendency Coleosporiaceae Dietel 02
Cronartiaceae Dietel 01
to omission of spore form in life cycles and
Melampsoraceae Schroeter 02
thus many different patterns exist, for exam-
Phakopsoraceae (Arthur) 11
ple, demi-cyclic, in which the uredial stage is Cummins & Hiratsuka
absent. In rust fungi, a widespread assump- Mikronegeriaceae Cummins & 01
tion is that parasitism and host specializa- Hiratsuka
tion are acquired at an early stage of rust Chaconiaceae Cummins & 10
fungus evolution. Nine types with 11 varia- Hiratsuka
tions are found in nuclear cycles associated Uropyxidaceae (Arthur) 10
in metabasidium development of microcy- Cummins & Hiratsuka
clic rust fungi (where only a telial stage with Pileolariaceae (Arthur) 03
Cummins & Hiratsuka
or without a spermogonial stage is formed
Raveneliaceae (Arthur) Leppik 14
on a plant throughout the season). Thus,
Phragmidiaceae Corda 10
rust species that produce only teleutospores Sphaerophragmidiaceae 06
that germinate without dormancy to initiate Cummins & Hiratsuka
new generations repeatedly in a single grow- Pucciniaceae Chevalier 15
ing season would be highly adaptive, e.g. P. Puccinosiraceae (Dietel) 09
pampeana Speg. on chillies (Capsicum spp.), Cummins & Hiratsuka
P. alyxiae Arthur on Alyxia spp., P. xanthii 99
Schw., M. machili (Hennings) T. Sato, E. aca- Unassigned genera 08
cia Hodges and Gardner. Microcyclic species
Total 107
exhibit two or more patterns of nuclear cycles
and different metabasidium development,
indicating that microcyclic lineages might
have evolved independently and repeatedly However, this dependence on teleutospore
from macrocyclic parental species. morphology has brought many unrelated
genera into the same family. Some of the
larger genera are:
Families of Rust Fungi 1. Puccinia Pers. ex Pers. (c.3000–4000
Based on Teleutospores spp.).
2. Uredo Pers. ex Pers. (c.3000 spp.).
Rust fungi comprise one of the largest and 3. Uromyces (Link) Unger (c.600–700 spp.).
best described groups of fungi and include 4. Ravenelia Berkeley (c.200 spp.).
The Rust Fungi 203
5. Melampsora Castagne (c.100 spp.). repeating like conidia, germinate very eas-
6. Hemileia Berk. & Br. (c.50 spp.). ily within 24 h at high temperatures and
7. Coleosporium Lev. (c.80 spp.). relatively higher humidity, but their viability
8. Phragmidium Link (c.60 spp.). is lost at very high temperatures. At lower
temperatures, spores remain viable for a long
In some cases, the original function has
time. They germinate mostly by germ tubes,
been changed irrespective of its basic nature
except in coffee rust, where they behave like
or structure; for example, the species of
uredino teleutospores.
Endophyllum, Monosporidium or Kulkern-
Urediniospores are most significant in
iella produce aeciospores morphologically
disease development and spread on epi-
in aecial cups, but they function like teleu-
phytotic scale (van der Plank, 1963, 1968).
tospores producing promycelium bearing
Hence, this spore state is also considered as
basidiospores and are thus called aecioid
the conidial state during sporulation and the
teleutospores. In H. vastatrix, urediniospores
release of spores form spore clouds in the
occasionally function like teleutospores and
air and serves as secondary and tertiary
are called uredinoid teleutospores. Of course,
inoculum within the crop in a favourable
in the first example, no teleutospores are
season. Rust diseases, due to their repeating
produced, while in the second example,
nature, are known as compound interest dis-
teleutospores normally are produced in the
eases. Log e(x/1 – x) where x is a proportion
life cycle. In the course of the evolution of
of infected susceptible tissue, if the patho-
rust fungi, there is a tendency to eliminate
gen is systemic. Urediniospores are light,
spores, narrowing the host range and sur-
airborne and travel long distances at various
viving in spite of unfavourable environmen-
heights; for example, wheat rust uredinio-
tal conditions or non-availability of the
spores travel from Mexico to Canada; in
required host. There are numerous exam-
Indian wheat rust, the rust originates in
ples in which a rust survives or continues
South India from the Nilgiri and Pulney
its life cycle by producing one type of
Hills and travel via Central India from the
spore, e.g. Aecidium, Uredo, Peridermium,
plateau of Mahabaleshwar and Panchgani to
Caeoma, etc.
North India.
Spore morphology
Rust physiology – urediniospore
In groundnut rust, the uredinospores are germination
one-celled, spherical to oval or angular,
stalked, mostly brown coloured, faint or Spore liberation is active and the terminal
dark, thick-walled with spiny, verrucose, velocity of fungal spores in the air is 0.05–
or a modification of these two, rarely 2.5 cm/sec. In calm weather, only 0.05%
smooth, bearing visible areas in the walls spores travel more than 100 m from their
through which germination takes place. source of origin. Spores of black stem rust
Tulasne and Tulasne (1847) observed for fall from a height of 1.6 km to the ground at
the first time, pores/oscules varying from a speed of 12 mm/sec and travel from place
2 to 20 in number. The germ pores may be to place at 11–32 km/h (Gregory, 1973).
distributed equatorially, zonal or scattered. Urediniospores of different rust species have
Cummins (1936) also recognized their phy- a different period of viability as they are
logenetic significance in the rust taxonomy. affected by environmental factors such as
There appears to be some correlation between RH, light intensity, as well as their own
the arrangement of pores and the shape of structural characteristics, namely wall
urediniospores; globoid spores usually thickness, etc. In northern India, uredinio-
have scattered pores, while ellipsoid, oblong spores are killed by high temperatures in
or asymmetrical spores usually have zon- the field and cannot serve as a source of
nate pores. Urediniospores are bi-nucleate, inoculum the following year.
Viability of urediniospores in rust fungi three groups are dominant, namely insects,
flowering plants and fungi. Among all these
Rust urediniospores show different periods plant pathogens, fungi are the most domi-
of viability at 20–40% RH and 23°C, e.g. P. nant and successful plant pathogens and are
graminis tritici 36 days, P. recondita 63 days, estimated to produce more than 25,000 dis-
P. coronata 87 days, P. menthae 173 days, P. eases. Among these, 8000 diseases of culti-
helianthi 185 days and U. pisi 75 days. Via- vated and plantation crops are extremely
bility of urediniospores decreases at higher damaging in the field every year. Kuhn,
or lower humidity. Spores germinate gener- (1858) wrote a book entitled Diseases of
ally at 90–100% RH, while the temperature Cultivated Plants. Rusts are complex; hence,
requirement varies greatly in different spe- it is difficult to understand how they dam-
cies. The period required for sporulation age standing crops in the field qualitatively
(urediniospores) in black stem rust of and quantitatively, creating problems of
wheat is found to be 5 days at 24°C. If the food crisis and insecurity; a global problem
temperature is lowered to 0°C, then sporula- today.
tion occurs after 85 days. Spore longevity
depends on light, temperature, relative humi-
dity, species of rust and type of spore. Basi- Epidemiological Studies
diospores and pycniospores are delicate
and have least viability. But if the spores are
Epidemiology is the science of epidemics or
kept at a low temperature, viability lasts for
diseases in plant population. Types of epi-
18 days. In the case of sunflower rust, rela-
phytotics are:
tive humidity is more important than tem-
perature. At 80% RH, only 5% aeciospores 1. Based on the rate of disease deve-
remain viable after 56 days. lopment:
Teleutospores spores are produced at (i) Tardiv (slow epiphytotics);
the end of a rust fungus’s life cycle, i.e. spores (ii) Explosive (rapid epiphytotics).
terminating the life cycle of rusts. They are 2. Area covered and time of development:
produced in telia in or on the host, are innate (i) Pandemic – developing on a conti-
or erumpent, covered or exposed in telial nental scale;
sori, in the leaves or in the stem. There is a (ii) Sporadic – seasonal and irregular
tendency in rusts to eliminate spore states incidence.
showing progressive reduction either due to
There are also secondary epiphytotics known.
non-availability of host or climatic condi-
Epiphytotics is also defined as ‘a host–
tions, for example, rusts in temperate regions
pathogen system, out of genetic balance in
on the family Liliaceae. Rust systematics, a
favour of the pathogen’. Such epiphytotics
dynamic science, is far from perfect; hence,
of crop plant diseases are known, in the his-
there is still a lot of work to be done on their
tory of plant pathology, to be followed by
taxonomy and pathology. Study of their
food famines:
host’s behaviour during development, vari-
eties, races, physiological forms and patho- 1. Wheat rust epidemics occurred in 1916
types is beyond the scope of taxonomists. in America and Canada; 1935 and 1937 in
America; 1951 in Europe; and 1827, 1907,
1947, 1949–1950, 1957, 1971–1972 in India.
Rust Diseases of Some Economically 2. Coffee rust epidemics occurred in 1867
Important Crops and 1875 in Sri Lanka; 1891 in the Philippi-
nes; 1891–1892 in Java; 1911–1913 in Cen-
tral Africa; 1871–1878 in South Africa; and
Plant pathology originated in Europe and
1970–1971 in Brazil.
migrated to North America, where it flour-
ished and spread to different parts of the The coffee rust famines influenced the coffee-
world. Among all known living organisms, drinking habit, which then changed to tea.
The Rust Fungi 205
Rusts are compound interest diseases The estimated annual crop losses world-
and an increase of infection at a compound wide (Agrios, 2005) are:
interest rate exponentially/logarithmically
increases the rate of compound interest of
disease by primary and secondary infection. US$
The compound interest equation can be
given as: Attainable crop 1–5 trillion
production (2002 prices)
X = XoeRT
Actual crop production 995 billion
where X = the amount of disease at time Production without crop 445 billion
T, Xo = initial amount of disease at O time, protection
R = infection rate, which is variable, and Losses prevented by crop 415 billion
e = 2.718 for cereal rusts. protection
The rate (R) of increase % per unit of Actual annual losses to 550 billion
time is a fundamental concept in epidemi- world crop production
ology, e.g. 12.5%/day in P. recondita and Losses caused by disease 220 billion
57%/day in Phytophthora infestans. Devel-
opment of epiphytotics is basically a trans-
port problem to get enough inoculum to the Rusts damage plants and plant products,
right place at the right time. Plant–pathogen– causing economic losses. Crop protection
environment is a triple interaction and may measures result in increased prices of pri-
be complicated by vectors and humans; mary products to consumers and pollution
according to van der Plank (1963, 1968), the of the environment. Rusts are also patho-
pathogen must be virulent. To express viru- genic to animals and humans. Diseases are
lence quantitatively, the disease reaction responsible for minor aesthetic losses – in
type is expressed in numerical values as: domestic gardens, avenues and forests. There
are six vital processes in plants and, corre-
R (resistant) = 01, MR (moderately
resistant) = 02, S (susceptible) = 03 spondingly, six ways in which rusts affect
their hosts adversely. Rusts as pathogens
Aggressiveness corresponds to disease seve- damage foliage, the main organ of photosyn-
rity on a 0–9 score scale: thesis, destroy seedlings, impair growth and
0 = absent, 9 = more than 75% leaf interfere in the metabolism of the hosts.
area in 12 days after inoculation Rusts keep hosts alive and active for their
own growth, development and spread. Hence,
therefore pathogens and hosts have coevolved. At the
VI (virulent index) = [1 + (virulence × same time, rusts are a useful means of con-
aggressiveness) × latent period] trolling weeds as their infection results in
thinning of plants in the field. Artificial infec-
VI = [1 + VAL – 1]
tion of rust fungi in fodder grasses brings
where VI = virulent index, A = aggressive- about an increase in protein content.
ness and L = latent period.
Green Revolution and Grain
Crop Losses Production in India
Conservative estimates of total annual losses In India, a green revolution began in 1960.
in crop production by diseases, insects and In 1965, hybrid varieties were introduced,
weeds worldwide are 220 billion US$ cor- followed by an increased consumption of
responding to 31–42% of all losses, of which fertilizers (N, K, P). From the 53% of total
diseases are 14.1%, insects 10.2% and weeds area under cereal cultivation, hybrid cultivars
12.2%, while 6–12% losses are postharvest have been introduced in 16% of the area.
losses. These innovations in agricultural practices
revolutionized grain production in India 4. Rust of jowar (Sorghum bicolor (L.)

from 1900 to 1971. From 1900/01 to 1910, Moench):
grain production was 67.6 Mt. It then (i) P. purpurea Cke.;
remained stable until 1948, in the pre- (ii) P. levis Arthur;
independence era. During 1948–1949, there (iii) P. nakanishiki Dietel.
was a wheat famine. During the first three of 5. Rust of pearl millet or bajara (Pennisetum
the Five Year Plans (1950–1965), the rate of glaucum (L.) R. Br.):
grain production increased by 2.5%. In the (i) P. substriata Ell. and Barth var. indica
fourth Five Year Plan, grain production was Ramachar and Cummins, India;
approximately 100 Mt, i.e. grain production (ii) P. substriata Ell. and Barth. var. de-
increased by 5%. The food crisis provided crospora Eboh, Nigeria;
not only a warning but also an opportunity (iii) P. substriata Ell. and Barth. var. peni-
for new thinking. Still, there is some hope cillaris Ramachar and Cummins.
as the International Grains Council has fore- 6. Rust of maize (Zea mays L.):
cast a 7% increase in global wheat produc- (i) P. sorghi Schw. (common corn rust);
tion. India is the world’s second largest (ii) P. polysora Underw. (southern corn
producer of wheat and rice and the expected rust);
wheat harvest this year is 76.8 Mt and rice (iii) Physopella zeae (tropical corn rust).
production is 95.7 Mt. Among the major
cereal crops, wheat is the most suitable due
to its superior quality of grain, coupled with Oilseed crops
its wide adaptability for cultivation under
varied conditions; humans and wheat will
1. Soybean rust (Glycine max (L.) Merr.):
survive in any environment. Even today,
Malupa sojae (P. Henn.) Ono, Y. et al.
the wheat rust management mission is still
or Malupa state of P. pachyrhizi H. and
incomplete and awaiting novel solutions to
P. Sydow.
increase the yield of quality grains. Lord
2. Groundnut rust (Arachis hypogaea L.):
John Boyd Orr, the first Director of the FAO,
P. arachidis Speg.
said in 1948, ‘a lifetime of poor nutrition
3. Sunflower rust (Helianthus annuus L.):
and actual hunger is the fate of at least 2/3rd
P. helianthi Schwein.
of the world’s population’. This is still true,
4. Safflower rust (Carthamus tinctorius L.):
even today.
(i) P. carthami Corda;
(ii) P. caleitrapae var. centaureae (DC.)
Cummins.
Diseases of Crop Plants and 5. Linseed/flax rust (Linum usitatissimum
Associated Pathogens L.): M. lini (Ehrb.) Lev.
Grain crops
Plantation crops
1. Wheat rusts (Triticum spp.)
(i) Black stem rust: P. graminis Pers. 1. Coffee leaf rust (Coffea arabica L. and
tritici Eriks. and Hennen; other spp.):
(ii) Brown rust: P. recondita Rob. ex (i) H. vastatrix Berk. and Br.;
Desm.; (ii) H. coffeicola (reported only from
(iii) Yellow or stripe rust: P. striiformis Cameroon, West Africa).
West. 2. Mulberry rusts (Morus alba L. and other
2. Leaf rust of rye (Secale cerealis L.): spp.):
P. graminis Pers. secalis. (i) A. mori Barclay;
3. Leaf or crown rust of oat (Avena sativa (ii) Cerotelium fici (Butler) Arthur.
L.): P. coronata Corda and P. graminis Pers. 3. Dalbergia rust (Dalbergia spp.): Sphaero-
avenae Fraser & Ledingham. phragmium dalbergiae Dietel = U. dalbergiae
The Rust Fungi 207
P. Henn. (1895) = U. sisso Syd. & Butl. 2. Rust of Vitex spp.:

(1906). (i) O. fimbriata (Mains) Cumm. & Hirat.;
4. Teak rust (Tectona grandis L.): Olivea (ii) O. scitula H. Sydow;
tectonae (Ramkr., T.S. and K.) Mulder = (iii) O. viticis Ono, Y. and Hennen.
Chaconia tectonae Ramkr., T.S. and K. 3. Rust of Vinca major L.: P. vincae Berk.
Pulses and vegetables Ornamental plants
1. Green gram rust (Cicer arietinum L.): 1. Rose rusts (Rosa spp.): Phragmidium
Uromyces ciceris arietini (Gron.) Jack. spp. (10 spp.).
2. Rust of Phaseolus sp: U. appendicula- 2. Gladiolus rust (Gladiolus spp.): P. glad-
tus var. appendiculatus (Pers.) Unger. ioli (Duby) Cast.
3. Cowpea rust (Vigna sp.): U. vignae 3. Tulip rust (Tulipa spp.): P. prostii
Barclay. Moug. (on wild species).
4. Bean rusts (Pisum, Vicia, Lens, Lathy- 4. Canna rust (Canna spp.): P. thalie Dietel.
rus spp.): 5. Chrysanthemum rust (Chrysanthemum
(i) U. viciae-fabae (Pers.), Schroeter, spp.): P. chrysanthemi Roze.
autoecious rust in Europe and America; 6. Saxifraga rust (Saxifraga spp.): P. saxi-
(ii) U. pisi (Pers.) Wint., heteroecious fragae Schlecht.
rust in Europe, rarely in India. The key to species and varieties of Puccinia
5. Chilli rust (Capsicum annuum L.): P. and Uromyces producing rust diseases can
pampaeana Speg. (Mexico, Peru, Brazil, be found in the Appendix at the end of this
Columbia and Gautemala). chapter.
Forage crops (Fabaceae) Rust of wheat (Triticum spp.)
1. Bersim/clover rust (Trifolium spp.). In India, wheat is cultivated on 16m ha,

2. Lucerne (Medicago spp.). but the average yield is very low, that is,
3. Alfalfa rust (M. sativus L.): U. striatus 810–1000 kg/ha as compared to the wheat
Schroeter. yield in other countries, namely Argentina
4. Clover rusts (Trifolium spp.): 1210 kg/ha, America 1610 kg/ha, Belgium
(i) U. trifolii-repentis Liro; 3700 kg/ha and Denmark 4000 kg/ha. The
(ii) U. fallens (Arthur) Barth. five major wheat-growing areas in India are
in the north-western zone, the north-eastern
zone and the central, peninsular and north-
Fibre crops ern hilly zones. Gene exchange of the new
allopolyploids inevitably would have resulted
1. Cotton rusts (Gossypium spp.): through hyphal fusion, nuclear exchange and
(i) P. gossypi (Arthur) Hiratsuka = genetic recombination in urediniospore pop-
U. gossy = C. desmium; ulation. This results in widening the host
(ii) A. gossypi, an aecial state of P. range of the hybrid rusts because of gene
cacabata Arthur & Holway. diversity and increases the value of survival
2. Linseed/flax rust (Linum usitatissimum and nutritional status of the rust. Continu-
L.): M. lini (Ehrb.) Lev. ous introduction of hybrid cultivars in the
field through plant breeding serve as new
hosts to rust. Three species of wheat and their
Medicinal plants cultivars are used mainly for cultivation of
durum wheat (T. durum Desf.) or macaroni
1. Adhatoda zeylanica Nees. rust: Chryso- wheat, which covers about an 85% area.
celis butteri (Dietel and Sydow) Laundon. Bread wheat (T. aestivum L.) covers a 14%
area and emmer wheat only about a 1% land plant is H. vastatrix Berk. and Br., which
area. Rusts of wheat have many physiologi- develops very serious disease on foliage,
cal races and wheat cultivars are recom- leading to defoliation.
mended by plant breeders in specific regions
for cultivation to avoid rust disease devel-
opment. Epidemiological studies of three Rust of groundnut (Arachis hypogaea L.)
rusts have shown that collateral hosts have
a restricted role, while alternate hosts virtu- Groundnut is the world’s second largest
ally have no role at all. The survival of these source of edible oil and ranks 13th in pro-
rusts in India is primarily through uredinio- duction among world food crops. India is the
spores that survive on self-growing plants largest producer of groundnut. Groundnut is
or volunteer plants. The airborne uredinio- cultivated in 26m ha of land worldwide and
spores favoured by wind and rain due to produces 34.5 Mt/year. Groundnut is culti-
tropical cyclones in the months of October vated in India on 7.6m ha and produces
and November get dispersed and deposited 7.8 Mt/year. The major states in India culti-
over Central India from the Nilgiri Hills in vating groundnut are Gujarat, Maharashtra,
South India. Brown and black stem rusts Tamil Nadu and Andhra Pradesh. This crop
become established there and then subse- is attacked by 55 pathogens. Among all the
quently spread to the eastern and northern diseases, three diseases, namely groundnut
states of India over the Indo-Gangetic Plain. rust and early and late leaf spot diseases, are
Brown rust appears first in the Himalayan more serious. They generally develop simul-
foothills, eastern Uttar Pradesh and north taneously and pod yield decreases by up to
Bihar in the month of January. 10–70% (Ghewande and Savalya, 1999). Rust
The western lines associated with dis- is caused by P. arachidis Speg.; this perpetu-
turbances and rain spread the pathogen to ates by uredinia, the only spore state through-
the north-western states of India, along with out the world except teleutospores, which
yellow rust. The Nilgiri and Pulney Hills were recorded in Paraguay only once. It is
are the primary focal point providing the not known how groundnut rust perpetuates
source of inoculum, that is urediniospores and it occurs regularly every year in India
migrating upward with air currents towards without having a telial state, alternate or col-
the north via Central India, periodically lateral host. It is said that groundnut rust
trapped and studied by Mehta (1929, 1952). develops first in South India and then
The Mahabaleshwar and Panchgani plateaus migrates to North India. This disease, along
also serve as a focal point for the secondary with early and late leaf spot disease, renders
source of inoculum (Joshi et al., 1986). the crop uneconomical in the rainy season,
which is the major period of groundnut cul-
tivation in India.
Rust of coffee
Coffee rust is a disease of the coffee planta- Rust of jowar (Sorghum spp.)
tion crop, namely Coffea arabica L., C. libarica
and C. canephora, cultivated for berries to The genus Sorghum Moench has 23 species
produce coffee, a well known non-alcoholic (Simon, 1993). The crop is damaged by four
drink like tea, in Ethiopia, Yemen, Sri Lanka, different fungal diseases: seed and seedling
South and Central Africa, Cameroon, Baha- disease, foliage disease, head disease and
mas, Brazil and India. The world production root and stalk disease. Sorghum rust is a foli-
of coffee is 3.16 Mt/year. In India, coffee is age disease which infects almost all species
cultivated mainly on the hill slopes of Kar- of Sorghum. High temperatures (75–80°F)
nataka, Tamil Nadu and Kerala. Coffee pro- and humid weather is favourable for disease
duction is estimated to be 964,000 t/year on development. The species of Puccinia that
a worldwide basis and its production in infect Sorghum (Cummins, 1971) are P. pur-
India is 230,000 t. The pathogen of the coffee purea, P. levis and P. nakanishiki. The most
The Rust Fungi 209
prevalent rust of jowar throughout the world is Rust of flax/linseed

P. purpurea Cooke, which is heteroecious and (Linum usitatissimum L.)
its aecial host is O. corniculata L. But the aecial
stage plays a negligible role in rust disease. This crop is cultivated mainly for oil and
The rust infection and host reaction results fibre. It is affected by rust in most of the
in the formation of bright purple-coloured linseed-growing areas of the world such as
spots on the leaves. There are 32 races in Asia, America and Europe. The rust appears
cultivated Sorghum distributed in South- in India in February. This rust, M. lini (Ehrb.)
east Asia (11) and Africa (21). Lev., also infects wild species of Linum. It is
an autoecious rust and infects all the green
parts of the plant. Telia develop late on stems
Rust of maize (Zea mays L.) and form crusts covered by epidermis. Aecia
of this rust are caeomoid. Flor (1956) studied
Maize rust or leaf rust of maize, P. sorghi this rust and differentiated 179 races from
Schw., is an American rust. However, America alone. Eighteen races are reported
maize is susceptible to two more rusts, i.e. from India. L. mysorense L., a wild host, has
P. polysora, southern corn rust, and P. been reported to harbour the rust from India.
zeae, tropical corn rust. It is a heteroecious
rust and its aecia are produced on species of
Rust of pea (Pisum sativum L.)
Oxalis, namely O. stricta, according to
Arthur (1929). Aecia are more common in
this rust than in Sorghum rust. However, Pulse crops are affected by two rusts, namely
Mishra (1962) has claimed that the alternate U. pisi (Pers.) Wint., a heteroecious rust
host of maize rust is O. corniculata, on reported from Europe only and rarely in India,
which aecia were collected from Nepal. The while U. viciae-fabae (Pers.) Schroeter, an
rust infects all types of maize with a varying autoecious rust, is found in Europe, America
degree of severity. and Asia. It was found that aeciospores played
a major role in the dissemination of lentil rust
during the active growing season. It was also
suggested that secondary aecia are produced
Rust of bajra/pearl millet at low temperatures (17–22°C), while higher
(Pennisetum spp.) temperatures induced uredinia. Teleutospores
are dormant spores, survive for 2 years and
The genus Pennisetum Rich. has c.80 spe- remain viable at low temperatures (3–18°C).
cies and is distributed throughout the trop-
ics. Pearl millet (P. glaucum (L.) R. Br.) is a
staple food crop of the semi-arid tropical Rust of gram (Cicer arietinum L.)
parts of the world, mainly Asia and Africa.
There are about 14 rusts reported on bajra Gram rust is caused by U. ciceris-arietini
(Cummins, 1971). However, only two are (Gron.) Jack. It is a heteroecious rust, but no
well-known, namely P. substriata Ell. and alternate host or pycnia and aecia have been
Barth. var. indica Ramachar and Cummins collected. The same rust has been collected
in India and P. substriata Ell. & Barth. var. on wild species of Trigonella polycerata, a
decrospora Eboh., recently reported from weed of Fabaceae growing at higher altitudes.
Nigeria. The first rust is predominant in It is claimed to be the source of uredinio-
India and is heteroecious. The alternate spores, the primary inoculum.
aecidial host is the species of Solanum. Rust
infection produces pustules on both sides of
the leaf with necrotic spots, due to which Rust of bean
premature drying of leaves may result.
Occasionally, pustules also develop on leaf Beans belong to different genera of the family
sheaths and stem. Fabaceae, namely Phaseolus, Vicia, Lathyrus,
Pisum, Dolichos and Vigna. Their commer- Rust of soybean (Glycine max (L.) Mill.)
cial cultivars are a source of vegetables,
pulses and forage crops. They are cultivated Soybean as an oilseed crop is cultivated all
extensively all over the world as kharif and over the world as kharif. About 25 diseases
rabi crops. These crops are infected in the are known on soybean crop. Among these,
field by many rusts. The pathogens are U. there are 19 predominant fungal diseases. In
appendiculatus (Pers.) Unger var. appen- fungal pathogens, rust is the most serious in
diculatus, U. vignae Barclay, U. viciae-fabae India. The rust entered India in 1970 from
(Pers.) Schroeter and U. pisi (Pers.) Wint. the New World to Japan via Nepal in north-
The first three pathogens are autoecious and ern India and spread to the south-western
the fourth is heteroecious. Many races of parts of India up until 1995. The rust was
these pathogens are known. Heavy infection first reported from Taiwan. The fungus incit-
of leaves results in defoliation and poor ing soybean in Asia was first described as
productivity. U. sojae P. Henn. from Japan in 1903. It was
subsequently described and renamed by
Sydow, as U. sojae H. and P. Sydow. How-
Rust of rose (Rosa spp.) ever, this was erroneous due to the host not
being soybean but Mucuna spp. (Butler and
All rusts of roses belong to the genus Phrag- Bisby, 1931). Moreover, U. sojae P. Henn. is
midium and ten species infect roses world- not considered as an anamorph of U. mucu-
wide. However, seven species are very naei Rabenh. The anamorph and teliomorph
common. The most common species is P. connection was first proved by Sawada
disciflorum, which perpetuates on hybrids (1931) and named as P. sojae Sawada. In
of R. canina and R. gallica, but is less the two rusts of soybean, namely P. pachy-
likely to attack climbing or rambling roses rhizi and P. meibomaiae (Arthur) Arthur,
like R. multiflora. The rusts are autoecious the former species shows wide geographical
and all spore types, except pycniospores, distribution in Asia, Australia and Africa,
are equally harmful to the foliage. As a while the latter is restricted to America. In
result of infection in some seasons, severe India, soybean rust does not produce telia
defoliation ensues and plants are greatly and perpetuates only by uredinia, possibly
weakened. Keeping a garden clean is the due to environmental factors.
most effective method of keeping roses
healthy.
Rust of fig (Ficus carica L.)
Rust of cotton (Gossypium spp.) Rust of fig and other species of Ficus is pro-
duced by C. fici (Butler) Arthur. It is a com-
mon rust found throughout tropical and
The genus Gossypium is known by 4–5 spe-
subtropical parts of the world. Telia have been
cies. Use of cotton fibre in India is found in
observed only in India on F. glomerata Roxb.,
‘Rig Veda’. Cotton cultivation is mainly for
an evergreen shade tree. In the commercial
fibre and oil from seeds. Cotton is cultivated
fig, F. carica L., this rust appears late in the
as a cash crop in 80 countries of the world.
season (monsoon) and does not affect the
The cotton crop is infected heavily by a
quality of the fruits, but defoliation exposes
large number of pathogens, including rusts.
them to sunburn. It also reduces host vigour.
Among all the rusts, P. gossypi (Arthur)
It is possible to have a cell-free culture
Hiratsuka is the most troublesome to cotton,
using a complex culture medium to culture
not only in cultivated varieties but also in
rust urediniospores. The method was first
perennial cotton. The rust disease seriously
used successfully in black stem rust, P.
damages the cotton crop and is responsible
graminis tritici, for sporulation. Nowadays,
for c.20–70% financial loss to cotton grow-
compounds like kinetin and benzimidazole,
ers annually.
The Rust Fungi 211
which exert a cytokinin effect, are used to ‘Deities – rust gods, Robigan and Robigus’.
culture detached leaves in solution in test Thomas Knight, the English plant physiolo-
tubes for up to a month to determine the gist, gave experimental proof of the ability
races of rusts. of aeciospore to infect cereals and after that
same year, voluntary eradication started in
Denmark.
Rust Disease Management Strategies
There is a need for effective disease forecast- Strategy of management of plant

ing and warning systems, as well as a dis- diseases (Sharvelle, Strategy of
ease calendar for each crop. Crop protection/ Plant Disease Control, 1961)
management varies widely with different
crops, due to different factors such as: Most control measures either reduce the ini-
tial inoculum or rate of spread of plant
● varietal susceptibility of crops pathogens (van der Plank, 1963). It is impor-
● soil types tant to reduce and delay the initial infection
● agronomic practices and as much as possible, by disease forecasting
● cropping patterns. in advance so that farmers can protect the
crop plants to avoid monetary losses. Shar-
The study of diseases, disease development, velle (1961) classified the strategies for plant
disease outbreak, pathogens, varieties, races, disease control into two categories: (i)
biotypes, ecotypes, pathotypes, specializa- immunization; and (ii) prophylaxis (to erad-
tion, host plants, their hybrids, cultivars, icate the pathogen).
fluctuating factors like soil, water, fertiliz-
ers, pesticides, host–pathogen complex, etc.
Strategy of plant disease control
is very vast and difficult. The outbreak of
disease on an epiphytotic scale resulting
from the interaction of pathogen, host and Immunization
environment can be represented through 1. Genetical resistance.
disease progress curves (DPC). The problem 2. Induced resistance.
becomes more serious due to a pathogen
having high pathogenicity, which includes: Prophylaxis
● virulence and 1. Protection:

● aggressiveness (vigorous races). (i) Chemical prophylaxis;
(ii) Environmental manipulation.
Today, virulence, based on evidence, can 2. Eradication:
often be considered to be oligogenic, in which (i) Crop rotation;
a few genes are involved, as suggested by (ii) Sanitation;
van der Plank (1968), while aggressiveness (iii) Alternate host elimination;
is generally polygenically inherited. Viru- (iv) Chemical eradication.
lence is conditioned due to gene diversity 3. Legislation:
and aggressiveness by variation in the doses (i) Quarantine;
of enzymes. Hence, disease reaction is a (ii) Regulatory measures.
chemical process which entails changes in
the host and parasite cell metabolism. Black
stem rust is found to be more severe on Developing New Strategies for
wheat than barley and yellow rust on barley Disease Management: Role of
than wheat. To avoid heavy losses, early Oxidative Burst
sowing was recommended by Pliny. In
Greek and Roman civilizations, the appear- The molecular biology of interactions between
ance of plant diseases was attributed to disease-resistance genes, defence genes and
their role in genetic engineered disease- 2. Cladosporium spp.

resistance elicitor (signal) molecules has been (i) C. aecidiicola Theum.;
detected in fungal, bacterial and viral patho- (ii) C. exobasidii Jaap;
gens. These molecules serve as signals to (iii) C. uredinicola Speg. on P. recondita
elicit defence mechanism of the host. Host (UK).
resistance genes may function as receptors 3. Verticillium spp.
of these signals. Only a few disease-resis- (i) V. hemileiae Steyaert;
tance genes have been cloned from plants. (ii) V. lecanii (Zimm.) Viegas.
Analysis of these genes shows the presence V. hemileiae and V. lecanii, as a virtue of
of leucine-rich repeats (LRRS), leucine zip- their growth on uredinia of coffee rust in a
pers and nuclear localization signals moist environment, produce a chitinase en-
(NLS). LRRS are involved in protein–pro- zyme to weaken the wall of the spores, as a
tein interactions of the signal transduction result of which the spores burst. Even the cul-
pathway. Several defence genes are widely tural filtrates are effective (Ellis, 1971, 1976).
found in both resistant and susceptible 4. Sphaerellopsis filum (Biv. – Bern. ex.
plants and are involved in the production of Fr.) B.C. Sutton = Darluca filum (Biv.) Cast.
antimicrobial compounds, namely phenols, This hyperparasite was considered by Tarr
phytoalexins and pathogen-related (PR) pro- (1972) as an ecologically balanced myco-
teins; PR-1, 2,3,5,6 and 8, co-enzyme reductase parasite on rust fungi, especially the ured-
(HMGR), transgenic plant expressing pheny- inia of the species of Puccinia and Uromy-
lalanine ammonial-yases (PAL) showed ces of grasses. It is distributed in the
enhanced disease resistance (Vidyasekaran, tropical and subtropical moist regions of
1997). the world. However, it has not been used
commercially.
5. Tuberculina costaricana H. Sydow.
Fungicides in the Control 6. Olpidium uredinis, an endoparasite in
urediniospores.
of Rust Diseases
The different fungicide dosages recommen-

ded for rust disease control are shown in Plant breeding
Table 16.1.
1. Cultivation of hybrid cultivars recom-
mended by plant breeders in different re-
Other Methods of Plant gions for different rusts. The hybrids have
Disease Management high resistance coupled with good quality
and high productivity. Some rust-resistant
Biological methods cultivars are ‘Maris Ranger’, ‘Heines VII’,
‘Fenman’, ‘Hybrid 46’, ‘Minster’, ‘Opal’, CS
2D/2M, T. spelta 391, T. spelta G652 (ICAR-
Biological control using different microbes
DA), CIM 25, ‘Dove’ (CIMMYT), HD 4502,
is the most popular and ecologically safe
‘Arkan’, ‘Blueboy II’, ‘Centurk’, ‘Chris’ (USA),
method, but is not used practically due to
‘Banks’ and ‘Egret’ (Australia).
many constraints. The following are some
2. Use of defence activators – spray of sal-
potential and promising parasites of rusts
icylic acid (SA), ferric chloride (FeCl3) and
which can be used in biological control in
dipotassium hydrogen phosphate (K2HPO4).
the future.
3. Adult plant resistance (APR) – there
1. Aphanoderma album (Preuss.) W.Gams has been an increasing interest in adult plant
This Hyphomycetes is characterized to pro- resistance, especially against leaf rust or
duce a metabolite which switches off sporu- brown rust pathogens of wheat in North
lation of urediniospores to teleutospores, thus India, because of its widespread occurrence
terminating the life cycle of rust. in germplasm and its durability.
The Rust Fungi 213
Table 16.1. The various fungicide dosages recommended for different diseases.
I – Sulphur fungicides
Sulphur 2–4 kg/ha Spray or dust 2–3 times Rust of beans – Uromyces
appendiculatus var.
appendiculatus, U. fabae
Lime – sulphur 0.75 g/100 g Sprays Bean rust – U. fabae
Ziram 0.2% solution Spraying at the outbreak Bean – U. appendiculatus or
of disease and repeated U. phaseoli
at weekly intervals Mint – Puccinia menthae
Sunflower – P. helianthi
Ferbam 0.15% solution Sprays as required Rose – Phragmidium
mucronatum
0.3% solution Seed treatment Safflower – P. carthamii
Thiram 0.15–0.2% spray Sprays Apricot – Tranzschelia discolor
Plum – T. discolor
0.2–0.3% solution Dry seed dressing Safflower – P. carthamii
0.5% solution Two sprays at 10–15-day Beet – U. betae
intervals
Zineb 0.2% solution Applied at 4-week intervals Almond and apricot – T. discolor
from petal fall
0.2% solution 3 sprays at 14-day intervals Barley – P. striiformis
0.15% solution Sprays at disease outbreak Chrysanthemum –
and repeat after 10–15-day P. chrysanthemi
intervals
0.2% solution 5 sprays at 10-day intervals Garlic – P. allii
0.2% solution Applied at 4-week intervals Peach – T. discolor
from petal fall
0.2% solution 3 sprays at 15-day intervals Phalsa – Dasturiella grewiae
starting in last week of
September before disease
outbreak
0.2% solution 5 weekly sprays in the Soybean – Phakopsora
growing season pachyrhizi
0.2% solution Spray at disease outbreak Wheat – P. recondita and
and repeat at c.10-day P. graminis tritici
intervals as necessary
Maneb/ 0.2% solution Sprays at disease outbreak Bean – U. appendiculatus
mancozeb and repeat at 7–10-day var. appendiculatus
intervals
0.25% solution Sprays at 14-day intervals Groundnut – P. arachidis
along with benomyl or
carbandazim
0.2% solution 4 sprays at 10-day intervals Peas – U. fabae
from disease outbreak.
Add triton at rate of 2 ml/l
suspension
0.3% solution Seed treatment Safflower – P. carthamii
0.2% solution 4 sprays at 12-day intervals Sorghum – P. purpurea
400 ppm Sprays Sunflower – P. helianthi
2–3 kg/ha Dusting 3–6 applications Wheat – P. graminis tritici and
at 10-day intervals P. recondita
Nabam 0.15–0.25% Sprays 4–5, beginning when Wheat – P. graminis tritici and
solution disease appears P. recondita
continued
Table 16.1. continued.
II – Copper fungicides
Bordeaux 5:5:1/2:50 Sprays, 5–10 Coffee – Hemileia vastatrix

mixture applications, add urea
and ZnSO4
Copper 1.8 kg/400 l Sprays Beans – U. appendiculatus
oxychloride Colloidal copper 1500 ml/400 l Sprays Beans – U. appendiculatus
0.35% solution (kocide 110) Sprays in March–April Coffee – H. vastatrix
and 0.5% (cupravit) 300 l/ha
III – Mercury fungicides
Agrosan GN 0.3% solution Seed treatment Safflower – P. carthamii
IV – Heterocyclic nitrogenous compounds
Captafol 0.3% solution Seed treatment Safflower – P. carthamii

0.2% solution 3 sprays at 10-day intervals Brown rust of wheat:
as soon as disease appears P. recondita
V – Systemic fungicides
Benzimidazole 1.5 kg/ha Dust or spray with carbendazim or Groundnut – P. arachidis

carbendazim (0.07%) plus mancozeb Speg. and early and late
(0.15%) leaf spot disease
1 g/kg Seed dressing with benomyl Safflower – P. carthamii
Oxathiin-carboxin 2.5 g/kg Seed treatment/foliar spray and Bean –
soil treatment U. appendiculatus
Oxycarboxin 2630 g/100 kg Seed treatment Stripe rust of wheat –
P. striiformis
1.12 kg/ha Granules in soil Leaf rust wheat –
1.68 kg/ha Spray P. recondita
3 kg/ha Spray – single in Europe or Black stem rust –
2–3 sprays common P. graminis tritici and
P. recondita
15 kg/ha Soil treatment Safflower (seedling)
stage – P. carthamii
Fig rust – Cerotelium fici
Soil treatment with benomyl spray Peanut – P. arachidis
Soil treatment with benomyl as spray Sunflower – P. helianthi
VI – Benzanilide derivatives
Benzanilide 1.87 kg/ha Foliar spray Stripe rust of wheat – P. striiformis

and barley – P. hordei
Benodanyl/ 500 mg/ml 2 sprays or soil drenching Effective against most of the
Vitavax rusts of cultivated plants
0.75 l/ha Spray/seed treatment Stripe rust of wheat – P. striiformis
0.3% 4 sprays at 15-day intervals Leaf rust of wheat – P. recondita
250 ml/l 2 sprays or soil drenching Sunflower – P. helianthi
Oxycarboxin 0.1125% Sprays at 6–9-day intervals Sunflower – P. helianthi
100 mg/l 2 sprays or soil drenching Sunflower – P. helianthi
Plantavax w.p. 10 g/kg Spray/seed treatment Stripe rust of wheat – P. striiformis
0.2% Spray/seed treatment Black stem rust – P. graminis tritici
The Rust Fungi 215
Integrated disease management Acknowledgement
1. Cultural methods include early sowing. The authors express their gratitude to The
2. Spraying of micronutrients such as Principal, Agriculture College, Kolhapur,
Na2B4O7, CuSO4 increases resistance in India, for providing the facilities of their
plants. library.
References
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Research, India, 65 pp.
Cummins, G.B. (1936) Phylogenetic significance of the pores in the Uredinales. Mycologia 23, 1–102.
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national, Wallingford, UK.
Kuhn, J.G. (1858) Diseases of Cultivated Plants. 312 pp.
Mehta, K.C. (1929) Annual recurrence of rust on wheat in India. Presidential Address (Botany section) at
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their species on Leguminosae. Mycological Research 96, 825–850.
Rajendran, R.B. (1967) Atypical and typical germination of uredinoid teliospores of Hemileia vastatrix.
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Appendix 1
Key to species and varieties of Puccinia and Uromyces producing rust diseases to
cultivated crops which are very common but confusing to identify
Wheat rusts
I. Uredinial infection produces chlorotic streaks with halos on leaves -----------P. striformis
I’. Uredinial infection does not produce chlorotic streaks with halos on leaves ----------- II
II. Urediniospores with 5–6 germ pores arranged equatorially -------------P. graminis tritici
II’. Urediniospores with 4–5 germ pores, scattered ----------------------------------- P. recondita
Jowar rusts
I. Uredinia aparaphysate ---------------------------------------------------------------------------- P. levis
I’. Uredinia paraphysate -------------------------------------------------------------------------------------II
II. Urediniospores with 5–8 germ pores, scattered ---------------------------------P. nakanishiki
II’. Urediniospores with 3–5 germ pores, equatorial ---------------------------------- P. purpurea
Bajra rusts
I. Infection mostly hyphophyllous, teleutospores measure 21–49 µm long, 2-celled, pedi-
cel coloured and short ---------- P. substriata Ell. & Barth. var. indica Ramachar & Cummins
I’. Infection amphigenous, teleutospores large, up to 5-celled ---------------------P. substriata
Ell. & Barth. var. decrospora Eboh.
Maize rusts
I. Teleutospores stalked and 2-celled -------------------------------------------------------------------II
I’ Teleutospores sessile, in chain and innate -------------------------------------------------------- P. zeae
II. Telia exposed/erumpent, urediniospores 26–31 µm long (aecia on Oxalis stricta L.)------
--------------------------------------------------------------------------------------------------------------- P. sorghi
II’. Telia covered, urediniospores 29–30 µm long (aecia not known) -------------- P. polysora
Rusts of Phaseolus and Vigna spp.

I. Urediniospores measure 24–29 × 17–19 µm, germ pores 4, equatorial (on Phaseolus
spp.) ------------------------------------------- U. appendiculatus (Pers.) Unger var. appendiculatus
I’. Urediniospores measure 29–32 × 20–22 µm, germ pores 2, equatorial (on Vigna spp.)
-------------------------------------------------------------------------------------------------------------- U. vignae
Rusts in forage crops

I. Urediniospores have 4–7 germ pores, scattered (on red clover) ------------------U. fallens;
T. pratens (only uredia and telia)
I’. Urediniospores have 2–4 germ pores, equatorial (on white clover) ---------------------------
------------------------- U. trifolii-repentis T. repens and other spp. autoecious and macrocyclic
Rusts on vegetable and pulse crops
I. Autoecious, occurs in America and Europe ------------------------------------ U. viciae-fabae

I’. Heteroecious, occurs only in Europe (Arthur, 1929) on P. sativum L. -------------- U. pisi
17 Etiology, Epidemiology and
Management of Fungal Diseases
of Sugarcane
Ayman M.H. Esh

Biotechnology and Tissue Culture Laboratories, Sugar Crops Research Institute,
Agricultural Research Center, Giza, Egypt
Abstract
Sugarcane (Saccharum officinarum L.) is one of the most important commercial crops in many coun-
tries of the world. It contributes nearly 70% of world sugar and provides the base materials essential
for many other industries. Sugarcane crop is attacked by numerous foliar and root pathogens. Some of
these diseases cause serious quantitative and qualitative losses which have negative effects on sugar-
cane production, as well as in the sugar industry.
About 56 diseases of sugarcane have been reported so far from different parts of the world. Of
these, 40 are caused by fungi, several of which can cause economic losses. The major sugarcane fungal
diseases in different tropical and subtropical regions are: smut disease (Ustilago scitaminea); rust dis-
ease (Puccinia melanocephela); red rot (Glomerella tucumanensis [Colletotrichum falcatum]); eye
spot disease (Bipolaris sacchari), pokkah boeng disease (Fusarium moniliforme); and pineapple dis-
ease (Ceratocystis paradoxa). This chapter includes the major fungal diseases of sugarcane and the
various control practices used against them.
Introduction Due to its wide range of adaptability, it

supplies more than 60% of world sugar
Sugarcane (S. officinarum L.) is a mono- demand and basic raw material in many
cotyledonous plant from the family Poaceae industries, which makes it one of the most
of the subfamily Andropogoneae (Cox et al., important cash crops that plays an enor-
2000) and is considered as one of the oldest mous role in the economy. Various biotic
cultivated crops known to man. Sugar, along and abiotic factors are responsible for yield
with honey, is the oldest natural sweetener reduction and economic losses. Among these
(Peng, 1984; Naik, 2001). Sugarcane is grown factors, fungal diseases are the major cause.
in the tropical and subtropical regions of Over 100 fungi, 10 bacteria, 10 viruses and
the world and is cultivated in nearly about 50 species of nematodes are pests of
60 countries as a commercial crop, with sugarcane in different parts of the world
Brazil, India, China, Cuba, Thailand and (Singh and Waraitch, 1981). Sugarcane,
Pakistan as the major sugarcane-growing being a long duration crop (10–12 months),
countries (FAO, 2005). remains in the field for several years (ratoons

218 A.M.H. Esh
remain in the field for up to more than Causal agents

6 years). A serious drawback of this prac-
tice, however, is that pathogens may build The fungus belongs taxonomically to Phylum:
up within the field and be disseminated dur- Basidiomycota; Class: Ustilaginomycetes,
ing propagation of new seed cane. The patho- (Bisby et al., 2007). Classification of U. sci-
gen within the seed pieces is transmitted taminea H. & P. Sydow, the causal agent of
easily into young plants, which in turn serve sugarcane smut disease, is based mainly on
as sources of inoculum for secondary infec- differences in spore morphology and the
tions in adjoining healthy plants. Several characteristics of germinating spores (Lee-
management strategies have been developed Lovick, 1978).
as a result of research and development Smut races have been reported subse-
work. The endless struggle between varieties quently based both on observations and
and the complexity of disease have led cor- inoculation studies (Gillaspie et al., 1983).
respondingly to the development of a vari- Usually, races are suggested when a cultivar
ety of approaches for control. The role of succumbs to smut after being grown for sev-
fungicides in modernizing and changing the eral years without being infected (James,
condition of agriculture is quite significant 1976). U. scitaminea races have been
(Mehta, 1971; McFarlane et al., 2006). reported in Hawaii, Pakistan, the Philip-
pines and Taiwan; the presence of the actual
number of races and their prevalence are
Sugarcane Smut Disease unknown (Ferreira and Comstock, 1989).
Pathogenic races of sugarcane smut have
Sugarcane smut, caused by the Basidiomy- been observed in several countries, includ-
cetes fungus Ustilago scitaminea Syd., is ing two races A and B from Hawaii (Com-
cosmopolitan in distribution and has been stock and Heinz, 1977) and three races (1, 2,
an important disease in nearly every sugar- 3) reported in Taiwan (Leu and Teng, 1972;
cane-producing country of the world. It can Lee et al., 1999). However, Ferreira and
reduce crop yields by over 50% and make Comstock (1989) considered the true preva-
ratoon crops unprofitable to maintain. It is lence of races to be controversial. Many
highly infectious and even developed coun- claims are based on the reaction of the same
tries have been unable to stay smut free with cultivar in different countries, but the inter-
the use of appropriate quarantine measures pretation of these claims is confused by test-
(Antony, 2008). The disease was first noted to-test variation and the use of different
in South Africa in 1877, then in the early inoculation methods. Two international
1930s it caused severe problems in India collaborations have attempted to standardize
and other countries in Asia. Years later, the race typing. Gillaspie et al. (1983) performed
disease started to establish and cause seri- race typing under glasshouse conditions to
ous problems in different parts of the world: standardize the environment and six races
1943, Argentina, (Cross, 1960); 1950, Brazil were identified. Grisham (2001) coordinated
and Paraguay; 1957, Bolivia; 1960 and 1971, a race typing study in nine countries using
Hawaii (Byther et al., 1971); and 1974, Guy- local isolates tested against a standardized
ana (James, 1976). By 1981. the disease had set of 11 differential cultivars. On the molec-
been found in most of the Caribbean and ular level, many researchers have studied
North, South and Central America (Ferreira the genetic diversity among U. scitaminea
and Comstock, 1989). In 1998, the disease isolates, either between local isolates or
was reported for the first time in the Ord between isolates collected from different
River area of Western Australia (Riley et al., parts of the world (Braithwaite et al., 2004a,b;
1999). Australia is a major exception since Xu et al., 2004; Singh et al., 2005). Genetic
the disease is present only in Western Aus- variation estimated from 12 AFLP primer
tralia. The sugar industries of eastern Austra- combinations showed that, overall, there
lia, Fiji and Papua New Guinea are still free was little variation in the smut population
of the disease (Braithwaite et al., 2004b). across the world. However, isolates from
Fungal Diseases of Sugarcane 219
the Philippines, Taiwan and Thailand form dikaryotic mycelium develops after fusion
a distinct cluster; it is therefore suggested of compatible sporidia. This dikaryotic myce-
that genetic variation is limited between the lium is infectious, penetrates behind bud
isolates and the phylogeny of U. scitaminea scales and invades the meristematic zone of
is poorly understood (Braithwaite et al., the bud. Entry into the meristem in the bud
2004b). occurs between 6 and 36 h after the telio-
spores are deposited on the surface (Alexan-
der and Ramakrishnan, 1980). Finally, the
Disease symptoms apical meristem of smut-infected cane pro-
duces a long whip-like structure bearing
billions of teliospores (i.e. sorus).
Smut-infected plants are distinguished by
Sugarcane smut is spread by spores
the emergence of a ‘smut whip’. The whips
which have an aerial dispersal mode. The
are the flowering structures of the patho-
whip serves as a source of spores that release
gen which produce teliospores. The flow-
approximately one billion spores/whip/day
ering structures transform into a whip-like
into the air to infect the buds of the standing
sori that grows out between the leaf sheaths.
sugarcane. The infected buds remain dor-
At first, it is covered by a thin silvery
mant until the cane is cut for seed. The
peridium (this is the host tissue), which
spores mixed with the soil of cropped or
peels back easily when desiccated to expose
newly prepared fields also become a source
the sooty black-brown teliospores. Whips
of infection to the disease-free seed pieces.
begin emerging from infected cane by
Under normal soil moisture, the spores only
2–4 months of age, with peak whip growth
survive for a short time in the soil. On the
occurring at the 6th or 7th month. Spindle
other hand, several species of insects have
leaves are erect before the whip emerges.
been associated consistently with smut
Affected sugarcane plants may tiller pro-
whips; this suggests insects could play a
fusely, with the shoots being more spindly
role in spore dispersal (Ferreira and Com-
and erect with small narrow leaves (i.e. the
stock, 1989; Agnihotri, 1990).
cane appears ‘grass-like’). Less common
symptoms are leaf and stem galls and bud
proliferation (Ferreira and Comstock, 1989;
Agnihotri, 1990). Disease control
The best control method is to use resistant

Pathogenesis cultivars. There is a strong genetic basis
for resistance and resistant varieties have
Ustilago scitaminea produces diploid spores been readily available and used to control
called teliospores. When teliospores germi- outbreaks of smut in several countries
nate, they undergo meiosis, which gives rise (Churchill et al., 2006). Disease-free plant-
to a septate promycelium bearing four hap- ing material usually can be obtained by sub-
loid sporidia (basidiospores). U. scitaminea, jecting seed to hot water treatment. Hot
like most parasitic Heterobasidiomycetes, water treatment, however, may not be prac-
has a diallelic bipolar mating system (Alex- tical on a large scale and its effectiveness
ander and Srinivasan, 1966; Leu, 1978; may be subject to varietal differences
Moosawi-Jorf et al., 2006) in which only (McFarlane et al., 2007).
sporidia of opposite mating types conjugate. Several fungicides (triadimefon, fludio-
Of the four initial sporidia or basidiospores xonil:mefenoxam:azoxystrobin, mancozeb,
from each teliospore, two have a positive metalaxyl + carboxin + furathiocarb, pyroqui-
mating allele and two have a negative mat- lon, benomyl and chlorothalonil) have been
ing allele. U. scitaminea can thus both self- used to control sugarcane smut when used
and outcross, but the frequency of natural as pre-planting fungicidal dips of planting
selfing versus outcrossing is unknown. A setts (Wada et al., 1999; Wada, 2003).
220 A.M.H. Esh
Sugarcane Rust Disease visible on both leaf surfaces. The spots

(Common and Orange Rust) increase in size up to 1.5 mm in diameter and
usually turn brown to orange-brown or red-
Throughout the world, the important leaf brown. The lesions occur irregularly and
rust disease causes severe losses in sugar- typically range from 2 to 10 mm in length,
cane fields (Magarey et al., 2008). In 2000, but occasionally reach 30 mm. The spots
sugarcane rust was once considered a minor are raised and are surrounded by a pale yel-
pathogen in the Australian sugar industry. low halo (Raid and Comstock, 2000). The
In 2000, it devastated most plantations of raised pustules are formed predominantly
the cultivar Q124 in Australia, causing yield on the undersurface of the leaves and the
losses of up to 40% (Apan et al., 2003; Braith- urediospores formed therein are orange to
waite et al., 2004a; Magarey et al., 2008). In orange-brown. On a highly susceptible vari-
the USA, the yield loss caused by a rust epi- ety, considerable numbers of pustules may
demic due to cultivar CP 72-1210 in 1987 occur on a leaf, coalescing to form large,
was 20% (Raid and Comstock, 2000). irregular, necrotic areas. High rust severities
Sugarcane rust is caused by two species may even result in premature death of young
belonging to the genus Puccinia, P. melano- leaves. Severe rust has caused reductions in
cephala and P. kuehnii, the former causes both stalk mass and stalk numbers (Rao
common rust disease, while the later causes et al., 1999; Raid and Comstock, 2000).
orange rust. Common rust caused by P. mel-
anocephala H. & P. Syd. was first reported
on sugarcane in 1949 in the Deccan area in
Causal agent
India (Patel et al., 1950). The disease has
been also reported from: Japan (Ohtsu, 1975
[cited by Muta, 1987]); the Philippines (Serra Puccinia melanocephala Syd. and P. Syd.
et al., 1983); Australia (Egan and Ryan, (common rust) and P. kuehnii Butler (orange
1979); Taiwan (Hsieh et al., 1977); Domini- rust) are reported to cause rust diseases
can Republic (Presley et al., 1978); Jamaica on sugarcane (Butler, 1914; Cummins and
(Burgess, 1979); Puerto Rico (Liu, 1979); Hiratsuka, 1983; Shine et al., 2005; Ido et al.,
Cuba (Sandoval et al., 1983); Carribean and 2006; Comstock et al., 2008; Ovalle et al.,
Central America (Purdy et al., 1983); Hawaii 2008).
(Comstock et al., 1982); and Angola, Kenya, The two obligate parasitic fungi belong
Madagascar, Tanzania, Uganda, Zambia, to Phylum: Basidiomycota; Class: Uredinio-
Zimbabwe, South Africa, Mozambique and mycetes, Order: Uredinales. The causal agents
Malawi (Egan, 1980; Sivanesan and Waller, of common and orange rust cannot be clearly
1986). distinguished based on colour of lesions
Orange rust caused by P. kuehnii Butl. and uredinia and the size of urediniospores.
was first reported on sugarcane in Java in However, they are distinguishable based on
1890 (Ryan and Egan, 1989). The disease the presence or absence of abundant capi-
has been reported from Japan (Ito, 1909), Aus- tate paraphyses in uredinia, echinulation,
tralia, Indonesia, the Philippines, Taiwan, colour and wall thickness of urediniospores,
Pacific Islands, Sri Lanka, Malaysia, Thailand, colour of the telia and colour and wall thick-
New Caledonia, China (Egan, 1980; Sivane- ness of teliospores. P. melanocephala has
san and Waller, 1986) and India (Mukerji abundant capitate paraphyses in uredinia
and Bhasin, 1986). and urediniospores with dense echinula-
tion, darker brown and uniformly thick
walls. They also have dark brown to blackish
telia with brown to dark brown teliospores
Disease symptoms with apically thickened walls. P. kuehnii
has morphologically indistinct paraphyses
The initial symptoms of common rust are in uredinia and urediniospores with
small, elongated yellowish spots, which are moderate echinulation, lighter brown and
sometimes apically or uniformly thickened resistant cultivars are threatened by the

walls (Virtudazo et al., 2001). establishment of new races of the pathogen.
For example, cultivar CP 78-1247 was con-
sidered to be resistant or moderately resis-
Pathogenesis tant until 1988, and then it exhibited
extremely high rust susceptibility through-
out south Florida (Raid, 1989). However,
The life cycle of sugarcane rust is simple,
resistance has not been stable or durable on
with the urediniospore being the only known
certain varieties, presumably because of rust
infectious spore. These are produced in,
variants. For this reason, it is highly recom-
and are released from, pustules that develop
mended that growers should diversify their
on the underside of sugarcane leaves. The
varietal holdings (Raid and Comstock,
development of substomatal vesicles, infec-
2000). Chemicals like propiconazole/manco-
tious hyphae, haustoria and subsequent
zeb, cyproconazole, triadimefon and triadi-
infection processes are similar to other Puc-
menol have been used for the control of
cinia spp. Urediniospore production occurs
sugarcane rust. Several soil factors influ-
8–18 days after the initial urediniospore
ence rust infection levels on sugarcane sig-
lands on a leaf, depending on varietal sus-
nificantly. Studies have shown that rust
ceptibility and environmental conditions
levels are higher on sugarcane grown on
(CABI CPC, 2006).
low pH soils, high soil moisture and high lev-
Spread of rust disease occurs primarily
els of phosphorus and potassium nutrients
by wind and water-splash movement of
present in the soil (Johnson et al., 2007).
urediniospores. The movement of diseased
vegetative parts of sugarcane, contaminated
equipment and workers from one location
to another may also provide a means of Red Rot Disease of Sugarcane
spread. The expression of the disease is influ-
enced by the interaction of genetic, environ- Red rot is one of the oldest known diseases
mental (primarily air temperature and leaf of sugarcane. It occurs in most cane-growing
wetness) and physiological (age of infected countries. The disease was first described
plants) factors. The infection may occur from Java by Went (1896) and then the dis-
within the temperature range of 5–34°C; how- ease was reported from Australia, India,
ever, the optimal temperatures for spore ger- Hawaii and the USA. It is clear that the
mination are between 15° and 30°C. Heavy disease was widely distributed before the
rains tend to remove spores from the atmo- knowledge of its impact on sugarcane crop
sphere, rendering them infective if they (Singh and Singh, 1989).
land on the soil (Egan, 1964; Comstock and
Ferreira, 1986). On the other hand, it has
been found that rains favour the development Symptoms
of orange rust but inhibit the development of
common rust (Croft et al., 2000). Sugarcane
The pathogen, Colletotrichum falcatum
plants appear to be most susceptible at
Went, can attack any part of the sugarcane
3–6 months old (Ryan and Egan, 1989).
plant – stalk, leaf, buds or roots – but it is
usually considered a stalk and a seed-piece
disease. C. falcatum completes its life cycle
Disease control on the sugarcane leaf and usually the dam-
age to the leaf does not pose a serious threat
The best control of sugarcane rust is use of to cane or cause much harm to the plant
resistant sugarcane varieties. The develop- (Singh and Singh, 1989; Raid, 2006).
ment of resistant cultivars has decreased The most damaging phase of this dis-
the economic losses caused by this disease ease occurs when the pathogen attacks the
(Ryan and Egan, 1989). Nevertheless, existing stalk. Depending on the age of the stalk,
222 A.M.H. Esh
time of infection and susceptibility of the in the host environment. Heterokaryosis is

cane genotype, it produces different types the mechanism through which the fungus
of symptoms. The typical stalk symptoms, collects and consolidates two or more genet-
that is, presence of white spots in otherwise ically different nuclei in the hypha and
rotten (dull red) internodal tissues and derives the benefit of the introduced genetic
nodal rotting, appear when the crop is at the material. These newly gathered nuclei also
fag end of the grand growth phase in sub- multiply in tandem with the native nuclei
tropical areas. These white patches are spe- (Duttamajumder, 2008).
cific to the disease and are of significance in
distinguishing red rot from other stalk rots.
At a later stage, some discoloration of rind Pathogenesis
often becomes apparent when internal tis-
sues have been badly damaged and are fully
The pathogen mainly infects the stalks
rotten (Singh and Singh, 1989; Raid, 2006;
through the nodes. Once the infection is
Duttamajumder, 2008).
established in the stalk, the fungal myce-
In susceptible varieties, the red colour,
lium grows intracellularly and is sparse in
sometimes along with some grey colour,
the reddened areas. The dead cells are packed
may be seen throughout the length of the
in white patches with profuse hyphae. The
stalk. The infection is confined largely to
size and number of these white areas are
the internodes in resistant varieties. On the
correlated with the susceptibility of the
leaves, the pathogen may produce elongated
variety. The lesions become dark red, nar-
red lesions on the midribs, reddish patches
row and sharp margins, with a few white
on the leaf sheaths and, infrequently, small
spots in resistant varieties, while in suscep-
dark spots on the leaf blades. Eventually,
tible varieties the lesions become wide,
the lesions may develop a straw colour in
light red and ill-defined margins with prom-
the centre. In seed pieces, the entire seed
inent white spots (Singh and Singh, 1989).
piece may become rotten and the internal
One of the major sources of inoculum is
tissues turn various shades of red, brown or
midrib lesions. Also, diseased stalks and
grey (Singh and Singh, 1989).
crop debris and infected plant material are
important sources of inoculum and cause
secondary infections. Wind, rain, heavy
Causal agent dews and irrigation water play a role in the
dispersal of the inoculum. The pathogen
spores washed into the soil may produce
The fungus causing red rot of sugarcane is
infection in planted seed pieces. Climatic
commonly known by its imperfect state, i.e.
factors affect both the spread and severity of
C. falcatum Went (Glomerella tucumanen-
red rot. In newly-planted cane, the disease
sis). The perfect state of the fungus belongs to
is favoured by excessive soil moisture,
Phylum: Ascomycota; Class: Ascomycetes;
drought conditions and low temperatures.
Family: Glomerellaceae; Genus: Glomerella
(Bisby et al., 2007).
Conidia are falcate (but not markedly
so), fusoid, apices obtuse, 15.5 (25–26.5)– Disease control
48 µm × 4 (5–6)–8 µm and contents are
granular and sometime contain oil globules. The use of resistant varieties is the most effec-
At least two races have been identified. The tive method of prevention and control of
variations in the asexual state of the fungus sugarcane red rot disease (Singh and Singh,
(Colletotrichum state) may originate through: 1989; Raid, 2006; Singh et al., 2008b). Man-
(i) heterokaryosis; (ii) by recombination agement of the disease by the use of disease-
through parasexual mechanism; and (iii) by free seed canes for planting is impractical
the universal mechanism of mutation, selec- due to the difficulty in diagnosing dormant
tion and adaptation in response to the changes infections of the fungus in seed canes under
field conditions (Viswanathan and Sami- Disease symptoms

yappan, 2002).
It is difficult to manage red rot through Typical mature eye spot symptoms are char-
chemotherapy because the impervious acterized by a reddish-brown elliptical lesion
nature of rinds and fibrous nodes at cut (0.5–4.0 mm long, 0.5–2.0 mm wide) with
ends does not allow sufficient absorption in yellowish-brown margins. Reddish-brown
setts (Agnihotri, 1990). However, better crop to yellowish-brown streaks, sometimes
stands have been achieved from enhanced called ‘runners’, extend upward from indi-
germination obtained by treating seed vidual lesions toward the leaf tip. These
pieces with a fungicide before planting streaks are 3–6 mm wide and 30–90 cm
(Raid, 2006). long. The entire leaf eventually may become
Thermotherapy (moist hot air or hot necrotic (Comstock and Steiner, 1989; Com-
water) is thought useful for inactivation of stock and Lentini, 2005).
red rot pathogen, but it is difficult to remove
deep-seated infections. It is limited in check-
ing secondary infections (Singh, 1973; Singh
and Singh, 1989; Raid, 2006). In India, Causal agent
extensive studies about the possibilities of
using biological control to control sugar- Eye spot disease is caused by Bipolaris
cane red rod disease have been carried out sacchari (Butler) Shoemaker. The fungus
(Mohanraj et al., 1999). Seventy-five per belongs taxonomically to Phylum: Ascomy-
cent of canes may be protected against sec- cota; Class: Ascomycetes; Order: Pleospo-
ondary infection of red rot by dipping the rales. B. sacchari is the teleomorphic stage
setts for 15 min in 2.5% culture filtrate of of Helminthosporium sacchari Butler. The
Trichoderma harzianum (Th 38) and also name H. sacchari is still used occasionally.
by applying Trichoderma multiplied cul-
ture in press mud 20 kg/ha beneath the setts
in furrows. Besides the biological control of
red rot, the growth in improved resulting is Pathogenesis
to enhanced yield by 15.4 t/ha (Singh et al.,
2008a,b). Sugarcane eye spot fungus B. sacchari (H.
sacchari) produces a host-specific toxin
(HST). HSTs are a group of structurally
complex and chemically diverse metabo-
Sugarcane Eye Spot Disease lites produced by plant pathogenic strains
of certain fungal species and function as
Eye spot has been reported in many essential determinants of pathogenicity or
sugarcane-growing areas of the world. The virulence. HSTs are referred to as ‘host
disease was first described by van Breda de selective’ because they are typically active
Haan in Java (1892, cited in Comstock and only toward plants that serve as hosts for
Lentini, 2005). The disease is prevalent and the pathogens that produce them and dis-
is found in 66 sugarcane-growing countries ease never occurs in the absence of toxin
(Agnihotri, 1990). Generally, the disease production (Wolpert et al., 2002). The HST
has a minor economic impact on sugarcane is a mixture of three isomers (A, B and C)
yield in most areas because of the use of (Lesney et al., 1982; Livingston and Schef-
resistant varieties (Comstock and Lentini, fer, 1984) and the HST produced by the sug-
2005). In India, in 1976, the disease was in arcane red rot pathogen is responsible for
epidemic form and affected 1600 ha sugar- the disease symptoms (Steiner and Byther,
cane crop in Mandya district of Karnataka 1971). The fungus causes eye-shaped lesions
only (Kumaraswami and Urs, 1978). The dis- on the leaves, followed by the development
ease can reduce sugarcane yield by 15–20% of reddish brown streaks or ‘runners’
(Sharma et al., 2004). extending from the lesions toward the tip of
224 A.M.H. Esh
the leaf. The toxic compound from the fun- a ladder-like appearance. These lesions some-
gus causes the runner (Steiner and Byther, times break through the surface of the rind,
1971). causing curvature and distortion of the stalk.
Eye spot spores, which are produced Exaggerated versions of these depressions
abundantly on leaf lesions, are dispersed by may look like neatly made ‘knife-cuts’ in
wind and rain. High humidity and dew for- the stalk. In the most advanced stage of pok-
mation are the favoured conditions for kah boeng, the entire top (growing point) of
spore germination. The disease is not trans- the plant dies (referred to as ‘top rot’). The
mitted by seed pieces and mechanical trans- ladder-like lesions are due to rupturing of
mission by equipment and by humans is the diseased cells that cannot keep up with
unimportant. the growth of the healthy tissue (Martin
et al., 1989; Raid, 2009a).
Disease control
Causal agents
The only practical and efficient method of
control of eye spot disease is with resistant The disease is caused by the fungi, Fusarium
clones. Chemical control using foliar fungi- moniliforme (G. fujikuroi) and F. moniliforme
cides is not practical (Comstock and Lentini, var. subglutinans (G. subglutinans). The per-
2005). ithecia of G. fujikuroi occur only on dead plant
material, while the perithecia of G. subglutin-
ans are rarely formed in nature; thus, the per-
ithecia of these two pathogens are rarely
Pokkah Boeng Disease associated with infected sugarcane plants
(Martin et al., 1989). The pathogens have a
Pokkah boeng, which is a potentially wide host range, i.e. rice, corn, sorghum and
destructive disease of sugarcane, is caused many other grasses. These fungi also cause
by Gibberella moniliformis (Sheldon) Wine- other diseases, such as seedling blight, scorch,
land. There have been many reported out- stalk rot, root rot and stunting in different
breaks of the disease which have been severe, crops (Martin et al., 1989; Raid, 2009a).
like the Java outbreak in 1896, but they have
caused little economic loss (Martin et al.,
1989).
Pathogenesis
The pathogens of pokkah boeng disease are

Disease symptoms transmitted by the movement of spores from
one locality to another by air currents (Martin
In the early stages of infection, the symp- et al., 1961; Raid, 2009a,b). Pokkah boeng
toms of the disease are chlorotic areas at the disease of sugarcane may also spread from
base of young leaves, distortion (wrinkling seeds contaminated with the fungus (Martin
and twisting) and shortening of the infected et al., 1961). It appears to be favoured by
leaves and finally stalk death in severe cases. dry climatic conditions being followed by a
The infected leaves can be distinguished by wet season. Cane that is 3–7 months old and
their narrow base. Irregular reddish stripes growing vigorously appears to be most sus-
and specks develop in the chlorotic parts ceptible (Martin et al., 1989).
which appear in mature leaves. The pathogen spores enter the spindle
The infection is present in the stalk and along the margin of a partially unfolded
dark reddish streaks may be found extend- leaf, then germinate and grow into the inner
ing through several internodes. Also, in tissue of the spindle leaves. The conidia ger-
the internodes, the infection may form long minate and the mycelium can pass through
lesions with cross-depressions that give them the soft cuticle of young leaves to the inner
tissues because the epidermal tissues are acetate content in the infected tissue may
still fragile and not protected by the plant rise up to 1%, which is sufficient to inhibit
system (Dillewijn, 1950). The mycelium the germination of buds (Kuo et al., 1969).
spreads to vascular bundles of the immature As a result, gappy stands are evident and
stem and blocks the vessels, which eventu- young crops have a patchy and uneven
ally leads to growth distortions and rupture, appearance. In the early stages of rotting, the
and this development shows the ladder-like disease may be diagnosed by a strong odour
lesions (Holliday, 1980; Martin et al., 1989). of overripe pineapple. Although pineapple
disease is not considered important in stand-
ing cane, infection may occur if the stalks
Disease control are physically damaged or stressed (Wismer
and Bailey, 1989).
The only efficient control for pokkah boeng
disease is the use of resistant varieties. Sug-
arcane resistance to pokkah boeng has been Causal agents
shown to be highly heritable (Martin et al.,
1989). Pineapple disease is caused C. paradoxa.
The fungus belongs to Phylum: Ascomycota;
Class: Ascomycetes. The fungus produces
Pineapple Disease of Sugarcane two types of imperfect spores, conidiospores
6–24 um × 2–5.5 µm (thin-walled cylindrical
conidia) and chlamydospores 10–25 um ×
Pineapple disease is an economically impor-
7.5–20 µm (thick-walled and oval). These
tant sugarcane disease that is widely dis-
spores are produced intensively on the
tributed in almost all the regions where
internal tissues of the infected seed pieces.
sugarcane is grown (Wismer and Bailey,
They are released into the soil on seed piece
1989). In India, the disease has been noticed
decay. The spores may survive for several
on different sugarcane varieties (Singh et al.,
years in the soil, serving as a source of inoc-
1990). The disease is caused by the fungus
ulum for the next crop (Wismer and Bailey,
Ceratocystis paradoxa, which induces seed-
1989). The perfect stage of the fungus has
piece decay following planting. The affected
been reported and it occurs naturally on
setts emit a smell resembling that of the
cacao (Dade, 1928) and sugarcane (Kuo
mature pineapple fruit (Went, 1896).
et al., 1969). The pathogen also causes dis-
eases of pineapple, banana, cacao, coconut
and oil palm (Wismer and Bailey, 1989).
Disease symptoms
The disease affects sugarcane setts in the first Disease control

weeks after planting. The fungus spreads
rapidly through the parenchyma and colo- Control is a priority, especially where the
nizes all the internal tissue of the seed piece, soil inoculum level is high through the ame-
which turns red and eventually black. The lioration of conditions that favour the ger-
black coloration results from the production mination of buds and the emergence of
of fungal spores within the seed piece. Nodes young shoots (e.g. good quality cuttings,
act as partial barriers to the spread of rotting, adequate irrigation, right planting time and
but with susceptible varieties, entire seed depth). The ends of cuttings are dipped in a
pieces may become colonized by the fungus fungicidal solution, either as a cold dip or
(Wismer and Bailey, 1989). The pineapple the fungicide may be incorporated into the
odour resulting in the decayed seed pieces water tank at the time of the hot water treat-
is due to ethyl acetate, formed by the meta- ment (Antoine, 1956). Other fungicides
bolic activity of the pathogen. The ethyl used to control pineapple disease include
226 A.M.H. Esh
benomyl, propiconazole carbendazim, etc. of seed pieces containing at least three

(Autrey, 1974). Systemic fungicides are found nodes increases the likelihood that buds
more efficient than non-systemic fungicides closer to the centre will germinate (Wismer
(Vijaya et al., 2007). and Bailey, 1989).
Since pineapple disease is a soilborne For disease control, sett treatment with
disease, crop rotation or a fallow period chemical fungicides before planting is an
between cane crops may prove to be of some effective method and is widely followed.
benefit in reducing its impact. Seed-piece Systemic fungicides, benomyl and car-
infection by pineapple disease frequently bendazim, were found more efficient than
proceeds from the exposed cut ends to the non-systemic fungicides such as captan and
centre of the seed piece. Therefore, the use mancozeb (Vijaya et al., 2007).
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18 New and Emerging Fungal Pathogens
Associated with Leaf Blight Symptoms on
Wheat (Triticum aestivum) in Argentina

CIDEFI (Centro de Investigaciones de Fitopatología) – CONICET
(Consejo Nacional de Investigaciones Científicas y Técnicas),
Facultad de Ciencias Agrarias y Forestales de la Universidad
Nacional de La Plata, La Plata, Provincia de Buenos Aires, Argentina
Abstract
Regional surveys are being conducted at the CIDEFI to investigate the presence of wheat (Triticum
aestivum L.) pathogens on leaves and seeds across the Argentinian cropping area. During the past
5 years in the wheat cropping area of Buenos Aires Province, Entre Ríos and Santa Fe Provinces,
Argentina, several unusual diseases have been found on wheat leaves. From the symptomatic tissues,
the fungi were isolated and identified. To test pathogenicity and fulfil Koch’s postulates, inoculations
of different wheat cultivars under greenhouse conditions were carried out; disease symptoms and the
causal agents are described.
Introduction Ríos and Santa Fé). Most symptoms were

observed on the upper leaves at growth
Wheat ranks as a primary source of food and stage 69 (anthesis complete) and at growth
livelihood for hundreds of millions of people stages 80 (early dough) to 85 (soft dough)
globally, especially in developing countries. according to the scale of Zadok et al. (1974).
Several serious foliar diseases caused by Samples of 20–40 plants of each disease
necrothrophic pathogens occur in this crop area were used for late laboratory identifica-
in Argentina. Among them, Septoria tritici tion of leaf-spotting fungi.
leaf blotch and tan spot caused by Drechslera The frequency of the pathogens was
tritici-repentis are the most important. Both different among localities and cultivars over
can cause serious yield and quality losses the past 10 years. Pyrenophora tritici-repentis
under the right conditions. Among biotrophic was predominant, followed by Mycosphaer-
diseases, leaf rust is a very dangerous one. ella graminicola. In a total of 193 and 240
Samples were obtained from different infected leaf samples collected in 2006–
wheat cultivars from 2001 in 13 different 2007, D. tritici-repentis was observed in 63
locations of the main wheat-growing area of and 77% of leaf samples, respectively, sug-
Argentina (northern, central and eastern gesting an increasing trend in incidence
regions of the Buenos Aires Province, Entre over the years. This may be explained in

232 A.E. Perelló
part due to the adoption of reduced-tillage and Rao, 1979). Some of these metabolites
cropping practices. Surprisingly, a signifi- are powerful mycotoxins not yet character-
cant increase in the frequency of Alternaria ized in Argentina.
spp. isolates was observed, with 41 and 53% During routine investigations across
of the samples harbouring tan spot in the the wheat (T. aestivum)-growing area of the
same period. The isolates were identified as Buenos Aires Province, diseased leaf sam-
belonging mostly to the A. infectoria species ples were collected from different wheat
group. Moreover, mapping of pathogen dis- cultivars. Discoloured oval lesions appear on
tribution during the past years in different lower leaves. The disease progresses upwards,
agroclimatic zones of Buenos Aires Prov- lesions enlarge and coalesce to irregular, dark
ince shows that A. infectoria is a widespread blotches, often with chlorotic margins.
pathogen that is gaining prominence as an Severely infected seeds are discoloured and
emerging wheat pathogen in latter years. shrivelled.
Also, it was found, together with A. alter- Necrotic tissue fragments were surface-
nata, on almost 70% of wheat seed samples. sterilized and plated on potato dextrose agar
Other diseases were sporadic, isolated (PDA), from where Alternaria specimens
or minor and included foliar blight or spots were isolated. Morphobiometrical and cul-
caused by the fungus A. triticina, A. infecto- tural features of the fungus were examined
ria species group complex, Bipolaris soroki- on potato carrot agar (PCA). Conidia were
niana, Cladosporium herbarum, Phoma irregularly oval, ellipsoid conical, gradually
sorguina, Ascochyta hordei, Pyricularia gri- tapering into a beak, 15–92 × 8–35 µm, with
sea, Cephalosporium gramineum and others. 1–10 transverse septa and 0–5 longitudinal
Some disease symptoms are characteristic septa, light brown to dark olive buff, becom-
and obvious. Other diseases, however, may ing darker with age. All isolates obtained
be difficult to diagnose without microscopic were identified as A. triticina following the
or laboratory analysis. Since different dis- morphological descriptions by Anahosur
eases require different control strategies, (1978) and confirmed by comparison with
their accurate diagnosis is essential. reference strains of CABI Bioscience (IMI
289962 and IMI 178784) kindly sent by
Dr D. Mercado (Université Catholique de
Louvain, Unité de Phytopathologie, Bel-
Leaf Blight of Wheat Caused by gique). One of the isolates has been lodged in
Alternaria triticina in Argentina the culture collection of La Plata Spegazzini
(LPSC) (accession number 798). Pathogenic-
Alternaria species are perhaps the most ity tests were conducted in the greenhouse.
common fungi encountered by mycologists Susceptible wheat cultivars were inoculated
working in plant pathology. As plant patho- at tillering and heading stages with a conid-
gens, over 4000 Alternaria/host associations ial suspension (2 × 105 conidia/ml). After
are recorded in the USDA Fungal Host Index 10 days, typical leaf blight symptoms devel-
and the genus ranks 10th among nearly 2000 oped and A. triticina was recovered from
fungal genera listed based on the total num- the lesions. No symptoms appeared on the
ber of host records. In Argentina, this genus control plants.
has been studied on wheat plants since the Alternaria triticina causes significant
last decade (Perelló, 2007; Perelló et al., 1992, yield losses in wheat on the Indian subcon-
1996, 2002, 2003, 2005a,b, 2008; Perelló and tinent, from where it originates and has
Sisterna, 2005, 2006). Several Alternaria spe- spread throughout the world (Agarwal et al.,
cies and numerous uncharacterized Alter- 1993). Although A. triticina has been detected
naria taxa have been found associated with previously in Argentina on wheat leaves
leaf blight symptoms. Alternaria species are and seeds (Perelló et al., 1992), it has prob-
some of the most prodigious producers of ably existed as a minor pathogen for many
toxic secondary metabolites, producing over years without being noticed. The recent
70 compounds of varying toxicity (Kumar increase in the severity of leaf blight may be
New and Emerging Fungal Pathogens 233
due to new cultural practices such as con- in colour. The lesions develop progressively
servation tillage, nitrogen fertilization, irri- from lower to upper leaves and blighting
gation, use of new germplasm, as well as may extend to heads and leaf sheaths. Symp-
favourable weather conditions. As A. triticina toms appear on leaves, seeds and spikelets.
is a quarantine pathogen in many countries, Severely infected seeds are discoloured and
it is important to investigate the incidence shrivelled (Rault et al., 1983). Under humid
and importance of this disease in Argentin- conditions, the lesions support visible clus-
ean wheat areas. This is the first published ters of dark, powdery conidia. Lesions are
record of A. triticina on wheat in Argentina difficult to distinguish from those caused by
and on any host in this country. Helminthosporium spp. Alternaria leaf blight
is likely to develop near irrigation ditches,
in low areas, or wherever humidity and soil
Alternaria leaf blight moisture are high. It develops rapidly once
wheat plants are 6–8 weeks old, and espe-
cially as the crop approaches maturity.
Alternaria triticina Prasada & Prabhu causes
Bread and durum wheat, barley and triticale
significant yield losses in wheat in the
are the primary hosts.
Indian subcontinent, from where it origi-
nated and has spread throughout the world.
The disease was first reported in 1924 but
remained incompletely characterized for Causal organism
several years. Early studies associated Alter-
naria spp. with the disease, but the causal
Isolations from leaf lesions routinely yield
organism, A. triticina, was not identified until
Alternaria spp., many of which are sapro-
1962 (Prasada and Prabhu, 1962). From
phytic and mask the pathogen. Alternaria
1960 to 1964, leaf blight damaged all com-
triticina is distinguished by its wheat-specific
mercial cultivars on the Indian subconti-
virulence and cultural characters. The
nent (Prabhu and Prakash, 1973; Bhowmik,
mycelium and conidia of A. triticina are ini-
1974; Sokhi, 1974). It developed on plants
tially hyaline and later olive buff. Conidio-
approaching maturity, caused premature
phores are septate, usually unbranched but
death of the uppermost leaves and heads and
occasionally branched, erect, single or fas-
reduced yield significantly. Today, durum
ciculate, emerging through stomata, genicu-
wheats, their derivatives and introduced
late, straight, length variable, between septa
Mexican wheats are considered most suscep-
17–28 µm, 3–6 µm wide. A chromogenic
tible (Frisullo, 1982). In addition to wheat,
variant in A. triticina was studied (Jain and
the disease affects triticale in India and other
Prabhu, 1976). Conidia of A. triticina are
graminaceous hosts in the Middle East and
acrogenous, borne singly or in short chains
Nigeria (Chaudhuri et al., 1976).
(two to four spores). They vary from 8 to
35 µm in width and 15 to 92 µm in length, are
dark, ellipsoid to conical, tapering to a beak.
Symptoms A. triticina grows on a variety of simple
media (Rao and Subrahmanyam, 1974). Col-
The pathogen may infect all foliar parts. onies on PDA are discrete or effuse, dark
Alternaria leaf blight is characterized by blackish brown to black, margin smooth
small, chlorotic, oval- or elliptical-shaped and entire. Growth is optimal between 20°
lesions scattered on lower leaves. As the plant and 24°C, with limits near 5° and 35°C.
matures, the disease progresses upwards and Physiological specialization of the pathogen
lesions darken to brown-grey, enlarge and exists and six races have been characterized
coalesce to irregular, dark blotches, irregu- and reported using 15 differentials. Non-
lar in shape and may have a yellow margin. specific phytotoxins produced by the
The chlorotic borders of the lesions may pathogen apparently play a role in wheat
become diffuse and turn light to dark brown pathogenesis.
234 A.E. Perelló
Alternaria infectoria Complex the cultivars Buck Arriero, Buck Charrúa,

Associated with Black Point and Buck Granar, Buck Poncho, Buck Yatasto,
Leaf Blight Symptoms in Argentina Klein Cacique, K. Estrella, ProINTA Cinco
Cerros, ProINTA Elite and Pro INTA. Sig-
nificant differences between cultivars, iso-
Among the field fungi found in cereals, Alter-
lates and the interaction isolate × cultivar
naria is the dominant genus and, within this
were shown according the ANOVA results.
habitat, taxa of the A. infectoria species group
Analysis of severity means (Tukey’s test)
predominate by far. Information available
showed cultivar Buck Charrúa as the one
on the A. infectoria species group is limited
with the best behaviour against all the iso-
as the taxa it comprises have often been mis-
lates tested, and cvs. Pro INTA Cinco Cerros
identified as other small-spored Alternaria
and Pro INTA Elite as the most susceptible.
species, due to the use of insufficient meth-
Symptoms observed were: chlorosis and/or
ods for identification. Members of the A.
apical or general necrosis (blight), or elon-
infectoria species group are morphologically
gated necrotic spots surrounded by a chlo-
distinguishable from other small-spored
rotic halo. Additionally, 20 samples of
species of Alternaria by their long secondary
wheat seeds from different localities in the
conidiophores and formation of white or
Buenos Aires Province were analysed by
grey colonies on dichloran rose bengal yeast
blotter test (ISTA) (Neergaard, 1979). After
extract sucrose media (DRYES) (Andersen
7 days incubation (20 ± 2°C and cycles of
et al., 2002).
12 h light plus NUV light), the microorgan-
Furthermore, this species group is the
isms developed were identified and the A.
only one among Alternaria where the teleo-
infectoria complex in particular was charac-
morph, Lewia, has been identified in Argen-
terized according to its morphobiometrical
tina. To date, the A. infectoria species group
features on PCA. A prevalence (samples
comprises the known species A. arbusti, A.
infected/samples analysed) of 55% and
conjuncta, A. infectoria, A. oregonensi, A. trit-
infection values of 37% of A. infectoria spe-
icimaculans, A. metachromatica, A. viburni,
cies group members was registered.
A. intercepta and A. novae-zelandiae, as well
Twenty isolates were tested in a com-
as an unknown number of distinct taxa yet
parative analysis of five isoenzyme pat-
to be described. Members of the A. infecto-
terns (phosphatase, peroxidase, A-esterase,
ria species group produce a range of unique
B-esterase, glutaminotransaminase) and total
secondary metabolites that are useful for
proteins. Mycelium for electrophoresis in
metabolic profiling and chemotaxonomy of
polyacrylamide gel was obtained from mono-
Alternaria. Until now, four different profiles
sporical cultures of the fungus on PCA over
have been identified, suggesting that there is a
10 days. The results obtained revealed dif-
potential risk of Alternaria mycotoxins in
ferences between the strains in the isozyme
wheat in Argentina (Pich et al., 2007).
banding patterns. Each isolate had a charac-
Black point and leaf blight caused by A.
teristic electromorph, showing different
infectoria species group complex is a new
main bands of enzyme activity and some
disease of wheat in Argentina. Four hun-
minor bands varying in intensity for all the
dred and ten isolates collected from 17 dif-
patterns assayed. Isozyme data corroborated
ferent geographical zones of the Argentinian
the morphological and pathogenic variabil-
cropping area were tested for their morpho-
ity observed previously on A. infectoria iso-
logical variation. The isolates differed in
lates collected from wheat. These results
their morphobiometrical and cultural char-
support the usage of isoenzymatic patterns
acteristics on PCA. On this basis, they were
for the characterization of isolates of A. infec-
categorized into four morphotypes. Further,
toria complex associated with black point
20 isolates were characterized according to
and leaf blight symptoms on wheat, as a
their pathogenic and biochemical variabil-
valuable additional tool to aid the tradi-
ity. Pathogenicity tests were conducted under
tional taxonomy, base on morphocultural
greenhouse conditions on wheat plants of
characters only.
Detection of Lewia infectoria 180 belonged to the Alternaria genus. Single

and its Alternaria Anamorph spore cultures were obtained on PCA. Based
from Wheat in Argentina on morphological characters like the conid-
ial sporulation pattern and the prominence
of their secondary conidiophore structure,
Occurrence of L. infectoria (Fuckel) Barr &
most of the strains were identified as mem-
Simmons (teleomorph of A. infectoria) devel-
bers of the A. infectoria species group. The
oped in culture is described, illustrated and
cultures were stored on slants of PCA at 4°C
reported for the first time. Monosporic iso-
in darkness. Numerous conidia appeared on
lates, obtained from infected wheat plants,
the surface of the agar within a week and
produced conidia within a week and asco-
groups of fruiting bodies within 7 months.
mata with fully mature ascospores within
To determine their stage of development,
7 months when stored on slants of PCA at 4
the fungal fruiting bodies were placed on a
degrees in darkness. The anamorph exhib-
microscope slide, stained with 0.25% Try-
ited the sporulation pattern of A. infectoria
pan blue in lactid acid:glycerol:water (1:1:1)
species group and was identified on the
and examined with a light microscope
basis of axenic colony morphology and by
(× 400). After 7 months, groups of fertile
the prominence of their secondary conidio-
ascomata (pseudothecia) with septate hya-
phore structure. Critical examination of the
line mature ascospores developed in isolates
teleomorph proved it to be L. infectoria. The
obtained from the cultivar Klein Estrella,
importance in interpreting the teleomorph–
from a particular field in the locality of Bal-
anamorph pair is discussed.
carce (Buenos Aires Province). There were
A. infectoria species group, causing leaf
detectable differences between isolates with
blight and black point of wheat, was not sig-
regard to pseudothecial density and speed of
nificant for many years, but currently this
ascospore maturity. In some cases, the pro-
constraint has become a new problem (Per-
duction of immature asci was observed.
elló and Sisterna, 2006). During 2005, wheat
The morphobiometrical and cultural
samples were collected and typical symp-
features of these ascomata on PCA allowed
toms of tan–dark brown leaf spot were
the identification of the teleomorph of A.
observed on several cultivars from the crop-
infectoria, L. infectoria (Fuckel) Barr & Sim-
ping area of Buenos Aires Province. Different
mons. Its description is as follows: ascomata
isolates of a fungus with characteristics of
ellipsoid, 400–500 × 150 µm, with a short,
Alternaria were obtained from this material.
papillate beak, dark, thin-walled at maturity.
In laboratory conditions on PCA, cultures of
Asci 105–125 × 13–16 µm, subcylindrical,
this fungus produced conidia within a week.
straight or somewhat curved. Ascospores 8,
Then, these isolates were stored to maintain
18–22 × 7–8 µm at full development, broadly
a fungal collection. Within 7 months, asco-
elliptic, muriform, becoming 5-septate (3 pri-
mata with fully mature ascospores of a previ-
mary septa), only end cells not longitudi-
ous undescribed genus were observed in
nally septated, constricted, yellow-brown.
connection with this anamorphic state.
Although most Alternaria species do
Based on morphological characters (Sim-
not have teleomorphic affinities, a number
mons, 2002), the teleomorph proved to be a
of anamorphically defined taxa within the
Lewia species, described here as L. infectoria
Pleosporaceae have recognized teleomorphs
with its Alternaria anamorph.
and most are not commonly encountered
Wheat leaves exhibiting necrotic symp-
(Simmons, 1986, 2002). These teleomorphs
toms were collected during September and
are representative of nearly all major lineages
October in 2005 from different cultivars of
within the Pleosporaceae. An evaluation of
farmers’ fields and research stations of eight
the teleomorphic characters of well-known
localities of Buenos Aires. Lesions on leaves
Pleospora spp. with anamorphs of Alternaria,
were viewed through a stereoscope at × 12
namely P. infectoria and P. scrophularieae,
and specific morphological characteristics
revealed that Pleospora spp. with Stem-
of pathogens were recorded. Fungi were cul-
phylium anamorphs were morphologically
tured on PDA. After screening of the cultures,
236 A.E. Perelló
distinct from Pleospora spp. with Alternaria is reported on wheat as a pathogen of minor
anamorphs, particularly in the size of the economic importance but there are some
ascomata and ascospores. This resulted in reports pointing out that high humidity con-
the designation of the genus Lewia for ditions could favour the occurrence of out-
Pleospora-like fungi with Alternaria teleo- breaks of the disease (Scharen and Krupinsky,
morphs (Simmons, 1986). Evidence of pro- 1971).
duction of Alternaria-related teleomorphs During September–October 2002, leaf
in axenic culture was previously reported spot symptoms on wheat cultivar Baguette
by Bilgrami (1974) in L. infectoria, Simmons 10, growing in farmers’ fields in Tandil,
(1986) in L. photistica, Kwasna and Kosiak eastern area of Buenos Aires Province, were
(2003) in L. avenicola and Kwasna et al. commonly observed. The leaves showed
(2006) in L. hordeicola. Other described symptoms similar to those described for
Lewia species, like L. chlamidosporiformans, other necrotrophic foliar pathogens (D. tritici-
L. ethzedia, L. intercepta, L. sauropi, L. viburni repentis) and Stagonospora nodorum, sug-
and L. eureka, usually produce ascomata on gesting that any of these might have been
tissues of infected plants (Simmons, 1986, involved. Ascochyta was commonly isolated
2002; Vieira and Barreto, 2005). During the from affected tissues of the samples col-
present study, ascomata of L. infectoria were lected. A. tritici Hori & Enj. is generally
produced in vitro, in axenic culture on PCA accepted as the cause of Ascochyta leaf spot,
slants, in connection with the anamorph. but A. graminicola Sacc. is cited in some
However, the fungus does not often literature (Zillinsky, 1984). Punithalingam
produce both anamorph and teleomorph on stated the status of A. tritici was uncertain
the same slant. Crossing between isolates is but it might be a synonym of A. hordei (Farr
evidently not necessary for production of the et al., 1989). Sprague and Johnson (1950)
teleomorph since single-ascospore axenic also stated that A. tritici was close to A. hor-
cultures continued to produce ascomata on dei Hara, differing mainly in the symptoms
PCA. These results were similar to observa- on barley. The identity of the culture of
tions made by Kwasna and Kosiak (2003) for Ascochyta isolate A1102 of this study was
L. avenicola. In addition, its finding in Argen- determined as A. hordei Hara var. europaea
tina has provided an important framework by experts of the Centraalbureau voor
for hypothesis testing in advanced studies Schimmelcultures (CBS), The Netherlands,
on Alternaria/Lewia epidemiology and patho- and deposited in the CBS culture collection
genicity variability on wheat plants. under the number 112525.
Lewia infectoria forms pseudothecia on Ascochyta was not previously reported
wheat straw in the field under determined on wheat and other grasses in Argentina. In
weather conditions. This could play an this sense, the first occurrence of this fun-
important role as a source of inoculum in gus as a member of the leaf spotting com-
Alternaria/Lewia disease able to infect wheat plex on wheat plants in the Argentinian
and wild grasses as a result of the dispersal cropping area is significant. Diseased leaves
of airborne ascospores. The discovery of the were collected, stored in paper bags and
sexual stage in nature may have a large transported to the laboratory. The pathogen
influence on localized development of the was isolated from typical necrotic symp-
diseases in different regions of the country. toms. Morphobiometrical and cultural stud-
ies of the fungus were studied.
Inoculation experiments to confirm
pathogenicity were performed in the green-
Occurrence of Ascochyta hordei house at 15–25°C and 80% relative humid-
Hara var. europaea Punith. ity on 16 wheat cultivars: Buck Arriero,
on Wheat Leaves in Argentina Buck Yatasto, Buck Poncho, Buck Charrúa,
Buck Halcón, ProInta Granar, ProInta Cinco
Ascochyta leaf spot is often overlooked in Cerros, Desimoni Caudillo, ProInta Impe-
association with other leaf spot diseases. It rial, ProInta Puntal, ProInta Guazú, ProInta
Colibrí, ProInta Elite, Klein Estrella, Klein had low disease severity. Buck Arriero, Buck
Cacique and Klein Dragón. Plants were Charrúa, Buck Halcón, Buck Poncho, Buck
grown in plastic pots 12 cm in diameter (4 Yatasto and Klein Dragón showed the most
seeds/plot in all samples) with a standard severe symptoms of the disease (between
potting mix. Plants were inoculated when 12–45% of the necrotic leaf area) 20 days
they had reached the third expanded leaf after inoculation. The rest of the cultivars
stage. Inoculum was prepared from 10-day- showed little evidence of infection with
old cultures of A. hordei var. europaea (iso- only a 10% necrotic foliar area, except Pro-
late A1102) growing on PDA and was Inta Imperial and ProInta Puntal, which
obtained by flooding each sporulating plate showed no evidence of infection, indicating
with sterile distilled water and gently scrap- that the disease pressure on Buck cultivars
ing the fungal colony with a flame-sterilized was much higher than on the rest of the wheat
scalpel to dislodge conidia. The conidial sus- cultivars examined. These observations are
pension was filtered once through a single consistent with other reports (Wiese, 1977),
layer of cheesecloth and spore concentra- which indicates that the fungus is appar-
tion was determined with a haemocyto- ently of little economic consequence as a
meter. The inoculum consisted of 12 × foliar pathogen of wheat. Inoculation stud-
106 conidia/ml. Twenty seedlings of each ies proved that A. hordei var. europaea was
cultivar were used for the inoculation. the cause of this outbreak on wheat in Argen-
Leaves were sprayed to runoff with a manu- tina. New cultural practices (reduced tillage,
ally operated sprayer. The inoculated plants nitrogen fertilization, irrigation), the use of
and controls were kept in a moist chamber new germplasm and favourable environmen-
for 48 h. The first symptoms appeared 92 h tal conditions could have contributed to cre-
after inoculation. Between 40 and 60% were ating ideal conditions for the increase and
necrotic 18 days after inoculation. In natu- spread of inoculum, not only of A. hordei
ral field infections, it was observed that var. europaea but of the foliar complex of
plants were affected rather severely, espe- necrotrophic pathogens in general. Other
cially the basal leaves. Lesions at first are wheat cultivars may also be susceptible to
distinct, chlorotic, ellipsoidal or round and isolates of the pathogen.
1–5 mm across. Later, they become diffused
and grey-brown internally. Pycnidia some-
times form and appear as black dots within
necrotic lesions. They are submerged in host Phoma sorghina (Sacc.) Boerema,
tissues, except for a papillate projection. Dorenbosch & van Kesteren
In culture on PDA, pycnidia measured in Wheat Leaves in Argentina
142.5–225 × 93.75–206.25 µm. Conidia
(pycnidiospores) are straight, hyaline and Phoma sorghina (Sacc.) Boerema, Dorenbo-
oblong, 3.75–5.60 × 15–18.70 µm, typically sch & van Kesteren is plurivorous, ubiquitous
with one median septum. and common in the tropics and subtropics,
All wheat plants inoculated with A. hor- causing diseases of cereals and other Grami-
dei var. europaea in the greenhouse devel- neae and forage crops (Punithalingham, 1985;
oped symptoms identical to those observed Manavolta and Bedendo, 1999; Kumar and
on naturally infected plants in the field. The Kumar, 2000). Although P. sorghina has been
amount of damage to seedlings was mea- found causing leaf spots in different hosts
sured as per cent necrotic leaf area from the such as Agave americana, Gossypium hir-
first leaf of 15 plants per each inoculated sutum, Lycium halimifolium, Lycopersicum
cultivar in comparison with controls. Culti- esculentum, Oryza sativa, Populus nigra,
vars Buck Arriero and Buck Poncho showed Sorghum spp. and Zea mays, the disease it
the most conspicuous symptoms 9 days after causes to aerial parts of plants is of minor
inoculation, with a disease severity rating of importance (White and Morgan-Jones, 1983).
between 8–35% of necrotic leaf area. The It causes considerable loss of seedlings of
rest of the cultivars showed no symptoms or Macroptilum, Stylosanthes and Sorghum
238 A.E. Perelló
through pre- and post-emergence death. The leaf surface spotted. The cvs. ProInta Cinco
fungus has been found on or associated with Cerros, ProInta Elite, ProInta Granar and
sorghum grains in the humid Argentinian ProInta Imperial were slightly spotted, with
Pampa (Gonzalez et al., 1997), but there are 1–5% of their foliage covered by spots. The
no previous reports of its presence on wheat cvs. Klein Estrella, ProInta Guazú and Pro-
plants in Argentina and therefore the confir- Inta Puntal were free of spots. Elongated
mation of this fungus as a foliar pathogen of necrotic yellowish to light-brown lesions
wheat in Buenos Aires Province is signifi- could be observed on the upper surfaces of
cant. The first occurrence of leaf spot dis- affected leaves of wheat cvs. Leaf spots later
eases of wheat by P. sorghina was in the coalesced to form large irregular spots with
Buenos Aires Province of Argentina in 2002. yellow margins. Under high humidity, pyc-
The fungus was detected on samples from nidia developed within spots on leaves after
two localities, Olavarría and Los Hornos, on 21 days. A gelatinous spore mass was extru-
experimental field plots with wheat culti- ded in cirri from pycnidia. No symptoms or
vars, Buck Poncho and Buck Diamante. spots were seen on the control plants. All
Diseased leaves were collected, stored wheat plants inoculated with P. sorghina in
in paper bags and transported to the labora- the greenhouse developed symptoms iden-
tory. The pathogen was isolated from typi- tical to those observed on naturally infected
cal necrotic lesions on PDA Petri dishes. plants in the field. In culture, the fungus
Fifteen plants of each of the cvs. Buck developed dark, greyish colonies with dense
Arriero, Buck Poncho, Klein Cacique, Klein aerial mycelium with abundant, solitary or
Estrella, ProInta Cinco Cerros, ProInta Elite, sometimes aggregated pycnidia with char-
ProInta Granar, ProInta Guazú, ProInta Impe- acteristic beaks. Conidia were globose to
rial and ProInta Puntal were grown in plastic ovoid or shortly cylindrical, usually straight,
pots 12 cm in diameter and containing a hyaline, unicellular 4–7 × 2 µm. Abundant
potting mix of clay 21.2%, lime 56%, sand chlamydospores and dictyochlamydospores
22.8%, soil organic matter (SOM) % = 3.35; were observed. Newly formed chlamydo-
C % = 1. Plants were inoculated at the third spores quickly became covered with a black
expanded leaf stage and heading stage. Inoc- coating that obscured their brown colour.
ulum was prepared from 10-day-old cultures The identification was confirmed by
of P. sorghina growing on PDA by flooding the Centraalbureau voor Schimmelcultures
each sporulating plate with sterile distilled (CBS), Utrecht, The Netherlands. One repre-
water and gently scraping the fungal colony sentative isolate of P. sorghina has been
with a sterile scalpel to dislodge conidia. lodged in the CBS culture collection with
The resulting suspension was filtered once the accession number 112525.
through a single layer of cheesecloth and the
spore concentration was determined with a
haemocytometer. The spore concentration in
inoculum was adjusted to 1 × 106 conidia/ml. Cephalosporium gramineum
Control plants were sprayed with sterile dis- Nisikado & Ikata on
tilled water. Leaves were sprayed to runoff Wheat Leaves in Argentina
with a manually operated sprayer. The inoc-
ulated plants and controls were kept in a Cephalosporium stripe is a disease of cere-
moist chamber for 48 h and observed daily. als that is sporadic in its distribution and
The first symptoms appeared 72 h after occurrence but can cause severe yield losses
inoculation under greenhouse conditions when it occurs. The disease is found most
and 15–50% of plants showed necrotic consistently in areas where frost heaving,
lesions 10 days after inoculation. The wheat resulting from fluctuating winter tempera-
cvs. showed different degrees of suscepti- tures, heavier soils and higher soil moisture
bility to the pathogen. The cvs. Buck Arri- damages roots (Bruehl et al., 1976). Cepha-
ero, Buck Poncho and Klein Cacique became losporium stripe is caused by Hymenula cere-
severely infected with up to 40% of their alis (synonym C. gramineum). This fungus
is slow growing in culture and probably in 2% PDA Petri dishes. Wheat plants of the
nature, too. It produces tiny conidia on same cultivar were inoculated with a patho-
sporodochia in the saprophytic stage on gen conidial suspension by a manual sprayer
wheat straw, but as a parasite it invades the under greenhouse conditions. Similar symp-
vascular system, where it interferes with toms developed from 7 to 21 days after the
water movement. It is the only true vascular inoculation.
parasite known to attack wheat.
Occurrence of Cladosporium
Hosts
herbarum on Wheat Leaves
in Argentina
C. gramineum attacks most winter cereals,
but especially wheat. It invades several
Cladosporium on wheat was reported to be
grasses (Bromus, Dactylis, Poa) and proba-
a common and mild parasite affecting dead
bly was indigenous to the region in native
or half-dead plant tissues in association
grasses. Until now in Argentina, it has been
with some other fungi. It often appears on
detected on Bromus and wheat plants only.
the ear heads, causing a greenish black
mouldy growth on the affected parts (Wiese,
1987). It was not found to cause severe symp-
Disease symptoms toms on leaves and stems of wheat, but there
were some reports pointing out that moist
Cephalosporium stripe is first observed in and shady conditions could favour the
the spring as distinct yellow stripes on leaf occurrence of outbreaks of the disease on
blades, sheaths and stems. The stripes may leaves (Arya and Panwar, 1955).
contain thin brown streaks (necrotic vascu- During the past 5 years, leaf spot symp-
lar tissues) surrounded by yellow. Fre- toms on wheat cultivars Buck Pingo, Buck
quently, a yellow stripe on the leaf blade Biguá, Buck Brasil and Buck Poncho grow-
continues as a single brown line down the ing in the north-east of the Buenos Aires Prov-
leaf sheath. ince, were commonly observed. Infected leaf
Nodes are darker than normal on dis- samples were collected during September–
eased plants and, when cut lengthwise, the November in an extensive survey conducted
inner nodal tissue is brown in colour. Plants in 2002. Samples were collected from differ-
are stunted and the heads are white and ent cultivars in farmers’ fields and one
sterile. If any seed is set, it is usually shriv- experimental research station across the
elled. Diseased plants have a scorched wheat region of Buenos Aires and Entre
appearance when hot weather accentuates Ríos Provinces, in five of the eight sites sur-
moisture stress. The fungus survives for as veyed (Los Hornos, Nogoyá, Olavarría,
long as 4–5 years in undecomposed infested Tandil and Victoria). In most of the plants,
straw. leaves showed symptoms sufficiently simi-
In 2004, on a non-tilled wheat assay lar to those described for the complex of
sown at the Julio Hirschhorn Experimental necrotrophic foliar pathogens, i.e. D. tritici-
Station, belonging to the Facultad de Cien- repentis (Died.) Shoem., S. tritici Rob. in
cias Agrarias y forestales de la Universidad Desm., A. triticimaculans Simmons & Per-
Nacional de La Plata, Argentina, chlorotic elló and B. sorokiniana (Sacc.) Shoem., sug-
stripes, which became necrotic, were obser- gesting that any of these might have been
ved on leaves of wheat cultivar Buck Biguá. involved. C. herbarum was commonly iso-
Samples were collected and remitted to the lated from affected tissues of the samples
laboratory at the CIDEFI. Morphocultural collected and it has emerged as a wide-
and morphobiometrical characteristics allo- spread and serious foliar disease. The fun-
wed identification of the fungus as C. gus was previously reported in Argentina
graminearum. The fungus was cultured on (Marchionatto, 1948) as C. herbarum Link.
240 A.E. Perelló
var. cerealinum Sacc. on leaves and spikes basal leaves rather severely. They often con-
of wheat and other grasses without describ- fluenced and elongated, developing pro-
ing the symptoms in detail. Since then, gressively from lower to upper leaves. The
there have been no other reports of this dis- margin of top leaves became brittle when
ease on wheat leaves in Argentina. dried and the tissue tore. When lesions
Diseased leaves were collected, stored spread over the leaf surface, they caused the
in paper bags and transported to the labora- death of the entire leaf. A velvety olivaceous
tory. The pathogen was isolated from typi- grey mould of spores and mycelia devel-
cal necrotic symptoms. On PDA Petri dishes, oped on the surface of the infected tissue,
morphobiometrical and cultural studies of forming dense tufts. Microscopic examina-
the fungus were conducted on single spore tion revealed the presence of conidiophores
colonies grown in Petri dishes containing more or less erect, septate, sparsely branched;
PDA, cultured at 20 ± 2°C under cool-white the spores are often in chains of 2 or 3, sub-
fluorescent light supplemented with near cylindric, pale olive, 1-(2-3) septate, 10–15 ×
UV with a 12 h photoperiod. 4–7 µm. The teleomorph, M. tulasnei (Jancz.)
Inoculation experiments to confirm Rothers was not seen.
pathogenicity were performed in the green- All wheat plants inoculated with C.
house at 15–25°C and 80% relative humid- herbarum in the greenhouse developed
ity. Fifteen plants of each of the cultivars symptoms identical to those observed on
Buck Biguá, Buck Brasil, Buck Pingo and naturally infected plants in the field. No dif-
Buck Poncho were grown in plastic pots ferences in degree of infection were noted
(12 cm diameter) with a standard potting among the cultivars. Nevertheless, adult
mix. Plants were inoculated when they had plants showed more severe symptoms than
reached the third expanded leaf stage and younger ones. No symptoms were observed
heading stage. Inoculum was prepared from in the control non-inoculated plants. Isola-
10-day-old cultures of C. herbarum (isolates tion from symptomatic tissue has consis-
Ch101 and Ch500) growing on PDA and was tently yielded cultures of C. herbarum. The
obtained by flooding each sporulating plate fungus sporulated on the diseased tissue in
with sterile distilled water and gently scrap- the Petri dishes. Comparison of morpholog-
ing the fungal colony with a flame-sterilized ical characteristics of C. herbarum isolates
scalpel to dislodge conidia. The conidial sus- revealed no differences between field- and
pension was filtered once through a single glasshouse-produced spores, according to
layer of cheesecloth and spore concentra- the shape and size of conidia.
tion was determined with a haemocyto- The isolates of C. herbarum have been
meter. The inoculum consisted of 2 × 105 lodged in the culture collection of the CIDEFI
conidia/ml. Control plants were sprayed (Centro de Investigaciones de Fitopatología),
with sterile distilled water only. Leaves Facultad de Ciencias Agrarias y Forestales
were sprayed to runoff with a manually de la Universidad Nacional de La Plata,
operated sprayer. The inoculated plants and Buenos Aires, Argentina, with the accession
controls were kept in a moist chamber for numbers 111-01, 209-02, 210-02, 212-02
48 h. The plants were observed periodi- and 215-02.
cally. The first symptoms appeared between Inoculation studies proved that C. her-
72 and 92 h after inoculation. All cultivars barum was the cause of this outbreak on
showed susceptibility to both of the isolates wheat in Argentina. In the past few years,
tested. Between 12 and 75% were necrotic the increased incidence of the disease may
10 days after inoculation. Reisolation from be related to new cultural practices (reduced
leaves with lesions was performed and the tillage, nitrogen fertilization, irrigation), the
isolates were compared morphologically with use of new germplasm and favourable
those used for inoculation to fulfil Koch’s weather conditions. This contributed to a
postulates. In natural field infections, amphi- major spread, not only of C. herbarum but
genous, irregular yellowish brown spots also of the foliar complex of necrotrophic
were observed that especially affected the pathogens in general.
The fact that other wheat cultivars apart on the northern, central and southern prairies
from those checked may also be susceptible of the Buenos Aires Province, a dramatic dif-
to the pathogen shows the importance of ference was observed between fungal dis-
conducting thorough research to determine eases. A high incidence of D. tritici-repentis
the reactions of those cultivars currently was commonly observed in all locations
used in the Argentinian cropping area. analysed.
Tan spot, caused by the fungus P. tritici-
repentis (Died.) Drechs. (anamorph D. tritici-
Pyricularia grisea on repentis) (Died.) Shoem., is a major disease
Wheat Leaves in Argentina of wheat (T. aestivum L.) worldwide (Wiese,
1977; Hosford, 1975). The disease has a fast
growth in the Southern Cone region of South
During 2006/2007, P. grisea (Cooke) Sacc.
America including Argentina, where it was
was detected for first time in the north-east
found for the first time affecting wheat crops
region of Argentina, a non-traditional, mar-
in the north-central region of the Buenos
ginal culture area. Plants of wheat cv. Klein
Aires Province in the early 1980s (Annone,
Chajá presented spots or blight symptoms
1985). Subsequently, tan spot has gained
on all aerial parts. Isolates and pathogenic-
predominance among other wheat diseases
ity tests confirmed the presence of P. grisea
in most wheat-growing areas in the country
associated to blight symptoms on leaves,
(Kohli et al., 1992; Annone, 1997; Carmona
sheets and spikles. Pyricularia grisea also
et al., 1999; Perelló et al., 2003). Tan spot
affects rice and other gramineous sponta-
was often observed throughout the growing
neous species in the region. Simultane-
season and was the most common leaf dis-
ously, during 2007, the fungus was isolated
ease observed each year, in 72.6% of all
from wheat plants cvs. Cronox, Baguette
wheat fields in 2001 and 90.4% in 2002.
11, ACA 304 and BioINTA from Bragado,
Additionally, strains of Alternaria spp. from
Baradero, Rojas, Alberti and 9 de Julio
wheat plants with symptoms of leaf blight
localities from the typical wheat area in
sufficiently similar to those described for
Argentina.
tan spot were collected from 11 localities.
All isolations corresponded to the A. infec-
toria species group (Simmons, 1994; Sim-
Conclusion mons, personal communication, 2001; Perelló
et al., 2002). Leaf blight of wheat caused by
Wheat (T. aestivum L.) cultivars currently Alternaria spp. isolates was not significant
grown in Buenos Aires Province, Argentina, in complex of wheat foliar diseases in
are susceptible to different leaf spotting Argentina for many years but, currently,
fungi. Surveys conducted over several years this pathogen has become a new problem in
in Argentina have determined that the main the Buenos Aires Province. Symptoms are
fungi involved in this disease complex are often difficult to distinguish in the field
M. graminicola (Fuckel) Schroet. in Cohn from those caused by D. tritici-repentis
(anamorph S. tritici Roberge in Desmaz.) (Died.) Shoem.
(leaf spot), Cochliobolus sativus (Ito & Other pathogens for wheat, like C. her-
Kuribayashi) Drechs. ex Dastur (anamorph barum, P. sorghina, C. gramineum, Pirycu-
B. sorokiniana (Sacc.) Shoemaker (spot laria oryzae and A. tritici were registered for
blotch) and P. tritici-repentis (Died.) Drechs. the first time in Argentina (Perelló, 2007).
(anamorph D. tritici-repentis (Died.) Shoe- The pattern of diseases produced by these
maker) (tan spot). A. triticimaculans Sim- phytopathogens in some areas of cultiva-
mons & Perelló was first described on wheat tion is changing drastically due, among
in Argentina in 1996 and commonly observed other causes, to new market trends that
since then, like others members of the infec- induce changes in agricultural practices
toria complex. During surveys of wheat and the introduction of new crops. Moni-
commercial fields from 2001 to 2002 to now toring these changes is important in order to
242 A.E. Perelló
take appropriate and timely action to pre- efforts to manage the spotting wheat leaf
vent disease dispersal. Information on the complex in order to avoid future epidemics.
most common leaf spotting fungi would
help to identify appropriate benchmarks for
selecting for disease resistance in different Acknowledgement
environments. It would also help breeders
to set priorities in the incorporation of dis- The author is grateful for the financial sup-
ease resistance to the leaf spotting complex port of Project 11/A142 ‘Patógenos fúngicos
into adapted wheat cultivars. del trigo y su posibilidad de biocontrol con
Moreover, widespread occurrence of microorganismos antagonistas en el marco de
these fungal diseases in the major wheat- una agricultura sustentable’ of the Programa
growing region of Argentina described warns de Incentivos a la Docencia e Investigación de
regional breeders and pathologists to increase la Universidad Nacional de la Plata.
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19
Diseases of Fenugreek
(Trigonella foenum-graecum L.) and
Their Control Measures, with Special
Emphasis on Fungal Diseases
S.N. Acharya,1 J.E. Thomas,2 R. Prasad1,2 and S.K. Basu1,2

1Agriculture and Agri-Food Canada Research Centre, Lethbridge, Canada;
2Department of Biological Sciences, University of Lethbridge, Lethbridge, Canada
Abstract
Fenugreek (Trigonella foenum-graecum L.) is an annual legume crop cultivated in India, the Mediter-
ranean region, China, parts of Africa, Europe and Australia and, in recent years, in North America.
Although traditionally used as a spice crop, fenugreek has important medicinal and nutraceutical
properties and is also grown as a forage crop in some countries. This multi-use crop has the potential
to expand into new areas, as well as increase in the area where it is traditionally grown. Therefore, its
reaction to biotic and abiotic factors that can limit its production deserves special attention. Although
this review contains a discussion on all fenugreek diseases and insect pests, the main focus is on the
causal organisms, symptoms and corresponding control measures for all of the major and minor fungal
diseases affecting its productivity. It is interesting to note that only a few diseases have been reported
to affect this crop adversely. The two major fungal diseases that affect fenugreek are powdery mildew
caused by Erysiphe polygoni and Cercospora leaf spot caused by Cercospora traversiana. However,
disease problems may change as this crop is grown more widely and with larger acreages outside of its
natural area of adaptation. Ongoing vigilance in disease monitoring and development of new resistant
varieties is needed to ensure productivity and usefulness of this crop in the future.
Introduction and white flowers, which typically produces

golden yellow seeds (Basu et al., 2008). It
Fenugreek (T. foenum-graecum L.) is an has two morphological forms of flowering
annual crop belonging to the legume fam- shoots, the common one bearing axillary
ily Fabaceae. Although widely cultivated flowers and an indeterminate growth habit,
in India, China, northern and eastern whereas plants with blind shoots possess
Africa, parts of Mediterranean Europe, both axillary and terminal flowers with a
Argentina and Australia (Acharya et al., more determinate growth habit (Busbice
2006a), it was only recently introduced to et al., 1972; Fehr, 1993; Acharya et al., 2008;
North America (Acharya et al., 2006b). Fen- Basu et al., 2008). Although both closed and
ugreek is a dicotyledonous, self-pollinated open type flowers are reported, the majority
plant with trifoliate leaves, branched stems of fenugreek flowers belong to the closed

246 S.N. Acharya et al.
type (Petropoulous, 2002; Acharya et al., are also effective in the treatment of hyperc-
2008). holesterolaemia (McAnuff et al., 2002).
Fenugreek is capable of fixing atmo- Fenugreek galactomannans appear to aid in
spheric nitrogen in the soil. The plants require the control of type 2 diabetes in both ani-
a minimal amount of nitrogen for growth, mals (Raju et al., 2001; Tayyaba et al., 2001;
reducing the need for nitrogen fertilizers to Puri et al., 2002; Vats et al., 2002, 2003) and
supplement crop growth and facilitating humans (Sharma et al., 1996; Puri et al.,
use of the plant in crop rotations. Fenugreek 2002). The amino acid isoleucine is a precur-
is considered a dryland crop; water require- sor of 4-hydroxyisoleucine, which is known
ments are low, making cultivation of fenu- to regulate the secretion of insulin in animals
greek an increasingly attractive alternative (Broca et al., 2000) and also making it poten-
to producers in regions with limited water tially useful in the control of diabetes.
supply. Use of fenugreek in arid and semi- Since fenugreek can be used for multi-
arid environments can reduce the cost of ple purposes (e.g. as a spice, forage crop,
irrigation, reduce the potential for eutrophi- eco-friendly dryland crop and for medicinal
cation of surface water and limit contamina- and nutraceutical applications), there is
tion of groundwater sources (Basu et al., interest in cultivation of the plant in new
2004; Acharya et al., 2008). According to biogeographical areas of the world (Acharya
Acharya et al. (2004), dryland adaptation of et al., 2008; Basu et al., 2008). Acharya et al.
fenugreek was a major consideration for (2006b) have described fenugreek as a tradi-
introducing it as a forage crop for growth in tional ‘Old World’ crop with significant
the temperate climates of western Canada. potential for use in the ‘New World’. ‘Tristar’
Well-drained, loamy soils are most favour- is the first North American variety of fenu-
able for the crop (Rosengarten, 1969; Acha- greek released by a research group in Can-
rya et al., 2008), while heavy and wet soils ada (Acharya et al., 2007c). As a result of its
are known to restrict fenugreek growth increased economic and industrial impor-
(Petropoulos, 1973; Acharya et al., 2008) tance, those involved in fenugreek produc-
and increase susceptibility of the plant to tion need to become more aware of the
disease. diseases that can affect both yield and quality
Although fenugreek is known primarily of the plant adversely. In many fenugreek-
as a spice crop used especially in India and growing areas, infectious and non-infectious
Mediterranean regions for cooking, the spe- diseases are becoming an important produc-
cies name foenum-graecum refers to ‘Greek tion constraint because of their ability to
hay’, highlighting its use as a forage crop in cause variation in crop yield and quality
early years (Acharya et al., 2006b). Fenu- (Basu et al., 2006a). When fenugreek is
greek also has been referred to as a medici- introduced to a new biogeographical area,
nal herb, both in Indian Ayurvedic and new diseases may emerge that can cause a
traditional Chinese medicines (Tiran, 2003). reduction in productivity, and even crop
Its leaves and seeds have been used exten- losses (McCormick and Hollaway, 1999;
sively to prepare extracts and powders for Fogg et al., 2000). This review looks at major
medicinal use like wound healing and pro- and minor diseases of fenugreek reported
motion of lactation in weaning mothers worldwide, as well as emerging fungal dis-
(Basch et al., 2003; Tiran, 2003; Acharya eases that are increasing in importance, to
et al., 2007a). The medicinal value of fenu- use of the plant commercially.
greek comes mainly from three chemical
constituents; i.e. steroidal sapogenins, galac-
tomannans and isoleucine (Acharya et al.,
2006a, 2007a, 2008). Steroidal sapogenins Diseases of Fenugreek
are often used as a raw precursor for the
production of steroidal drugs and hormones Fenugreek production is affected by both
such as testosterone, glucocorticoids and biotic and abiotic agents. Abiotic diseases
progesterone (Fazli and Hardman, 1968) and or disorders are non-infectious and are often
Diseases of Fenugreek 247
caused by a deficiency in nutrients, extremes Bacterial diseases

in temperature, moisture, soil acidity or
alkalinity, an excess of certain micronutri- McCormick and Hollaway (1999) found that
ents within the soil and toxic impurities infection of fenugreek with Pseudomonas
in the atmosphere (Petropoulos, 2002; syringae resulted in bacterial blight. First
Acharya et al., 2008). For example, Sins- reported in Victoria, Australia, infection
kaya (1961) reported yellowing of some with these bacteria caused small isolated
fenugreek plants under field conditions patches, to entire crop loss in the field. Fogg
due to mineral deficiencies in boron, mag- et al. (2000) reported the same disease on
nesium, manganese or potassium. Physio- fenugreek in New Jersey, USA. It has also
logical diseases resulting from abiotic been suggested that the bacterium Xanthomo-
agents can lead to premature death of the nas alfalfa can infect fenugreek (Petropou-
plant and loss of forage and seed yield. In los, 2002), leading to loss in productivity
western Canada, exposure of fenugreek (Table 19.1).
crops to very dry and hot conditions has
resulted in stunted growth and yellowing,
with occasional loss of leaves from the
Nematode diseases
plant.
Diseases caused by living or biotic
agents (pathogens) are often infectious (Acha- Various nematodes, typically not identified
rya et al., 2008). The most important diseases as a problem for other crops, can damage
of fenugreek are caused by plant pathogenic fenugreek roots (Weiss, 2002; Jongebloed,
fungi. Bacterial diseases are next in degree 2004). The soilborne nematode Meloidog-
of importance, followed by viral diseases yne incognita has been shown to cause root
(AAFRD, 1998; Fogg et al., 2000; Prakash rot and the death of immature fenugreek
and Sharma, 2000; Petropoulos, 2002; Weiss, plants in Australia (Jongebloed, 2004)
2002; Jongebloed, 2004). While information (Table 19.1). However, it is interesting to
on specific pests and diseases damaging fen- note that fenugreek has also been reported
ugreek is limited, in general, insects and to have some anti-nematicidal properties.
pathogenic organisms that attack other com- Zia et al. (2003) reported that decomposed
mon legume crops grown in the vicinity of seeds of fenugreek caused a marked reduc-
fenugreek, such as alfalfa, can also attack tion in population densities of the soil nem-
fenugreek (Basu et al., 2006b). atode M. javanica, which causes root-knot
development in mungbean. Decomposed
seed and aqueous extracts of fenugreek were
also able to enhance plant height and shoot
Viral diseases fresh weight in mungbean.
Bean yellow mosaic virus, alfalfa mosaic

virus, cowpea mosaic virus, soybean mosaic Insect pests
virus, pea mosaic virus, potato virus A and
Y and clover vein mosaic virus are all com- In Australia, insects such as thrips, pod-
mon viral infections of fenugreek (Petro- borers and Heliothis can cause serious dam-
poulos, 2002). Bhasker and Summanwar age to forage yield in fenugreek (Lucy, 2004).
(1982) reported mosaic wilt on fenugreek. Basu et al. (2006b) reported that in southern
Flexuous rod-shaped viruses like bean yel- Alberta (Canada), a low level of insect pests
low mosaic potyvirus (Singh, 1969) and pea such as Lygus bugs and, to a lesser extent,
streak carlavirus (Hagedorn and Walker, alfalfa plant bugs and aphids had been
1949) have also been reported on fenugreek. observed in fenugreek fields. In addition,
These viral diseases have been associated the researchers reported that western flower
with moderate losses of fenugreek seed and thrips (especially severe under greenhouse
forage yield (Table 19.1). conditions), alfalfa looper, alfalfa weevil
Table 19.1. The major non-fungal diseases of fenugreek reported worldwide.
Tolerant
Disease Country varieties/
groups Causal organisms reported genotypes References
Viral Bean yellow mosaic virus England Fluorescent, Brunt, 1972; Petropoulos,
diseases Ethiopian 1973, 2002
Potato virus A NA* NA Schmelzer, 1967;
Anonymous, 1968
Cowpea mosaic virus NA NA Vidamo and Conti, 1965;
Anonymous, 1968
Potato virus Y NA NA Schmelzer, 1967
Tobacco etch virus NA NA Petropoulos, 2002
Pea streak virus NA NA Hagedorn and Walker,
1949; Anonymous, 1968
Pea mosaic virus NA NA Petropoulos, 2002
Soybean mosaic virus NA NA Quantz, 1968; Schmelzer
and Wolf, 1971
Alfalfa mosaic virus NA NA Quantz, 1968; Schmelzer
and Wolf, 1971; Latham
and Jones, 2001
Tomato black ring virus NA NA Quantz, 1968; Schmelzer
and Wolf, 1971
Clover vein mosaic virus NA NA Quantz, 1968; Schmelzer
and Wolf, 1971
Bacterial Pseudomonas syringae Australia NA McCormick and Hollaway,
diseases pv. syringae 1999; Fogg et al., 2000
Xanthomonas alfalfa NA Petropoulos, 2002
Nematode Meloidogyne incognita Australia NA Jongebloed, 2004
diseases
Insect- Lygus keltoni, L. elisus, Canada Tristar Basu et al., 2006a
mediated L. borealis and L. lineolaris
diseases Adelphocoris lineolatus Canada Tristar Basu et al., 2006a
Acyrthosiphon pisum Canada Tristar Basu et al., 2006a
Frankliniella occidentalis Canada Tristar Basu et al., 2006a
Sitona sp. Canada Tristar Basu et al., 2006a
Hypera postica Canada Tristar Basu et al., 2006a
Autographa californica Canada Tristar Basu et al., 2006a
Aphis craccivora India, NA Weiss, 2002
West Asian
countries
Myzodes persicae India, NA Weiss, 2002
West Asian
countries
Scirtothrips dorsalis Australia, NA Weiss, 2002; Lucy 2004
India, the
Mediterranean
region
Tetranychus cucurbitae India NA Weiss, 2002
Pachymerus pallidus Sudan NA Weiss, 2002
Diacrisia oblique India NA Weiss, 2002
D. orichalcea India NA Weiss, 2002
Prodenia litura India NA Weiss, 2002
Maruca testulalis India NA Weiss, 2002
Note: *NA = not available.

and Sitona sp., were attracted to standing Bohra, 1999). Time of sowing can influence
fenugreek crops under field conditions in the damage caused by an infection. For
western Canada. Aphis craccivora and example, in Haryana, India, seed sown in
Myzodes persicae have caused damage to mid October as compared with the end of
fenugreek crops from west Asia to India, November exhibited a 30% reduction in
while various Thysanoptera (thrips), includ- damage caused by E. polygoni and Leveil-
ing Scirtothrips dorsalis, have been found lula taurica (Sharma, 1999). Downy mildew
on almost all fenugreek crops grown from caused by Peronospora trifoliorum and
the Mediterranean to India (Petropoulos, spring black stem and leaf spot caused by
2002; Weiss, 2002). There have also been Phoma pinodella have recently become more
reports of mite (Tetranychus cucurbitae) common (Lakra, 2002, 2003; Bretag and Cun-
attacks on fenugreek in India (Weiss, 2002). nington, 2005). Several leaf diseases causing
Pachymerus pallidus, a seed beetle, which varying degrees of damage generally or in
attacks a wide range of crops, is a major pest specific seasons, including rust due to
of fenugreek in the Sudan (Weiss, 2002). A Uromyces anthyllidis, have been reported
number of polyphagous caterpillars belong- in India (Weiss, 2002). Root and collar rots
ing to the order Lepidoptera, including Dia- caused by Rhizoctonia spp., typically R.
crisia oblique, D. orichalcea and Prodenia solani and Alternaria spp., often A. alter-
litura, and especially the mung moth (Maruca nata, can damage individual crops (Weiss,
testulalis), have been reported to affect fen- 2002).
ugreek in India (Weiss, 2002) (Table 19.1). Antifungal activity for fenugreek has
also been reported in the primary litera-
ture (El-Gizawy et al., 2000). Lupin and
fenugreek seed extracts significantly sup-
Common Fungal Diseases pressed Pythium damping-off of cucumber
of Fenugreek and tomato seedlings, as well as radish
damping-off caused by R. solani. Moreover,
The two most common fungal diseases infect- application of seed extracts had a significant
ing fenugreek are Cercospora leaf spot and positive effect on seedling growth of the veg-
powdery mildew (AAFRD, 1998). Powdery etables tested (El-Gizawy et al., 2000). A
mildew on fenugreek, caused by E. polygoni, detailed description of major and minor
can seriously reduce crop yield (Prakash fungal diseases of fenugreek reported all
and Sharma, 2000; Jongebloed, 2004) and across the globe and their prescribed con-
has the potential to affect biomass and seed trol measures are outlined individually in
yield in crops grown under moist agrocli- the following sections.
matic conditions in North America. In Aus-
tralia, yield of fenugreek was seriously
affected by blight caused by C. traversiana
and wilt caused by Fusarium oxysporum Cercospora leaf spot
and Rhizoctonia solani (Jongebloed, 2004).
The pathogen C. traversiana is spread by Cercospora leaf spot is a seedborne fungal
contaminated seed and is now found in many disease, considered to be one of the most
countries, ranging from India to Europe, east- serious threats to fenugreek. This disease is
ern Africa including Ethiopia and in several capable of causing considerable economic
countries in South America; it is slowly loss (Leppik, 1959, 1960; Khare et al., 1981;
becoming a major fenugreek disease concern Zimmer, 1984; Ryley, 1989). The Cercospora
(Weiss, 2002). Other well-known fungal dis- leaf spot of fenugreek has been reported all
eases observed to be associated with fenu- across the world and is most common in
greek are collar rot, leaf spot and pod spot Australia, several eastern European coun-
diseases (Petropoulos, 2002) (Table 19.2). tries, South America, North America, in the
In India, 27 species of fungi have been Near East and India (Voros and Nagy, 1972;
isolated from fenugreek seeds (Prabha and Cook, 1978; Khare et al., 1981; Ryley, 1989).
Table 19.2. The major fungal diseases of fenugreek reported worldwide.
Name of the Pathogenic fungal Country Tolerant varieties/

disease species reported genotypes References
Cercospora Cercospora traversiana India, NA Leppik, 1959, 1960;

leaf spot Australia, Khare et al., 1981;
Canada Zimmer, 1984;
Ryley, 1989
Collar rot Rhizoctonia solani India TG-18, UM-20, Hiremath et al., 1976;
Pusa Early Hiremath and
Bunching Prasad, 1985; Raian
et al., 1991; Petro-
poulos, 2002; Datta
and Chatterjee, 2004
Leaf spot Ascochyta sp. UK Fluorescent, Walker, 1952;
Ethiopian Petropoulos, 1973
Powdery mildew Oidiopsis sp. Israel, Fluorescent Palti, 1959; Rouk and
Ethiopia, Mangesha, 1963;
England Petropoulos, 1973
Downy mildew Peronospora trigonellae India HM-346, HM-350, Lakra, 2002, 2003;
HM-444 HAU, 2008
Powdery mildew Leveillula taurica Israel NA* Palti, 1959
Leaf spot Pseudoperiza NA Glaeser, 1961
medicaginis
Spring black stem Phoma pinodella Australia NA Bretag and
and leaf spot Cunnington, 2005
Powdery mildew Erysiphe polygoni Israel, HM-350, Petropoulos, 1973;
Ethiopia, HM-444, Zimmer, 1984;
India, Fluorescent Prakash and
Canada Saharan, 2000;
Basu et al., 2006a;
HAU, 2008
Rust Uromyces trigonellae Israel NA Ubrizsy, 1965
Pod spot Heterosporium sp. UK Kenyan, Petropoulos, 1973
Moroccan
Charcoal rot Macrophomina Pakistan NA Haque and
phaseolina Ghaffar, 1992
Root rot Sclerotinia trifoliorum UK NA Petri, 1934
Fusarium wilt Fusarium oxysporum India, NA Borg, 1936;
Sudan, Komaraiah and
Malta Reddy, 1986;
Hashmi and Thrane,
1990; Bansal and
Gupta, 2000
Note: *NA = not available.
The causal organism for this disease is C. C. traversiana are dark, paler towards the tip,
traversiana, a member of the Ascomycetes unbranched, rarely geniculate and rarely
(Agrios, 1997). Several researchers have septate. These conidiophores develop in fas-
suggested that C. traversiana is the only spe- cicles of 3–5 conidiophores per fascicle, with
cies of the Cercospora infecting fenugreek a length of up to 420 µm and width ranging
(Cook, 1978; Ryley, 1989). Conidiophores of from 3 to 5 µm (Ryley, 1989). The conidia are
hyaline, acicular, straight or slightly curved, the apex of the plant (Agrios, 1997). Stem
apex rounded, base truncate and multicellu- and pods also can become infected. Disease
lar. The main source of overwintering inocula symptoms on pods include discoloured
is plant debris, where sclerotia or stromata infected areas, as well as severely infected
can form. Conidia germinate best at a high areas that can become shrunken and twisted
relative humidity and at a high temperature. (Zimmer, 1984). The life cycle of the patho-
They are dispersed mainly by rain-splash gen is shown in Fig. 19.1.
and to some extent by wind (Agrios, 1997).
Cercospora leaf spot initially presents Control measures
itself as circular, sunken lesions that appear
bleached in colour, with narrow (1–2 mm) As the pathogen is often seedborne, seed treat-
chlorotic halos on the surface of the leaves. ment before planting has been an effective
These lesions expand rapidly as the infec- control measure in some cases (Leppik, 1960;
tion progresses, producing necrotic areas. Khare et al., 1981). However, selection of
Each area of infection is sharply defined, healthy seeds as planting material may also
with most lesions surrounded by a character- provide an effective control (Cook, 1978).
istic yellowish halo. Lesion size is increased Rotation with crops outside of the host
significantly on mature leaves, where sporu- range for the fungal pathogen C. traversiana
lation becomes evident, giving the lesions a may also be useful. It appears that preven-
whitish, velvet-like appearance (Zimmer, tion of seed contamination by treating plants
1984). Severely infected plants are reported when the pathogen is first detected will
to have only a few leaves situated towards likely be the best approach to limiting spread
Lesions surrounded by yellowish

halo and lesion size increases Mycelia giving rise to conidiophores
considerably: advanced symptoms and conidia
Lesions spread as infection advances

Conidia disseminated by rain-splash
and wind
Appearance of circular, sunken

bleached lesions on leaves: initial
symptom
Life cycle of Cercospora leaf
spot on fenugreek host plant
Infected seed giving rise to an

infected plant Conidia infecting healthy plants
and healthy leaf tissue
Fungus overwintering in plant debris

may produce sclerotia or stromata
Fungus overwintering in
non-treated seeds
Fig. 19.1. The life cycle of Cercospora traversiana on fenugreek host plant.
of this pathogen. Spraying the plants with control the infection effectively. The Gram
fungicides such as benomyl, chlorothanolin, positive bacterium Bacillus subtilis can also
Bordeaux mixture, mancozeb and maneb has be used effectively as a biological control
been suggested as an effective chemical agent for R. solani (Tschen and Kou, 1985;
control measure (Agrios, 1997). Tschen, 1987). Prasad and Herimath (1985)
demonstrated that carbendazim could be
used as a seed and dry soil mix fungicide
Collar rot and that captan also could be used to drench
the soil and kill the fungus.
Collar rot is another important fungal dis-
ease of fenugreek and has been reported in
all parts of India (Hiremath et al., 1976; Leaf spot
Hiremath and Prasad, 1985; Raian et al.,
1991). The causal organism for this disease Leaf spot is another seedborne disease of
is a member of the Basidiomycetes. R. solani fenugreek that is caused by fungal patho-
reduces yield of fenugreek causing foot-rot gens of the Ascochyta sp. belonging to the
and damping-off where freshly emerged Ascomycetes (Walker, 1952; Petropoulos,
seedlings fall over and die (Petropoulos, 2002). The fungus attacks the leaves, stems
2002). The vegetative mycelium of R. solani and pods of fenugreek, reducing both yield
is colourless when young but turns brown on and quality severely. It can survive in the
maturity. The mycelium consists of hyphae soil, on infected seed and on crop residues.
partitioned into distinct individual cells by a The pathogen is disseminated by both wind
septum consisting of a doughnut-shaped pore and rain-splash (Agrios, 1997; Petropoulos,
(Ogoshi, 1987; Alexopoulos et al., 1996). R. 2002). Irregular brown to black spots with dis-
solani survives as sclerotia in the soil and on tinct margins are detected on infected leaves.
plant tissue, and as mycelia by colonizing soil As the disease progresses, the leaves on the
organic matter as a saprophyte. Sclerotia and/ plant may die and fall off. Infected seeds have
or mycelia present in the soil and/or on plant round, dark brown lesions. Seedlings from
tissue germinate to produce fungal hyphae infected seeds start rotting from the point of
that can attack the subsequent year’s crop seed attachment and rotting advances towards
(Alexopoulos et al., 1996). The pathogen pri- the stem and taproot; subsequently, the young
marily attacks below-ground plant parts such seedlings die (Petropoulos, 2002). Cool,
as the root system, but is also capable of infect- moist weather is favourable for rapid dis-
ing other parts such as green foliage, seeds semination and growth of the fungus (Anon-
and hypocotyls. The most common symptom ymous, 1970; Agrios, 1997).
of the disease is damping-off (Petropoulos,
2002). Most of the severely infected seed- Control measures
lings may die at pre- or post-soil emergence
stages. The infected seedlings may develop Cultivation of tolerant genotypes is a good
reddish-brown cankers on roots and stems idea to avoid rapid infestation of the fungus
at or near ground level (Anderson, 1982; (Agrios, 1997). To protect fenugreek plants
Adam, 1988; Agrios, 1997). from primary infection, seeds can be treated
effectively with benlate, while to prevent
Control measures secondary infection, use of a frequent foliar
spray containing benlate is also recom-
Cultivation of resistant varieties has been mended (Petropoulos, 2002).
suggested as the best control measure for
the disease (Prasad and Hiremath, 1985).
According to Haque and Ghaffar (1992), Fusarium wilt
seed dressing and soil drenching with
Rhizobium meliloti, Trichoderma banatum, Fusarium wilt of fenugreek is caused by the
T. harzianum and T. pseudokonongii can fungus F. oxysporum, an Ascomycete that
has been reported by several investigators infections. Fungal spores produced within
across the world (El-Bazza et al., 1990; Borg, leaf spots during the growing season are
1936; Hashmi, 1988; Bansal and Gupta, spread by splashing rain (Petropoulos, 1973).
2000; Petropoulous, 2002). The pathogen F. Symptoms of the disease become visible at
oxysporum is both seed and soilborne the third stage of pod development and can
(Komaraiah and Reddy, 1986; Hashmi and be seen as dark brown to black spots on the
Thrane, 1990; Bansal and Gupta, 2000; Pierre pods that extend to produce a dark olive,
and Francis, 2000). The pathogen can remain velvet-like cover. Initially, localized spots
in infested soils for up to 10 years. Dissemi- of infection elongate transversely to the pod
nation of the pathogen occurs through seed, axis but with time spread over the pod sur-
soil and infested plant parts (Pierre and face and transform into more rounded to
Francis, 2000). Fusarium wilt first appears as oblong lesions. These spots are also visible
a slight clearing in veins found on the outer on the stems, but are rarely found on the
portion of younger leaves, followed by down- plant leaves. Petropoulos (2002) suggests
ward drooping of the mature leaves. At the that the fungus does not enter into the seeds
seedling stage, plants infected by F. oxyspo- as the mycelium of the fungus is not buried
rum may wilt and die soon after the symp- deeply in the epidermis of the pod and that
toms appear. In mature plants, vein clearing contamination of fenugreek seeds by this
and downward drooping of the leaf are often fungus takes place specifically during the
followed by stunting, yellowing of the lower threshing process.
leaves and subsequent wilting of leaves and
young stems. Marginal necrosis of the infected Control measures
leaves, rapid defoliation and finally death of
the entire plant typically follow (Agrios, Hot water treatment of the seeds before
1997). Browning of the vascular tissue is planting is efficient to remove the fungus
strong evidence of Fusarium wilt infesta- from the seeds (Pirone et al., 1960). Resistant
tion. Furthermore, symptoms become more cultivars tolerant to that fungus have been
apparent on mature plants during the period suggested as the best way to restrict rapid
between blossoming and fruit maturation dissemination of the disease on a standing
(Jones et al., 1982; Smith et al., 1988). crop effectively (Petropoulos, 2002).
Control measures
Some effective means of controlling F. oxy- Spring black stem and leaf spot
sporum include disinfection of the soil and
planting of the seeds with thiram or captan, This disease of fenugreek has been reported
crop rotation with non-hosts of the fungus, in Australia by Bretag and Cunnington
or use of resistant cultivars (Singh, 2001). (2005). These investigators also identified P.
pinodella, an Ascomycete previously known
as A. pinodella (Jones, 1927), as the causal
agent of the disease. This observation is sup-
Pod spot ported by another investigation conducted
by Boerema et al. (2004). Phoma sp. has been
Petropoulos (1973) first investigated and isolated from the seeds of fenugreek in
described this disease in fenugreek and Egypt, India, Nepal, Pakistan, Sri Lanka,
identified Heterosporium sp., an Ascomy- Sudan and Syria (Hashmi, 1988), suggesting
cete, as the causal agent. Heterosporium that the organism is not new to fenugreek
medicaginis is the only species of Hetero- crops. The pycnidia of the fungus are more
sporium that has been reported to be patho- or less globose, glabrous, ovoid to ellipsoid
genic to legumes (Karimov, 1956). The fungus and usually aseptate (Punithalingam and
overwinters on dead leaves. Spores spread Gibson, 1976; Bretag and Cunnington, 2005).
from old plant debris to initiate new plant Onfroy et al. (1999) reported that the length
of conidia could range from 7.3–9.6 µm. The affecting both biomass and yield (Petropou-
pathogen persists as pycnidia and mycelia los, 2002; Basu et al., 2006a). Powdery mil-
in plant debris. It is dispersed mainly by dew is most commonly found in hot and
splashing rain and to some extent by wind. humid tropical and subtropical areas, as well
Numerous small, irregular-shaped, dark as in temperate to subtemperate regions (Palti,
brown to black leaf lesions surrounded by 1959; Rouk and Mangesha, 1963; Prakash and
small chlorotic areas often appear as disease Saharan, 2000; Basu et al., 2006a). On the
symptoms on the leaves, petioles and stems basis of recent observations, Basu et al. (2006a)
of the growing plant. Elongated black lesions suggested that powdery mildew could become
may also develop on the taproot (Bretag and a serious disease problem in North America,
Cunnington, 2005). Infected plants become where fenugreek is a recent crop introduction.
stunted with a mild chlorosis. In cases of Although some investigators have reported
severe infection, most of the leaves turn com- Oidiopsis sp. as the causal organism (Rouk
pletely yellow, wither and the taproot system and Mangesha, 1963; Petropoulos, 1973),
becomes completely girdled with sharp the majority of investigators from across the
lesions (Bretag and Cunnington, 2005). globe consider E. polygoni (an Ascomycete)
as the causal organism for the disease (Zim-
Control measures mer, 1984; Prakash and Saharan, 2000; Bretag
and Cunnington, 2005; Basu et al., 2006a).
Bretag et al. (2006) suggested practising
The conidiophores of the fungus are simple
crop rotation, destruction of infected plant
and erect and the corresponding conidia are
portions and chemical seed treatments to
unicellular, hyaline in colour, ellipsoidal to
control primary infections by the disease
cylindrical in shape (Agrios, 1997; Nyvall,
efficiently. When the fungus was restricted
1999; Basu et al., 2006a) (Figs 19.2 and 19.3).
effectively at the primary infection level, it
The conidiophores vary in size from 32.5 to
did not spread to advanced stages in most
65.6 µm × 9.7 to 13.6 µm, whereas dimen-
disease trials conducted (Bretag et al., 2006),
sions of the conidia are 22.6–48.4 µm × 12.4–
suggesting that restricting primary infection
20.8 µm (Basu et al., 2006a) (Fig. 19.2). The
is the key to control of the disease.
pathogen survives mostly by developing
cleistothecia in diseased plant debris. They
survive in soil until the next season. Asco-
Powdery mildew spores are released after the disintegration
of the wall of the asci. The ascospores first
Powdery mildew is one of the most common infect the lower and older leaves in the
and serious fungal diseases of fenugreek, next season. The spores are carried by the
(a) (b)
Fig. 19.2. Light microscopy images of Erysiphe polygoni conidiophores (a) and conidia (b).
(a) (b)
(c) (d)
Fig. 19.3. Scanning electron microscopy (SEM) images of healthy fenugreek upper leaf surfaces [Top,
(a) and (b)] compared to powdery mildew (caused by Erysiphe polygoni) infected upper leaf surface
[Bottom, (c) and (d)]. Left images were magnified 500×, while the right images were magnified 1000×.
wind to new hosts. The pathogen is also lower surfaces of the leaves (Fig. 19.4), on
known to survive as a mycelium (Sharma, pods but rarely on flowers, and by the strong
2005). odour emitted by the infected plants. Dur-
Powdery mildew is one of the easier ing the initial stages of an infection, fungal
diseases to identify on plants as its symp- patches appear isolated or in scattered
toms are quite distinctive. The disease can patches which coalesce as the infection pro-
be identified easily by the presence of white gresses. At first, leaves near ground level are
to grey powdery masses or distinct circular infected, after which the whole plant can
to ellipsoidal patches on both the upper and become covered with the fungus over a
Fig. 19.4. Comparison of healthy fenugreek upper leaflet surfaces (centre) with infected leaflets from
the same plant.
short period of time (Fig. 19.5). The upper hence, use of resistant varieties has been
surface of the leaves typically bears more strongly recommended to avoid disease infes-
fungal structures and spores than the lower tation. Basu et al. (2006a) demonstrated that
surface (Fig. 19.4). application of tilt 250E-propiconazole or
Severely infected leaves become irregu- milgo-ethrinol (28% at 2.5 ml/l) and captan-
lar in shape, dry and shrivelled, resulting in captane (50%) or benlate-benomyl (50% at
stunted growth of the whole plant (Basu 2.0 g\l) could control the disease at a satis-
et al., 2006a) (Figs 19.4 and 19.5). Although factory level, whereas Petropoulos (1973)
Zimmer (1984) first identified powdery mil- showed that spraying with dinocap (8–10
dew infecting fenugreek in North America, oz a.i/acre in 100 gals) could also control
Basu et al. (2006a) reported the first major the disease.
in-depth investigation of powdery mildew
as a major disease of fenugreek in North
America based on trials that were conducted
at different locations and under different Conclusions
physico-geographic conditions and variable
climatic factors on the west and east coast Fenugreek is affected mostly by seedborne
of North America and the mid interior of fungal diseases. From our experience, and
Canada. The life cycle of the pathogen is other reports of fenugreek disease, it is clear
presented in Fig. 19.6. that powdery mildew and Cercospora leaf
spot are the two most important diseases
Control measures currently affecting this crop. These diseases
can reduce the production and quality of
Petropoulos (1973) and Avtar et al. (2003) fenugreek crops significantly all across the
reported variation in the sensitivity of globe. Other minor fungal diseases of fenu-
fenugreek genotypes to powdery mildew; greek, namely collar rot, leaf spot, Fusarium
Fig. 19.5. Spread of powdery mildew

infection on a fenugreek potted plant in
the greenhouse at Lethbridge Research
Center, Agriculture and Agri-Food
Canada, Lethbridge, Alberta, Canada.
wilt, pod spot, spring black stem and leaf (Laroche, 2007) could be a good strategy to
spot, and downy mildew, have the potential prevent emergence of new fungal diseases
to become major fenugreek diseases in many for this crop. Susceptibility of a plant to dis-
areas, including subtemperate climatic zones. ease is determined by the genetic relation-
Use of resistant cultivars and application of ship between the plant and the pathogen.
suitable chemical agents are suggested by The relationship between genes of the host
most research groups as potential control and the pathogen can determine disease
measures against infection and spread of expression in the host. Genetic resistance in
fungal diseases. Although certain Internet plants is considered a major form of biologi-
sites do make widely optimistic claims cal control of disease and is possibly the
about effective biological control of fenu- most cost-effective and environmentally
greek fungal diseases, they do not have friendly way to control crop diseases. Resis-
strong evidence from multi-location and tant cultivars have been used effectively to
multi-year trials to support their claims and control diseases in many crops. However,
so are not included in this review. development of resistant cultivars takes
Fenugreek is being cultivated in many time and so work should continue in the
new areas as it becomes more widely recog- interim to find chemical and other biologi-
nized as a multiple-use crop. Development cal control agents to protect the crop from
of new fenugreek cultivars and improve- disease and other pest damage. It should
ment of existing cultivars with disease also be noted that disease control measures
resistance using conventional plant breed- should not only be cost-effective but also
ing methods (Acharya et al., 2007b) and need to be environment friendly and socially
advanced plant biotechnological approaches acceptable.
Healthy green tissue infected,

most prominent symptoms
on leaves and shoots
Ascospores and conidia disseminated by air
Conidia Asci containing ascospores
Cleistothecium
Mycelia finally Production of cleistothecia on plant parts

generates condiophores
bearing conidia
Life cycle of powdery mildew

Infected buds giving rise to on fenugreek host plant
shoots and leaves completely
covered by fungal mycelia
Fungus overwintering in
Young plant infected
non-treated dormant buds
and seeds
Fig. 19.6. The life cycle of Erysiphe polygoni on fenugreek host plant.
Acknowledgements SEM images, Mr Doug Friebel, Technician,

Forage Lab, AAFC, LRC for his help with field
The authors express their sincere thanks to trials. The authors also extend their gratitude
Mr Byron Lee, Research Technician, Electron to the School of Graduate Studies, University
Microscopy and Image Analysis Laboratory, of Lethbridge, Alberta Agriculture Research
Lethbridge Research Centre (LRC), Agricul- Institute (AARI) and AAFC matching grant
ture and Agri-Food Canada (AAFC) for his initiatives for graduate student assistantships
help in taking all the light microscopy and and project funding, respectively.
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20 Fungal Diseases of Oilseed Crops
and Their Management
S.S. Adiver and Kumari

Oilseeds Scheme, Main Agricultural Research Station,
University of Agricultural Sciences, Dharwad, Karnataka, India
Abstract
During the past few years, pathogens in oilseed crops have been recognized as major forces causing
economic losses, with identification of certain important ones based on their symptoms, etiology and
also ecological zones. Recent research has helped by developing new resistant varieties and other
effective management strategies. This chapter describes the causal organisms, symptoms and manage-
ment of diseases of oilseed crops like castor, groundnut, safflower, sesame and sunflower. Cultural
practices for managing certain diseases have been pinpointed. Critical stages for growth of some foliar
diseases, namely rust, early and late leaf spot of groundnut, blight and mildew of sunflower, fusarial
wilt of safflower and castor, have been identified. Recommendations are given on controlling various
diseases by chemical, botanical and other effective and eco-friendly methods. Oil is an essential house-
hold commodity required for food and daily use. Certain oils are used as therapeutic agents and are in
much demand for their conversion into energy or potential biodiesel. Losses to the tune of 20% in
certain oilseed crops need our utmost attention. Various fungal diseases of groundnut, sunflower, saf-
flower, sesame and castor are described. Disease management with fungicides and other available
methods are illustrated.
Groundnut Early leaf spot caused by

Cercospora arachidicola Hori
Groundnut, known as poor man’s almond,
contributes about 38% to the oilseed pool of The perfect stage of the fungus is Mycospha-
India. India is the second largest producer erella arachidis. In India, losses in yield
of groundnut after China. The crop is sub- due to leaf spots have been estimated to be
jected to attack by numerous pests and in the range of 15–59%. Besides the loss in
pathogens. Among foliar fungal diseases, pod and kernel yield, the value of fodder is
early and late leaf spots, commonly called also affected adversely. Lesions are subcir-
‘Tikka’ disease, and rust are economically cular in shape and measure 1 to over 10 mm.
important. On the upper surface of the quadrifoliate
264 S.S. Adiver and Kumari
leaves, the lesions appear dark brown, while are important measures in reducing the pri-
on the lower surface they are a lighter shade mary source of infection. Resistant/tolerant
of brown. The early leaf spot usually has a varieties like Girnar-1, RG-141, IGV-87160,
light to dark brown centre and a yellow halo. ICGV-86590, ICGV-86325, R-8808, GPBD-4,
These are oval to elongate in shape and have Kadari-4, Co-3,4, M-335 and BG-3 can be
more distinct margins than the late leaf spot grown wherever late leaf spot is severe. Foliar
lesions. The early leaf spot pathogen sur- spraying of carbendazim (0.05%) + mancozeb
vives through conidia on affected plant (0.2%), chlorothalonil (0.2%), difenacon-
debris in soil, or through conidia being car- azole (0.1%) or hexaconazole (0.1%) is
ried on the pod shell. recommended 2–3 times at 2- to 3-week
intervals, starting from the initiation of the
Disease management disease (Adiver et al., 1995).
Tolerant varieties like GPBD-4, ICGV-86590,

ICGS-44, M-335, BG-3 and M-522 can be
Rust caused by Puccinia arachidis Speg.
grown wherever early leaf spot is severe.
Intercropping pearl millet/sorghum with
groundnut (1:3) is useful in reducing the Rust of groundnut is prevalent throughout
intensity of early leaf spot. Foliar spraying India; however, it is more severe in the
of carbendazim (0.05%) + mancozeb (0.2%), southern states. In India, losses in yield due
chlorothalonil (0.2%), difenaconazole (0.1%), to rust alone have been reported in the range
tebuconazole (0.1%) or hexaconazole (0.1%) of 10–52%, depending on the variety. Rust
is recommended 2–3 times at 2- to 3-week can be recognized readily as orange-coloured
intervals starting from the initiation of the pustules (uredinia) that appear on the lower
disease (Adiver et al., 1995). leaflet surface and rupture to expose masses
of reddish-brown urediniospores. Pustules
appear first on the lower surface and, in
highly susceptible cultivars, the original
Late leaf spot caused by Phaeoisariopsis pustules may be surrounded by colonies of
personata (Burk. and Curt.) Van Arx secondary pustules.
The perfect stage of the fungus is M. berke- Disease management

leyii W.A. Jenkins. Late leaf spot is more
severe in the southern and central parts of Early sowing in the first fortnight of June is
India. Dark brown to black, circular to sub- recommended to avoid incidence. Use of
circular lesions, measuring 1–6 mm diameter resistant/tolerant varieties like Girnar-1,
appear on the lower surface of the quadrifo- ICGV-87160, ICGV-86590, DRG-12, ALR-2,3,
liate, where most sporulation occurs. The Co-4, ALR-1, ICGS-5 and DRG-17 is recom-
lesions are black in colour and fruiting mended. Spraying mancozeb (0.2%), tride-
structures occur in concentric rings on the mefon (0.1%), chlorothalonil (0.2%),
lower leaflet surface, giving lesions a slightly difenaconazole (0.1%), tebuconazole (0.1%),
rough appearance. The ambient tempera- hexaconazole (0.1%) or cyproconazole
ture required is between 25 and 30°C. Pro- (0.1%) 2–3 times at 2- to 3-week intervals
longed leaf wetness hours and high relative starting from the initiation of the disease
humidity (> 80%) favour infection and dis- helps to control the disease.
ease development. Conidia are dissemi-
nated by the wind and insects, leading to
secondary infection.
Seed and seedling diseases of groundnut
Disease management
Pre-emergence seed rot and post-emergence
Deep burying of crop residues in the soil seedling mortality are of common occur-
and removal of volunteer groundnut plants rence. The disease develops either from the
Fungal Diseases of Oilseed Crops 265
fungi already present in the seed or result in both dry weight and oil content of ground-
from direct invasion of seeds and seedlings nut kernels. The first symptom is partial or
by soil fungi. Among seedling diseases, col- complete wilting of the stem or branch that
lar rot, root rot and stem rot are of economic is in contact with the infected soil. The
importance and are known to reduce yields leaves turn brown and wilt, but remain
by 25–50%. attached to the plant. The pathogen has a
wide host range. S. rolfsii can colonize
either living plant tissue or plant debris.
Collar rot caused by Deeply buried sclerotia survive a year or
Aspergillus niger van Tiegh less, while those near the soil surface remain
viable for many years. Disease development
occurs when soil moisture is 40–50%.
In India, collar rot, also known as crown rot
Generally, when the temperature remains
or seedling blight, is prevalent in almost all
between 29 and 32°C during the day and
groundnut-growing states, causing 28–50%
seldom drops below 25°C during the night,
losses. The diagnostic symptoms are pre-
the disease develops more favourably.
emergence rotting of seeds and rotting of
hypocotyls, but the most common cause of
loss is early post-emergence seedling blight. Disease management
The first symptom in emerged seedlings is Deep ploughing, early sowing and close
usually a rapid withering of the entire plant planting is recommended and rotation of
or its branches. Lesions develop on the stem groundnut with cotton, maize, sorghum and
below the soil and spread upwards along pearl millet. Seed treatment with T. viride/
the branches. The dead and dried branches T. harzianum at 4.0 g/kg seed or seed treat-
are easily detached from the disintegrated ment with carbendazim/captan at 2–3 g/kg
collar region. seed is suggested.
Disease management
Avoiding deep sowing (not more than 5 cm), Sunflower
mixed cropping with moth bean in alternate
rows, deep tillage and early sowing of crop Sunflower (Helianthus annuus var. macro-
is recommended. Soil application of neem carpus (DC) Cockerell) is an important edi-
cake/castor cake at 500 kg/ha, seed treatment ble oilseed crop. It belongs to the family
with Trichoderma harzianum/T. viride at Asteraceae. The sunflower head is com-
4.0 g/kg seed, bacterization of groundnut posed of about 1000–2000 individual flow-
seeds with strains of fluorescent pseudo- ers. The fertile disc florets bear the seed,
monads or seed treatment with carbendazim which is white, black or striped grey and
(1.0 g/kg), mancozeb (2.0 g/kg seed) or chlo- black. The seeds contain 40–50% oil and
rothalonil/captan (2.0 g/kg) is suggested. 50–55% meal, which contain high protein
(35%), calcium, phosphorus, iron, potas-
sium and vitamin E. The sunflower is a
Stem rot caused by native of North America, where it is used in
Sclerotium rolfsii Sacc. dyes, food preparations and medicines.
The stem rot pathogen has a very wide host

range. In India, stem rot, also known as Alternaria leaf spots and
Sclerotium wilt, occurs in all groundnut- blight of sunflower
growing states and is particularly severe in
Maharashtra and Gujarat. In India, 27% or Different species of Alternaria, namely A.
more yield loss has been reported. S. rolfsii alternata, A. helianthi, A. zinniae, A. helian-
also causes indirect losses such as reduction thiacola, A. leuconthemi and A. tenuissima
have been reported to cause the disease. stage, when the crop attains a dense canopy.
Among these, A. helianthi (Hansf.) Tubaki The disease appears in the form of small
and Nishihara are economically important. cinnamon brown-coloured uredia on the
The disease has been reported to cause lower surface of the lower leaves. In severe
a huge grain yield loss in Australia, where conditions, younger leaves, stems, petioles
yield potential of 1.25 t/ha of the crop was and floral parts are also infected. When the
reduced to 0.1 t/ha (Allen et al., 1981). In crop reaches physiological maturity, most
Karnataka, India, the disease occurred in of the uredia are converted to telia and are
epidemic form in 1987, with a disease inci- dark brown in colour.
dence of 95–100% (Hiremath et al., 1990).
The disease is caused by A. helianthi
Disease management
(Hansf.) Tubaki and Nishihara. The myce-
lium of the fungus is septate, rarely Altering the date of sowing reduces disease
branched, brown and 2.5–5.0 µm in breadth. pressure. Removal of self-sown plants, crop
Conidiospores are cylindrical and yellow to rotation for at least 3 years and deep sum-
black grey, with one to 11 transverse septa mer ploughing are recommended to reduce
and a few longitudinal septa. The conidia the inoculum level in the soil. Use of resis-
measure in the range of 40–110 × 8–28 µm, tant varieties like SH-41, SH-187, PH-1, 2, 3,
with an average of 74 × 19 µm. 4, 7 and 8, ICI-306, 331, PAC-36, 9128 and
systemic fungicides containing triazoles,
Disease management namely hexaconazole and cyproconazole
(0.1%), are found suitable under field
Summer deep ploughing reduces the inocu- conditions.
lum level in the soil, which is present in
plant debris as dormant mycelium; altering
the date of sowing in order to reduce dis-
ease pressure and sowing during August– Downy mildew of sunflower
September onwards is suggested. Following
spacing 60 × 30 cm under irrigated and Downy mildew causes heavy yield losses in
45 × 20 cm under rainfed conditions is rec- sunflower-growing countries of the world.
ommended. Use of resistant varieties like A serious outbreak (80–90%) of the disease
GP-145, AH-303, BSH-1 is suggested. Foliar was recorded in the Red River Valley of
sprays of mancozeb (0.2%), chlorothalonil North Dakota and Minnesota (USA) during
(0.2%), difenaconazole (0.1%) or tebucon- 1970, resulting in a reduction of about 50%
azole (0.1%) can prevent the crop from yield, with a loss of about US$0.5m. Later,
Alternaria blight. it spread to many European countries, then
to Asia. This spread was mainly through the
seed trade. In India, the disease first appeared
during 1984, in experimental plots of the
Rust of sunflower Regional Research Station, Latur, particu-
larly during September–October. Later, it
The pathogen Puccinia helianthi Schw. is a spread to many areas of Maharashtra (Mayee,
macrocyclic, autoecious fungus and it pro- 1989), Karnataka and Madhya Pradesh
duces all the five stages on sunflower only. (Agarwal et al., 1991). Causal organisms are
The disease has been reported to cause vari- Plasmopara halstedii, P. perennis and P.
able yield losses in the crop, depending on patens. The sporangiophores, measuring
variety, environmental conditions and time 150–750 µm, are monopodially branched
of the outbreak of the disease in the crop almost at right angles and bear zoosporangia
season. Early infection of the variety ‘Sun- singly at the tips of the branches. Zoospo-
rise’ and S-37-338 showed 17% and 68% rangia produced from leaves are elliptical
less yield, respectively. Under field condi- with an apical papilla and measure
tions, the disease usually starts at flowering 17–30 × 15–21 µm. The zoosporangia from
roots are uniform, pyriform to oval with 1–3 soft and pulpy, with superficial whitish to
papillae and 36–66 × 39–40 µm. The sporan- blackish mycelium on the head. Under severe
gia germinate at 5–28°C, with the optimum conditions, rotting spreads to the flower stalk
temperature being 16–18°C. Zoosporangia and the head drops off. Sometimes, the seeds
formed at 27°C show 86–95% germination. from the rotted head shed and those that
Oospores are formed in the intercellular remain on the head have a bitter taste.
spaces of roots, stem and seeds and measure
27–32 µm. The fungus causes damping off, Disease management
systemic infection and local lesions on
leaves and basal root or stem galls, depend- Management of insects by spraying endo-
ing on the stage of infection during plant sulphan or diazinon at the onset of bloom
growth. Damping-off occurs either as pre- or and spraying of fungicide, i.e. carbendazim
post-emergence under damp and cool (0.1%), on completion of the flowering stage
weather at seedling stage and gives poor is effective in controlling the disease.
plant stand (Goosen and Sackston, 1964).
Systemically infected plants remain stunted
with chlorotic leaves. Safflower
Disease management Safflower (Carthamus tinctorius L.) is one

of the rabi season oilseed crops cultivated
Regulatory measures: the pathogen is seed- in medium to heavy textured soils, mainly in
borne and exhibits races; therefore, a quar- Maharashtra, Karnataka and Andhra Pradesh,
antine measure has been imposed to check India. Being a crop mostly of the poor small-
the movement of virulent races from endemic holder, it came to be recognized as an edible
areas to other countries. Use of the resistant oilseed crop because of its superior role over
variety LDMRSH-1 and seed treatment with animal fats and other vegetable oils, result-
metalaxyl MZ 72 WP at 5–6 g/kg or apron 35 ing in a boom in the cultivated area under
SD at 5–6 g/kg seed is suggested. the crop.
Rhizopus head rot of sunflower Alternaria leaf blight of safflower
The disease is caused by three different Rhizo- Leaf blight caused by A. carthami Choud-
pus spp., namely R. nigricans, R. arrhizus and hary is the most destructive disease of saf-
R. oryzae. The fungal colony is cottony-white flower in India, appearing in a severe form
to brown in R. arrhizus, while it is cottony- wherever the crop is grown and causing up
white turning brownish-grey to blackish-grey to 90% reduction in crop yield and oil con-
in R. oryzae and R. nigricans. The optimum tent of affected seeds. However, the patho-
temperature for the growth of R. arrhizus, R. gen is reported to increase significantly the
oryzae and R. nigricans is reported to be 37°C level of free fatty acids in the seeds (Heaton
(thermophyllic), 30°C and 22°C, respectively. et al., 1978). Mycelium of the pathogen A.
The disease causes severe yield losses, par- carthami is septate, inter and intracellular
ticularly in wet weather conditions. The dis- and dark coloured on maturity. Conidio-
ease has no effect on seed size but it reduces phores are septate, unbranched, erect and
seed weight. Affected seeds become scurfy brown to olivaceous brown, pale near the
with discoloration of the hull and partial to apex, measuring 15–85 µm × 6–10 µm, aris-
complete discoloration of the nut meal and ing through the epidermis or stomata singly
the quality of the oil is affected because of or in clusters. Conidia are light brown to
off-flavours. The disease first appears as translucent in shade, with/without a long
brown, water-soaked irregular spots on the beak, showing constrictions at the septa and
back of the ripening head, usually adjacent to borne singly or in short chains. The disease
the flower stalk. The spots enlarge and turn appears in seedlings on hypocotyls and on
cotyledons as dark necrotic lesions up to 1993). The mycelium of the pathogen F.

5 mm in diameter, which may sometimes oxysporum f.sp. carthami Klisiewicz and
result in damping-off. Spots, having concen- Houston is septate and branched conidia,
tric rings up to 2 cm in diameter, light to dark straight or curved, often pointed at the tip
brown with the centre lighter in colour, are with a rounded base and measure up to
observed in mature plants on leaves and fre- 10–36 µm × 3–6 µm. Microconidia are oval
quently coalesce into large irregular lesions. to elliptical, one-celled and measure up to
5–16 µm × 2.2–3.5 µm in size. Chlamy-
Disease management dospores are single celled, smooth, faintly
coloured, single or in chains and 5–13 µm ×
The disease can be managed by using seeds 10 µm in size. Four biotypes of the patho-
from early sown dry land crops and treating gen have been identified on the basis of the
them with thiram and TPTH, captan 0.3% reaction of safflower differentials to its iso-
(Siddaramaiah et al., 1980). Hot water treat- lates (Sastry and Chattopadhyay, 1999).
ment at 50°C for 30 min is also found useful
(Sastry, 1996). Bulb extract (1.0% w/v) of Disease management
Allium sativum also shows promise in check-
ing the disease. Varieties like EC-32012, JLA- Seed treatment with carbendazim (1.0 g/kg),
1753, C-2603, Co-1, C75-7218, HUS-524, captan (2.0 g/kg), thiram (2.0 g/kg) or
476, 305, 260, SSF-112, CTC-251, 248, 252, Trichoderma (4.0 g/kg) helps to avoid infec-
etc., are reported to exhibit a variable degree tion of the plant by the pathogen. Crop rota-
of tolerance to A. carthami infection. tion with legumes like chickpea, cowpea
and pigeon pea helps to manage the disease
(Sastry and Jayaraman, 1993). Use of toler-
Fusarium wilt of safflower ant varieties HUS-3234, 3123, 305, BSF-3,
CTV-53, etc., is recommended.
Wilt of safflower is caused by Fusarium
oxysporum f.sp. carthami. Fusarial myco-
toxins capable of causing mycotoxicoses Phytophthora root rot of safflower
have been reported as being produced in
sufficient quantities on infested seeds of saf- The mycelium of the pathogen Phytophthora
flower in storage (Ghosal et al., 1977). The drechsleri is hyaline, aseptate, branched
disease manifests at all growth stages. It may and 4.5 µm wide. Sporangia are hyaline to
cause pre-emergence death or delayed ger- faint in colour, thin-walled, non-papillate,
mination of seeds. Symptoms on seedlings pyriform to ovate, 34–38 µm × 15–24 µm in
during post-emergence are blackening at the size and having zoospores measuring
collar region; chlorotic, small brown spots 10–20 µm in diameter. Oospores are spheri-
appear on cotyledonary leaves, which then cal, smooth, thick-walled, yellow to bright
shrivel, become brittle, sometimes get rolled brown and are 16–45 µm in diameter (Klisie-
and droop downwards; finally, the seedlings wicz, 1977).
bend and die. Plants grown from infected Root rot of Safflower caused by P.
seeds rarely survive beyond the seedling drechsleri Tuck is reported to cause about 3%
stage. In mature plants, lateral branches on losses on average, although 80% losses have
one side may be killed, while the other half been observed in a few instances, particu-
of the plant shows no disease symptoms. larly when grown under surface irrigation
Such plants show partial recovery, but symp- (Sastry, 1996). Safflower is affected by
toms may reappear later. Sporodochial pro- Phytophthora root rot at any stage from
duction on stems may also be visible. Flower pre-emergence to maturity. Symptoms on
head size is reduced in severely affected seedlings of 2–3 weeks of age appear as
plants, less seeds are formed and many of water-soaked lesions with softening and
them are small, distorted, black and chaffy collapse of cortical tissue of the lower stem,
(Chakrabarti, 1980; Sastry and Jayaraman, whereon the plants lodge, shrivel and die.
Disease management in light soils during kharif and in heavy

soils during the early rabi season. It occu-
Draining out excess water from beds after pies an area of 17.50 hundred thousand ha
irrigation and avoidance of monocropping in India, with production of 587.1 thousand
may help to control the disease (Kolte, 1965). t. The overall productivity of this crop in
Use of resistant varieties US-10, Gila, Frio India is 335 kg/ha. About 72 fungi have
and VFR-1 is recommended. been reported on this plant in India (Vyas
et al., 1984).
Rust of safflower
Phytophthora blight of sesame

Uredosori of the obligate, autoecious, het-
erothallic, macrocyclic pathogenic fungus
P. carthami (Hutz) Corda contain numerous Phytophthora blight is caused by P. para-
globoid or broadly ellipsoid echinulate, light sitica var. sesame. It was first reported from
chestnut brown uredospores measuring India by Butler (1918). Now, it has become
21–27 µm × 21–24 µm, thick-walled and 3–4 an important disease of sesame and has
equatorial germpores (Singh, 1998). Saf- been reported from the Dominican Republic
flower rust caused by P. carthami, is an (Ciferri, 1930) and Argentina (Frezzi, 1950).
important disease in India. It causes a stand In India, it was severe in Madhya Pradesh
loss of 55–97% in susceptible varieties with Rajasthan, Uttar Pradesh and Gujarat
considerable yield loss, particularly if the (Vasudeva, 1961; Verma, 2002). This disease
infection starts early in the crop growth. The has caused 66% losses in Gujarat (Kale and
first pathological phase of the safflower rust Prasad, 1957) and 79.8% in Central Madhya
is seen in the seedling stage of the crop, when Pradesh (Singh et al., 1976). It may cause
orange to yellow spots representing pycnia even 100% loss under the most favourable
appear on cotyledons, which ultimately conditions for infection to occur severely at
leads to drooping and wilting of the plants. seedling stage. Disease occurs on all the
With the development of uredospores and aerial plant parts. The symptoms of the dis-
teliospores, the colour of the spots later ease appear as brown, water-soaked spots
changes to brownish black. The second on the leaves of seedlings at a very early
pathological phase of the rust is uredia devel- stage. Gradually, the spots increase in size.
opment on leaves, flowers and fruits, where Under favourable weather conditions, the
teliospores are formed later towards crop whole leaf rots and becomes black. Rotting
maturity when the atmospheric temperature progresses further and the whole stem is
rises (Schuster and Christiansen, 1952). rotted. Frequently, the attack on the seed-
ling starts at the collar region and gives
Disease management damping-off like symptoms. The cottony-
white growth of the fungal mycelia appears
Destruction of the infected host, crop debris on the lower side of the leaves and on pods
and collateral host Carthamus oxycantha under humid condition.
(Pohli weed) and crop rotation checks the
disease to some extent. Three sprays of tride- Disease management
morph (0.5%), thiophanate methyl (0.15%)
or tridimefon (0.1%) are effective against saf- Intercropping with soybean, castor, maize,
flower rust (Singh et al., 1997). Use of resistant sorghum and pearl millet in the ratio of 1:3
varieties APPR-1 and APPR-3 is suggested. or 3:1 shows a low incidence of the disease,
with a higher yield. Application of FYM
alone or neem cake with inorganic fertilizer
Sesamum (N60, P40, K20) reduces the disease as com-
pared to without FYM. Application of the
Sesame (Sesamum indicum L.) is an impor- species of Pseudomonas, Bacillus and Strep-
tant oilseed crop of India. It is grown mainly tomyces, which are most active at 25–27°C
at field capacity moisture level, can be sup- F. oxysporum f.sp. sesami completely (Hyun
pressive to Phytophthora species in soil. Seed et al., 1999).
treatment with vitavax (1.0 g/kg) and captan
(2.0 g/kg) controls seedling disease effectively.
Captan 75D is the best fungicide for reducing Alternaria leaf spot of sesame
the disease, followed by thiram 75D.
The pathogen is A. sesame (Kawamura)
Mohanty and Behera. The conidiophores of
Fusarium wilt of sesame
the pathogen are pale brown, cylindrical,
erect, not rigid and arise singly with a size
Fusarium wilt of sesame is quite serious of 30–54 × 4–7 µm. Conidiophores produce
wherever the crop is grown. In India, it has conidia at the apex, which are in chains of
been reported from all the sesame-growing one to two. The conidia are straight or
areas, such as Madhya Pradesh, Maharash- slightly curved, obclavate, yellowish brown
tra, Andra Pradesh, Rajasthan, Haryana, Pun- to dark brown in colour and measure
jab, etc. The disease is quite serious when it 30–120 × 9–30 µm. The disease affects all the
starts in the early stages of crop growth. The aboveground plant parts. The initial symp-
causal organism is F. oxysporum f.sp. ses- toms appear as small, brown, round to irregu-
ami. The fungus produces profuse light pink lar spots on the leaf blade. Later, the spots
mycelial growth on PDA. Microconidia are enlarge and turn dark with concentric rings.
hyaline, ovoid to ellipsoid, unicellular and On the lower surface of the leaves, spots are
produce abundantly even on the medium light brown in colour. The appearance of the
and are about 8.5 × 3.25 µm in size. The disease at the seedling stage can cause post-
macroconidia are produced abundantly in emergence damping-off. On capsules, small,
sporodochia and size ranges from 35 to brown spots appear which result in the for-
49 × 4.5 µm. The chlamydospores are glo- mation of shrivelled and deformed seeds.
bose to subglobose, smooth or wrinkled and
about 7–16 µm in diameter. The pathogen
Disease management
grows at a temperature range of 10–25°C,
with an optimum temperature of 26°C and a Application of Bordeaux mixture (0.1%)
pH of 5.6. The initial symptoms of the dis- and zineb (0.1%) has been reported to be
ease appear as yellowing of the leaves, which effective. Application of mancozeb (0.2%)
later droop and desiccate. On the infected at the time of disease initiation is effective
plant, the leaves may show inward rolling of in managing the disease.
the edge and eventually may dry up. If the
disease appears at the later stages of crop
growth, the symptoms may appear on one
side of the plant, resulting in partial wilting. Powdery mildew of sesame
Discoloration of the vascular system is con-
spicuous in the roots. This disease is common, especially in South
India. It has been reported that powdery
Disease management mildew of sesame is caused by Oidium ery-
siphoides, Leveillula taurica (Lav.) Trnaud,
Seed treatment with benlate (1.0 g/kg) and Sphaerotheca fuliginea (Schlecht) Pollacci
vitavax (1.0 g/kg) is most effective against and Erysiphe cichoracearum DC. The dis-
wilt. Application of conidial dust of Gliocla- ease causes considerable losses in yield,
dium virens gave better disease control. Sim- depending on the time of its appearance, as
ilarly, application of T. harzianum and T. well as the intensity of the disease. Powdery
viride in the field also reduced the incidence mildew causes a loss of 42%; every 1%
of wilt significantly. Soil drenching with increase in disease intensity results in a yield
antibiotic KB-8A isolated from B. polymyxa loss of 5.63 kg/ha. Four different fungi have
at a concentration of 13 µm/ml inhibited been reported to cause powdery mildew,
but in India E. cichoracearum is predomi- were best in controlling the disease. Resis-
nantly prevalent. Both conidia and ascospores tant varieties recommended are BIC-7-2,
on germination give rise to an abundant Sidhi-54, Rewa-114 and Seoni Malwa.
superficial mycelium of uninucleate cells,
which form a white coating on the leaf and
send haustoria into the host. The disease nor- Castor
mally appears after 45–60 days. The initial
symptoms appear as dirty whitish fungal Castor (Ricinus communis L.), belonging to
patches on the upper surface of the leaves. the family Euphorbiaceae, is the most
Later, these specks coalesce to cover the entire important non-edible oilseed crop of arid
leaf and result in premature defoliation. Gen- and semi-arid regions of India. Castor oil finds
erally, it affects the leaves but in severe cases, its application in the manufacture of a wide
the disease spreads to petioles and other plant range of ever expanding industrial products,
parts. In severe infection, pods or capsules are such as nylon fibres, jet engine lubricants,
shrivelled and produce smaller seeds. hydraulic fluids, dyes, detergents, soaps, oint-
ment, greases, paints, varnishes, cosmetics
Disease management and perfumes, etc. (Pathak, 2003).
Castor is grown in tropical and subtrop-
Two sprays of wettable sulphur (0.3%), ical climates; the major growing countries
dinocap (0.1%) or hexaconazole (0.1%) are India, China and Brazil. India occupies
at 15-day intervals can help to control the about 57% of the world castor acreage, but
disease. produces about 62% of world production.
The major castor-growing states in India are
Gujarat, Andhra Pradesh, Tamil Nadu and
Cercospora leaf spot/white Orissa. Productivity is highest in Gujarat
leaf spot of sesame state because more that 90% of the cultivated
area is covered by castor hybrids under irri-
Mycelium of Cercospora sesami Zimmer- gation. There are a number of diseases occur-
man is yellowish-white in colour and pro- ring on castor and the important ones are
duces profuse conidiophores in culture. explained below.
The conidiophores are olivaceous, septate,
usually single but sometimes up to 10, epi-
phyllous, nodulase, thickened towards the Alternaria blight of castor
tip, conidia with 7–10 septa and measure
about 90–135 × 3–4 µm. Generally, the symp- This is caused by A. carthami (Yoshii) Han-
tom of the disease appears at the time of flow- sford. The disease appears on leaves, stem,
ering, but the disease may also appear after inflorescence and capsules. At seedling
30–40 days after sowing. The initial symp- stage, light brown spots first appear on coty-
toms of the disease are circular spots scattered ledonary leaves, which become angular with
on both leaf surfaces. These spots enlarge rap- age. Severe infection results in the death of
idly and become up to 5 mm in diameter. The young seedlings or foliar blight. Symptoms on
spots are initially brown in colour with a adult plant leaves are brown, zonate and vari-
whitish centre, but later they may be brown to able in size and usually surrounded by yellow
dark brown in colour. The symptoms on peti- halos. In the case of severe infection, prema-
oles are visible as elongated lesions, whereas ture defoliation occurs. Sunken spots develop
on capsules they are more or less circular and on capsules on one side, which gradually
brown to dark brown in colour. enlarge to cover the whole capsule with fun-
gal growth. Such capsules are smaller in size
Disease management and have underdeveloped or wrinkled seeds
with little oil content. In heavily infected
Three sprays either of carbendazim (0.05%) field crop, all the young racemes and even
and topsin M-70 (0.2%) at 10-day intervals flower primordia are killed.
Disease management collar rot, root rot and twig blight. The dis-
ease appears at different phases as collar
Foliar application of mancozeb (0.2%) at rot, stem blight and root rot. Initially, the
intervals of 15 days starting from the appear- infected plant shows signs of water short-
ance of the disease is beneficial. Judicious age. Within a week, the leaves and petiole
use of nitrogenous fertilizers also reduces droop and finally, within a fortnight, the
the development of the disease. entire plant dries up and can be pulled up
easily. Collar rot phase is observed 30–40
days after sowing. Dark black discolorations
Botrytis grey rot of castor are seen at the collar region of the plant,
which gets sunken and later becomes abnor-
This is a very serious disease of castor as it mal. The affected tissue becomes shredded
affects the flowers and capsules directly and and weak and finally shows sign of wilting.
the entire crop may be lost if there are con- Stem blight symptoms appear slightly later,
tinuous rains during capsule formation. The due to aerial infection, as straw-coloured or
disease is confined to only a few states in brown depressed small lesions on the stem,
India and is serious in Andhra Pradesh and usually at the nodes. The lesions increase in
Tamil Nadu. It is caused by Botrytis ricini size by both upward and downward exten-
Godfrey. The disease is confined to spikes sion of the infection, resulting in a 2–20 cm
or racemes. Generally, pale to olive grey oval-shaped necrotic area. The surface of
coloured woolly growth of the fungus is the infected stem shrinks at this region and
observed on flowers or capsules. The disease the plant breaks easily at this point. The
appears initially as small blackish spots, affected spikes are discoloured, turn black
exuding a drop of yellow liquid. Fungal and dry up in the course of time. Infected
infection from these spots further spreads to capsules become discoloured and drop off
racemes. The infected flowers appear soft easily. In the case of the root-rot phase, the
due to the profuse growth and sporulation taproot shows signs of drying and the root
of the pathogen. This later turns to grey bark shreds off easily. Rotting sometimes
masses covered with dusty powder, result- spreads partly above the ground. At an
ing in the rotting of capsules. The unripe advanced stage, sclerotial bodies may be
seed becomes soft and mature ones hollow, seen as minute black dots on the surface of
resulting in a discoloured seed coat and loss woody tissues and in the pith region.
in seed weight.
Disease management
Disease management Crop rotation with non-host crops and
Adoption of wider spacing with varieties mixed cropping with moth bean can be
having open racemes reduces the severity of helpful in reducing the disease. Infected
the disease. Two prophylactic sprays of car- plant material should be collected and
bendazim (0.05%), one at 50% flowering burnt. Application of thiram (2.0 g/kg) or
and the other soon after the appearance of carbendazim (1.0 g/kg) as seed dresser along
the disease, reduces incidence of the dis- with spray and soil drench is recommended.
ease effectively. Topsin M-70 has also been found effective
for controlling root-rot disease in castor.
Macrophomina root rot of castor

Wilt of castor
Macrophomina phaseolina (Tassi) Goid is
reported to cause different symptoms on Wilt of castor is caused by F. oxysporum
castor, namely seedling blight, dieback due f. sp. ricini Nanda and Prasad. The extent of
to aerial infection, spike blight, stem blight, disease incidence has been up to 80% in
Russia (Moshkin, 1986). Losses in yield were plants, only one side of the root system is
realized in all cultivated castor hybrids in observed as being blackish and necrotic; the
Gujarat and up to 85% incidence of the dis- other side of the root system remains healthy.
ease has been reported in North Gujarat When the stem of the wilted plant is split
(Dange et al., 1997). Young seedlings at the open, a white cottony fungal growth is
two- to three-leaf stage exhibit discoloration observed in the pith region, which then
of hypocotyls and loss of turgidity, with or becomes blackish.
without change in colour. The mycelium
penetrates the vascular system of the roots, Disease management
stems and leaves causing necrosis, which
leads to wilting and finally death of the Use of healthy seeds, crop rotation, summer
plant. At the time of flowering and spike deep ploughing and field sanitation reduce
formation stages, the disease is character- the incidence of the disease. Use of bio-
ized by a gradual yellowing and shrivelling, agents like T. harzianum and T. viride have
with marginal and interveinal necrosis of been screened for their antagonistic activity
leaves. Infected plants rarely bear seeds and against castor wilt pathogen. Seed treatment
such seeds are deformed and light in weight. (1.0 g/kg) and pre-sowing soil application of
Roots of wilted plants show blackening and carbendazim at 3.0 kg a.i./ha with thiram
necrosis, while in the case of partial wilted (3.0 g/kg seed) is recommended.
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21 Occurrence of Pyrenophora
tritici-repentis Causing
Tan Spot in Argentina
M.V. Moreno1,2 and A.E. Perelló2,3

1Laboratorio de Biología Funcional y Biotecnología, CEBB, Facultad de Agronomía
de Azul, Universidad Nacional del Centro de la Provincia de Buenos Aires,
Buenos Aires, Argentina; 2Consejo Nacional de Investigaciones Científicas
y Técnicas (CONICET); 3Centro de Investigaciones de Fitopatología (CIDEFI),
Facultad de Ciencias Agrarias y Forestales, Universidad Nacional de La Plata,
La Plata, Provincia de Buenos Aires, Argentina
Abstract
Wheat (Triticum aestivum L.) is currently considered as one of the most important crops worldwide.
It can be affected by several diseases. However, only a limited number of them, like ‘tan spot’ resulting
from the fungus produced by Pyrenophora tritici–repentis, cause serious problems to the crop and may
be given special attention. Tan spot has significant economic consequences. In recent years, the
incidence of the disease has increased in many areas where wheat is cultivated, becoming a serious
problem by causing losses of up to 70%. It has been found in a lot of countries worldwide: North
Dakota, Nebraska and Kansas (USA), Canada, Australia, Asia, Pakistan, Czech Republic, Poland,
Ukraine, Hungary, France, Denmark and Belgium.
This disease has increased its incidence, prevalence and severity, particularly in the whole of the
South Cone region in the last few years: Argentina, Brazil, Bolivia, Colombia, Ecuador, Peru, Paraguay
and Uruguay. Tan spot is one of the most destructive and widespread problems of wheat production
in Argentina. In this chapter, we summarize the knowledge of many and diverse contributions and we
highlight what is known and unknown about the disease.
Introduction traded is greater than any other grain (Eikboir

and Morris, 2001).
Wheat is considered one of the most impor- Wheat has been one of the most impor-
tant crops of the world, along with rice, tant crops for the past 100 years in Argen-
maize and potato. Humans consume around tina. Between 2005 and 2006, 2m ha were
75% of worldwide production (Wiese, 1987; cultivated, giving a yield of 70–74 Mt (Inf.
Rajaram, 2001). In the period between 1970 Económico de Coyuntura No. 261, 2006).
and 2000, wheat yields rose at an annual The future of this production and South
rate of 2.3%; however, the area cultivated America’s participation in the international
remained the same. The volume of wheat market depended on how Argentina and

276 M.V. Moreno and A.E. Perelló
Brazil developed the crop. In Argentina, The disease began to affect wheat crops
planting in the field to exportation of the crop noticeably in the north-central region of the
was dependent on the introduction of new Buenos Aires Province in the early 1980s
technological developments. These devel- (Annone, 1985, 1996). Since then, tan spot
opments include new cultivar composition, symptoms have been detected in most
management of the crop and the manage- growing areas of the country. The disease
ment of future areas to expand yield (Eik- is particularly prevalent and intense in
boir and Morris, 2001). Fungal pathogens the northern area of the Argentine wheat-
are the result of a combination of these fac- producing region (central and northern
tors (Klein, 2001). Management of these dis- Buenos Aires, southern Santa Fe, south-
eases requires specific knowledge and an eastern Cordoba and Entre Rios Provinces),
increased ability to identify the fungus and where highly conducive environmental
techniques to reduce crop losses to a mini- conditions and increasing use of minimum
mum (Kohli, 1995). tillage have created a disease hotspot. In the
In the last few years, minimum tillage region, the pseudothecia of the pathogen are
has been considered advantageous to soil formed on wheat residue left on the soil sur-
conservation, but it leads to a loss of avail- face at crop sowing and/or early growth
able nutrients and a potential increase in stages. Conidia are formed and released
necrotic pathogens whose saprophytic stage soon after the development of the first symp-
lives in the straw of the crop (Annone, toms on leaves (Annone et al., 1994).
1985). Establishment of the crop under this Wright and Sutton (1990) observed that
management can be affected by pathogens of when P. tritici-repentis was introduced in
this type (Table 21.1). In Argentina, the an area of wheat, it was dominant over other
increased incidence in leaf spot since the leaf pathogens. In Argentina, tan spot is one
application of minimum tillage has been a of the most important diseases, along with
cause for concern (Annone and Kohli, 1996). rust and head blight (Annone, 2006). The
massive expansion of minimum tillage in
Argentina has encouraged the establish-
ment and development of this disease
Tan Spot (Annone and García, 2004).
The first time that tan spot was observed on

wheat was in the 1920s in Japan (Hosford,
1981). In 1954, the first loss (75%) was Importance
reported in Kenya (Gilchrist et al., 1984). In
1940, tan spot was reported for the first time Tan spot is frequently observed in most
in the USA (Barrus, 1942). At the present farmers’ fields, often affecting the upper
time, the name of the fungus causing tan leaves at flowering to early grain filling
spot is reported with high frequency in the stages. Yield losses of between 9 and 50%
wheat-growing areas of the world (Conners, have been observed by several authors (Hos-
1939; Tekauz, 1976; Watkins, et al., 1978; ford and Busch, 1974; Sharp et al., 1976;
Sim and Willis, 1982; Loughman et al., Rees et al., 1982; Rees and Platz, 1983). In
1998, Postnifova and Khasanov, 1998; Ali South America, yield losses of around 40%
and Francl, 2001a; Sarova et al., 2002). were observed by Mehta and Gaudencio
In South America, tan spot has been (1991) in Brazil and Kohli et al. (1992)
observed in Colombia, Ecuador and Peru reported wheat yield losses of between 20
(Dubin, 1983). It has recently gained pre- and 70% in Paraguay and Argentina. Esti-
dominance among wheat diseases in the mates of losses (10–20%) caused by the dis-
Southern Cone region of South America, ease have been made by comparing fungicides
comprising Argentina, Brazil, Chile, Para- protected with non-protected wheat plots
guay and Uruguay (Kohli et al., 1992; Lin- (unpublished data Annone, 1996). Similar
hares and da Luz, 1994). results were obtained by Galich and Galich
Tan Spot in Argentina 277
Table 21.1. Diseases of wheat and the agent cause (Annone and Kohli,
1996).
Pathogen Disease
Xanthomonas campestris pv. undulosa Bacterial stripe

Septoria tritici Spot blotch
Drechslera tritici-repentis Tan spot
Fusarium graminearum Head blight
Gaeumannomyces graminis var. tritici Take-all
(1994) in Marcos Juarez (Cordoba Province). with Winter that the presence or absence of
They determined that losses due to tan spot setae was not an important enough charac-
associated with Septoria tritici blotch ranged teristic to separate the two genera. He
between 6 and 13.5%. Tan spot is a complex emphasized the connection between Pyreno-
disease that is dependent on its geographic phora and the conidial stage Drechslera, as
location and the environmental conditions found by Fuckel for P. phaecomes. Drechsler
prevailing (de Wolf and Francl, 1998). too determined the connection between P.
teres and D. teres, P. tritici-repentis and D.
tritici-repentis, and P. bromi and D. bromi
The pathogen (Shoemaker, 1962). At the same time, Ito and
Kuribayaski (1931) connected five species
The tan spot fungus is an Ascomycota cur- of Pyrenophora with the conidial stage of
rently known as P. tritici-repentis (Ptr) Drechslera. In 1949, Wehmeyer worked on
(Died.) Drechs. It is a facultative pathogen the distinction in form and size of the Pleo-
whose asexual stage is Drechslera tritici- spora and Pyrenophora ascospores (Weh-
repentis (Dtr) (Died.). meyer, 1949).
P. tritici-repentis was isolated for the In 1930, Ito described the genera
first time from Agropyron repens in Ger- Drechslera. In 1809, Link described the gen-
many and it was named Pleospora trichos- era Helminthosporium, where species of
toma by Diecke. In 1928, it was isolated Drechslera were included (Ito, 1930). In
from wheat by Nisikado (Nisikado, 1928), 1902, Diedicke (Drechsler, 1923) determined
when it was named Helminthosporium H. tritici-repentis as formae of H. gramineum.
tritici-repentis (= Drechslera tritici-repentis) In 1923, Drechsler recognized H. teres, H.
(Hosford, 1981). bromi, H. gramineum and D. avenae as unique
The genera Pyrenophora Fr. was used species. In 1959, Shoemaker (1962) made the
frequently for some ascomycota parasitic on distinction between two subgenera, Cylindro-
cereals and other grasses (Diaz de Ackerman, Helminthosporium, in which all the species
1987). It was described by Fries in 1849 and have conidia germinating from all cells and
cited by Shoemaker in 1961 (Shoemaker, Eu-Helminthosporium, in which all the spe-
1962). In 1869, Fuckel noted the tendency cies have fusiform conidia germinating from
of P. phaecomes to mature only after over- end cells only. In 1930, Ito (Shoemaker, 1962)
wintering and found a Drechslera conidial proposed the name Drechslera for those spe-
stage of P. phaecomes (Shoemaker, 1962). cies with cylindric conidia germinating from
In 1883, Saccardo used the presence of setae all cells, using as a type D. tritici-repentis. He
on the ascocarp of Pyrenophora and the used the name Bipolaris for those species
absence of setae on the ascocarp of Pleo- whose conidia were fusiform, germinating
spora to separate these two genera. In 1885, from end cells only. In 1962, Shoemaker
Winter (Shoemaker, 1962) included the considered D. tritici-vulgaris as D. tritici-
species of both genera in Pleospora and in repentis. Currently, the teleomorphic nomen-
1934, Drechsler (Shoemaker, 1962) agreed clature of the fungus is P. tritici-repentis
and the anamorph of the fungus is unani- 9, 10, 11 and 12 (Lamari and Bernier, 1989a,b;
mously accepted as D. tritici-repentis. Mor- Lamari et al., 1995, 1998, 2003, 2005;
phological data can be found in Drechler Lamari and Gilbert, 1998; Ali and Francl,
(1923), Shoemaker (1962) and Wehmeyer 2001a,b, 2002a,b). Races 9 and 10 have been
(1954). identified in South America, which indi-
cates that the Ptr population is heteroge-
neous in this area (Ali and Francl, 2002b).
Host–Parasite Interactions In Argentina, the race population structure
is unknown and in 2007, Moreno observed
that isolates obtained from Argentina pro-
Symptomatology. On susceptible wheat leaves,
duced three reaction types on cultivars of
P. tritici-repentis(Ptr) produces characteristic
local and international wheat (Moreno,
oval to diamond-shaped lesions. However,
2007). Actually, the isolates were inocu-
newly formed tan spot lesions cannot be sep-
lated on different wheat sets to determine
arated reliably from those caused by other
the races present in Argentina.
necrotrophic pathogens. Later, lesions elon-
Ptr can also infect wheat seed during
gate and develop a tan colour with a chloro-
the grain-filling period (Schilder and Berg-
tic halo and a small dark brown infection
strom, 1994). This disorder is called red
site. Chlorotic areas tend to coalesce on heav-
smudge, because infected seed has a reddish
ily infected leaves, especially on young
discoloration (Valder, 1954).
plants, a symptom which leads to the disease
name, ‘yellow leaf spot’ (Fig. 21.1). On resis-
tant and partially resistant wheat, lesion size
is reduced and chlorosis and necrosis may Disease cycle
be absent (de Wolf et al., 1998).
Lamari and Bernier (1989a) identified Dispersal and infection by Ptr can develop
two different types of symptoms produced between 10° and 30°C with moisture
by the pathogen: tan necrosis and extensive between 6 h and 48 h (Larez et al., 1986;
chlorosis. However, they reported that the Hosford et al., 1987; Sah, 1994). These con-
pathogen isolates could be characterized by ditions are the reason why tan spot can
their ability to induce tan necrosis and/or occur all year round and which distin-
chlorosis. They grouped the isolates into guishes it from the white head disease, but
four pathotypes based on the production of they all depend on environmental condi-
different symptoms on different lines. In tions (Carmona, 2003).
this system, an unlimited number of isolates The disease cycle of tan spot (Fig. 21.2)
were designated as races 1, 2, 3, 4, 5, 6, 7, 8, provides a convenient framework on which
(a) (b)
Fig. 21.1. Pyrenophora tritici-repentis produces characteristic oval to diamond-shaped lesions.

Symptoms on leaf tissues
Primary infection
Conidia
Secondary host
Secondary infection
Primary infection
Seeds
Ascas
Fruiting bodies on stem
Fig. 21.2. Disease cycle of Pyrenophora tritici-repentis, agent cause of tan spot of wheat.
to explain our current understanding of the inoculum between growing seasons, as a

progress of the disease. The rate of progres- source of genetic variation and as a reser-
sion through the disease cycle depends on voir of a fungal population genetically dif-
the host and on temporal and environmen- ferent than that prevalent on wheat (de Wolf
tal components of the pathosystem (de Wolf et al., 1998). The tan spot fungus has been
et al., 1998). reported on many grass species from differ-
The seeds, straw and collateral hosts ent parts of the world, among which are
are the principal source of inoculum of tan Agropyron sp., Avena fatua, A. sativa, Echi-
spot. The primary inoculum can travel long nochloa sp., Elymus innovatus, Andropogon
distances through the wheat-growing areas gerardi, Alopecurus arundinaceus, Bromus
and is introduced into new areas by seeds. inermis, Dactilys glomerata, Lolium perenne,
In the seed, the pathogen lives in the Phalaris arundinaceae, Poa sp. and Secale
pericarp as mycelium and transmission cereale (Diedicke, 1902; Drechsler, 1923;
to the rest of the plant is non-systemic Conners, 1939; Dennis and Wakefield, 1946;
(Schilder and Bergstron, 1994). In Argen- Sprague, 1950; Andersen, 1955; Dickson,
tina, Barreto (1984, unpublished data) 1956; Shoemaker, 1962; Hosford, 1971; How-
observed infection on 2% of wheat seed. ard and Morral, 1975; Farr et al., 1989;
Future investigations are required to estab- Krupinsky, 1992c; Ali and Francl, 2002b).
lish the sanitatary management of seeds In Argentina, the host range is unknown.
(Carmona, 2003). Ascospores are generated in the pseudo-
Another source of primary inoculum is thecia that live in the wheat straw. The
wheat straw. Several authors consider straw conidia are formed in the straw containing the
as the principal source of the inoculum of pseudothecia and on the leaves of infected
Ptr (Rees and Platz, 1980). plants or the leaves of collateral hosts.
Collateral hosts of Ptr could play The ascospores of Ptr are dispersed pri-
an important role as a source of primary marily by wind, but the distance an ascospore
can travel is limited (Schilder and Bergstrom, 2003a). The growth stage seems to influence
1995). Limitations on ascospore dispersal tan spot severity and expression of resis-
distance have been attributed in part to tance (Hosford et al., 1990; Fernandez et al.,
short discharge distances from the pseudoth- 1994; Perelló et al., 2003a).
ecia. However, it is doubtful that the short Then, infecting the wheat leaves, conidia
discharge distance alone can account for are produced and the pathogen’s asexual
these short dispersal distances. Schilder cycle life develops by infecting new plants of
and Bergstrom (1992) proposed that move- wheat. Even so, conidiogenesis continues in
ment was limited during periods of high wheat straw. The production of conidia and
relative humidity when ascospores were the development of pseudothecia depend
discharged from the ascocarps (de Wolf on temperature and water potential. Stem
et al., 1998). Infested residue usually results colonization appeared to be the result of the
in significant disease severity at flag leaf progressive colonization of the leaf sheath
emergence and later growth stages due to and upper internode. No differences in sap-
secondary infections (McFadden and Hard- rophytic colonization were observed among
ing, 1989; Wright and Sutton, 1990; McFad- cultivars of varying resistance (de Wolf
den, 1991). et al., 1998). Numerous researchers have
Following liberation from the host, the investigated the factors affecting the initia-
conidia of Ptr can be sampled readily dur- tion and development of pseudothecia in
ing aerial dispersal and differentiated suc- laboratory experiments (Odvody et al., 1982;
cessfully from other fungi (Morral and Pfender and Wootke, 1987; Pfender et al.,
Howard, 1975; Rees and Platz, 1980; Wright 1988; Summerell and Burgess, 1988a,b,
and Sutton, 1990; Krupinsky, 1992b; Maraite 1989; Zhang and Pfender, 1993).
et al., 1992; Schilder and Bergstrom, 1992; The effects of water potential in wheat
Wolf and Hoffmann, 1993). Morrall and straw on pseudothecial development have
Howard (1975) reported that conidia num- also been studied in an outdoor environment
bers reached their highest levels late in the (Fernandes et al., 1991; Zhang and Pfender,
growing season and that the number of 1993). The number of ascocarps per gram of
conidia has a clear diurnal periodicity. The straw in near-soil straw was 32% and 42%
numbers of conidia of the pathogen decline of that found in mowed and no-till treat-
sharply with dispersal distance. Schilder ments, respectively. In addition, the num-
and Bergstrom (1992) reported that the high- ber of ascocarps produced in the lower
est number of conidia occurred within 3 m portion of standing stubble of no-till plots
of the inoculum source and that 60–100% was 12% of the number found in the upper
of the recoverable conidia were sampled portion.
within 25 m. Only a few conidia could be Reports of pseudothecia maturation in
recovered 100 m away from the inoculum an outdoor environment vary from region to
source, but this suggested that longer dis- region (Rees and Platz, 1980; Odvody et al.,
persal distances were possible. When the 1982; Summerell and Burgess, 1988b, 1989;
conidia were deposited on the leaf, their ger- Wright and Sutton, 1990; Wolf and Hoff-
mination was influenced by both tempera- mann, 1993). In most regions where wheat
ture and the availability of free moisture is grown, the pseudothecia of Ptr are initi-
(Mihtra, 1934). The conditions that contrib- ated when the crop has reached full matu-
ute to infection by Ptr in an outdoor envi- rity and begins to senesce (Odvody et al.,
ronment have also been studied (Ali, 1993; 1982; Wolf and Hoffmann, 1993). However,
Francl, 1998; de Wolf and Francl, 1997). The in colder climates, pseudothecia may not be
precise range of temperatures optimal for initiated until the following growing season
disease development varies with cultivar (Fernandez et al., 1998).
(Luz and Bergstrom, 1986). Leaf age affects In Argentina, the sexual stage of Ptr has
the severity of the disease caused by Ptr been detected in wheat straw, but it is
(Cox and Hosford, 1987; Lamari and Bernier, unknown in which regions and under what
1989a,b; Hosford et al., 1990; Perelló et al., conditions development took place.
Physiological Specialization differences in lesion length and percentage

of severity among isolates of Ptr obtained
The terms ‘pathogenicity’ and ‘virulence’ from Bromus inermis.
are likely to be used to describe the ability Lamari and Bernier (1989a) grouped
of an organism to cause disease. Pathogenic- the isolates of Ptr into three pathotypes on 11
ity is regarded as a general attribute of a spe- cultivars of wheat based on the type of reac-
cies, while virulence is an attribute reserved tion. Schilder and Bergstrom (1990) tested
for a particular strain of a pathogen in rela- 70 isolates obtained from Canada on 12
tion to a particular host genotype (Day, 1960). wheat cultivars and detected significant dif-
There exist virulent races of Ptr that interact ferences among the interaction of isolate ×
with wheat hosts in a highly specific man- cultivar. Some results were reported by Sah
ner. This suggests that host-specificity attri- and Ferhmann in 1992 for isolates originat-
butes are superimposed on the general ing from Brazil, Germany, India, Nepal and
pathogenic ability of Ptr. the USA. However, Krupinsky (1992a,b)
Variation in virulence in the popula- detected variation among levels of aggres-
tion of this pathogen is essential in under- siveness but he found no differences in lev-
standing the interaction of the genomes els of virulence. In 1992, Ali and Buchneau
involved in tan spot. Studies of the diver- observed physiological specialization based
sity of virulence within a pathogen popula- on the reaction type for isolates obtained from
tion should help in the development of a the USA. Mehta et al. (2004) tested 40 isolates
successful disease management programme, obtained from Parana (Brazil) on six wheat
particularly resistant cultivars. Several inves- cultivars; they observed low interaction for
tigators have described diversity among Ptr isolate × cultivar. In 2007, Moreno detected
isolated from different areas around the significant differences in isolate × cultivar for
world (Christensen and Graham, 1934; Misra isolates of Ptr obtained from wheat-growing
and Singh, 1972; Luz and Hosford, 1980; Gil- areas in Argentina.
christ et al., 1984; Krupinsky, 1987, 1992a,b; Races 1, 2, 3 and 4 of Ptr correspond
Diaz de Ackermann et al., 1988; Lamari and with those determined by Lamari et al.
Bernier, 1989a; Schilder and Bergstrom, (1995). Races 1 and 2 are predominant in
1990; Ali and Buchenau, 1992; Sah and Fer- North America (Ali and Francl, 2003). The
hmann, 1992; Brown and Hunger, 1993; greater part of isolates identified as race 5
Moreno, 2007). originate from North Africa, North America
In 1971, Hosford observed differences and Azerbaijan (Ali et al., 1990; Lamari
between the reaction type on wheat culti- et al., 1995, 1998; Strelkov et al., 2002; Ali
vars produced by isolates of Ptr. Misra and and Francl, 2003). Races 6, 7 and 8 were
Singh (1972) tested isolates originating from identified from collections originating from
India and they detected significant differ- Algeria, Caucaso and South America (Ali
ences in virulence, based on lesion size. and Francl, 2002a; Strelkov et al., 2002;
Some results were observed by Gilchrist Lamari et al., 2003). Finally, races 9 and 10
et al. (1984) when they tested isolates col- were identified from isolates originating
lected from Mexico on the wheat cultivar from South America (Ali and Francl,
Morocco. Luz and Hosford (1980) grouped 2002a,b).
the isolates tested into 12 races based on These studies indicate that variation in
statistical mean separation. However, Díaz the pathogen population can be detected by
de Ackermann et al. (1988) did not find any using either quantitative or qualitative rating
difference in virulence among the isolates scales (Table 21.2). Research using quantita-
tested by Luz and Hosford (1980). Hunger tive scales generally detected variation in
and Brown (1987) tested nine isolates origi- virulence on susceptible lines, but isolates
nating from the USA; these isolates showed in different studies produced an equal reac-
significant differences on the susceptible tion on resistant cultivars (de Wolf et al.,
cultivar TAM 105. Krupinsky (1987) showed 1998).
Table 21.2. Relationships between pathotypes, races and wheat cultivars.
Cultivars/lines of wheat
Races Glenlea Katepwa 6B662 6B365 Salomouni M3
1 N (Tox A) N (Tox A) R Cl (Tox C) R R

2 N (Tox A) N (Tox A) R R R R
3 R R R Cl (Tox C) R R
4 R R R R R R
5 R Cl (Tox B) Cl (Tox B) R R R
6 R Cl (Tox B) Cl (Tox B) Cl (Tox C) R R
7 N (Tox A) N (Tox A) Cl (Tox B) Cl (Tox B) R R R
8 N (Tox A) N (Tox A) Cl (Tox B) Cl (Tox B) Cl (Tox C) R R
Note: R, resistance; N, necrosis; CL, chlorosis; Tox A, presence of Tox A and production of Tox A; Tox B, presence of Tox
B and production of Tox B.
In Argentina, the race population struc- Strategies used for the control of tan
ture is unknown. Future research studying spot are the application of fungicides, cul-
physiological specialization in Ptr should tural control and the search for new germ-
consider collections originating in Argentina. plasms and their incorporation in Argentina
(Carmona, 2003).
Recently in Argentina, several biologi-
Disease Management Strategies cal antagonists of Ptr have been identified
(Pfender et al., 1989; Li and Sutton, 1995;
Perelló et al., 2003b; Annone, 2005).
From the point of view of the disease’s
development, its management is achieved
in different ways: by reducing or delaying
the disease early in the growing season or Genetic resistance
by reducing its rate of development during
crop growth (Zadoks and Schein, 1979). Genetic resistance is complex for diseases
This practice has helped to block the life such as head blight and leaf spot. The prin-
cycle of the pathogens, preventing the intro- cipal limitations are due to the changes
duction of inoculum and susceptible hosts, made by pathogen populations over the
eliminating certain pathogens (Palti, 1981). years to challenge new cultivars (Carmona,
Tan spot is one of a complex of necro- 2006).
trophic leaf diseases of wheat which over- Unfortunately, only a few of the cur-
winter on infested crop residue (Hosford rently grown cultivars have a high level of
and Busch, 1974; Loughman et al., 1998; resistance, while somewhat larger numbers
Carmona, 2003; Annone, 2006). The occur- possess a moderate level of resistance (Rees
rence of tan spot with other leaf spots, such and Platz, 1992). Kohli et al. (1992) reported
as septoria blotch, spot blotch and with the low presence in South America of culti-
rusts and mildews, can complicate disease vars resistant to Ptr. Several studies have
management practices (de Wolf et al., 1998; been conducted in Argentina to screen
Carmona, 2003; Annone, 2006). The man- breeding material for resistance (Galich and
agement of tan spot is based on integrated Galich, 1994; Annone, 1995).
management of diseases that use reasonable In Argentina, cultivars have either a
techniques and resources for sustainable moderate level of resistance or are suscep-
agriculture (Carmona, 2006). tible to tan spot (Simón, 2006).
Chemical protection typically increase in incidence and severity

(Rees and Platz, 1979; Mehta and Gauden-
Fungicides offer a complementary tool to cio, 1991; Kohli et al., 1992). The rotation of
the genetic resistance available. Its use in crops has a high impact on the sexual stage
direct seeding crops under-compensates for of this type of pathogen. Because sexual
the lack of genetic protection to facultative stage viability is minor or low when wheat
parasites. Fungicides are used as seed pro- straw is mineralized, the primary inoculum
tection and/or treatment coverage with is therefore low and reduces the severity of
ground or air equipment. tan spot (Carmona et al., 1999).
The majority of literature regarding the In relation to crop rotation, of relevance
use of fungicides to manage tan spot alone or was wheat as an antecessor of barley and
in combination with other leaf diseases has oats as an antecessor of wheat and barley as
focused on the timing of application and an antecessor of oats (Carmona et al., 2001).
comparative efficiencies. Research results Barley, wheat and oats are common
have been mixed, but it appears that in situ- hosts of Ptr and other pathogens, so there
ations where disease pressure is high and are therefore no alternative crops available
conditions favour further development of for crop rotation. Oats were not hosts to leaf
foliar disease, a single well-timed applica- spots specific to wheat, so crop rotation is
tion of an efficacious fungicide can reduce possible in Argentina. However, in Brazil,
disease severity, increase yield and improve Paraguay and Uruguay, where B. sorokini-
product quality (Sutton and Roke, 1986; ana is a relevant pathogen, rotation of these
Bockus et al., 1992; Duczek and Jones-Flory, crops should not be authorized (Carmona,
1994; Stover et al., 1996). 2006).
Fungicides such as tebuconazol, fru-
tiafol, fluzilazol propiconazol and prochlo-
raz reduced the intensity of lesions and
showed control of from 50 to 70%, depend- Biocontrol
ing on the cultivar and the density of wheat
straw infested (Annone et al., 1994; Car- Biological control using antagonistic micro-
mona, 1996). The most efficient fungicides bes alone or as supplements has become
are systemic triazoles and estrobirulinas more important in recent years in order to
(Carmona, 2003). minimize the use of chemicals (Annone,
2005). It is an additional tool available for
the design of more sustainable control strat-
egies of wheat diseases. While biological
Cultural control control is a widespread natural phenome-
non, it is often inadequate, especially in
Cultural practices alter the development of agricultural ecosystems in which condi-
foliar diseases of wheat, particularly those tions strongly favour pathogens and disease
caused by facultative pathogens. Tan spot, epidemics. It can also fail in natural ecosys-
representative of the latter group of dis- tems, especially against aggressive alien
eases, is affected by tillage practices in pathogens that are able to overcome the nat-
almost all wheat-growing regions of the ural biological buffering of these systems
world (Mehta and Gaudencio, 1991). (Sutton, 2005).
Retention of wheat residue on the soil Several biological antagonists of Ptr
surface generally results in increased tan have been identified (Pfender et al., 1989; Li
spot severity (Gough and Ghazanfani, 1982; and Sutton, 1995; Perelló et al., 2003b; Per-
Summerell and Burgess, 1988a,b; Schuh, elló et al., 2006, 2009). Luz et al. (1998)
1990; Bockus and Claasen, 1992; Stover found that treatment with Paenibacillus
et al., 1996; Carmona and Reis, 1998). maceruns or Pseudomonas putida reduced
In areas where zero tillage is practised, transmission of Ptr by seed to levels equiva-
tan spot and other debris-borne diseases lent to that of a fungicide seed treatment.
Some of the fungi found to be inhibitory to development of Ptr and the severity of dis-
pseudothecia development by Ptr were eases on wheat plants (Perelló et al., 2003b,
Limonomyces roseipellis, Myrothecium ror- 2006, 2008, 2009). No previous records of
idum, Acremoniun terricola, Stachybotrys antagonism between isolates of Trichoderma
sp. and Laetisaria arvalis (Gough and Ghaz- spp. and the necrotrophic foliar pathogen
anfani, 1982; Pfender et al., 1989). Assays have been found. On the other hand, there
in Argentina have demonstrated that some are increasing economic and social pres-
Trichoderma harzianum isolates are capa- sures to develop usable biological control
ble of suppressing growth, the mycelial strategies in Argentina.
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22 Epidemiological Studies on Septoria
Leaf Blotch of Wheat in Argentina
Cristina A. Cordo
Comisión de Investigaciones Científicas de la Provincia de Buenos Aires,
Centro de Investigaciones de Fitopatología (CIDEFI) – Facultad de Ciencias
Agrarias y Forestales, La Plata, Argentina
Abstract
This chapter introduces the detailed and novel contributions on the epidemiological spread of
Mycosphaerella graminicola over the wheat field and over time. The within-season and between-crop
methods of multiplication, survival and their environmental relations are reviewed. Genetic arguments
are given to demonstrate the influence of ascospores as the major source of movement of the pathogen
into new fields. Coupled with the evidence that populations worldwide are genetically very similar, it
does seem possible that a novel form of the pathogen has been spreading worldwide. This would raise
the interesting question as to what epidemiological characteristic confers the new form’s invasiveness.
There is a clear association between the evolution of the disease and weather conditions. Wheat cultivars
exhibit differential responses to infection by M. graminicola. Breeding for disease resistance is an impor-
tant tool in the integrated management of disease. Also, fungicide application and the use of biocontrol
organisms alone or in combination with fungicides is mentioned as other integrated action.
Introduction global average yield must increase from the

current 2.5 t/ha to 3.8 t/ha. In 1995, only 18
Cereals and the processed foods derived countries worldwide had an average annual
from them are still the principal sources of rate of growth over 2% between 1961 and
nutrition in many parts of the world. Bread 1994 (INTA-CIMMYT, 1996). In Western
wheat (Triticum aestivum L.) is the most Europe and North America, the annual
widely grown and consumed food crop. It is growth rate for yield was 2.7% from 1977 to
the staple food of nearly 35% of the world’s 1985, falling to 1.5% from 1986 to 1995
population and the demand for wheat will (Rajaram, 1999). Argentina, with a produc-
grow faster than for any other major crop tion of 16.11 Mt in the 2006/07 campaign
(Rajaram, 1999). The forecasted global demand (Encuesta Agrícola, DIEA-MGAP) and
for wheat in the year 2020 varies between 17.47 Mt in the 2007/08 campaign (OPYPA,
840 (Rosegrant et al., 1995) to 1050 Mt 2008), is an important wheat exporter with
(Kronstad, 1998). To meet this demand, a volume of 300.00 t and an average yield of
global production will need to increase by 2800 kg/ha (data from OPYPA yield and
1.6–2.6% annually from the present pro- production). The increased need for wheat
duction level of 560 Mt. For wheat, the export forces producers to improve soil

292 C.A. Cordo
management and plant cultivation strate- would be equivalent to the cost of fungicide
gies, including the application of crop pro- application. The threshold values estab-
tection agents to reduce losses, and is lished should represent infection limits
associated with the effective use of mea- beyond which economic losses are highly
sures designed to increase yield and ensure likely in the shorter or longer term, and the
quality. With the increased proportion of point at which the pathogen population
conservation tillage practice and with the reach this limits in the field crop determines
striving for optimal exploitation of the yield the time at which fungicide should be used.
and quality potential of cultivars with the The pathogen-specific thresholds must be
aid of appropriate cultivation and fertiliza- worked out within the framework of exact
tion measures, the importance of certain scientific investigations in relation to the
fungal diseases as yield-limiting factors has crop management methods used and the
increased considerably. The occurrence of environmental conditions prevailing during
fungal pathogens can not only limit cereal the growing period. Their development
production in temperate climates, but can requires extensive case studies (under out-
also jeopardize the requisite return on capi- door conditions) in order to establish how
tal under conditions of intensive farming. the population dynamics and detrimental
The epidemic development of pathogens, effects are influenced by the weather, wheat
which vary greatly in their ecological require- varieties, use of fertilizer, the preceding
ments, is strongly dependent on the weather crops and the level of inoculum. The pur-
and, together with the slightly different cul- pose of collecting all this information is to
tivation systems, this leads to differences in control individual pathogens with appro-
the type of infection (pathogen species) and priate products at appropriate application
in the level of infection (severity of the dis- rates, and to do this at a time at which one
ease) from one year to the next. Given these application is most likely with regard to
circumstances, the level of application of pathogen development and limitation of
fungicides in cereal cultivation in Argen- damage, using the lowest possible input.
tina has increased since 1996. Its applica- Epidemiology and population genetics
tion produced an increased yield of 20–32% are different but related subsets of popula-
in relation to the control test with respect to tion biology. Epidemiology focuses on dis-
time of application, fungicide molecular type ease progression, the increase in pathogen
and wheat variety (Annone et al., 1995). populations through time and the move-
Chemical crop protection generally has ment of pathogen populations through
been accepted in Argentina because it space (usually from plant to plant). Most
should only be used when circumstances epidemiology studies deal with a short
make it necessary to achieve the production timescale (e.g. 1–2 growing seasons) and
target and when all other options have been small spatial scales (e.g. disease develop-
considered. Crop protection measures should ment in a field or a plantation). Epidemiol-
be harmonized with the actual infection ogy involves mainly physical processes
situation prevailing in the crop and targeted such as distances of spore movement or
countermeasures must be initiated only effects of weather variables on latent peri-
where there is a real risk. Cortese et al. ods. It does not take account of the differ-
(1998) and Carmona et al. (1999) have fixed ences in behaviour or genetically distinct
the economic damage threshold (UDE) and individuals in a collection of individuals.
an action threshold (UDA) for different dis- Population genetics focuses on the pro-
eases on wheat. UDA represents the inci- cesses that lead to genetic changes, or evo-
dence value of the disease to decide the lution, in populations over time and space.
fungicide application on the crop to reduce Population genetics deals mainly with
the cost of application. UDE is based on the genetic processes such as genetic drift, gene
formula of Munford and Norton (1984) and flow, mating system, natural selection and
represents the value of the disease when mutation. Present study records that epide-
the yield losses produced by the pathogen miological investigations, based on disease
Septoria Leaf Blotch of Wheat in Argentina 293
evolution, resistance supply, early detec- information on resistance sources for diverse
tion of the disease, biological crop protec- pathogen populations under variable envi-
tion and genetic studies of the pathogen, ronmental conditions. Differences among
can be used to orientate the management of accession reaction were significant due to
disease under natural conditions. the rich composition of the selected sources
of resistance identified by CIMMYT and a
tentative group of differentials proposed by
The Disease as a Problem Eyal (Gilchrist et al., 1999) that were incor-
porated into the SMNs.
The inoculum for grain application was
Septoria tritici blotch (STB) is caused by M.
prepared in sterilized 500-ml flasks with
graminicola (Fuckel) Schroeter, in Cohn,
100 g of oat grains and 50 ml of a liquid
which is the teleomorphic stage of S. tritici
extract malt medium (Perelló et al., 1997).
Roberger and Desmazieres (anamorph
The grains were soaked with 10 ml of an
stage). Sanderson (1972) proved the con-
inoculum suspension (107 conidia/ml) of S.
nection between the two stages and the sex-
tritici isolate and incubated for 15–21 days
ual (teleomorph) form has been reported in
at 23 ± 2°C in darkness and shaken daily to
several countries (Hunter et al., 1999).
promote good fungal growth. After the incu-
Cordo and Arriaga (1990) reported the sex-
bation, the grains were colonized by a stro-
ual stage in Argentina. It is also known to
matic mycelium and were spread and dried
play a role in the disease’s cycle. It causes
on trays under laboratory conditions. The
most of the initial infection in winter wheat
covered grains were spread on to the soil
crops, during the autumn in the UK (Shaw
next to the plants during the tillering growth
and Royle, 1989) and USA (Schuh, 1990). In
stage (GS23, Zadoks et al., 1974). Plants in
Argentina, an increase in ascospores at har-
the plots were assessed for S. tritici infec-
vest time has been reported, suggesting that
tion at anthesis (GS60) and at the medium
the sexual stage may be important in initiat-
milk (GS75) stages.
ing the infection in the next growing season
The accessions (1-BOBWHITE S; 2-TIA.
(Cordo et al., 1999). Another possible means
2/4/CS/TH.CU//GLEN/3/ALD/PVN;
of spread within a crop during summer is by
3-CHIRYA.1; 4-CHIRYA 4; 5-CS7TH.CU//
airborne ascospores, which may play a more
GLEN/3/ALD/PVN/4/NANJING; 6-EG-A/H56
major role than previously recognized
7.71//4#EG-A/3/2#CMH79.243; 7-MH86.540-
(Hunter et al., 1999; Cordo et al., 2005).
A-1Y-3B-2Y-1B-1B-1B-1Y-1M-1Y; ALD/PVN//
YMI#6; 9-SHA5/BOW; 10-ENCOY 1582–1B;
11-BOBWHITE S as the other derivative line;
Studies on the Disease’s Evolution 12-DON ERNESTO INTA; 13-SERI M82; 14-
BETHLEHEM; 15-LAKHISH; 16-KAUZ; 17-
Several control methods, including the use PENJAMO; 18-ETIT 38; 19-GLENNSON M81)
of fungicides and other cultural practices, were sown in a factorial design experiment.
may reduce the effect of STB, but genetic The pulverization inoculum was pro-
resistance is the most cost-effective and duced using the same isolate as in the previ-
environmentally safe technique to manage ous year. The conidial concentration of the
the disease. suspension was adjusted to 1 × 107 conidia/
In Argentina, an inoculation technique ml. A comparison between the pulveriza-
using oat grains covered with the stromatic tion and the grain application methods was
mycelia of S. tritici were presented to check made in the field in 2000. The inoculum
the resistance of the Septoria Monitoring suspension was sprayed on to the leaves at
Nursery (SMN) set. The international set the tillering stage (GS23). After inoculation,
created by the CIMMYT provides informa- plants were kept moist by sprinkling water
tion on the interactions of pathogen × culti- several times a day over 3 days. The sever-
var on different regions of the world. The use ity of the infection was registered on the flag
of this set allows the generation of extensive leaf at the beginning of the flowering (GS60)
294 C.A. Cordo
and medium milk (GS75) stages using a ‘S’ germplasm and its derivative lines (in
modified double digit Saari–Prescott scale Argentina represented by Don Ernesto INTA)
(Saari and Prescott, 1975). The cut for resis- showed variable levels of resistance caused
tant behaviour was estimated as 5.3 (Gil- by its background with more than one genetic
christ et al., 1999). Weather variables (daily source and the presence of a low number of
temperature, relative humidity and rainfall) major genes (Cordo et al., 1994).
were recorded from the date of inoculation Plant height was not associated with
to anthesis. Plant height was evaluated. the resistant reaction. The negative associa-
To compare the inoculation techniques, tions were present when weather condi-
both the necrotic coverage percentage (NCP) tions were less conducive to the development
and pycnidial coverage percentage (PCP) of the disease. Non-conducive conditions
were scored on the upper three leaves of 15 and the further distance between leaves in
plants, 21 days after inoculation. The cut- tall cultivars could have reduced the rain-
off point between resistant and susceptible splash dispersal of pycnidiospores, thus
response classes was 16.8% NCP following causing this negative association (Arama
Eyal et al. (1985). The comparison between et al., 1999; Simón et al., 2005; Arraiano
pulverization and grain application showed and Brown, 2006); it could also depend on
that, except for the variety Bobwhite ‘S’ CM the presence of ascospores, which could
33203-K-10M-7Y-3M-2Y-1M-OM and the reduce the effect of plant height on the
line Tia.2/4/CSTH.CU//GLEN/3/ALD/PVN expression of the disease. In Argentina, the
CIGM88.734-1B-3PR-0PR-1M which reacted presence of the teleomorphic stage during
as in the observations of Gilchrist et al. the whole growing period has been reported
(1999), all genotypes were more susceptible (Cordo and Arriaga, 1990; Cordo et al., 1999,
under Argentine conditions. The higher 2005).
level of virulence of the Argentine isolates The modified double digit Saari–Prescott
and frequency of variation could explain scale was adopted for evaluation of this set
this behaviour (Eyal et al., 1985; Gilchrist (CIMMYT, rules for evaluation, Eyal et al.,
et al., 1999; Cordo et al., 2006). 1987). The separate analysis of digit 1 and 2
The results of the severity for NLP and allowed the relative height reached by the
PCP in this study are in agreement with pre- disease to be shown simultaneously with
vious research (Eyal, 1985; Gilchrist et al., the severity of the damage (PCP) (Table 22.1).
1999). The advanced resistant lines coming The differences observed for the first digit
from the crosses with a group of resistant Chi- in the accession response to the inoculum
nese lines did not show a high level of resis- concentration were attributed to the maxi-
tance (Ald/Pvn/YM#6, Milan/Sha#7, Catbird, mum level of attacked leaf (8th leaf) that
Talhuen INIA, Sha3/Seri/PSV/Bow and the was reached with the highest concentration
cultivar with Kavkaz/K4500 sources). of inoculum: 280 g. In contrast, the second
The resistant check Bethlehem was not digit did not show differences for either
resistant at CIMMYT or in our conditions. concentration. The lesions were restricted in
The bread wheat checks SeriM82 and extension, reaching only a maximum of 20%
Glennson M81 (with Veery ‘S’ germplasm) more of the PCP in the cases of highest sus-
and Lakhish were susceptible, as was ceptibility. This result confirmed that the
expected (Gilchrist et al., 1999). The durum level of resistance of tested materials was
wheat ETIT 38 and the resistant check Beth- adequate to maintain a low intensity of
lehem had the same level of susceptibility infection according to the objectives pro-
as bread wheat checks (SeriM82, Lakhish) posed by Eyal and Gilchrist at the beginning
and as was scored by Kohli (1995). The dis- of this project (Gilchrist et al., 1999).
ease resistance introduced from Brazilian Two factors were influencing the exp-
germplasm was detected on a short, early- ression of the disease on the leaves: the
maturing resistant line derived from IAS 20 concentration of the grain inoculum (120 g/
spring wheat and a more susceptible reac- m2 was optimum for differentiation between
tion on lines derived from IAS 58. Bobwhite susceptible and resistant accessions) and
Table 22.1. S. tritici infection average (digit 1 and 2) for different concentrations of inoculum and
different years.
Accession Inoculum concentration Years
Digit 11 Digit 21 Digit 11 Digit 2
n C1 C2 C1 C2 1997 1999 1997 1999
1 6.00 b2 6.50 b 2.50 b 1.00 a 6.50 b2 6.00 b 2.50 b 1.00 a

2 5.50 b 7.25 d 2.25 a 0.75 a 6.75 b 6.00 b 2.50 b 0.50 a
3 2.50 a 7.50 e 2.75 c 1.75 a 5.50 a 4.50 a 3.00 d 1.50 a
4 7.25 c 7.25 c 0.50 a 1.50 a 8.00 f 6.50 b 1.75 a 0.25 a
5 6.75 b 7.25 d 2.00 a 2.25 a 7.00 c 6.00 b 2.75 c 1.50 a
6 6.25 b 6.75 b 1.25 a 1.75 a 6.75 b 6.25 b 1.75 a 1.25 a
7 6.50 b 8.00 g 1.00 a 0.75 a 7.75 e 6.75 b 1.00 a 0.75 a
8 6.50 b 6.75 b 2.75 c 0.50 a 7.00 c 6.25 b 2.75 c 0.50 a
9 6.50 b 5.75 b 1.50 a 2.00 a 6.25 b 6.00 b 2.50 b 1.00 a
10 6.00 b 7.75 f 1.00 a 1.75 a 7.25 d 6.50 b 1.75 a 1.00 a
11 6.50 b 7.00 c 0.75 a 2.00 a 7.75 e 5.75 a 1.00 a 1.75 a
12 6.25 b 7.00 c 1.00 a 5.00 g 7.25 d 6.00 b 3.25 d 2.75 c
13 6.75 b 7.25 d 0.75 a 1.50 a 7.75 e 6.25 b 0.50 a 1.75 a
14 7.00 c 7.25 c 1.25 a 2.25 a 8.00 f 6.25 b 2.75 c 0.75 a
15 6.25 b 6.50 b 4.00 f 3.00 d 6.75 b 6.00 b 3.25 d 3.75 e
16 7.00 c 7.25 c 3.00 d 3.50 e 8.00 f 6.25 b 2.50 b 4.00 e
17 6.75 b 7.75 f 2.25 a 2.25 a 8.00 f 6.50 b 3.25 d 1.25 a
18 6.50 b 7.75 f 2.00 a 0.50 a 7.750e 6.50 b 1.25 a 1.25 a
19 6.50 b 7.75 f 2.61 c 1.50 a 7.50 d 6.75 b 3.25 d 0.86 a
Note: 1As assessed by a modified double-digit Saari–Prescott scale (1975). 2Mean values followed by the same letter
are not statistically different. LSD test (P < 0.01); C1 = 120 g/m2; C2 = 280 g/m2.
the wet environment. For the grain applica- practically stopped the development of the
tion treatment, the density of the plants was fungus at the end of GS60.
too important for rainfall to produce the The higher values of the disease in 1997
infection. If the rain regime was not frequent compared with those of 1999 were caused
and intensive, the pycnidiospores could not by the influence of the climatic conditions.
reach the higher leaves, making it difficult The temperature was not an important factor
for the inoculum to ascend. because there was no statistical difference in
In the pulverization treatment, the sur- 3 years of experiments. In 1998, high humid-
face covered by the inoculum included ity (30% more than the following year) and
more than one leaf stratum. A simultaneous increased rainfall (425.29 mm more than
proliferation of the pathogen was obtained the following year) were responsible for the
in all foliage levels, which, in addition to rapid increase of the disease compared with
the beneficial structure of the canopy, pro- results of 1999 (data not shown).
duced the highest values of severity. The Both inoculation techniques were
most susceptible varieties at the GS75 stage appropriated to monitor the behaviour of
were those that had a longer period of green the accessions of the SMN set. If the experi-
leaf during the growth cycle; but something mental field is under a good rain regimen
different occurred with ETIT 38 that did not from tillering to flowering, grain application
show any difference on NCP and PCP for is recommended. But if it is on a dry irri-
both growth stages. This could be explained gated area, pulverization with extra irriga-
by the quick senescence of the leaves that tion as a humidity chamber is suggested.
296 C.A. Cordo
Comparing the efficacy of each inocula- had the advantage that the incubation
tion technique, different symptoms produced period of the disease was maintained with
by each treatment at the beginning of the irrigation. In this case, a known weight of
disease have been associated with environ- inoculated grains was spread on to the soil,
mental conditions (Table 22.2). In relation between the rows, as primary inoculum. In
to the grain application treatment, the gen- this technique, pycnidia on stromatic myce-
eralized necrosis and pycnidial develop- lia formed on grains could release pycnidio-
ment on the lower leaves could have risen to spores over a long period if wetted. This
the upper leaves if irrigation or rainfall had process may be repeated several times if the
been present at this early stage of infection. grains are dried and wetted again. The
The density of the plants was too important splash dispersal effect can increase spore
for rainfall to produce the infection. The transport from a low to a high level of the
more compact density of the canopy helps crop and from plant to plant.
to maintain a microclimate for the progress There was a general correlation between
of the disease. The lack of germination that the reaction of some lines and the differen-
affected seeds of some accessions could tial varieties in relation to NCP and PCP.
have modified the canopy structure and the The most susceptible varieties at the GS75
inner microclimate; consequently, it could stage were those that had a longer period of
have delayed the movement of the pathogen green leaf (8 days more); therefore, the
to the upper leaves of the plant (Lovell et al., pathogen had a higher probability of prolif-
1997). It could explain the absence of rela- erating in the leaf. Something different
tion between the increase of inoculum con- occurred with Lakhish. In this differential
centration and the decrease of the severity on variety, NLC and PCP did not show any dif-
digit 2. In the pulverization treatment, the ferences for both growth stages. This could
surface covered by the inoculum included be explained by the quick senescence of the
more than one leaf stratum. A simultaneous leaves that practically stopped the develop-
proliferation of the pathogen was obtained ment of the fungus at the end of GS60.
in all foliage levels, which, in addition to The technique of grain inoculation pre-
the beneficial structure of the canopy, pro- sented in this research has the advantage of
duced the observed reactions. being simple to handle compared with the
The strong differences observed between installation of a barrier to infect wheat
treatments (pulverization and grain applica- plants artificially, as is necessary in the pul-
tion) could be explained by the different verization methods (Sanderson et al., 1986).
rates in the progress of the disease for each In the latter, it is necessary to plan the exact
treatment. In pulverization, the inoculum date of the previous sowing and the direc-
included more canopy levels. In agreement tion of this barrier in relation to the tested
with Lovell et al. (1997), the ascending move- wheat rows.
ment of the disease was facilitated, especially Comparing infectivity of different inoc-
in cultivars that, because of their compressed ulum concentrations in the grain applica-
canopy, maintained a more favourable micro- tion treatment, a gradient of infection was
climate. On the contrary, the delayed attack obtained and its effect was related to the
with grain application could be explained variation of humidity and rain regime. The
because only the rain dispersed the conidia differentiation between susceptible and
from the infected grains. If the rain regimen resistant entries of this set was possible
was not frequent and intensive, the spores using 120 g of oat grains/m2 covered with
could not reach the leaves, making it diffi- stromatic mycelia of S. tritici. With this, it is
cult for the inoculum to ascend. not necessary to use the highest concentra-
Although the pulverization and grain tion of oat grains. This type of inoculum has
application techniques were shown to be a long-lasting effect next to the plants, but
effective and could be recommended for the most important characteristics are that the
field trials, each one demonstrated different incubation period is produced without the
advantages. The grain application treatment installation of a wet chamber; it is simple to
Table 22.2. Necrotic and pycnidial coverage percentages caused by Septoria tritici.
Inoculum type Growth Stage
Pulverizationa Grain applicationa a GS60d GS75d

Nb Pc N P Nb Pc N P
Accessions %
Septoria Leaf Blotch of Wheat in Argentina

1 41.60 abc1+ 40.94 1+e 28.91 abcd1+ 28.02 def1+ 3.21 a2+ 18.31 cde2+ 51.28 cde2+ 50.66 cd2+
2 42.59 bc 42.68 de 37.33 defg 37.43 i 12.89 b 12.88 bc 67.03 ijk 67.24 f
3 62.45 fg 61.91 g 32.26 bcde 28.17 ef 30.08 bcd 30.08 fg 64.63 hij 60.00 e
4 27.96 a 11.67 a 25.32 ab 25.32 cd 1.59 a 0.93 54.88 efgh 37.93 a
5 31.92 a 26.98 b 40.91 efg 37.14 hi 20.12 b 12.74 bc 52.71 def 51.37 d
6 46.52 bcd 39.60 d 18.99 a 18.76 ab 21.84 b 21.70 de 43.67 abc 36.66 a
7 38.89 ab 33.34 c 26.14 abc 17.97 ab 22.48 bc 13.16 bc 42.55 abc 38.15 a
8 42.73 bcd 37.68 cd 36.93 def 30.61 fg 17.67 b 16.47 bcd 61.99 ghij 51.83 d
9 70.62 gh 67.48 g 47.88 g 48.56 j 29.93 bcd 30.55 fg 88.58 m 85.49 h
10 51.60 de 47.35 ef 35.07 cde 34.66 ghi 10.83 b 11.18 b 75.84 l 70.82 fg
11 48.73 cd 41.15 de 45.73 fg 40.59 ij 49.17 e 36.89 gh 45.29 bcd 44.86 bc
12 46.77 bcd 47.16 ef 25.63 ab 21.81 bc 11.48 b 42.07 h 60.93 fghi 56.90 de
13 50.64 cde 39.80 d 25.32 ab 20.23 abc 32.39 cd 16.93 bcd 43.58 abc 43.10 b
14 58.97 ef 51.89 f 34.75 cde 31.52 fgh 24.49 bc 17.64 cd 69.22 jkl 75.26 g
15 76.43 hi 63.16 g 44.35 fg 37.24 hi 33.84 d 17.70 cd 86.95 m 82.70 h
16 83.23 i 83.55 h 45.79 g 35.10 ghi 55.20 e 45.17 h 73.82 kl 73.48 g
17 62.21 f 61.88 f 40.54 efg 38.19 i 27.77 bcd 24.81 ef 74.97 l 65.77 f
18 66.24 fg 65.99 g 19.39 a 14.87 a 46.40 e 41.56 e 39.23 ab 39.30 a
19 33.79 a 38.58 cd 20.97 a 25.48 cdf 22.18 bc 23.02 de 32.57 a 41.03 ab
Mean 51.78 47.42 33.27 30.09 25.60 21.15 59.46 56.45
Note: atwo types of inoculum; bmean of necrotic coverage percentage; cmean of pycnidial coverage percentage; dtwo growth stages. 1+Each value is the average of the three upper
leaves with two inoculation methods; 2+each value is the average of the upper three leaves in two growth stages. LSD test (P < 0.01).
297
298 C.A. Cordo
transport over a long distance and to store for ciation between pycnidial coverage and
a long period of time (5 days at 5°C). days to heading.
In another experiment, Cordo et al.
(2007) related that the higher values of the
Climate Influences disease in 1997 compared with those of 1999
were caused by the influence of the climatic
After the initial infection or inoculation, the conditions from boot (GS 43) to hard ripen-
environment is one of the factors conducive ing (GS 87) stages, following Zadoks et al.
to the development of the disease. Different (1974). In 1997, high humidity (30% more
experiments have demonstrated which are than the following year) (Fig. 22.1) and
the weather conditions for a more favour- increased rainfall (425.29 mm more than
able expression of the disease (Simón et al., the following year) (Fig. 22.2) were respon-
2005, Cordo et al., 2006). In one experiment sible for the rapid increase of the disease
conducted in Argentina in 1998 (Simón compared with the results of 1999. For this
et al., 2005), the severity of the disease was experiment, temperature was not an impor-
highest in the early cultivars because pre- tant factor in the development of the disease
cipitation was higher and radiation lower for since in 3 years of experiments there were
these cultivars. Precipitation was 53.4 and no statistical differences in temperature
18.8 mm and radiation 3511 and 5127 Watt/m2 (mean temperature from the inoculation to
for a period of 15 days before evaluation for the end of the experiment was 17.10°C for the
the earliest and the latest cultivars, respec- first 2 years and 16.69°C for 1999). Related
tively. Also, these differences in weather to the inoculation process that is under dis-
variables in 1998 produced a negative asso- cussion, if the experiment is to be carried
100
% Relative humidity
80
60 1997
40 1999
20
0
7- 14- 21- 28- 4- 11- 18- 25- 2-
Oct Oct Oct Oct Nov Nov Nov Nov Dec
Weeks
Fig. 22.1. Histogram showing relative humidity during October–December of 1997 and 1999.
60
50
Rainfall (mm)
40
1997
30
20 1999
10
0
7- 14- 21- 28- 4- 11- 18- 25- 2-
Oct Oct Oct Oct Nov Nov Nov Nov Dec
Weeks
Fig. 22.2. Histogram showing rainfall during October–December of 1997 and 1999.
out on an artificially irrigated area, pulveri- 0.68; FL-1 = 0.69). This indicates that the
zation with an appropriate suspension of infection increased (correlated with AgU/
spores and 48 h of extra irrigation in a wet ml) throughout the different growth stages
chamber are suggested. However, if the area (Table 22.3). With a variance analysis, the
is under a good rain regime from tillering to antigenic units registered and the severity
flowering stages, the application of grains of the infection on three wheat cultivars
covered with sporulated mycelia is a feasi- coming from inoculated and protected treat-
ble option. ments were compared (Table 22.4). Highly
significant differences were observed between
inoculated and protected treatments for
Early Detection of the Disease severity and antigenic units into Flag leaf
and Flag leaf-1. A high correlation was calcu-
At the beginning of the wheat-growing sea- lated (C. coefficient = 0.56) between the aver-
son in 1997, Adgen Phytodiagnostic invited age per cent visual attack of a sample and the
the author to participate in a pilot project to measured antigenic units. Despite the good
identify and quantify S. tritici and S. nodo- correlation, the lower interval of the level of
rum by antibody-based immunoassays and attack scale gave the most confirmable anti-
also to compare with the visual method and genic unit values. This immunoassay has
the sensitive methods for the early detection demonstrated to be highly sensitive and quan-
of S. tritici. The objective of this work has titative, with antigenic unit concentrations
been to test the Adgen ELISA kit in a moni- being correlated with the severity of the dis-
toring process on lower to higher leaves dur- ease. The infection levels of S. tritici in Los
ing the wheat season in Argentina. Hornos samples could be determined with
A randomized complete block design significant precision. In addition, the speci-
with four replicates and a 1.4 × 1 m size ficity of the assay allowed accurate identifi-
subplot was used. Treatment consisted of cation of this pathogen, despite the presence
either an inoculated plot or a control treated of other foliar pathogens. In this assay, only
with a foliar fungicide spray programme. the presence of Alternaria triticimaculans
Plantvax and Tilt were applied at 500 cm3/ gave a cross-reaction.
ha. Ten main tillers were collected per sub- The Adgen Phytodiagnostic Septoria
plot using a uniform, randomized sampling ELISA kit detected and quantified the
pattern at GS10.1 (first spikelets just visible amount of S. tritici antigen in infected plant
28 October); GS10.3 (heading process 14 tissues. As our experience indicates, the use
November); GS10.5 (flowering 27 Novem- of this kit can be recommended in a moni-
ber); GS11 (ripening 8 December). Samples toring process for earlier reports of S. tritici
for testing consisted of ten leaves bulked for infection.
each layer of leaves, each replication and
each data of collection. At the same time,
the severity of the lesions on the sampled
leaves was noted. Samples were homoge- Checking the Ascendant
nized in 50 ml buffer and testing following Movement of the Inoculum
the protocols described for the DU PONT
enzyme-linked immunosorbent assays The ascendant movement of the inoculum
(ELISA) for S. tritici. ELISA results were was also checked with the diagnostic immu-
expressed as the number of S. tritici antigen noassay kit for S. tritici from the Adgen
units/ml of homogeneized plant tissue Company. Increased severity on different
(AgU/ml). AgU/ml values were averaged. levels of the canopy was registered from the
A good correlation was observed between latent period of the infection produced by
ELISA readings from infected leaves coming two types of inoculum application (grains
from different growth stages (GS10.1, GS10.3, covered by pathogen mycelium and pulver-
GS10.5) and the visual development on the ization) during 4 weeks from inoculation at
respective foliar level (C. coefficient = FL tillering stage (GS23).
300 C.A. Cordo
Table 22.3. Correlation between percentage of lesions covered by pychnidia and antigenic units during
the 4 weeks of study.
Grain application Grain application Pulverization Pulverization

Susceptible
cultivars Canopy level AU3 PCP4 AU PCP
1°a Y1 42 b 0.0 a 42 c 0.0 a

1°a B2 140 c 0.0 a 300 d 19.16 c
2°b Y 5.5 a 0.0 a 1.0 a 0.0 a
2°b B 34 b 3.91 b 120 c 20.8 c
3°c Y 3.6 a 0.0 a 4.2 a 0.0 a
3°c B 34 b 3.91 b 82 b 25 c
4°d Y 5.5 a 0.0 a 22 b 0.0 a
4°d B 54 b 6.5 b 54 a 6.5 b
Resistant
cultivars AU3 PCP4 AU PCP
1°a Y 2.45 a 0.0 a 3.10 a 0.0 a

1°a B 65 b 0.0 a 115 b 0.0 a
2°b Y 9.30 a 0.0 a 3.6 a 0.0 a
2°b B 8.5 a 0.0 a 180 c 15 b
3°c Y 65 b 0.0 a 130 b 0.0 a
3°c B 140 c 4.80 b 195 c 10 b
4°d Y 42 b 0.0 a 20 b 0.0 a
4°d B 80 c 1.28 a 80 c 1.28 a
Note: 1youngest leaf; 2the leaf below; 3antigenic units/ml; 4pycnidial coverage percentage; a, 14 October;
b, 9 November; c, 16 November; d, 23 November.
Table 22.4. Information summary for two populations of Septoria tritici from Argentina.
Los Hornos population Balcarce population
Total isolates 58 62
No. of genotypes 35 39
No. of alleles 24 22
Isolates having fingerprint data 55 58
No. of fingerprint patterns 14 13
Fingerprint pattern types A,E,F,G,M,N,O,P,R,S,U,V,W,X A,B,D,E,H,I,K,L,M,P,Q,R,V
Six leaves per canopy level were sam- and antigen units/ml for each week. An LSD
pled per week. Two levels of asymptomatic test was used to compare treatment means.
leaves (the youngest and the leaf below) were Correlation between per cent of lesions cov-
chosen from two varieties (Chirya 1 as resis- ered by pycnidia and antigenic units were
tant and Bethlehem as susceptible) belong- performed throughout the treatments and
ing to the 8th SMN set. The first sample was during the 4 weeks.
taken at GS30 (first node) stage and the fol- According to the analysis of variance
lowing were taken one per week for 3 more (Table 22.3), highly significant differences
weeks. Samples were homogenized in 50 ml were found between each level of the can-
buffer and tested following the protocols opy for AU and PCP throughout the weeks
described for the Adgen ELISA. An analysis and with the two inoculation techniques.
of variance was performed with the dates of There were significant differences for culti-
percentage of lesion covered by pycnidia vars. A significant correlation was found
between PCP and antigen units (C. coeffi- per cent varied from 1 to 21 times for the
cient = 0.56***) (calculated on 67 dates). Los Hornos population and from 1 to 9 times
The youngest leaves almost had the lowest for the Balcarce population. Genotype diver-
values of AU. On the leaves below, it had sity was greater in the Balcarce population
increased. The AU and PCP values were (Ĝ = 31.61 or 26.34% of the theoretical
higher in susceptible than in resistant culti- maximum of 120) than in the Los Hornos
vars. In general, on the 2nd or 3rd week population (Ĝ = 26.19 or 21.82% of the the-
after inoculation, the infection was detected oretical maximum of 120). As the mean
in the youngest symptomatic leaf, indicat- genetic diversity between populations was
ing that the infection was installed in an high for the 8 loci of RFLP, a significant dif-
ascendant level by splashing. ference existed between the populations of
the two localities.
Fifty-eight multilocus haplotypes and
13 fingerprint patterns were registered for
Population Studies of the Pathogen the Los Hornos population and 55 multilo-
cus haplotypes and 14 fingerprint patterns
The population structure and genotypic for the Balcarce population when they were
diversity of S. tritici from two crop field hybridized with pSTL70. Many isolates of
populations in Buenos Aires Province sepa- both populations had from one to several
rated by 500 km were studied with DNA haplotypes for each fingerprint pattern. In
restriction fragment length polymorphism. the Los Hornos population, the E fingerprint
From of the 137 isolates from different areas pattern was present on 14 different haplo-
of the Argentine wheat-growing region, only types, but it corresponded 3 times with the
120 were characterized using the RFLP tech- same 11112110611 haplotype. In the Bal-
nique with P32 labelled probes. The pSTL70 carce population, the same fingerprint was
fingerprinting probe hybridized many DNA present on 11 different haplotypes, but it
fragments of different sizes in isolates from corresponded 8 times with the
field populations of both locations. All leaf 11101010211 haplotype. This last result
samples were processed for isolation of the showed that there were clones in both pop-
fungus, followed by fungus culture, DNA ulations. Some genotypes were detected as
extraction, Pst1 enzyme digestion, radioac- shared across the populations. In other
tive hybridization and X-ray film detection. cases, several individuals in the two popu-
Some of the isolates did not yield good qual- lations had the same multilocus haplotypes
ity DNA for the restriction enzyme digestion but different DNA fingerprints, indicating
process. This explains the loss of 17 isolates that they were not the same clone.
in the samples of the populations. The alleles’ frequencies were signifi-
In total, 24 alleles were found for the cantly different from the 8 loci of RFLP. The
Los Hornos population and 22 alleles for Argentine population must be compared
the Balcarce population at the eight RFPL with other continental populations – Swiss
loci (Table 22.4). Despite the difference in and USA (Oregon) – as independent popu-
the number of alleles, Nei’s measure of lations. Over a total of 834 individuals,
genetic diversity across all loci was differ- there was a 40% gene diversity between
ent for both populations (0.2619 for Los native populations and the total population
Hornos and 0.3161 for Balcarce). Among differentiation was 11%, showing that dif-
the 58 isolates of Los Hornos and 62 of Bal- ferentiation between native and foreign
carce with complete data from individual populations exists. The average number of
RFLP loci, 35 multilocus haplotypes for the migrants was 3.68. This number meant that
first locality and 39 for the second locality 3–4 individuals would need to be exchanged
were registered. Seven new haplotypes (3a, across populations of each generation to
20a, 71a, 37a, 47a, 52a, 58a) were added to maintain the observed level of genetic simi-
the list published on the Internet (S. tritici larity. Moreover, the amount of gene flow
RFLP alleles). The haplotype frequency in
302 C.A. Cordo
between populations was high when all the The results of this contribution are in
populations were compared. agreement with Keller et al. (1997), who
The genetic distance was small when demonstrated that ascospores were the pri-
comparing the population of Los Hornos mary agent for unifying geographically sep-
with the other populations, showing a high arated populations on a regional scale.
level of similarity, but the genetic distance of Added to this, Cordo et al. (2005) showed
the Los Hornos and Balcarce populations was that ascospores were the most significant
major compared with the Oregon and Swiss component of the M. graminicola life cycle
populations. Salamati et al. (2000) suggested in the wheat-producing areas in Argentina.
that the similarity among populations on a Their release was registered in the vegeta-
regional basis was explained because the tive and debris wheat stages for the periods
gene flow was significant over spatial scales analysed. According to these experiments,
of at least several hundred kilometres. It was the high degree of gene flow among popula-
found that genetic distances among fields tions would be associated neither with the
within a region were small, while genetic dis- pycnidiospores presence as dominant in the
tances among different continents were larger life cycle of the pathogen nor the infected
for the Rhynchosporium secale populations. seeds that could act as a human dispersal
Genotypic diversity within populations mechanism (Keller et al., 1997). The Los Hor-
and similarity over regional spatial scale was nos population result was different because
explained because regular sexual recombina- the clonal lineages of S. tritici probably origi-
tion was occurring in S. tritici rather than in nated from the inoculations applied for the
R. secalis (Salamati et al., 2000), Stagono- resistance tests.
spora nodorum (McDonald et al., 1994) and If it is assumed that S. tritici had not
Phaeosphaeria nodorum (Keller et al., 1997) colonized Argentina recently, the high degree
populations. This was explained because the of similarity could be explained from the
ascospores from the teleomorph were dis- most likely centre of origin for this pathogen.
persed over distances of up to 100 km (Shaw Banke et al. (2004) demonstrated that the
and Royle, 1989; Cordo et al., 1990/1991). New World areas (where the South Cone is
The field populations of the fungus located) appeared less likely to represent
exhibited high degrees of gene and genotype ancestral populations because they had
diversity distributed on very small spatial lower diversity, whereas Israel and Europe
scales. Microgeographical-level observations appeared to be the ancestral populations
showed a higher variation of type and num- because they showed the highest genetic
ber of genotypes for the Balcarce than for diversity. This pattern is related to the fact
the Los Hornos population. In general, dif- that wheat has been grown in the Old World
ferent genotypes were often found within a for thousands of years, but in the New World
single lesion and most lesions on the same for only hundreds of years. Movement of
leaf also had different genotypes. This result the fungus from Israel into Europe could
demonstrated, in coincidence with Boerger have been from windblown ascospores or
et al. (1993), that a lesion might result due via transport on infected seed or straw.
to coinfection by two or more genotypes. Ascospore movement produced a natural
The genetic distance, for native popula- gene flow out of the possible centre of origin
tions, was very small considering that the and into European populations, which
geographic distances between them was could explain the finding that more haplo-
500 km; the North American and European types were found in European than in New
populations, separated by to 7000 km, had a World populations.
low increase of this genetic distance. Then, Another way of dispersion could be an
the high degree of similarity could be caused alternate host of S. tritici producing pycnidia,
by the gene flow on a regional scale and which constitute a continuous host popula-
between continents (Boerger et al., 1993; tion where ascospores (Boerger et al., 1993;
Zhan et al 2003; Banke et al., 2004; Banke Linde et al., 2002) would maintain a uniform
and McDonald, 2005). source of inoculum that infects the wheat
field each autumn. This way of transmis- pulverization and undercoated seed treat-
sion was not demonstrated in Argentina. ments. Two strains of Trichoderma sp. (Th5
and Tk11) were selected. Conversely, trials
performed during 2005 examined only plants
Disease Control with produced by seeds coated with Trichoderma
Alternative Techniques Th5 and Tk11 isolates. The T. koningii 11
strain was selected for the third experiment,
instead of the Th2 strain, because the necrotic
The most common approach to biological
coverage percentage of Tk11 was statistically
control consists of selecting antagonistic
different with respect to the control and with
microorganisms, studying their modes of
a higher value than the others (Table 22.5).
action and developing a biological control
Moreover, the value of pycnidial coverage
product. Despite progress made in the
percentage was also one of the highest that
knowledge of the modes of action of these
was statistically different from the control.
biological control agents, practical applica-
This work shows that T. harzianum, T.
tions often fail to control diseases in the
koningi and T. aureoviride reduce the leaf
field. One of the reasons for this failure is
blotch caused by S. tritici in greenhouse-
that biocontrol products are used in the same
grown wheat. The effect of T. aureoviride was
way as chemical products. Other methods
considered similar to that of T. harzianum in
include the choice of an appropriate crop
reducing the leaf blotch caused by S. tritici
rotation with the management of crop resi-
because, under the most effective application
dues, added to organic amendments and
method (seed coating), the pycnidial coverage
biological disinfestations of soils. In that
percentage was statistically different to the
sense, Cordo et al. (2007) evaluated the effi-
control, but not to that of T. harzianum. The
cacy and mechanisms of action of Tricho-
positive result of the immunochemical test
derma sp. for controlling leaf blotch in
applied on all asymptomatic leaf intercellular
wheat grown under greenhouse conditions.
fluid samples demonstrated the presence of
Because of their capacity to act as bio-
S. tritici on plants free of Trichoderma and
control agents, members of the fungal genus
plants coming from Trichoderma-coated
Trichoderma have been broadly studied (Bar-
seeds, both inoculated with S. tritici.
nett and Lilly, 1962; Tronsmo, 1986; Melo,
1991; Harman, 2000; Monte, 2001). Thus, T.
harzianum and T. aureoviride are known to
be effective antagonists against phylloplane Effect of Trichoderma on
pathogens (Perelló et al., 1997, 2001, 2003, Leaf Proteolysis
2006).
There were significant differences for Plants pretreated with Trichoderma Th5
necrosis and pycnidial coverage percentages and Tk11 isolates were selected to assess
for 2 years of experiment and for the behav- the balance between leaf apoplast pro-
iour of the 14 antagonists, each treated with teolysis and protease inhibitory capacity.
Table 22.5. Severity of necrosis and pycnidial coverage percentage in leaves with different
Trichoderma spp. isolates in 2005.
Trichoderma spp. isolates Necrotic coverage (%)* Pycnidial coverage (%)*
Th5 34.23 a 46.91 b

Tk11 41.62 a 57.50 a
Control 46.17 a 59.14 a
Note: *Each value is the mean of two replicates for necrotic and pycnidial coverage percentage. Means
followed by the same letter are not significantly different (P = 0.05) according to the LSD test.
304 C.A. Cordo
Compared to controls, leaf proteolytic activ- action in plants of susceptible cultivars

ity decreased by 40% 12 days after S. tritici (Segarra et al., 2002). Conversely, leaf blotch
inoculation. Conversely, it increased in symptoms decrease in susceptible cultivar
plants produced by Th5-coated seeds. This PRO INTA Molinero plants pretreated with
was visible within 15–22 days after sowing some Trichoderma isolates after challeng-
(Table 22.6). Moreover, the increased prote- ing with S. tritici. For this, the protease
olytic activity coincided with a decreased action in plants pretreated with isolates Th5
protease inhibitory capacity. Furthermore, and Tk11 with high and low biocontrol
the proteolytic activity remained higher in capacity, respectively, was tested. The apo-
plants produced by Th5-coated seeds chal- plastic protease activity increased only after
lenged with S. tritici. Proteolytic activity treatment with Th5. In order to know if this
did not increase when comparing wheat increase ocurred independently of the inoc-
plants grown without inoculation and ulation with the pathogenic fungus, the
plants produced by Tk11-coated seeds. kinetics of this phenomenon were analysed
The genus Trichoderma is a soilborne in plants pretreated with this isolate. The
fungus whose survival on the phylloplane proteolytic activity was controlled by the
environment is difficult (Perelló et al., 1997, leaf germin-like protease inhibitor already
2003). Thus, a Trichoderma spp. popula- described (Segarra et al., 2003).
tion applied over wheat leaf decreases rap- In plants, the apoplast forms a space
idly. Conversely, coating wheat seeds with the pathogens necessarily must cross to colo-
T. harzianum is the finest application tech- nize tissues. Therefore, it plays a central role
nique to control the leaf blotch caused by S. in defence strategies, being a place where not
tritici. To understand how T. harzianum acts, only signals for plant response originate, but
the general features of the biological control also where the proteins for defence mecha-
set up by Viterbo et al. (2002) were tested. nisms accumulate: glucanases, chitinases
Leaves of plants formed by pre-coated seeds and proteases among others (Bowles, 1990).
did not contain T. harzianum. This suggests Within proteases must be mentioned the
that its biocontrol of leaf blotch is indirect tomato P-69 induced by the citrus viroid (Vera
and able to produce morphological or bio- and Conejero, 1988; Tornero et al., 1996),
chemical changes. the tomato aspartil protease that degrades
As mentioned, inoculation with S. trit- proteins related to pathogenesis (Rodrigo
ici decreases the apoplastic serine protease et al., 1988), the specific race join protease
Table 22.6. Effect of S. tritici and Trichoderma spp. on leaf apoplast proteolytic and inhibitor activity.
Treatment Days after sowing Protease activity (%) Inhibitor activity (%)
T1 22 100 100
T2 22 61 +/– 15 150
T4 (Th5) 7 95 +/– 4 98
T4 (Th5) 15 167 +/– 25 87
T4 (Th5 22 140 +/– 20 33
T6 (Th5) 22 128 +/– 10 60
T4 (Th11) 22 70 +/– 10 –
T4 (Th11) 22 98 +/– 12 –
Note: In T1 (wheat plants without inoculum), the IWF (leaf intercellular washing fluid) was obtained 22 days after sowing.
In T2 (wheat plants inoculated with the pathogen) and T6 (wheat plants grown from Trichoderma spp. pre-coated wheat
seeds and inoculated with the pathogen), the plants were inoculated with S. tritici 10 days after sowing and the IWF was
examined 12 days after inoculation. In the case of plants grown from Trichoderma spp. pre-coated wheat seeds (T4), the
IWF was examined 7, 15 or 22 days after sowing. For all treatments, the proteolytic and the protease inhibition activity
was considered 100% in non-inoculated plants. Each value is the mean of two replicates.
that processes the AVR9 of the compatible that seek to incorporate resistance to S. trit-
reaction tomato–Cladosporium fulvum (de ici. In coincidence with Boerger et al. (1993),
Witt et al., 1985; Schaller and Ryan, 1996), our evidence of gene flow suggests that
two closely related subtilisin-like proteases plant breeders in Argentina are driving the
that are associated with the defence response breeding process well. They are testing the
of tomato and encoded by the P69B and resistance of their cultivars at many loca-
P69C genes (Jordá and Vera, 2000) and a tions away from the area of local adaptation.
unique 33-kDa cysteine protease mobilized The fine scale of patterns with genetic vari-
in response to caterpillar feeding in maize ability suggests that plant breeders should
lines that are resistant to feeding by several use a wide spectrum of pathogen genotypes
lepidopteran species (Pechan et al., 2002). when testing wheat cultivars resistant to
this pathogen in any location.
Throughout the genetic evidence on the
gene flow between continents, it is possible
Conclusions to affirm that lesions of leaf blotch of wheat
could arise from seed transmission or could
The relevant advances for Septoria leaf be attributed to a failure of isolation and a
blotch of wheat fall into two classes. First, stray ascospore. In the face of the high envi-
qualitative, as the conditions that allow inoc- ronmental contamination produced by agro-
ulum transfer, permit infection and encour- chemical products, new ecological alternatives
age sporulation; second, quantitative as, in are applied to control diseases in extensive
a given agroecosystem, what factors in prac- plant cultures. So, biological control is a
tice control pathogen regulation size. It was complementary strategy in the ecological
demonstrated that the distance between leaf management of wheat cultivation.
layers with and without infection varied These results suggest that the sapro-
greatly according to both the architecture of phytic fungus T. harzianum provokes a bio-
the wheat cultivar and the latent period of chemical plant defence response, as has
the pathogen on the cultivar. The interac- been reported previously. Immunochemical
tion of these factors, as was observed on the tests proved that although these leaves
SMN collection, caused great variation in looked asymptomatic, they contained S.
the potential for the spread of pathogens to tritici. Because T. harzianum does not meet
the upper part of the crop. DNA restriction leaves coming from pre-coated seeds, its
fragment polymorphism (RFLP) markers stimulation of leaf proteolytic activity might
labelled with radioactive compounds were be considered a systemic induced response,
used to assess the potential for gene and which is one of the different biochemical
genetic diversity and for gene flow between mechanisms of plant defence proposed by
geographically separated populations. Viterbo et al. (2002) and Hoitink et al.
The results on the genetic composition (2006). We conclude that prospects for the
of two populations separated by 500 km biological control of leaf blotch with T. har-
show shared haplotypes. This has significant zianum are auspicious. The results encou-
implications for wheat-breeding programmes rage trials under field conditions.
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Part VI
Alternative Control Strategies

23 Review of Thecaphora amaranthicola
M. Piepenbr., Casual Agent of Smut on
Amaranthus mantegazzianus Pass.
M.C.I. Noelting,1 M.C. Sandoval,2 M.M.A. Gassó1 and M.C. Molina1,3

1Instituto
Fitotécnico de Santa Catalina, Facultad de Ciencas Agrarias y Forestales,
UNLP, Llavallol, Buenos Aires, Argentina; 2Facultad de Ciencas Agrarias,
UNLZ, Llavallol, Buenos Aires, Argentina; 3CONICET (Consejo Nacional de
Investigaciones Cientificas Tecnicas)
Abstract
The amaranth (Amaranthus spp.) is becoming a socially and economically important crop due to the
high level of quality proteins in its seeds and leaves. Among the factors that could limit the expansion
of this crop are the smuts (Ustilaginales) that prevent normal development of the seeds. The objectives
of the project were to: (i) characterize the pathogen which is responsible for smut on A. mantegaz-
zianus, taking into account the cultural and morphobiometrical characters, and the germination of its
teliospores; (ii) assess the incidence of smut in two amaranth cultivars; (iii) analyse fast techniques for
the detection of the inoculum in seeds and plants cultivated in the field; and (iv) identify any possible
reservoirs of the pathogen on wild amaranths. The results obtained allowed the authors to determine
that: (i) Thecaphora amaranthicola is the causal agent of smut on A. mantegazzianus; (ii) the cultivar
Don Manuel was affected most by the smut (36% incidence); (iii) the residue and plastic card tech-
niques are fast and efficient for the detection of the pathogen inoculum; and (iv) the wild species A.
hybridus and A. retroflexus are hosts of the pathogen. This is the first report in the world of Theca-
phora amaranthicola as a pathogen of the A. mantegazzianus cultivated species.
Introduction The seeds and leaves of this plant have

a very high level of quality proteins. This
The amaranth is an ancestral precolumbine property makes the amaranth a valuable
crop that was cultivated by the Aztecs, resource, especially appropriate for a popula-
Mayas and Incas and which remained rele- tion which lives in areas that are considered
gated for a long time after being banned by marginal for the cultivation of traditional
the Spanish conquistadors. However, in the cereals.
past few decades it has been subjected to The amaranth can be affected by pests
numerous investigations with the objective and diseases. Among the diseases of fungal
of studying its nutritional value, improve- etiology which affect the normal develop-
ment and adaptation to new areas of culti- ment of its seeds are two species of smut,
vation (Afolabi et al., 1981; Kulakow, 1987; T. amaranthi (Hirschh) Vanky (syn. Glo-
Bressani, 1989; Espitia, 1991). mosporium amaranthi) (Vánky, 1994) and
312 M.C.I. Noelting et al.
T. amaranthicola M. Piepenbr. (Piepembring, Samples of teliospores obtained from infected

2000). The spores of both types of smut seeds of amaranth (A. mantegazzianus)
infect the ovaries, preventing normal devel- were used in order to study these charac-
opment of the seeds. On the inside of the ters. The seeds were harvested in an experi-
affected ovaries, hypertrophied sorus devel- mental field of the Instituto Fitotécnico de
opment takes place and these contain a mass Santa Catalina, situated in the locality of
of spores (teliospores). The species T. ama- Llavallol, in the south of the Buenos Aires
ranthi was described and reported in wild Province, Argentina, in 2003. The telio-
species of amaranth (Vánky, 1985; Hirschhorn, spores were observed in an optical micro-
1986) and in cultivated species (Alcalde, scope and measured with a micrometric
1995; Noelting and Sandoval, 2003). On the objective (Carl Zeiss Jena 7×) (n = 50).
other hand, T. amaranthicola was described In order to observe the germination, the
in only one wild species of amaranth col- teliospores were disinfected with a solution
lected in Ecuador (Piepembring, 2000). In of sodium hypochlorite (2%) for 5 min,
Argentina, the data on this species are in a rinsed three times with sterile distillate
report where the symptoms caused by this water and dried between sterile paper fil-
smut on a cultivated species of amaranth ters. To inoculate Petri plates, teliospores
are described (Noelting et al., 2005a). There were streaked on the surface of the agar
are no records of T. amaranthicola on culti- media to dilute the inoculum. The plates
vated species of amaranth in the rest of the were incubated at 25 ± 2°C with a 16 h pho-
world. Owing to the little information avail- toperiod. To assess the colony characteris-
able on the biological and epidemiological tics, sections 6 mm in diameter, obtained
aspects of this smut, and that its spreading from a colony in active growth, were trans-
could have a negative effect on the propaga- ferred to dishes with PDA. The material was
tion of the amaranth crop, it was decided to incubated in a chamber at 25 ± 2°C with a
start the present study, the objectives of 16 h photoperiod. After 14 days of incuba-
which were: tion, the cultural and morphological char-
acteristics (colour, edge, diameter) of the
1. To characterize the pathogen which is
developed colonies were described.
responsible for smut on A. mantegazzianus,
taking into account the cultural and mor-
phobiometrical characters, and the germi-
nation of its teliospores;
2. To assess the incidence of smut in two Incidence assessment
amaranth cultivars;
3. To analyse fast techniques for the detec- The incidence assessment was carried out
tion of the inoculum in seeds and plants; and in seeds of two cultivars of A. mantegaz-
4. To identify any possible reservoirs of zianus (cvs. Don Juan and Don Manuel)
the pathogen on wild amaranths. which had been harvested in 2004 and
presented spontaneous infections with
smut. Each panicle was harvested and
Materials and Methods threshed by hand. Once the material had
been threshed, samples of 200 seeds/pani-
cle were taken, determining the number
Morphological and cultural
of seeds infected with T. amaranthicola.
characterization of pathogen
The incidence was calculated by using the
formula:
The taxonomic identification of the fungus
was carried out by observing the teliospores,
the evolution of the germination process Number of
(Piepembring, 2000) and the characteristics infected seeds
Incidence (%) = × 100
of the cultivated colonies in vitro which Total number of
developed in potato dextrose agar (PDA 2%). analysed seeds
Review of Thecaphora amaranthicola M. Piepenbr. 313
Techniques for Detection of Inoculum Results and Discussion
In seeds Morphological and cultural

characterization of pathogen
Samples of four different cultivated species
of amaranth were used: A. mantegazzianus, Morphobiometrical characteristics
A. caudatus, A. hypochondriacus and A. cru- of teliospores
entus from Argentina, Bolivia and Mexico.
The technique applied consisted in deposit- The observations carried out in samples of
ing 10 g of seed samples from the different infected A. mantegazzianus seeds allowed
countries between pieces of reticulated cel- determination of the presence of spore balls
lulose paper and then submitting them to with the following characteristics: ochre
three cycles of manual pressure. Next, the colour, globose to subglobose (40.78 µm ×
seeds were taken out of the paper and the 34.23 µm) (Fig. 23.1a). Each spore ball was
residue contained in the reticules was formed by 8–23 teliospores, polyhedral to
observed with a stereoscopic magnifying cuneiform: spore walls were deeply corru-
glass (10×). The observations that tested gated on the central parts of the teliospores,
positive for the presence of teliospores were as seen by SEM (Fig. 23.1b). No individual
confirmed by means of a preparation, with a teliospores were observed in any of the sam-
solution of lactophenol and cotton blue, ples analysed. This phenomenon coincides
and observed with an optical microscope with the one observed in smuts that have
(450×). grouped spores and which develop on dif-
ferent kinds of plants (Barrus and Muller,
1943; Andrade et al., 2004).
The germination of the teliospores
In plants cultivated in fields
plated on PDA was initiated after 24 h of
incubation generating phragmobasidia, fol-
In order to determine the infection in the lowed by the development of lateral and
crop, 4 cm × 6 cm plastic cards were used, terminal basidiospores. Variations in the
with lithium grease as an adhesive on one number of germinated teliospores in each
side of the cards. Eighteen cards were dis- spore ball were observed (Fig. 23.2a,b,c). In
tributed randomly, 9 in each of the cultivars addition to this, multiple germinations
of A. mantezzagianus (cvs. Don Juan and occurred simultaneously (Fig. 23.2b,c).
Don Manuel), hanging from the plants for a The cultural characteristics of the colo-
month. Each card was later analysed with a nies grown in laboratory conditions were as
stereoscopic magnifying glass to detect the follows: velvety surface at the expense of the
teliospores. development of the mycelium, light beige
colour, slightly serrated edges and softly
lobated outline of the colony (Fig.23.2f).
Wild hosts of T. amaranthicola The growth of the colonies was slow, reach-
ing a maximum diameter of 34 mm after 14
To detect natural reservoirs of the inoculum days of incubation. In the colonies analy-
of this smut, a sampling that involved three sed, no yeasty type of development charac-
wild species of amaranth, A. retroflexus L., teristic of this type of fungus was found.
A. hybridus L. and A. viridis L., was carried According to analysis, T. amaranthicola,
out in 2006. The plants of these species belonging to the Basidiomycota Subking-
were located around a crop of A. mantegaz- dom, Ustilaginomycetes Class, Ustilagino-
zianus, as well as areas further away at a mycetidae Subclass, Ustilaginales Order,
distance of 1.5 km. The panicles of the col- Glomosporiaceae Family, was identified as
lected material were taken to the laboratory the causal agent of smut in A. mantegaz-
and observed with a stereoscopic magnify- zianus (Fig. 23.3). The morphological and
ing glass and an optical microscope. cultural data shown complement the
(a) (b)
Fig. 23.1. Teliospore balls of Thecaphora amaranthicola: (a) under the light microscope (scale bar
10 µm); (b) SEM (scale bar 10 µm).
pr bl
(a) (b) (c)
Coil
(d) (e) (f)
Fig. 23.2. Culture of Thecaphora amaranthicola on PDA: (a) single germination of a teliospore after
24 h; pr = probasidium; (b) and (c) multiple germination of teliospores, bl = lateral basidiospores;
(d) hyaline basidiospores; (e) mycelia formation, coiling of hyphae (coil); (f) colony of T. amaranthicola
developed on PDA after 17 days of incubation.
cb
(a) (b) (c)
Fig. 23.3. (a) Panicle of Amaranthus mantegazzianus; (b) healthy seeds (12×); (c) seeds of
A. mantegazzianus infected with T. amaranthicola (12×), seed coats show irregularities (cb).
preliminary information about the pathogen filtering seeds and is used for the detection
(Noelting et al., 2005a). of smuts in the seeds of many crops (ISTA,
1985). The inoculum (teliospores) was
detected on and among the seeds; therefore,
Incidence individualization of the teliospores with this
technique offered information about infection
The percentage of incidence varied between as well as contamination in amaranth seeds.
10 and 36.66% (average rates) for the Don Furthermore, the retrospective character of
Juan and Don Manuel cultivars, respectively. the analysis led to the conclusion that T. ama-
These results apparently indicate the exis- ranthicola was already present in cultivated
tence of resistance mechanisms, especially in species of amaranth in Argentina prior to its
the Don Juan cultivar. More studies would first report (Noelting et al., 2005a).
have to be carried out in the future in order to
learn more about said mechanisms. Neverthe-
less, it cannot be discarded that the relatively In plants cultivated in the field
high rates of incidence which were detected
may be so because the pathogen could have The employment of cards to detect aerial
been introduced by contaminated germplasms inoculum in plants cultivated in the field
in the region. With respect to this, it can be allowed the detection of teliospores (T.
stated that the growing interest in amaranth amaranthicola) in 55% of the cards analy-
cultivation crop has originated the incorpora- sed, as well as other fungal propagules. This
tion of seeds from several countries and, since technique is a sampling method by deposi-
this pathology had not been reported previ- tion or capture similar to those which use
ously in cultivated species of amaranth, there slides covered by an adhesive substance
are no controls for it in Argentina. (Bugiani and Govoni, 1991). The results
obtained from both A. mantegazzianus cul-
tivars indicate that the T. amaranthicola
Inoculum detection in seed samples teliospores are spread by the wind.
Teliospores of T. amaranthicola were found

in 50% of the analysed samples (Table 23.1). Wild host of T. amaranthicola
The technique applied is effective, fast and
simple (Noelting et al., 2005b) compared Seeds infected by T. amaranthicola from
with the test which consists of washing and panicles of A. hybridus and A. retroflexus
Table 23.1. Results of the analysis of amaranth seeds samples.
Thecaphora
Seed sample Locality Province Country Year amaranthicola
A. cruentus cv. Don Armando Anguil La Pampa Argentina 1995 x

A. cruentus cv. Don Guiem Anguil La Pampa Argentina 1997 x
A. hypochondriacus cv. G. Covas Anguil La Pampa Argentina 1999 x
A. hypochondriacus Se143 Anguil La Pampa Argentina 1999 x
A. caudatus Llavallol Buenos Aires Argentina 2003 –
A. hypochondriacus cv. G. Covas Luis Guillon Buenos Aires Argentina 2003 –
A. hypochondriacus – – Mexico 2004 –
A. caudatus – – Bolivia 2004 –
A. hypochondriacus Llavallol Buenos Aires Argentina 2004 –
A. cruentus cv. Se1MC Luis Guillon Buenos Aires Argentina 2004 x
A. mantegazzianus cv. Don Manuel Santa Rosa La Pampa Argentina 2005 –
A. mantegazzianus cv. Don Manuel Colonia 25 La Pampa Argentina 2005 –
de Mayo
A. mantegazzianus cv. Don Juan Llavallol Buenos Aires Argentina 2005 x
A. mantegazzianus cv. Don Juan Llavallol Buenos Aires Argentina 2005 x
plants were detected (Noelting et al., 2006). not only in Argentina but also in the world, of
The infections of a spontaneous nature smut as a pathogen. The inoculum detection
which were found in the two species of wild techniques applied to samples of seeds and
amaranth that affect many crops and which plants cultivated in the field are appropriate
grow in a vast region of Argentina are thought as a fast method for identifying the presence
to be of epidemiological interest. This is due of smut. Two species of wild amaranth, A.
to the fact that they may turn into ‘bridge’ hybridus and A. retroflexus, are hosts of T.
species for the entrance of the inoculum and amaranthicola. The interest in the amaranth
the spreading of smut to cultivated amaranth crop has originated an intense interchange of
species. germplasms among several countries of Amer-
ica, Asia and Europe. This situation suggests
the need to undertake a major study of the
Conclusions biological and epidemiological characteristics
of the seedborne pathogens that as the smuts
The presence of T. amaranthicola in A. man- (T. amaranthi and T. amaranthicola) have
tegazzianus (cultivated crop) is the first report, negative incidence in the crop.
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24
Population Biology and
Management Strategies of Phytophthora
sojae Causing Phytophthora Root
and Stem Rots of Soybean
Shuzhen Zhang1 and Allen G. Xue2

1Soybean
Research Institute, Key Laboratory of Soybean Biology of Chinese
Education Ministry, Northeast Agricultural University, Harbin, Heilongjiang, China;
2Eastern Cereal and Oilseed Research Centre, Agriculture and Agri-Food Canada,
Ottawa, Ontario, Canada
Abstract
Soybean is an important oilseed crop; it is also the richest source of protein. Root and stem rot patho-
gen Phytophthora sojae can reduce yield by up to 40%. Symptoms, disease cycle and genetic diversity
of P. sojae are described. The paper is a valuable document listing molecular markers and the role of
14 resistance genes located at eight genomic loci in the development of disease-resistant soybean vari-
eties. There have been only a few fungicides available for the control of P. sojae and their effects are
limited. The development of pathogen resistance to these fungicides is not known. Little information
is available on cultural and biological controls of P. sojae. More effective management of P. sojae will
require integration of all available strategies to address all stages of the disease cycle. The integrated
approach may prove a boon to growers using a variety of susceptible cultivars. Suitable cultural,
chemical and biological methods are recommended as alternative control strategies.
Introduction The disease has since been reported from

many countries, including Canada (Hildeb-
Phytophthora root and stem rot of soybean rand, 1959), Australia (Pegg et al., 1980; Ryley
(Glycine max (L.) Merr.), caused by P. sojae et al., 1998), Argentina and Brazil (Wrather
Kaufmann and Gerdemann, is a destructive et al., 1997), China (Shen and Su, 1991) and
disease throughout the soybean-planting the Republic of Korea (Jee et al., 1998). The
regions of the world (Schmitthenner, 1985). disease is more prevalent when soil is satu-
The symptoms were first discovered as an rated for prolonged periods of time and sus-
unknown etiology in the state of Indiana in ceptible cultivars are planted (Grau et al.,
the USA in 1948, and subsequently in Ohio in 2004). Diseased plants reduce yield by
1951, but the causal agent was not described 10–40%, or a total crop loss when infection is
until 1958 (Kaufmann and Gerdemann, 1958). severe (Anderson and Tenuta, 2003).
Phytophthora Root and Stem Rots of Soybean 319
Pathogen and Disease Symptoms any time from the first trifoliate to late R9
stage (Grau et al., 2004). Severely infected
P. sojae traditionally has been classified as a plants have few lateral roots, with almost no
fungus due to its outward resemblance of nodules and only a short portion of taproots
growth habits and nutritional requirements. left, and brown and drooping leaves remain-
In fact, it is very distant from ‘true’ fungi ing attached to the stem, even though the
evolutionarily and falls within the Kingdom plants die.
Stramenopila (Förster et al., 1990; Harper
et al., 2005), which constitutes a distinct
branch of the eukaryotic evolutionary tree
(Tyler, 2007). Disease Cycle
The pathogen may infect soybean at
any stage of plant development and cause P. sojae has a narrow host range and is
seed rot, seedling pre- and post-emergence restricted primarily to soybean, but there
damping-off and root and stem rots of soy- are reports that lupin, lucerne, bean and
bean (Kittle and Gray, 1979; Athow, 1987). sweetclover could be infected using artifi-
The seeds could be rotted by infection of P. cial inoculation in controlled environments
sojae in both heavy and light sandy soils, (Erwin and Ribeiro, 1996).
after periods of cool and rainy weather. The P. sojae has both asexual and sexual
damping-off, stem and root rot symptoms stages in the life cycle and produces sporan-
often appear shortly after emergence and dur- gia, zoospore and chlamydospore in the
ing early flowering when plants are under asexual stage and oospore in the sexual
stress (Anderson and Tenuta, 2003). The stage, as shown in Fig. 24.1 (Tyler, 2007).
infected seedlings have dull grey leaves and Oospores are produced by the fusion of
reddish, water-soaked lesions that occur from a female organ, called oogonium, and a male
the base of the stem and slowly advance up organ, antheridium. Chlamydospores are
the plant, and may collapse if the infection thick-walled spores that protect the organ-
is severe. Symptoms on older plants are isms surviving through periods of abiotic
characterized by chocolate-brown discolor- stress. P. sojae can survive for many years
ation extending from the soil line to the third in soil, mainly as oospores that are formed
or fourth node and into lower branches at in the roots and stems of infected soybeans
Secondary
Motile zoospore
zoospore
Zoosporangium
Cyst
Mycelium
Sporangium
(attached or detached)
Sexual
reproduction Germinated
Germination cyst
Oogonium
Oospore INFECTED
Antheridium
PLANT
Fig. 24.1. Phytophthora sojae life cycle.

320 S. Zhang and A.G. Xue
in large quantity and are released into the pathogen is often covered by bacteria and
soil when these tissues decompose (Ander- saprophytic fungi. The commonly used
son and Tenuta, 2003). methods of isolating P. sojae are the plant
Oospores serve as the primary inocu- stem lesion and soil methods described by
lum and germinate to produce sporangia Dorrance et al. (2008). The protocol for
under flooded conditions or infective hyphae plant stem lesion isolation is by surface-
(Anderson and Tenuta, 2003). Zoospores do sterilizing cut-off tissues of symptomatic
not have a cell wall and each has two fla- stems first and incubating on the selective
gella and are released by flooding. They can medium, e.g. PBNIC agar, to control bacte-
swim a short distance (1.0 cm or less) in ria and other fungi, such as Pythium. P.
saturated soil, but are disseminated primar- sojae has a distinct growth pattern on PBNIC
ily by moving flood water. At the end of the agar, showing white mycelium 2–3 days
motile period, which may last up to several after incubation. The mycelium is coeno-
days, zoospore movement becomes sluggish cytic and have branches almost at right
and jerky and encystment occurs (Schmit- angles, with curved tips. The asexual spo-
thenner, 2000). Zoospores can be attracted rangium looks like an inverted pear and the
towards the compounds excreted by soy- round oospores on solid culture media can
bean root tips (Morris and Ward, 1992; Tyler be seen 8–10 days later. Soil isolation is
et al., 1996). On reaching the root surface, usually done by grinding the soil to fine par-
the zoospores begin to encyst and germinate, ticles, flooding it for 24 h, then draining and
and the hyphae penetrate directly between air-drying the soil until it cracks or pulls
the cell walls of the epidermis (Beagle- away from the side of the container, although
Ristaino and Rissler, 1983). The infection it is still damp. The process is to break dor-
process can be completed in 30 min in opti- mancy and induce germination of oospores
mum conditions. On resistant cultivars, in the infested soil. The soil is then used for
hypersensitive response (HR) may occur, the planting a susceptible cultivar and P. sojae
pathogen is contained in numerous necrotic can be isolated readily from collapsed hypo-
or dead cells and there is no development of cotyls of emerging seedlings 5–6 days later
haustoria in the resistance interaction. How- using the same procedure described for
ever, there is no early HR reaction occurring plant stem lesion isolation.
in susceptible cultivar; the hyphae initially The common way to store P. sojae is to
grow intracellularly and then form many grow the pathogen on V8 juice agar slants
haustoria in root cells, which remain alive for 2 weeks, then cover the culture with
in direct contact with the pathogen after 2 ml of sterile deionized water and store the
infection for around 10 h (Enkerli et al., culture at 15°C. The fungus may be stored
1997) or approximately 12 h (Ward, 1990), in such conditions for up to 3 years without
when P. sojae is able to colonize host cells losing its virulence and aggressiveness. For
in an initial biotrophic phase of growth a longer-term preservation, P. sojae can be
without triggering any response from the stored in liquid nitrogen for at least 4 years
plant. After the initial infection, the patho- (Dorrance et al., 2008).
gen begins to enter a necrotrophic growth
mode and causes many host cells to die.
The hypha penetrates from the epidermal
cells of the root into the deep layers and Pathogenic and genetic
vascular tissues. diversity of P. sojae
The pathogenetic variation of P. sojae was

Isolation and Identification of first reported in 1958 (Kaufmann and Gerde-
Physiologic Races of P. sojae mann, 1958). During the interaction process
with the soybean varieties, the pathogenic-
P. sojae is known for the difficulty in isola- ity and virulence of P. sojae evolved rapidly
tion due to its slow growth. As a result, the and 55 physiologic races were identified
based on their differential reaction on a set between the base constitution of ITS1 and
of 8 or 13 differentials with a single resis- ITS2 among isolates. The 17 isolates were
tance gene. Of the 55 races, races 1 to 45 classified into three groups based on the ITS
were identified using a set of 8 differentials sequence and those isolated from the same
(Bernard et al., 1957; Morgan and Hartwig, region belonged to the same group, which
1965; Schmitthenner, 1972; Schwenk and showed the variation in geography. These
Sim, 1974; Haas and Buzzel, 1976; Lavio- studies demonstrated that molecular tools
lette and Athow, 1977; Keeling, 1979, 1982; could be used to disclose the intraspecific
Laviolette, 1983; White, 1983; Layton, 1986; diversity of P. sojae isolates both within and
Wagner and Wilkinson, 1992; Henry and among geographic origins.
Kirkpatrick, 1995; Abney et al., 1997) and
races 46 to 55 on a set of 13 differentials
(Ryley et al., 1998; Leitz et al., 2000). Management Strategies
Several DNA-based molecular markers
such as SSR (simple sequence repeat), rDNA-
The management strategies for prevention
ITS (rDNA-internal transcribed spacer),
against Phytophthora root and stem rot at
RFLP (restriction fragment length polymor-
present are mainly by deployments of culti-
phism) and RAPD (random amplified poly-
vars with race-specific or race non-specific
morphism DNA) have been used successfully
resistance, or a combination of the two.
to identify the genetic variation and diver-
Chemical methods and cultural practices
sity of P. sojae. Whisson et al. (1992) used
like crop rotation and tillage and integrated
RFLP to confirm sexual recombination of P.
management are used to a lesser extent.
sojae in vitro and studied the segregation of
avirulence genes. Föster et al. (1994) pro-
posed that occasional outcrosses had been a
major contributor to the origin of new phys- Screening for resources of race-specific
iological races of P. sojae, in addition to and race non-specific resistance
clonal evolution. Meng et al. (1999) used
the RAPD method to study populations of P. Race-specific resistance (Rps) genes in soy-
sojae from Indiana, Iowa and Minnesota bean have been used extensively to manage
(USA) and found no correlation of popula- P. sojae (Dorrance et al., 2003). New sources
tions with a geographic origin. Wang et al. of resistance to P. sojae have been reported,
(2003) analysed genetic diversity of 75 P. mainly from soybean varieties and germ-
sojae isolates from China using the RAPD plasm in China, where soybean was origin-
method and distinguished 12 genetic groups, ated. Lohnes et al. (1996) reported that the
but most of the isolates were clustered into Rps1d gene was common in accessions from
one group and no relationship between clus- Anhui and Jiangsu Provinces after they
tering and geographic origin was found. evaluated 517 soybean germplasms col-
Wang et al. (2006) studied the genetic varia- lected from several provinces in central
tion among P. sojae in the USA and China China. Kyle et al. (1998) investigated soy-
and found that there existed higher genetic bean accessions from southern China in
variations in populations in the USA com- response to several races of P. sojae and
pared to the Chinese populations based on demonstrated that germplasm from Hubei,
the RAPD analysis. Gally et al. (2007) exam- Jiangsu and Sichuan Provinces appeared to
ined by RAPD analysis the diversity of 32 P. be valuable multi-gene resistance sources.
sojae isolates of different geographic origins Lv et al. (2001) screened 956 soybean acces-
from Argentina and detected intraspecific sions from north-east China (Heilongjiang,
variability even among isolates of the same Jilin and Liaoning Provinces) and identified
geographic origin. Xu et al. (2007) detected 23 varieties with resistance to both race 1
17 P. sojae isolates from three locations in and race 25, the predominant and the most
Heilongjiang Province, China, and demon- virulent races of P. sojae in the region,
strated by sequence analysis the difference respectively. Zhang et al. (2007) evaluated
530 soybean germplasms including 280 of zoospores of P. sojae. Irwin et al. (1982)
native soybean accessions and 250 commer- reported a laboratory assay by inoculating
cial cultivars and found that the percentage soybean seedlings with dry P. sojae myce-
of resistance in native soybean varieties was lium for rapid determination of relative lev-
higher than that of the commercial culti- els of race non-specific resistance. Dorrance
vars. Similarly, Zhu et al. (2000, 2004), Li et al. (2008) described a layer test and a tray
et al. (2001), Huo et al. (2005) and Jin and test for screening soybeans for race non-
Zhang (2007) reported the identification of specific resistance to P. sojae. The layer test
P. sojae resistant germplasm with a number is done by placing inoculum of a 14-day-old
of Rps genes from wild soybean accessions P. sojae culture 5 cm below the seeds in
and soybean varieties from China. In addi- cups containing coarse vermiculite and the
tion, Dorrance and Schmitthenner (2000) amount of root rot and seedling death is rated
identified several soybean accessions with 3 weeks after planting. The tray test is
multi-gene resistance after evaluating 1015 assessed by wound inoculation of a myce-
plant introductions originated from the lial slurry on the root of 7-day-old seedlings
Republic of Korea. These single Rps genes, and root rot is rated after 7 days. Using the
however, have often been short-lived, with layer test, Jia and James (2008) identified
an effective ‘life’ of 8–15 years, due to the several accessions with high levels of race
emergence of new virulent races in response non-specific resistance compared with Con-
to selection pressure exerted by the conrade, the common known race non-specific
tinuous use of specific resistant cultivars resistant soybean variety to P. sojae.
(Schmitthenner and Van Doren, 1985; Fer-
guson, 1987; Schmitthenner et al., 1994;
Abney et al., 1997; Ryley et al., 1998).
With the known Rps genes defeated, Resistance genes and
race non-specific resistance or partial resist- marker-assisted selection
ance, described as the ability of plants to
survive root infection without displaying A single dominant resistance gene has been
severe disease symptoms such as death, widely explored since the first resistance
stunting or yield loss, is of great interest and gene (Rps1a) was identified by Bernard
gains more and more attention from soy- et al. (1957). With the Rps1a defeated and
bean breeders (Buzzell and Anderson, 1982; the emergence of new races in response to
Tooley and Grau, 1984; Schmitthenner and selection pressure exerted by the continuous
Van Doren, 1985). The strategy of the com- use of Rps1a, new Rps genes are identified. A
bination of race non-specific resistance with total of 14 Rps genes including Rps1a,
race-specific resistance is brought forward Rps1b, Rps1c, Rps1d, Rps1k, Rps2, Rps3a,
to provide long-term management of Phy- Rps3b, Rps3c, Rps4, Rps5, Rps6, Rps7 and
tophthora root and stem rot, as well as to Rps8 at eight genomic loci have been
avoid the boom-and-bust cycle of single reported so far (Bernard et al., 1957; Kilen
gene deployment, since it reduces the sever- et al., 1974; Laviolette and Athow, 1977;
ity of root rot and slows the rate of disease Mueller et al., 1978; Athow et al., 1980; Ber-
development (Buzzell and Anderson, 1982; nard and Cremeens, 1981; Athow and Laviol-
Burnham et al., 2003b). ette, 1982; Ploper et al., 1985; Anderson and
Race non-specific resistance commonly Buzzell, 1992; Burnham et al., 2003a). All of
had been evaluated under natural infection these loci have been placed on the soybean
in the field until recently, owing to the genetic map. Rps1 and Rps3 are mapped on
unavailability of a suitable laboratory pro- molecular linkage groups (MLG) N and F,
cedure. Jimenez and Lockwood (1980) first respectively (Diers et al., 1992; Demirbas
described a laboratory procedure for screen- et al., 2001; Burnham et al., 2003a). Rps2
ing race non-specific resistance by growing (MLG J), Rps4 (MLG G), Rps5 (MLG G), Rps6
soybean seedlings in cups that were placed (MLG G), Rps7 (MLG N) and Rps8 (MLG A2)
in plastic trays containing a specified number have also been mapped (Diers et al., 1992;
Lohnes and Schmitthenner, 1997; Demirbas Chemical, cultural, biological

et al., 2001; Burnham et al., 2003a). and integrated control
Molecular markers have been used to
facilitate selection for both single and multi- The most commonly used chemical in the
resistance genes (Bent and Yu, 1999; Kumar, prevention of P. sojae is metalaxyl, which is
1999). SSR and RFLP markers have been an acylalanine fungicide specific to oomy-
identified for Rps1 (Diers et al., 1992), Rps1a cetes. The fungicide is commonly applied
(Weng et al., 2001), Rps1b and Rps1c (Demir- as seed treatment (apron fungicide) and in-
bas et al., 2001), Rps1d (Sugimoto et al., furrow, spray or granule (ridomil fungicide)
2008), Rps1k (Kasuga et al., 1997; Bhatta- to reduce plant emergence loss and increase
charyya et al., 2005), Rps2, Rps3, Rps4, Rps5, yields of susceptible varieties (Anderson
Rps6 (Diers et al., 1992; Cregan et al., 1999) and Buzzell, 1982; Guy et al., 1989).
and Rps7 (Lohnes and Schmitthenner, 1997). Since soybean is the only host of P.
With the development of the molecular bio- sojae in the field, crop rotation is an effec-
technique, more and more markers closer to tive means of reducing the severity of the
the resistance genes will be used in marker- disease. Although a short-term crop rotation
assisted selection (MAS), which has been may not allow for reduction of inoculum, it
complementary to conventional breeding does prevent the immediate build-up of P.
programmes and to shortening the breeding sojae populations (Schmitthenner, 1985).
period. Because saturated soil favours the occur-
In some cases, genes for complete race- rence of P. sojae, tillage that could promote
specific resistance that have already been soil drainage is proven to be effective in
defeated by new races of P. sojae may con- reducing the infection period (Grau et al.,
tribute to race non-specific resistance (Geb- 2004). Oospores could also be buried deeper
hardt and Valkonen, 2001), which appears in the soil by tillage (Workneh et al., 1998).
to be controlled by several genes (Walker Biological control has been considered a
and Schmitthenner, 1984; Glover and Scott, more natural and environmentally acceptable
1998) and is more durable (Tooley and Grau, alternative to the existing chemical treatment
1984). Several QTLs have been mapped to methods (Cook and Baker, 1983; Baker and
linkage groups for race non-specific resist- Paulitz, 1996). Several bacteria and fungi have
ance to P. sojae. Burnham et al. (2003b) been identified as potential bioagents in dual-
used three recombinant inbred line (RIL) culture and greenhouse experiments.
populations with the cultivar Conrad as the There are inevitable shortcomings for
race non-specific resistance parent and each of the prevention measurements, that
identified two putative QTLs on MLG F and is, the Rps genes in cultivars can be defeated
D1b + W from Conrade in all three popula- by new races of P. sojae, and the varieties
tions. Han et al. (2008) used the RILs popu- with race non-specific resistance, seed treat-
lation of Conrade and OX760-6-1 as the race ment, rotation and tillage cannot provide an
non-specific resistance and susceptible par- effective control when disease pressure is
ent, respectively, and detected three QTLs, high. A combination of two or more strate-
i.e. QGP1, QGP2 and QGP3, for Phytoph- gies to prevent the infection of P. sojae is
thora root and stem rot tolerance. They fur- very essential. The disease could be best
ther confirmed that QGP1 was located on managed with integrated strategies in a com-
linkage group F and QGP2 in a different bination of deployment of cultivars incorpo-
interval on linkage group F and QGP3 on rated into race-specific and race non-specific
linkage group D1b + W. Furthermore, an RIL resistance genes, fungicide treatments, impro-
population of a cross between Conrade and ved soil drainage and biocontrol. These man-
Hefeng 25 was constructed and four mark- agement tactics can reduce inoculum in fields
ers on three linkage groups, MLG D1b + W, and limit the amount of water available for
MLG F and MLG A2, were identified as the pathogen to germinate and infect, there-
being associated significantly with race fore minimizing disease damage and increas-
non-specific resistance (Li et al., 2008). ing soybean production efficacy.
Conclusions To our knowledge, limited research has

been carried out on the expression of genes
Owing to the shift of P. sojae races, use of which are possibly involved in soybean
race-specific resistance may quickly become resistance to P. sojae, notwithstanding that
inefficient in management of the disease. 14 dominant Rps genes at 8 loci have been
More efforts are needed in the identification identified and resistance gene mapping and
and incorporation of multi-gene resistance quantitative trait loci have been explored.
and race non-specific or partial resistance Moy et al. (2004) reported the patterns of
into new soybean cultivars in the future for gene expression on infection of soybean
prevention against Phytophthora root rot plants by P. sojae race 2 and demonstrated
and stem rot. Soybean cultivars with a com- that genes identified as strongly upregulated
bination of two types of resistance would be during infection included those encoding
long-lived and more desirable by the soy- enzymes of phytoalexin biosynthesis and
bean industry. Conventional breeding will defence and pathogenesis-related proteins.
still be the main method of resistance breed- A better understanding of the resistance
ing in the foreseeable future but, with the mechanism in the P. sojae–soybean interac-
rapid development of molecular technol- tion at the molecular level is needed for
ogy, there will be more and more molecular effective gene deployments and resistance
markers closely linked to the Rps genes breeding. Research into these new disease
mapped, which could be useful in MAS to management strategies is required with trans-
hasten the breeding and cultivar develop- genic soybean cultivars, in order to provide
ment process. solutions for future needs when transgenic
technologies will be more acceptable.
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25 Management of Fungal Pathogens –
A Prerequisite for Maintenance of Seed
Quality During Storage
Anuja Gupta
Indian Agricultural Research Institute, Regional Station, Karnal, Haryana, India
Abstract
Storage of seed is essential for any seed programme to sow the crop in the next season, to maintain
buffer stock as an insurance against crop failure in times of drought, excessive rainfall or other natural
calamities, to maintain parental lines for the production of hybrid seed, to conserve germplasm for
breeding purposes and for seed trade at national and international levels. Availability of good quality
seed at the right time and place is a basic prerequisite for sustaining agriculture. While maintenance
of seed germination is of utmost importance to any seed person, preservation of seed quality in terms
of its health status is equally important. A quality seed should have high genetic purity, physical
purity, seed germination, seed vigour and good health status.
Introduction with the traders, are far from satisfactory.

Farmers and traders are not fully aware of
Seed health is being recognized as one of the large savings that can be obtained by
the important criteria in evaluating seed proper storage and preservation techniques.
quality. Seed health refers primarily to the The important factors that determine the
presence or absence of disease-causing longevity of seeds are seed moisture, the type
organisms such as fungi, bacteria and viruses, of storage container and storage environ-
or animal pests such as nematodes and ment. These factors generally interact, lead-
insects, or physiological disorders due to ing to a number of physiological and
deficiency of trace elements. One of the biochemical changes in the stored seeds,
major problems associated with crop pro- which result in deterioration of seed both in
duction in India is the maintenance of the quality and quantity, especially in tropical
prescribed level of seed vigour and viability and subtropical countries. According to a
from seed harvest till the next sowing sea- current estimate, 10% of food grain is lost in
son. In our plans for attaining self-sufficiency storage due to microbial spoilage and insect
in food grains, preventing their loss in stor- attack. The damage caused by rodents and
age is as important as the various measures insects is visible and therefore remedial
to increase production. About 60–70% of measures are adopted for their control, but
the annual output is retained by the farmer. microbial spoilage of seeds/grains cannot be
Storage facilities with the farmer, as well as seen easily.

330 A. Gupta
Microbial Spoilage of of fatty acids, glycerol, sugar and amino acids

Seed during Storage in radish seeds infected with Aspergillus fla-
vus. Dube et al. (1988) reported changes in
Seeds are the end product of a series of steps starch, fatty acids and sugars in wheat grains
that include sowing, growing, harvesting infected with A. flavus and A. niger. Mishra
and threshing, wherein it becomes vulnera- and Dharam Vir (1991) observed higher mill-
ble to various pathogens/saprophytes. Among ing losses ranging from 34.0 to 58.6% in
the different microbes, fungi form a major discoloured rice grains. Joshi et al. (1988)
group of organisms that infest seeds. Nearly reported 73% reduction in starch content in
150 species of fungi have been found associ- stored pearl millet seeds infected with stor-
ated with grains and seed in storage (Dharam age fungi and an increase in the amount of
Vir, 1974). Mechanical damage in the seeds, reducing sugars and phenolic contents. Bil-
cracks, breaks or scratches in the pericarp grami and Sinha (1983) have reported afla-
or seed coat developed during threshing toxin contamination in maize, groundnut
and processing substantially facilitate inva- and a variety of agricultural foods and feeds.
sion by fungi, which find their way to the Vaidehi (1997) showed that storage fungi
storage warehouses. lowered the quality of maize grains due to
The fungi found associated with seeds the biochemical changes they brought about.
during storage are known as storage fungi. Storage fungi may be present as dormant
They can grow without free water, on spores or mycelium on the seed surface or
media with high osmotic pressure, at below the pericarp, which activate and mul-
RH = 70–90%. Some common storage fungi tiply at a phenomenal rate under favourable
include species of Aspergillus, Penicillium, storage conditions.
Rhizopus, Fusarium, Cladosporium, Alter-
naria, Mucor, Chaetomium, Epicoccum, etc.
Discoloration and distortion of seeds is a
major degrading factor because of seedborne Seed Mycoflora
infection. Other common manifestations are
reduction in seed size, seed rots, shrivelling The initial mycoflora of the seeds can give
of seeds, seedling decay and pre- and post- an idea of the type of fungi that can initiate
emergence mortality and abnormalities. The the process of deterioration in storage.
fungi primarily invade the embryo and in With an increase in the storage period,
the early stages of infection, the seed may there is an increase in the incidence of
appear normal but, due to well-established storage fungi and a decrease in seed germi-
infections, these embryos are killed and the nation (Gupta and Singh, 1990, 1993). Loss
seeds appear darkened. These fungi are in germination due to storage fungi may be
responsible for a decrease in market value, attributed to several factors. A toxin pro-
germinability and nutrition of the produce, duced by A. ruber kills the tissues in the
making the grains unfit for human con- embryonic axes of pea seeds in advance of
sumption and reducing the viability of the infection (Harman and Nash, 1972). Wheat
seed. Excessive fungal growth may also seed infected with Aspergillus spp. and
result in heating, caking and decay. The imbibed with water becomes a jelly-like
seed may thus become totally spoiled, dark- mass, suggesting that cell wall degrading
ened or charred by prolonged exposure to enzymes may be involved. In contrast, pea
the heat generated during storage, which and squash embryos are killed without
brings about biochemical changes leading physical invasion by fungi, indicating the
to the production of toxins and loss in seed involvement of diffusible toxins (Harman
weight. and Pfleger, 1974). Mitochondria isolated
The invasion by fungi leads to physical from the embryonic axes of A. ruber-
and chemical changes in the seeds. Prasad infected pea seeds were less active than
et al. (1990) observed changes in the amount those from non-infected seeds, suggesting
Management of Fungal Pathogens 331
that mitochondria damaged by fungi play a Seed Mycoflora and Seed Viability
role in seed deterioration (Harman and
Drury, 1973). Gupta et al. (1989) observed The incidence of fungal flora associated
a decrease in the amount of volatile alde- with different seeds is low initially, but it
hyde compounds with increased levels increases with an increase in the duration of
of fungi on the seed and treatment with storage and subsequently there is a decrease
benomyl increased these compounds, in seed viability. With the advancing stor-
indicating control of fungi and increased age period, the field fungi become limited
germination. and the produce becomes infested with stor-
An increase in seed mycoflora is corre- age fungi. A significant negative correlation
lated directly to an increase in FFA content (r = –0.793) between seed viability and seed
and leaching of solutes (especially electro- mycoflora has been observed with advanc-
lytes and water-soluble sugars) with advancing storage period (Table 25.1), and conse-
ing storage period (Gupta, 2003). Agarwal quently a decrease in seed viability.
(1980) demonstrated that seed deterioration
in okra, carrot and onion seeds was accom-
panied with leakage of sugars. Analysis of Treatments and seed mycoflora
exudates from the seeds showed that the per-
meability of the membrane increased with Seed treatments, especially with fungicides
the deterioration of seeds during ageing like captan, thiram or mancozeb, restrict
(Dadlani and Agarwal, 1983). According to the growth of mycoflora on the seeds and
Chen et al. (1998), with an increase in the maintain better seed viability (Gupta, 2003).
fatty acid contents of different seeds like Moreno et al. (1985) suggested the use of
wheat and brassicas, storage potential decrea- fungicides to protect the viability of corn
sed. Ramamoorthy and Karivaratharaju (1986) seeds. In another study, Moreno and Ramirez
also found that with an increase in the stor- (1983) recorded that after 330 days of stor-
age period, the oil and protein content in age at 26°C with 75% RH, the germination
groundnut seeds decreased gradually, while of untreated corn seed was only 61%, while
free fatty acid content increased, accompa- it ranged from 68 to 90% in seeds treated
nied by a loss in seed viability under ambi- with different fungicides either singly or in
ent storage conditions. Thus, during storage, combination. The incidence of storage fungi
especially under an ambient environment, was also very low in treated seeds.
seeds produce changes due to fungal activ- Kushwaha and Raut (1994) reported
ity, resulting in deterioration of their qual- that seeds treated with thiram and stored in
ity (Zagrebenyer and Bern, 1998; Gupta and poly-lined bags suppressed most of the
Aneja, 2004). fungi. Asalmol and Zade (1998) also observed
Table 25.1. Influence of seed mycoflora on seed viability and seed moisture during storage.
Storage period (months

after seed treatment) Seed germination* (%) Seed mycoflora* (%) Seed moisture* (%)
0 92.0 1.05 7.8

2 86.5 2.4 5.9
4 92.8 3.4 6.6
6 85.3 2.7 9.9
8 50.8 5.9 8.6
10 44.3 4.6 8.9
12 38.3 5.0 8.8
Correlation coefficient (r) –0.872 0.35
Note: *Average of 20 treatments.

332 A. Gupta
that pre-storage seed treatment helped to seed and stored in 700 gauge polythene bags
improve the shelf life of seeds and checked maintained seed quality. The disease, ginger
seed mycoflora during storage. Fungicide yellow, was controlled effectively by seed
seed treatments were found to restrict the treatment with 0.1% carbendazim (Rana and
growth of mycoflora on different vegetable Sharma, 1995).
seeds (Gupta and Singh, 1993). Sandhu (1989) reported seed dressing
The incidence of Colletotrichum demain the form of slurry with benlate at 1 g/kg
tium associated with chilli seed at the time and dry seed treatment with brassicol at
of storage was 5%. Seed treatment with cap- 2.5 g/kg resulted in 100% and 93.9% inhi-
tan controlled the pathogen just after its appli- bition of germination of pea seeds after their
cation. Other fungicides like thiride and storage for 1 year. However, captan (0.25%)
carbendazim controlled the pathogen after 5 and bayletan (0.1%) treatments enhanced
months of storage, whereas in the untreated germination by 5.21 and 11.88%, respec-
control the pathogen persisted up to 7 tively. He also found that in steam-sterilized
months in a cloth bag and up to 15 months soil, germination of poor quality seeds of
in an airtight container (Gupta et al., 1992). pea variety Punjab-87 was enhanced from
Thiram, bavistin and captan could con- 17 to 56%. They were further enhanced up
trol more than 96, 93 and 90% of the fungi to 69% in seeds treated with captan.
associated with paddy seed as against 72% Van Toai et al. (1986) observed that only
and 65% in hinosan and emisan + strepto- reduced quality seed of soybean responded
cycline treatments, respectively, after 17 to fungicide seed treatment under prolonged
months of storage under ambient conditions storage. The accelerated ageing germination
(Fig. 25.1). Mancozeb (78.6%) was most effec- (AAG) results of the 24-month-old methanol-
tive in the control of seed mycoflora on soy- washed seeds were lower than the AAG
bean seed during storage, followed by thiram results of the unwashed seeds (the fungi-
(65.1%), bleaching powder (13.1%) and nim- cides were removed from the treated seeds
becidine (10.1%) during storage (Fig. 25.2). by methanol prior to the AAG test), but sig-
Onion seed variety Phule Safed dried at nificantly higher for all cultivars than the
6–7% moisture content and treated with AAG values of the untreated seeds. The
0.2% carbendazim could be stored safely in fungicidal seed treatments, in addition to
700 gauge polyethylene bags for 32 months, protecting the seeds and seedlings during
as against 24 months in untreated seed imbibition and germination, also helped to
(Mahajan et al., 2001). Pumpkin seed treated maintain the seed quality of soybean during
with iodine-based halogen mixture at 3 g/kg storage.
100
Fungal inhibition (%)
80
60
40
20
0
Thiram Bavistin Captan Hinosan Emisan +
streptocycline
Seed treatments
Fig. 25.1. Effect of different seed dressings on the control of seed mycoflora on paddy seed during
storage.
Occurrence (%) Inhibition (%)

100
Occurrence/inhibition (%)
80
60
40
20
0
Mancozeb
Thiram
Neembicidine
Bleaching
powder
Untreated
Seed treatments
Fig. 25.2. Influence of seed dressings on the occurrence and inhibition of seed mycoflora on soybean
seed during storage for 15 months.
Treatments and seed viability stored in poly-lined cloth bags (polythene

bags of 400 gauge kept inside a cloth bag)
The influence of seed treatments on seed remained above the prescribed standard of
viability and vigour is not apparent during certification (70%), even on the 15th month
the early period of storage, but becomes sig- of storage after seed harvest, whereas germi-
nificant on prolonged storage. In some crops nation of both treated and untreated seeds
like mung bean, mustard, muskmelon, etc., stored in cloth bags fell below the certifica-
the effect of seed treatments on seed germi- tion standards on the 11th month of storage.
nation is insignificant during storage, but in Seed treatment with mancozeb and thiram
other crops like cowpea, sorghum, chillies, resulted in significantly better root and
fenugreek, spinach and soybean, etc., seed shoot lengths of seedlings as compared to
treatments have a significant effect on main- other treatments. Seedling vigour measured
taining seed viability for longer duration in terms of seedling dry weights and/or
under ambient storage conditions. seedling lengths closely follow the pattern
Mung bean cv. PS-16 retained seed dor- of seed germination. The results of treating
mancy for up to 6 months of storage and mustard seeds with different seed dressings
viability for more than 36 months of storage are shown in Table 25.3.
(Table 25.2). Chickpea cv. P-256 treated The seed germination of wheat variety
with fungicides like thiram and ABC dust HD-2329 remained above the prescribed
retained more than 85% germination for up standard of certification (85%) for up to
to 24 months of storage. Thiram-treated 20 months of storage under ambient condi-
seed had better germination compared to tions when treated with carboxin, as against
ABC dust treatment, and re-treatment with 16 months in untreated seeds. The stacking
thiram further enhanced seed germination. of seed bags (8 bags of 40 kg each stacked
The germination of soybean seeds, cv. one above the other) in the seed warehouse
P-16, treated with mancozeb or thiram had an insignificant effect on the viability
334 A. Gupta
Table 25.2 Treatments to enhance storability of legume seeds under ambient storage.
Effective seed Storability in months

Crop seeds treatment at 2 g/kg seed after seed harvest** References
Cowpea Captan/thiram*/mancozeb 28 Gupta and Singh, 1990

Mung bean *** > 36 Gupta and Singh, 1990
Chickpea Thiram* Gupta and Singh, 1990
Note: *Seed treatment at 2.5 g/kg seed; **period that seed viability remained above the prescribed standards of
certification; ***germination in treated and untreated seeds on a par.
Table 25.3. Treatments to enhance storability of oilseeds under ambient storage.
Effective treatments
Seed treatment Storability in months

Crop seeds at 2 g/kg seed Storage container after seed harvest**
Soybean Mancozeb/thiram* Poly-lined bag 15

Mustard *** Poly-lined bag 24
of both treated and untreated seeds. How- results of fungicidal seed treatments of sor-
ever, varietal differences were observed ghum cv. PC-9 are shown in Table 25.4.
with respect to the storability of the seeds. Among vegetable seeds, fungicidal seed
Wheat seed of the HD-1553 variety main- treatments also influenced germination sig-
tained more than 85% seed germination for nificantly in spinach cultivar Pusa jyothi
up to 15 months of storage, irrespective of and fenugreek (Pusa kasuri) seeds (Table
seed treatments. Germination in untreated 25.5). However, the effect of seed treatments
seeds of the HD-2285 variety fell below the was insignificant in brinjal (Pusa kranti)
certification standard on 9 months of storage and muskmelon (Pusa madhuras) seeds.
as against seeds treated with captan, where The germination of brinjal and palak seeds
germination remained above 85% for up to remained above the certification standards
15 months of storage. In the HD-2009 variety, (70% and 60%, respectively) for up to 18
the germination of seeds treated with car- months of storage after seed harvest. Spinach
bendazim, captan or thiram was above the seeds treated with fungicides like captan or
prescribed standard, even after 21 months mancozeb had higher seed germination.
of storage as against 15 months in untreated Muskmelon seeds were stored for 42 months
seeds. The decline in germination started at after seed harvest without any substantial
a faster rate after 21 months of storage and loss in seed viability under ambient storage
reached zero level after 39 months, irrespec- conditions. Fenugreek seeds retained via-
tive of variety or fungicide seed treatment bility for up to 30 months and treatment
when stored under ambient conditions. improved seed germination as against
In paddy, seed germination remained untreated seeds.
above the prescribed standard of certifica- Fungicide treatments improved seed
tion (80%) for up to 20 months after seed germination by about 5–7% on the 30th
harvest and both the seed treatments and month of storage after seed harvest, but there-
storage containers had insignificant effect after their influence on seed germination was
on the viability of the stored seeds. The negligible. The germination of chilli seeds
Table 25.4. Treatments to enhance storability of cereal seeds under ambient storage.
Effective seed treatment Storability in months

Crop seeds at 2 g/kg seed after seed harvest**
Wheat Carboxin* 20
Sorghum Captan/thiram*/brassicol/carbendazim/mancozeb 21
Paddy *** 20
Table 25.5. Treatments to enhance storability of vegetable seeds under ambient storage.
Effective treatments
Storability in months
Crop seeds Seed treatment at 2 g/kg seed Storage container after seed harvest**
Spinach Mancozeb/brassicol 18
Brinjal *** 18
Chilli Thiram* Airtight 19
Muskmelon *** 42
Fenugreek Thiram*/captan/carbendazim 30
remained above the minimum prescribed the parental lines (Fig. 25.3). The germina-
standard (60%) for 19 months in airtight tion of paddy seeds of both the parental
containers as against 10 months when stored lines stored in poly-lined bags (76.62%)
in cloth bags, irrespective of fungicide treat- was significantly higher than seeds stored
ments. However, thiram gave higher seed in cloth bags (72.05%). Seed treatment with
germination. thiram and captan also improved seed ger-
Adverse effects of copper-oxychloride mination as against the untreated control
fungicide (CuO) have been reported on veg- under both storage conditions (Fig. 25. 4).
etable seeds during storage (Gupta et al., The different treatments also influenced
1996). Fenugreek, brinjal and muskmelon the germination of parental lines of pearl
seeds treated with CuO recorded 37, 50 and millet (MS841A, MS841B and D23) during
36% germination as against 69, 83 and 80% storage (Gupta, 2007). Seeds of MS841A,
in untreated seeds after 18, 30 and 24 MS841B and D23 retained germination above
months of storage, respectively. However, minimum seed certification standards (MSCS)
in spinach seed, the CuO treatment retained (75%) for up to 16, 16 and 20 months after
germination on a par with other seed dress- seed harvest, respectively. As in paddy, the
ings. germination of pearl millet seeds (Fig. 25.3)
Paddy seeds of parental lines of paddy stored under controlled conditions (82.25%)
IR58025A and IR58025B retained seed lon- was significantly higher than seeds stored
gevity above the prescribed standards (80%) under ambient conditions (66.43%). Thus,
for up to 5 years after seed harvest when storage under low temperature can prolong
stored under controlled conditions (temper- the longevity of the precious seeds of the
ature = 15°C; RH = 30%), as against 2 years inbred parental lines of paddy and pearl
under ambient storage conditions in both millet.
336 A. Gupta
Controlled conditions A Controlled conditions B

Ambient conditions A Ambient conditions B
100
80
Germination (%)
60
40
20
0
0 8 12 16 24 28 36 48 60
Storage months
Fig. 25.3. Effect of storage conditions on seed germination in parental lines of paddy during storage.
Thiram Captan Untreated
100
90
80
70
Germination (%)
60
50
40
30
20
10
0
0 8 12 16 24 28 36 48 60
Storage months
Fig. 25.4. Effect of seed treatments on seed germination in parental lines of paddy during storage.
Treatment of pearl millet seeds of differ- millet than the seeds stored in cloth bags
ent parental lines with bioagent T. viride (Fig. 25.7).
maintained 78.30% germination during stor-
age that was on a par with thiram (78.34%),
captan (78.16%) and carbendazim (77.37%),
as against 74.27% in the untreated control Role of Storage Environment in the
and 59.58% in P. fluorescence treatment (Fig. Perpetuation of Fungi
25.6). Also, the seeds stored in polythene-
lined cloth bags maintained higher seed The lifespan of seed is highly influenced by
germination in both paddy and pearl storage conditions, especially temperature
Effect of storage conditions on germination in bajra seeds
Controlled storage Ambient storage

100
Germination (%)
80
60
40
20
0
0 8 16 20 24 32
Storage months
Fig. 25.5. Effect of storage conditions on seed germination in pearl millet.
Bavistin Captan
Thiram Trichoderma viride
Pseudomonas fluorescence Untreated
90
80
70
Germination (%)
60
50
40
30
20
10
0
0 8 16 20 24 32
Storage months
Fig. 25.6. Effect of seed dressings on seed germination in pearl millet.
and relative humidity (RH). The effects of tissues, from where it moves to the surface
temperature and RH (and its subsequent effect and evaporates and is dependent on the RH
on seed moisture) of the storage environment of the atmosphere. At different RH levels, the
are highly interdependent. Most crop seeds equilibrium moisture content (MC) varies
lose their viability when RH is about 80% with different seeds. The MC of seeds ranges
and the temperature varies from 25 to 30°C. from 3 to 7, 6 to 10 and 9 to 14% at 20, 45
This hot and humid environment is conge- and 75% RH, respectively. According to Har-
nial for the activity and growth of microor- rington’s rule of thumb (Harrington and Doug-
ganisms, which leads to deterioration in seed las, 1970):
quality. The expression of the mycoflora
● For each 1% decrease in seed moisture
depends essentially on the temperature and
content, the storage life of the seed is
humidity conditions of seed warehouses
doubled.
and also on the intergranular atmosphere of
● For each 10°F (5.6°C) decrease in seed
the seed. The moisture in the seed is present
storage temperature, the storage life of
either on the seed surface or in the internal
the seed is doubled.
338 A. Gupta
Poly-lined bag Cloth bag

90
80
70
Germination (%)
60
50
40
30
20
10
0
0 8 16 20 24 32
(a) Storage months
Jute bag Poly bag

100
80
Germination (%)
60
40
20
0
0 8 12 16 24 28 36 48 60
(b) Storage months
Fig. 25.7. Effect of storage containers on seed germination in pearl millet (a) and paddy (b) seed
during storage.
● The arithmetic sum of the storage tem- rioration of soybean seeds in 6 months of
perature in degrees F and the per cent storage (Nkang and Umoh, 1996).
RH should not exceed 100, with no Under normal storage conditions, the
more than half the sum contributed by temperatures and relative humidity of the
the temperature. seed warehouse fluctuates with the environ-
ment. The temperature range in a seed ware-
However, this rule is valid only when the house located in Karnal varies from 13 to
seed moisture varies from 5 to 14%. 33°C and 18 to 38°C (Fig. 25.8). The relative
Storage fungi are unable to grow and humidity of the seed warehouse varies from
multiply if the MC of the stored produce is 51 to 75%. Since seed moisture is a function
12% or less. The optimum moisture for many of RH, so it changes with variations in the
types of seed is 6–8%, at which even damage RH of the seed warehouse. Seed dressings
by insects reduces (Dharam Vir, 1996). It was do not affect the MC of seeds appreciably,
observed that the per cent of seed viability but storage containers do seem to affect it. It
was highest at low temperatures and RH and is higher in seeds stored in cloth bags as
short storage periods, but it decreased with against those in poly-lined cloth bags because
increased storage period. Temperatures cloth bags, being pervious, allow a free flow
above 35°C are reported to cause rapid dete- of air from the surrounding atmosphere.
Min. temp. (°C) Max. temp. (°C) RH (%)

40 80
35 70
Relative humidity (%)

30 60
Temperature (°C)
25 50
20 40
15 30
10 20
5 10
0 0
January April June September December
Months
Fig. 25.8. Ambient conditions in a seed warehouse at Karnal during the year.
Although seed MC, type of storage ent on the seed, inhibit the fungi strongly,
container and storage temperature are inter- while others exhibit weak or almost nil
related, high temperatures hasten the deteri- inhibition. The storage container also seems
oration of high-moisture seeds by increasing to influence the residual activity of the
the metabolic activity of hydrolysed sub- chemical on treated seeds during storage.
strates and enzymes. Hence, maintaining Vyas and Nene (1971) reported minimum
these factors at low levels in the seed ware- loss of thiram on the seeds of cowpea, maize,
houses can improve the longevity of the paddy and soybean in tin boxes as com-
seeds. pared to polyethylene bags, polyethylene-
lined cotton bags or hessian bags. Gupta and
Chatrath (1983) found that the quantity of
Persistence of seed thiram on soybean seeds decreased gradu-
dressings during storage ally with increase in the storage period and
fungicide degradation was maximum when
Suitable dressing at proper dosage and its the seeds were stored in cloth bags, followed
uniform distribution on the seed is equally by paper and alkathene-lined jute bags. Sas-
important for proper effect of seed treat- try and Chatrath (1984) correlated the persis-
ments. This also goes a long way in enhanc- tence of carbendazim on wheat seeds with
ing the storage life of seeds, especially under storage conditions and type of container.
ambient conditions in the seed warehouses. The quantity of fungicide on seed decreased
Dharam Vir (1977) observed that organo- with the increased storage period, but the
mercurials retained their bioefficacy for a least loss was of seed stored in polythene-
longer period as compared to antibiotics lined jute bags followed by polypropylene
and dithiocarbamates, which degrade and polyethylene, cloth and jute bags, and
become biologically ineffective after storage storage at 30°C resulted in more degrada-
of treated paddy seeds for 1 year. tion as compared with lower temperatures.
The persistence of fungicides on the Lakshmi and Gupta (1997) reported a sig-
seed is of paramount importance as it deter- nificant reduction in the quantity of thio-
mines the longevity of effective seed treat- phanate methyl on soybean seed with an
ment during storage. Certain fungicides, extended period of storage. Maximum per-
irrespective of the amount of dressing pres- sistence of fungicide was found on seeds
340 A. Gupta
stored in polythene-lined jute bags, fol- before the crop is harvested. It is essential to
lowed by polypropylene polyethylene bags. keep the crop healthy and disease free. Use
Gupta (2002) observed a loss of 10–100% in of pathogen-free/certified seed material is
the activity of different chemicals during the most effective method of disease-free
storage. The loss was more in treated seeds seed production. Disease-free seed produc-
stored in cloth bag packaging compared to tion should be planned in safe areas and
treated seed stored in a polythene bag inside seasons where disease development is
a cloth bag. restricted or absent. Preharvest sprayings
The loss in the activity of the chemicals with suitable chemicals, namely fungicide
apparently may be due partly to evapora- or biocontrol agents, and harvesting the
tion of the active compound of the chemical crop at proper maturity also help to main-
and partly to diffusion of the compound tain seed quality during storage. Stress con-
into the seed. Raju and Chatrath (1978) ditions during plant growth also influence
reported that the process of the degradation seed longevity.
of fungicides was influenced by storage con- Discoloration of seeds by fungi occurs
ditions. Besides, other factors such as envi- when the crop is in the field. Govindrajan
ronmental changes or physiological changes and Kannaiyan (1982) observed reduction
may also be responsible for the depletion of in grain discoloration of rice through pre-
the activity of the chemicals. However, this harvest spraying with copper oxychloride.
loss in the activity of the chemical can also Seed discoloration in paddy increased with
be correlated to the presence of seed myco- higher levels of nitrogen and phosphorus
flora during storage. The loss of activity is and decreased with larger spacing in the
lower, the incidence of fungal flora is lower field (Misra and Dharam Vir, 1992). Accord-
and seed germination is higher. ing to Deka et al. (1996), application of
maneb at boot leaf stage, followed by spray-
ing with common salt, was highly effective
in reducing discoloration in paddy grains.
Management of Storage The association of fungi is likely to be
Fungi to Preserve Seed Longevity greater in regions where the produce is har-
vested in the wet season. Indira and Rao
Seed longevity can be maintained either by (1968) observed higher association of stor-
reducing or preventing the fungal inocu- age fungi in samples obtained from areas
lum, or by creating unfavourable conditions with high humidity. Misra and Kanaujia
for their growth. Management strategies (1973) considered the presence of antifun-
should include practices both at preharvest gal substances in the seed coat of some
and postharvest stages. Proper storage and oilseeds to be the reason for less storage
application of safe chemicals as postharvest fungi. Nair (1982) reported fewer fungi on
treatments can control seed mycoflora effec- seeds of Luffa acutangula because of their
tively and reduce losses due to storage fungi thick and hard seed coat, which has a low
considerably. Seed treatment is one of the moisture-holding capacity. Varietal differ-
most effective, safe and economic technolo- ences with regard to their susceptibility to
gies which protects the seed from microbial fungal attack during storage has been
deterioration, and thus improves its health observed by Sheeba and Ahmed (1994),
status during storage and also ensures better who found higher fungal incidence on seeds
field emergence and seed yield. of high-yielding varieties of paddy as com-
pared to local cultivars.
Preharvest management strategies

Postharvest management strategies
For seed to remain healthy during storage,
management strategies need to be followed Initial seed quality, seed moisture, storage
from the time the crop is in the field, i.e. temperature and RH play an important role
in determining seed longevity. It is essential where the material has to be kept is clean,
to avoid mechanical injuries to seed during dry, cool and properly aerated. The seed
harvesting/threshing. The produce needs to material should be packed in clean and, if
be dried properly to safe moisture levels possible, new containers. If old containers
before storage. The maximum drying tem- are being used, they should be disinfected
perature recommended for vegetable crop or fumigated properly to avoid any carry-
seed is 35°C. Sun drying of seeds can be over pathogens.
practised at the farmer’s level. The seeds are Storage structures should not permit
usually packed in gunny bags or cloth bags. entry of water by seepage from the ground
Moisture-proof containers, hermetically or walls. Low temperature retards the devel-
sealed cans, polyethylene pouches or poly- opment of storage fungi on seeds and so is
lined aluminium foil packets are usually advisable, especially for low-volume, high-
used for high-value, low-volume seeds, but value seeds. It is also essential to ensure
it is essential to dry the seed to 5–6% mois- that the seed material meant for storage
ture level before packing in these containers should be of high quality. The material
because moist seeds tend to deteriorate should be stacked on wooden pallets, main-
faster in sealed containers in comparison to taining a proper distance from the walls and
ordinary containers. ceilings. The material should be checked
Pre-storage seed treatment improves regularly for the development of any pests
the shelf life of the seed, protects it from and efficient remedial measures must be
microbial deterioration and ensures better employed immediately to keep them under
seed germination and better field stand. control. Thus, disease management of stored
Many horticultural crops are propagated by grains requires optimum storage conditions
stems, roots, leaves, tubers, corms, rhizomes, and deployment of treatments that do not
suckers, grafts and other vegetative stocks pose any health hazards to consumers.
besides seed. This propagative material may
carry several pathogens which cause differ-
ent diseases, thereby affecting their field Conclusions
establishment. The pathogens present in the
soil may also hamper field establishment of Seed storage is highly influenced by several
these propagules. Adopting proper seed intrinsic and extrinsic factors. Among them,
treatment technology can reduce most of genotypes, seed treatment and storage con-
these problems. This technology is benefi- tainers assume a prime role in successful
cial as it involves less wastage of chemicals, seed storage. Studies are required to identify
greater control over application, less envi- disease-free and disease-prone areas for seed
ronmental pollution, low risk for operators, production, as healthy seed produces healthy
minimum man power, is independent of crops. Certification standards for many seed-
weather conditions and has less deleterious borne diseases need to be developed. The
effects on the treated material. development of suitable cost-effective pack-
Lal (1975) reported propionic acid and aging material for safe and prolonged stor-
potassium metabisulphite as effective against age of seeds is also needed. Identification of
A. niger, A. flavus, P. oxalicum and A. alter- suitable pre-storage treatments with suit-
nata on wheat and maize grains. Acetic acid able agrochemicals and botanicals for the
and propionic acid has proved effective seeds is another important field which
against A. flavus and C. lunata on groundnut needs further work.
kernels. According to Vaidya and Dharam Some research has been initiated on an
Vir (1986, 1987), sodium metabisulphite eco-friendly approach against diseases in
and propionic acid checked the growth of the field, but their efficacy during storage
Aspergillus and Penicillium sp. on ground- needs elucidation. The methods available
nut kernels. After ensuring the quality of for the proper application of seed/soil dress-
seed material meant for storage, it is also ings also need further refinement. The
essential to ensure that the seed warehouses application of microorganisms as agents for
342 A. Gupta
the biocontrol of plant diseases in agricul- are artificially encapsulated propagules

ture is an important alternative to chemical used for sowing as a seed and they possess
fungicides. Halogenation of seeds has also the ability to convert into a plant under in
proved a better seed storage treatment for vitro or in vivo conditions. The preservation
prolonging seed viability (Dharmalingam of viability and vigour of somatic embryos
et al., 2000). Invigoration treatments, namely and synthetic seeds is one of the problems
the effect of hydration–dehydration or invi- which has to be solved prior to applying
goration with different salt solutions on synthetic seed technology practically. One
substandard seed lots for microbial growth, of the future uses of synthetic seeds would
need elucidation. Mid storage hydration– be in germplasm conservation through
dehydration treatment helps to maintain cryopreservation. With the introduction of
vigour, viability and productivity of crop transgenic crops, it has become all the more
seeds (Basu, 1994; Mandal et al., 2000). important to ensure the quality of seeds that
Priming improves biological seed treatment, are traded across borders. The genetically
where the primed seeds of pea and French modified seeds need to be assessed very
bean show superiority over other treat- carefully for any contamination with seed-
ments (Rawat and Kumar, 2003). Pelleting borne pathogens, which can be accom-
of seeds is also advantageous, as the appli- plished by using the modern tools of
cation of pesticides, micronutrients, biofer- biotechnology. Although Bt genes have
tilizers or plant leaf powders can be proved to be quite effective in short-term
incorporated into the seed for improve- protection against insect damage, there are
ment in germination. concerns that widespread use of Bt varieties
Some biotechnological approaches may will accelerate the development of resis-
also be exploited by incorporating genes for tance to Bt in target pests. Thus, to facilitate
better storability of seeds. The production seed trade, strict quarantine measures at
of artificial seeds has unravelled new vistas in national and international level are neces-
plant biotechnology. These synthetic seeds sary in restricting high-risk diseases.
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26 Controlling Root and Butt Rot
Diseases in Alpine European Forests
Paolo Gonthier
Department of Exploitation and Protection of Agricultural and Forestry Resources
(DIVAPRA), Plant and Forest Pathology, University of Torino, Grugliasco, Italy
Abstract
Alpine European forests comprising of conifers and broadleaf trees at lower altitudes are facing major
problems of poor regeneration and occurrence of numerous fungal diseases. Fungal organisms like
Armillaria mellea and Heterobasidion annosum are taking their toll on a large number of conifers.
These two pathogens are responsible for most of the root and butt rot diseases in natural forest stands.
Diagnosis of disease can be done by macro/micromorphology of basidiomata. The wood-inhabiting
fungi can also be identified by taxon-specific primers using PCR. This chapter deals with various bio-
logical and cultural control strategies and the promotion of disease-tolerant plants, which can reduce
the occurrence of diseases. Integrated disease management plans are suggested for different species as
found suitable in the Aosta Valley of the western Italian Alps.
Introduction landscape and nature conservation. Sub-

stantial economic and social changes in
Mountains and uplands cover approxi- mountain areas over the past few decades
mately one-fifth of the earth’s surface and have modified forest use drastically. Tradi-
about one-tenth of the world’s population tional forest functions (i.e. wood produc-
lives in mountain regions (Ives et al., 1997). tion) have been abandoned, while the
Mountain forests have drawn growing atten- importance of other functions has grown.
tion in the past few decades in both North Human activity has transformed mountain
America and Europe. It has been hypothe- forests radically in various ways: large for-
sized that if no forests existed in the Alps, est areas have been destroyed and the natu-
humans would not inhabit most of the val- ral composition of forests has been modified
leys (Motta and Haudemand, 2000). Forests through logging and thinning (Motta and
protect cities and villages against ava- Haudemand, 2000).
lanches, landslides, debris flows and rock- European alpine forests comprise
falls. They fix surface soil, prevent erosion mostly of conifers, while broadleaves are
and play an essential role in water resource generally widespread at lower altitudes,
management. They influence climate and where they may form significant stands.
air quality. At present, the main functions Only eight native coniferous tree species are
afforded by alpine forests are protection, present in the alpine region (Ozenda, 1985):
tourism and recreation, wood production, Abies alba Miller (silver fir), Picea abies (L.)
346 P. Gonthier
Karsten (Norway spruce), Larix decidua Although a large number of lignicolous fun-
Miller (European larch), Pinus cembra L. gal species are reported on conifers in Euro-
(Swiss stone pine), P. sylvestris L. (Scots pean mountain areas, including the Alps
pine), P. uncinata Miller (mountain pine), P. (Breitenbach and Kränzlin, 1986, 1991,
mugo Turra (dwarf mountain pine) and P. 1995; Bernicchia, 2005), only a few of them
nigra Arnold (Austrian pine). These conifers are aggressive organisms having a signifi-
can grow in pure or mixed stands, depend- cant impact on forests (Table 26.1). All these
ing on the site. fungi belong to the Basidiomycota and are
Most forests in the Alps are naturally necrotrophic tree pathogens causing wood
regenerated and current guidelines of forest decay. They are facultative pathogens, being
management are aimed at maintaining ade- able to survive saprotrophically on dead
quate levels of natural regeneration. Another wood. They may be classified either as
trait differentiating alpine forests from other white rot or brown rot agents depending on
forests located in flat or even in mountain the component of the plant cell wall they
areas is that in alpine forests, given their are able to utilize, i.e. lignin or cellulose,
prominent protective function, clear cut fol- respectively.
lowed by artificial planting is generally for- The infection biology of wood decay
bidden. Furthermore, mixed, irregular or fungi in living trees has been reviewed pre-
uneven-aged stands are supported locally viously (Rayner and Boddy, 1986). In gen-
and maintained through appropriate silvi- eral, primary infections occur by means of
cultural practices (e.g. selective cutting, airborne meiospores, which allow for the
‘forêt jardinée’) where such features fulfil infestation of new forest areas. Some of
particular functions better (i.e. protection, these fungi may also operate a secondary,
landscape). Several forests in the Alps, most vegetative spread, allowing for the expan-
of which are protected forests, are affected sion of individuals established through
by major problems (Mayer, 1982), including primary infection. Depending on the tree
a lack of regeneration, a scarcity of medium- pathogen, this expansion may occur vegeta-
aged trees, insufficient stability and increas- tively through root grafts or contacts, lead-
ing vulnerability to natural disturbances. ing to a tree-to-tree contagion, or by free
Plant pathogens, including those caus- growth of the fungus in the soil through rhi-
ing root diseases or butt rots, may behave as zomorphs or mycelial cords. In some patho-
natural disturbances. In addition to causing systems, insect vectors are essential for the
severe economic losses, they are reported to transmission of wood decay fungi (Slippers
influence patterns and processes in forest et al., 2002).
ecosystems and to be affected by forest The relative importance of primary and
development and landscape characteristics, secondary infection is significant not only
as well as by human activities (Castello et al., for our understanding of the epidemiology
1995; Hansen and Goheen, 2000). This chap- and population biology of these fungi, but
ter reviews the significance and the epidemi- also for control and management purposes.
ology of the most important and widespread Pathogens like Armillaria spp. and Heteroba-
root and butt rot diseases of alpine forests, as sidion spp. are able to spread secondarily.
well as the most effective and promising When this happens, there is a carry-over of
control strategies to fight them. the pathogen into new generations; in this
case, novel attacks are not necessarily caused
by new primary infections, but by the inocu-
lum established at that site previously.
Root and Butt Rot Pathogens, Most root and butt rot agents are wound
Their Significance, Ecology and pathogens able to gain entry into the trees
Infection Biology through wounds or lesions. Some of them
are obligate wound pathogens (e.g. Stereum
Root and butt rot fungi are important com- sanguinolentum), while others are facultative
ponents of forest ecosystems worldwide. wound pathogens (e.g. Heterobasidion spp.).
Table 26.1. Summary of characteristics of the most important root and butt rot fungi present in alpine forests.
Fungi Main hosts1 Rot type Disease/symptoms Infections Reference2
Armillaria mellea Several conifer and White, fibrous, Root rot and mortality; Root contacts; active Guillaumin and
(Vahl:Fries) broadleaf species wet, often with occasionally heart decay: pathogenesis through Legrand, 2005
P. Kummer sensu lato black zone lines butt rot rhizomorphs
Controlling Root and Butt Rot Diseases

Climacocystis borealis Norway spruce, White, mottled rot Heart decay: butt rot Through wounds3 on Solheim, 2006
Kotl. & Pouzar silver fir, larch, pines roots and bole
Fomitopsis pinicola Silver fir, Norway Brown, cubic Decay: butt and stem rot By spores through Butin, 1995;
(Sw.) P. Karst. spruce, pines stem wounds Bernicchia, 2005
Heterobasidion Conifers; mostly White, pocket rot Root rot and mortality; heart By spores through fresh Asiegbu et al., 2005
annosum (Fr.) Bref. pines, Norway decay in roots, butt and stem stump surfaces or
sensu lato spruce, silver fir wounds; root contacts
and grafts
Laetiporus sulphureus Larch, Norway spruce; Brown, cubic Heart decay: butt and stem rot; By spores through Bernicchia, 2005;
(Bull.) Murrill sensu oaks and chestnut root rot and mortality when wounds Butin, 2005
lato among broadleaves decay reaches the root system
involving sapwood
Onnia tomentosa (Fr.) Norway spruce, White, pocket rot Root rot and mortality By spores through deep Tainter and
P. Karst (= Inonotus Scots pine root wounds; vegetatively Baker, 1996
tomentosus) through root contacts and
grafts
Phaeolus schweinitzii Norway spruce, Brown, cubic Heart decay: butt and stem rot Vegetatively in the roots Barrett and Greig,
(Fr.) Pat. silver fir, pines 1985; Butin, 1995
Stereum sanguinolentum Norway spruce Pale brown, Butt and stem rot By spores through wounds Vasiliauskas et al.,
(Alb. & Schwein.) Fr. stringy rot 1996; Solheim,
2006
Note: 1Species are listed based on their in-field susceptibility, the most susceptible listed first; 2reference for the infection biology; 3wounds are considered of mechanical origin.
347
348 P. Gonthier
Only a few root and butt rot disease agents distinguishable from those of typical cubi-
do not need wounds to gain entry into the cal brown rots in that they are much finer
tree (e.g. Armillaria spp.). At the same time, (1–2 mm). The fungus is rarely lethal, but
most root and butt rot fungi are weak, sec- has been reported to cause significant eco-
ondary pathogens, being unable to attack nomic losses locally and to amplify the
vigorous trees. However, some of them are mechanical instability of trees during storms,
not (i.e. Heterobasidion spp., Onnia tomen- especially in mountain, mature Norway
tosa) and behave as primary pathogens, spruce stands (Rigling et al., 2005).
which may cause significant disease with or Fomitopsis pinicola and Laetiporus sul-
without pre-existing tree stresses. Weak- phureus sensu lato are two powerful wood-
ened physiological conditions caused either destroying fungi, responsible for brown,
by primary or by secondary pathogens may cubic rots. In the alpine region, the former is
then trigger off attacks by other, secondary associated mostly with severely damaged
parasites (i.e. bark beetles) (Tainter and silver fir and Norway spruce trees. The sec-
Baker, 1996; Jakuš, 2001). ond species, which recently was investigated
Wood decay fungi may rot standing phylogenetically (Vasaitis et al., 2009), attacks
trees in two ways, either starting from the mostly larch trees in alpine forests (Butin,
cambium and then proceeding inward (sap- 1995). They are wound pathogens, the sec-
wood decay), or by decaying the central ond being able to progress towards the root
portion of roots, bole and stem (heart decay) system and here producing root rot and sap-
(Rayner and Boddy, 1986). When the cam- wood decay. Phaeolus schweinitzii is another
bium, functional xylem or outer sapwood widespread brown rot agent. It is reported
are involved, several physiological func- on all coniferous tree species growing in the
tions in trees may be altered. This is partic- Alps and it causes a heart decay of the roots
ularly true for decays affecting the root and the bole, spreading up to 1–2 m into the
system or the collar, which generally lead to stem (Bernicchia, 2005). The infection biol-
a relatively rapid death of the host. In the ogy of this fungus is still largely unknown.
second type of decay, only the smaller The pathogen is believed to infect the roots
woody roots are killed, whereas the larger through the mycelium (Barrett and Greig,
ones, the bole or the stem may remain phys- 1985; Bernicchia, 2005), which is unable to
iologically functional for a long time (Rayner extend freely in the soil over long distances
and Boddy, 1986; Gonthier et al., 2003). (Barrett and Greig, 1985). Thus, spores do
Very few root and butt rot diseases have not play any primary role in the infection.
been studied in detail, for instance those However, they are an important source of
caused by A. mellea or H. annosum species soil infestation (Barrett, 1985). It should be
complexes (reviewed in Shaw and Kile, noted that other ways of infection, such as
1991; Woodward et al., 1998a; Fox, 2000; mechanical butt wounds or root contacts
Asiegbu et al., 2005; Guillaumin, 2005). with diseased trees, have also been suggested
Current knowledge on the other pathosys- for this pathogen (Tainter and Baker, 1996).
tems here described is still very limited. There is very scanty information on the
For instance, only scanty information is significance of O. tomentosa in alpine for-
available on Climacocystis borealis. This ests. Its presence is probably overlooked. In
fungus is reported as a saprophyte and sec- fact, despite differences in the rot type, the
ondary pathogen (Bernicchia, 2005), being fungus can be confused easily with P. sch-
able to cause a typical heartwood rot in the weinitzii since the basidiomata of the two
roots and the bole, which seldom reaches species display similar macroscopic traits
more than 2–3 m in height (Solheim, 2006). (Butin, 1995). O. tomentosa was found on
Sometimes, the sapwood is also colonized. Norway spruce and Scots pine trees. This
The borealis rot is a characteristic white fungus infects trees by spores through deep
mottle rot (Bernicchia, 2005) which, on a root wounds and is also capable of second-
closer look, is cubic with white mycelium ary spreading through root contacts and
in between (Solheim, 2006). Cubes are easily grafts (Tainter and Baker, 1996).
Controlling Root and Butt Rot Diseases 349
S. sanguinolentum is incapable of sec- A. borealis to produce rhizomorphs and to

ondary spreading but is a very strong wound infect trees in this way is variable (Guil-
colonizer, especially on Norway spruce. laumin and Legrand, 2005).
Every wound, from root to top, is vulnerable In Europe, H. annosum sensu lato com-
to infection, even the oldest ones (Vasiliaus- prises three species, responsible for losses
kas et al., 1996). Very important factors for estimated at more than 800m/year (Wood-
infection are wound size and depth (Sol- ward et al., 1998b). H. parviporum Niemelä
heim, 2006). Wound rot is initiated by inju- & Korhonen primarily causes butt rots in
ries caused by bark-stripping red deer, as Norway spruce, but it has also been reported
well as by harvest-induced injuries (Cermák to kill Scots pine saplings and attack exot-
et al., 2004). In Norway spruce, the potential ics. H. abietinum Niemelä & Korhonen is
economic losses caused by cut-off waste commonly associated with root or butt rots
wood or low-quality logs are of considerable in trees of the genus Abies, while H. anno-
magnitude and wound rot affects the trees’ sum sensu stricto is associated typically with
stability negatively (Cermák et al., 2004). root rot and mortality of trees in the genus
Several taxa in the A. mellea and H. Pinus, but it can also be found on Picea, Juni-
annosum species complexes are responsi- perus and even on deciduous trees (Kor-
ble for most of the root and butt rot diseases honen et al., 1998a). All the three species of
of conifers in natural forest stands and planta- the fungus are widespread in alpine conifer-
tions throughout the northern temperate ous forests (Korhonen et al., 1998a; Gonthier
regions of the world (Kile et al., 1991; Asiegbu et al., 2001) and they are extremely perva-
et al., 2005). A. mellea sensu lato encom- sive locally. For instance, levels of disease
passes about 40 biological species of vary- incidence of up to 95% were reported in
ing geographic distributions, host ranges some subalpine Norway spruce stands in
and virulence (Pegler, 2000), seven of which the western Alps (Gonthier et al., 2003). In a
are present in Europe (Marxmüller and recent study, it was discovered that a large
Guillaumin, 2005): A. mellea (Vahl: Fries) majority of gaps and mortality centres in
P. Kummer sensu stricto, A. ostoyae (Romag- mountain pine forests of the Swiss Alps was
nesi) Herink, A. borealis Marxmüller and caused by a Heterobasidion species rather
Korhonen, A. gallica Marxmüller and Romag- than by pathogenic Armillaria species (i.e.
nesi, A. cepistipes Velenovský, A. tabescens A. ostoyae) or other factors (Bendel et al.,
(Scopoli) Emel and A. ectypa (Fries) Lam- 2006). Heterobasidion primarily infects its
oure. This last species is only marginally hosts by means of airborne meiospores, nor-
important since it is a non-lignicolous, non- mally through freshly cut stumps or wounds,
parasitic species. All the European species and is capable of secondarily spreading
may be found in the alpine area, although from tree to tree through root grafts and con-
some of them (i.e. A. tabescens, A. mellea), tacts (Asiegbu et al., 2005). Airborne infec-
being thermophilic, are more common at tion through thinning stumps not only may
low elevations and in the Mediterranean result in a rapid and heavy infection of a
region (Marxmüller and Guillaumin, 2005). healthy stand in areas where Heterobasid-
There is general agreement on the fact that ion is common (Pratt and Greig, 1988; Swed-
basidiospores play a marginal role in wood jemark and Stenlid, 1993), but also may aid
colonization and infection (Guillaumin and the spread of the fungus into new areas
Legrand, 2005). Armillaria root disease may (Berry and Dooling, 1962). Thus, stumps
spread either through root contacts or rhizo- play a crucial role in the epidemiology of
morphs, depending on the Armillaria spe- this forest pathogen, as confirmed indirectly
cies. Root contacts are essential for the by the positive relationship between the
spread of A. tabescens and A. mellea, which incidence of disease in residual trees and
is characterized by fragile and short-lived rhi- the intensity of earlier thinnings, as well as
zomorphs, while the less pathogenic A. gal- the proportion of thinning stumps infected
lica and A. cepistipes generally infect through (Rishbeth 1957; Vollbrecht and Agestam,
rhizomorphs. The ability of A. ostoyae and 1995).
350 P. Gonthier
Effects of Silviculture and and it may be able to spread into the sur-
Land Management rounding trees more easily through non-
grafted root contacts. Such an increase in
With very few exceptions, root and butt rot spreading ability after cuttings could also
fungi are opportunistic pathogens being able occur with other heart rot fungi.
to take advantage of habitat modifications for Logging operations are likely to increase
their establishment and spread. Thinning the probability of attack by most root and
and logging, as well as other forest manage- butt rot fungi, since new infection courts,
ment activities, appear to increase the dam- i.e. wounds, are created. As an example, the
age caused by root and butt rots (Garbelotto, wound rot caused by S. sanguinolentum,
2004). It has been suggested that the current which is a relatively recent problem, seems
high incidence of H. annosum in unman- to be the result of increasing mechanization
aged mountain pine forests of the central of forestry (Butin, 1995). With the use of
Alps is due to intense logging in the past heavy machinery for the extraction of thin-
(Bendel et al., 2006). Large amounts of tim- nings, large bark wounds occur much more
ber and fuel were needed to support mining frequently. This may be crucial for the
activities, which were thriving in the area establishment of fungi, like S. sanguinolen-
between the 14th and 17th centuries. Simi- tum, that need wounds larger than
larly, the high disease severity recorded in 10 × 10 cm to infect trees (Butin, 1995). In
spruce forest stands of the western Alps general, wounds play an important or funda-
(Gonthier et al., 2003) could also be associ- mental role in the infection biology of fungi
ated with mining activities in the past. In that are able to produce infective airborne
some areas of the western Alps, intensive inoculum. Nevertheless, it has been proposed
cutting occurred during the 17th and 18th that wounds could also trigger attacks by
centuries and this led to the creation of fungi unable to infect by spores, i.e. Armil-
stumps over large surfaces (Nicco, 1997). At laria spp., because they can induce a lowering
the same time, the current high level of of tree defences (Popoola and Fox, 1996).
infestation of subalpine forests (Gonthier
et al., 2003) might also have a human ori-
gin. Cuttings have been performed regularly Diagnosis and General
at the upper edge of forests to conserve Control Strategies
alpine grasslands for summer grazing.
As previously stated, the creation of Management options are based on our knowl-
stumps is particularly important for H. anno- edge of the ecology, epidemiology and infec-
sum, as stumps behave as main infection tion biology of the causal agent. Hence,
courts for primary infections. It has been before planning control, there is good rea-
reported that thinnings also promote the son to perform an accurate diagnosis and
tree-to-tree vegetative spread of the pathogen identification of the causal agent.
in infected Norway spruce stands (Bendz- The type of rot may aid in the diagnosis
Hellgren et al., 1999; Piri and Korhonen, but in general it is not an exhaustive trait for
2008). The growth rate of the fungus in roots the identification of wood decay fungi (Ber-
increases after the felling of infected trees nicchia, 2005; Solheim, 2006). Tradition-
(Bendz-Hellgren et al., 1999). In living spruce ally, diagnosis is based on the macro- and/
roots, Heterobasidion is confined typically to or micromorphology of basidiomata and it
dead heartwood. Hence, the transfer of the may be achieved through the use of mycologi-
fungus between living trees may be limited cal keys (Eriksson et al., 1984; Breitenbach
to functional root grafts, which enable the and Kränzlin, 1986, 1991, 1995; Bernicchia,
fungus to grow from the xylem of infected 2005; Gonthier and Nicolotti, 2007). When
roots into the xylem of healthy roots. After performing field diagnosis, a pathologist
the tree is cut, Heterobasidion begins to should consider that basidiomata of wood
expand outwards from the centre of the root decay fungi usually emerge at advanced
stages of the fungal infection and they may the identification of the most important root
be rarely or sporadically visible. Further- and butt rot fungi. A summary of them is
more, basidiomata of some species (e.g. P. given in Table 26.2. Some techniques allow
schweinitzii) are short-lived (Butin, 1995) for the identification of rots directly from
or may be found only during particular peri- wood. For instance, polymerase chain reac-
ods of the year (i.e. Armillaria spp.); thus, tion (PCR) with taxon-specific primers pro-
the timing of diagnosis is also important. vides reliable fungal diagnostics from both
Most root and butt rot fungi can be cultured pure culture and environmental samples.
easily from decayed wood. A few of them Theoretically, a pathogen can be controlled
(i.e. Heterobasidion spp., L. sulphureus) during all stages of its life cycle, starting from
develop a fast-growing conidial stage in cul- primary infection and early establishment,
ture or when colonized wood is incubated in through spreading inside the host, to forma-
a damp room. Usually, asexual mitospores of tion, spread and survival of its propagules
these fungi do not play any significant role (Holdenrieder and Greig, 1998). Unfortu-
in the infection biology. Nevertheless, they nately, root and butt rot diseases are virtually
may have a diagnostic value. Identification impossible to eradicate once they are estab-
of wood-inhabiting fungi, non-sporulating lished. They may be controlled successfully
in pure culture is also possible by using only when pathogens have a small biomass
appropriate keys (Nobles, 1965; Stalpers, and are therefore weakly competitive.
1978). Pure culture analysis, however, is In general, when dealing with root and
difficult and time-consuming. butt rot diseases characterized by abundant
A number of molecular techniques have primary infection events (i.e. H. annosum,
been developed and are now available for S. sanguinolentum, etc.), forest management
Table 26.2. Taxon-specific primers developed for the identification of some of the most important root
and butt rot fungi present in alpine forests.
Fungi Forward primer Reverse primer Amplicon size Reference
A. mellea ARM-1 (agggta ARM-2 (ggaaagctaa 660 bp Schulze et al., 1997

sensu lato1 tgtgcacgttcgac) gctcgcgcta)
ITS3 (gcatcgat Armi2R (aaacccccat 184 bp Guglielmo et al., 2007
gaagaacgcagc) aatccaatcc)
H. annosum HET-7 (cttctcac HET-8 (caggtccccca 400 bp Bahnweg et al., 2002
sensu lato aaactcttcg) caatcg)
H. annosum MJ-F (ggtcctgtc MJ-R (ctgaagcacac 100 bp Hantula and
sensu lato tggctttgc) cttgcca) Vainio, 2003
H. parviporum KJ-F (ccattaac KJ-R (gtgcggctcattc 350 bp
ggaaccgacgtg) tacgctatc)
H. annosum MLF (taaaaatttaa Mito7 (gccaatttatttt 230 bp Garbelotto et al.,
sensu stricto attagccataa) gctacc) 1998; Gonthier et al.,
H. abietinum Mito5 (taagaccgctata 195 bp 2001, 2003
H. parviporum MLS (aaattagcca waccagac) 185 bp
tattttaaaag)
L. sulphureus 25sF (tggcgaga LaetR (ccgagcaaac 146 bp Guglielmo et al., 2007
sensu lato gaccgatagc) gaatgcaa)
O. tomentosa It-ITS-209-f (gcta It-ITS-700-rc (agga 491 bp Germain et al., 2002
aatccactcttaacac) gccgaccacaaaagat)
Stereum spp. ITS3 (gcatcgatg Ste2R (gtcgcaacaa 234–240 bp Guglielmo et al., 2007
aagaacgcagc) gacgcactaa)
Note: 1Some Armillaria species may be distinguished through PCR-restriction fragment length polymorphism (RFLP)
(Harrington and Wingfield, 1995; Schulze et al., 1997; Sierra et al., 1999).
352 P. Gonthier
should focus on minimizing those activities infected trees are isolated. It is usually quite
likely to create good primary infection courts, difficult to determine whether or not trees
e.g. wounds on roots, stems and stumps. Care are infected; colonization of the root sys-
should be taken in order to avoid harvest- tems may go undetected for long periods of
induced injuries. Containment of wildlife time. Trenching is a very impractical con-
populations, especially of bark-stripping trol method and its use is advisable only
deers, may also have some effects on wound when pathogen inoculum is very localized.
rot severity (Cermák et al., 2004). It should be noted that in the case of
Lowering stand density may regulate pathogens that are also able to spread aeri-
secondary infection events, especially for ally, trenching would offer limited protec-
pathogens spreading through root grafts and tion. In the case of H. annosum, for instance,
contacts. In addition, a regulation of stand observational data suggest that instead of
density aimed at reducing tree competition controlling the disease, trenching could
may prevent or reduce infections by a wide actually promote the spread of the fungus
range of weakness pathogens, regardless of by breaking and injuring roots (Korhonen
their mode of transmission (i.e. Armillaria et al., 1998b).
spp., F. pinicola, etc.).
The management of mature alpine for-
ests that are diseased or under the risk of
root and butt rots could be achieved through Tree and stump removal
several other and more specific methods,
which are reviewed below. Some of them Secondary mycelia of most root and butt rot
are hardly applicable in mountain areas or fungi will survive and produce basidiomata
may be justified only locally. Some others for a long period of time on colonized wood.
are currently used over large areas, includ- Thus, infected trees should be removed
ing the alpine region. An integrated disease from the stand promptly in order to reduce
management approach could be more effi- the airborne inoculum of fungi spreading
cient than single methods in controlling through spores. As most of these fungi can
these diseases. attack timber, asymptomatic felled trees
should also be removed. In general, timber
is unselective or less selective than standing
trees to infection by wood decay fungi
Trenching (Rayner and Boddy, 1986). As an example, S.
sanguinolentum, which commonly attacks
Digging isolation trenches around diseased Norway spruce trees, is reported to colonize
trees to prevent the secondary, vegetative felled wood of spruce but also of pine and
spread of root and butt rot fungi is one of the silver fir, in which it causes a red streaking
most traditional control methods recom- (Butin, 1995). Thus, attention should be
mended against several pathogens including given not only to preferential hosts, but also
H. annosum sensu lato and A. mellea sensu to other tree species that can become sapro-
lato (Korhonen et al., 1998b; Kliejunas et al., phytically colonized.
2005; Legrand et al., 2005; Eyles et al., 2008). Removing stumps and roots from the
The trench should be at least 70–100 cm soil has been recommended for controlling
deep. Instead of an open trench, it could be both H. annosum sensu lato and A. mellea
more practical to bury a plastic sheet in a sensu lato (Korhonen et al., 1998b; Legrand
vertical position into the soil (Korhonen et al., 2005). Benefits of this method have
et al., 1998b; Legrand et al., 2005). As sug- been reviewed recently by Vasaitis et al.
gested by Eyles et al. (2008), the effective- (2008). It should be noted that de-stumping
ness of this method depends on the regular can show effectiveness not only against dis-
maintenance of trenches, to prevent the re- eases spreading from tree to tree through
establishment of root contacts, and on the root grafts and contacts, but rather it may be
proper siting of the trenches to ensure all effective against a wide range of wood decay
agents. In fact, for instance, basidiomata of on the pathogen might result in the infec-
P. schweinitzii growing on stumps are tion of plants normally considered as non-
reported as sources of soil infestation for host (Garbelotto, 2004).
long periods of time (Barrett, 1985). Stump This method of control might show
removal is an expensive, time-consuming some effectiveness in controlling Heteroba-
control method (Korhonen et al., 1998b; sidion root and butt rots. In a recent unpub-
Legrand et al., 2005) that requires the use of lished study conducted in the western
machines (Omdal et al., 2001). This method Italian Alps, and based on the analysis of
can be adopted optionally in certain artificial about 2300 recently felled trees, it was
plantations after clear-felling. It is rarely used found that disease incidence was signifi-
in mountain forests or after selective cuttings. cantly higher on Norway spruce (43% of
Furthermore, de-stumping contrasts with cur- average incidence) than on other native tree
rent trends in forest management since it may species (Table 26.3). Silver fir was also
have negative effects on biodiversity. rather susceptible, while larch and, espe-
cially, Scots pine trees were more tolerant.
Moreover, with the exception of H. anno-
Promoting tolerant species sum sensu stricto, a strict host preference of
H. annosum species has been reported in
If a forest is heavily infested by a root or butt the Alps (Gonthier et al., 2001). Thus, based
rot agent with restricted or defined host on these data and on other observational
range, control may be achieved by increas- data (Table 26.3), it is likely, for instance,
ing the proportion of trees more resistant to that the establishment of deciduous tree
the pathogen. In principle, a rotation of a species would have beneficial effects in
resistant tree species can clean the site of most Heterobasidion-infested forests, but
the pathogen inoculum (Korhonen et al., not necessarily in H. annosum sensu stricto
1998b). The concept of forest rotation has infected stands (Table 26.3). Also, the con-
been widely advocated for the management trol of H. abietinum in heavily infected sil-
of root rots of forest trees, with contrasting ver fir forests could be achieved either by
results (Korhonen et al., 1998b; Lygis et al., promoting Scots pine trees or Norway
2004). The pathogen inoculum usually per- spruce trees. The disease in severely dam-
sists in stumps and roots for decades after fell- aged Norway spruce stands can be lowered
ing, and this was reported not only for H. by favouring the more resistant larch.
annosum or A. mellea species complexes A. mellea sensu lato species display a
(Korhonen and Stenlid, 1998; Guillaumin and lower degree of host preference with respect
Legrand, 2005) but also for other root and butt to H. annosum sensu lato species. Never-
rot fungus, i.e. P. schweinitzii (Barrett, 1985). theless, variation of tree species composi-
Changes in tree species composition, tion could have some beneficial effects for
turning a susceptible forest into a more tol- this pathosystem also. For instance, Swiss
erant one, can be achieved in a relatively stone pine trees have been reported as very
short period of time through clear-felling susceptible to A. ostoyae in subalpine forest
only, followed by artificial plantation. In nat- (Anselmi and Lanata, 1989). Cuttings pro-
urally regenerated, uneven-aged or irregular moting the regeneration and establishment
alpine forests, shifts in tree species com- of the most tolerant larch could be effective
position are more difficult to obtain and in the management of Armillaria root rots in
they should be driven by appropriate silvi- these high elevation forests.
cultural practices. Obviously, in such con-
ditions, for a reduction of the pathogen
inoculum to occur, several decades, or even
centuries, may be necessary. As for most Timing of thinning and cutting
plant diseases, it could be advisable to avoid
the complete removal of the susceptible The timing of logging and harvesting may
species, since the resulting selection pressure have a strong impact on the incidence of
354 P. Gonthier
Table 26.3. Susceptibility of native alpine forest trees to H. annosum species based on the author’s
experience and on previously published (Gonthier et al., 2002, 2003) and unpublished data. The list does
not include susceptibility and symptoms of seedlings.
Average
Heterobasidion H. annosum sensu
Tree species incidence1 H. parviporum H. abietinum stricto
Abies alba 17% H +++

Picea abies 43% H ++++ H ++
Larix decidua 12% H ++ H +
Pinus cembra 15% H ++ H ++
P. sylvestris 4% M ++
P. uncinata ? M ++?2
Broadleaves ? – +
Note: 1Based on the analysis of about 2300 recently felled trees from 22 forest stands in the western Italian Alps;
2symptoms and tentative susceptibility according to Bendel et al., 2006. Symbols: +, recorded; ++, occasionally
diseased; +++, susceptible; ++++, very susceptible; M, root rot and mortality; H, heart rot; –, saprophyte.
airborne infectious diseases. As a rule, oper- the hazard of stump infection is not always
ations should be done preferably when envi- described accurately by spore loads on
ronmental conditions are unfavourable to woody traps (Driver and Ginns, 1969), recent
the pathogen. For H. annosum sensu lato, unpublished data confirm the above seasonal
winter thinning and logging operations have patterns of spore deposition and indicates
been used in Fennoscandia to take advan- that the highest risk of stump infection
tage of the low inoculum pressure during the occurs in autumn (Gonthier and Nicolotti,
cold season (Brandtberg et al., 1996; Piri and unpublished).
Korhonen, 2008). In that area, infections fol- Winter operations may not be feasible
low a bell-shaped curve with very low spore at all sites in the Alps. However, Heteroba-
deposition rates in winter; an average of only sidion primary infections would be con-
2% of Norway spruce stumps was infected trolled successfully by planning logging
following thinning in November–February and thinning in most of spring and in early
compared with 34% in June–July (Brandt- summer. Although the above timing may be
berg et al., 1996). somewhat impractical, its advantage in Nor-
Seasonal patterns of spore deposition way spruce includes limiting infection
of Heterobasidion species have been stud- through wounds not only by H. annosum
ied in the alpine region recently with the sensu lato but also by S. sanguinolentum,
aid of woody traps (Gonthier et al., 2005). whose annual basidiomata are produced in
Here, the airborne inoculum of this patho- autumn (Solheim, 2006).
gen, although present starting in February at
most sites, is higher in August–October,
reaching a peak in September. A relative peak,
lower than the late summer one, appears in Biological and Chemical Control
late spring (Fig. 26.1). Thus, despite the
development of perennial basidiomata, in Several biological and chemical methods
the Alps the inoculum production of Heter- have been tested for the control of root and
obasidion spp. is concentrated largely in a butt rot fungi, especially of H. annosum sensu
period of 2–3 months. Spore inoculum in lato and A. mellea sensu lato (reviewed in
winter, spring and early summer is gener- Holdenrieder and Greig, 1998; Pratt et al.,
ally low (Gonthier et al., 2005). Although 1998; Guillaumin et al., 2005a,b). Most
100
90
80
70
Infected traps (%)
60
50
40
30
20
10
0
JAN FEB MAR APR MAY JUN JUL AUG SEP OCT NOV DEC
Months
Fig. 26.1. Average percentage of woody traps infected monthly by Heterobasidion spores in four forests
of the western Alps from 1998 to 2000. Re-elaborated from data published by Gonthier et al. (2005). Bars
show standard errors.
experiments were conducted in vitro. Cur- higher colonization frequency of spruce

rently, only a very few control approaches stumps with respect to the Rotstop® strain
are recommended in practical forestry and (Cech et al., 2008).
they are all devoted to H. annosum sensu lato. Several chemicals proved to be effec-
Stump treatment with appropriate biological tive as stump protectants against Heteroba-
or chemical products immediately after fell- sidion airborne infections (Pratt et al., 1998),
ing may prevent H. annosum primary infec- the most known of which are urea in Europe
tions, therefore reducing timber losses. and borax in North America. Both com-
A number of fungi have been tested on pounds, as well as other chemicals, were
stumps as competitors or antagonists through- tested in the western Alps on spruce stumps
out North America and Europe (Holdenrie- and they showed very good results, compa-
der and Greig, 1998), including in the alpine rable to those obtained with the Rotstop®
forests (Nicolotti et al., 1999). Only Phlebi- treatment (Nicolotti et al., 1999; Nicolotti
opsis gigantea (Fr.) Jül is used currently, and Gonthier, 2005). The effectiveness of
with good results over large areas (Holden- urea was dependent on the concentration of
rieder and Greig, 1998; Thor, 2003; Berglund the water solution: the best results were
and Rönnberg, 2004; Thor and Stenlid, 2005). obtained with a 30% concentration. The
Three distinct products based on this sapro- rise of pH of stump surfaces, which occurs
trophic fungus have been developed: PG commonly during hydrolysis after treat-
Suspension® in the UK, PG IBL® in Poland ment, rather than a toxicity of urea or urea-
and Rotstop® in Fennoscandia. Rotstop® derivate compounds per se (e.g. ammonia
showed a very good effectiveness in alpine and ammonium ions), is responsible for the
Norway spruce stands heavily infected inhibition of Heterobasidion germination
by Heterobasidion (Nicolotti et al., 1999; and growth (Johansson et al., 2002). Such a
Nicolotti and Gonthier, 2005). In a recent high urea concentration allows high pH
comparative study performed in Austrian values on stumps to be maintained for at
alpine protection forests, strains of Phlebi- least the length of time these remain suscep-
opsis gigantea from Poland resulted in a tible to infection, i.e. approximately 1 month.
356 P. Gonthier
Because the costs of registration are very methodologies to reduce parasites are more
high, it is unlikely that any biological or effective and even cheaper than single con-
chemical product will be registered in the trol methods. In the field of forest trees,
near future for stump treatments in the integrated pest management (IPM) systems
alpine area. However, urea and borax are have been developed especially for the pro-
currently classified as fertilizers and their tection of forests against insects or nurseries
use is mostly unregulated for forestry pur- against diseases (Volney and Mallett, 1998;
poses (Nicolotti and Gonthier, 2005). Urea South and Enebak, 2006). Appropriate IPM
could be preferred for its long history in systems may be developed only with a good
stump treatment in Europe (Nicolotti and understanding of the pathogen biology and
Gonthier, 2005; Oliva et al., 2008) and for disease epidemiology. Except for a few
its moderate effects on non-target organisms pathosystems (i.e. H. annosum sensu lato,
inhabiting stumps (Table 26.4). Further- A. mellea sensu lato), our current under-
more, urea is effective on stumps of several standing of the epidemiology of root and
native alpine tree species (Gonthier and butt rot diseases is still limited to allow the
Nicolotti, unpublished). development of efficient IPM systems. How-
ever, the biology and epidemiology of H.
annosum sensu lato are well known and
Integrated Disease Management some weak points exist in its life cycle (i.e.
and Forest Protection: stage of infection by spores).
A Concluding Example An integrated management system was
designed to control H. annosum root and
It is generally agreed that systems combin- butt rots in the Aosta Valley, western Italian
ing cultural, biological, chemical or other Alps. The system is based on stump treatment
Table 26.4. A summary of the effectiveness and impact on non-target fungi of biological and chemical
treatments against Heterobasidion airborne infections on Norway spruce stumps in the western Alps.
Effectiveness
Antagonist/competitor or Application method or against Impact on
active ingredient commercial product Heterobasidion1 non-target fungi2
Biological
Hypholoma fasciculare Wheat mash Low Low (after 2 years)
Phanerochaete velutina Wheat mash High Low (after 2 years)
Phlebiopsis gigantea Rotstop® High High
Vuilleminia comedens Wheat mash Low Low (after 2 years)
Verticillium bulbillosum Culture filtrate Medium Very low
V. bulbillosum Conidial and mycelial Medium Very low
suspension
Trichoderma harzianum Conidial and mycelial Low Very high
suspension
Chemical
Copper oxychloride Azuram® High Low (after 2 years)
Propiconazole TILT (25% emulsion) High Low (after 2 years)
Sodium tetraborate Borax powder High Very high
decahydrate
Urea Water solution 10% conc. Low Very low
Urea Water solution 20% conc. High Low (after 2 years)
Urea Water solution 30% conc. High Low (after 2 years)
Note: 1Categories (low, medium, high) were designed based on previously reported results (Nicolotti et al., 1999;
Nicolotti and Gonthier, 2005); 2categories (very low, low, high, very high) were designed based on previously reported
results (Varese et al., 1999; 2003a,b).
with urea at 30% concentration, combined potential dispersal range of Heterobasidion

with an appropriate timing of thinning and spores (Gonthier et al., 2001), with spore
logging operations, and with practices densities undergoing huge dilution after the
aimed at promoting tolerant species (Fig. first metres (Stenlid, 1994), the migration of
26.2). In the Aosta Valley, forest manage- even a few spores may be significant for
ment activities are planned yearly by the areas still not colonized by the pathogen
Regional Forest Administration, who (Garbelotto, 2004).
decides the stands that need to be thinned Winter operations may be possible
each year. Every forest harvesting team is in locally, in low elevation stands. Sanitation
charge of thinning a variable number of fellings are advisable in Scots pine forests to
stands (3–8). While planning the timing of reduce bark beetle attacks. Depending on
thinnings, priority is given to the most sus- the forest function and on the economic
ceptible (see Table 26.3) and heavily injury level, practices such as de-stumping
infected stands, which are thinned prefera- and, especially, the transformation of heav-
bly in spring and early summer, when the ily infected susceptible forests into more
risk of stump infection is still limited. The tolerant ones can be arranged locally and
average minimum air temperature of a they may be suited. For instance, increasing
4-week period has been identified as a suit- the larch component in subalpine spruce
able predictor for modelling Heterobasidion forests would have positive effects not only
primary infections in the Alps (Gonthier in reducing Heterobasidion incidence, but
et al., 2005) and may be used for an accurate also in improving general forest stability
estimation of the seasonal risk of stump (Motta and Haudemand, 2000). At lower
infection for each forest stand. Remaining, elevations, the regeneration of diseased
less susceptible and uninfected stands are spruce forests with silver fir or Scots pine
thinned in summer or autumn. Stump treat- could be advisable since H. parviporum
ment is necessary during summer and very seldom attacks mature firs or pines
autumn thinnings and is strongly recom- (Korhonen et al., 1998b).
mended whenever dealing with uninfected Obviously, any integrated disease man-
or susceptible stands, regardless of their agement system to fight root and butt rots of
location and distance from an infection forest trees should fit and meet the require-
source. In fact, despite a general limited ments of the general forest management
Winter Spring Summer Autumn
ST Stump treatment
?
Highly susceptible Non-susceptible Heavily infected Uninfected stands

stands stands stands
Promote Sanitation cuttings (DT, WC)

tolerant tree species de-stumping (WC)
Fig. 26.2. Diagram of the integrated disease management system developed to fight Heterobasidion
root and butt rots in the Aosta Valley, western Italian Alps. Arrows indicate the appropriate timing of
thinning. Stump treatment is performed with urea at 30% concentration. Symbols: ?, where possible;
ST, stump treatment; DT, dead trees; WC, where convenient.
358 P. Gonthier
system of the area (Tainter and Baker, 1996). pest management systems, for instance those
Within an integrated forest protection appro- designed for the control of Ips typographus
ach, the integrated disease management L. or other bark beetles threatening alpine
system here described could combine other forests.
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27 Some Important Fungal Diseases and
Their Impact on Wheat Production
Aakash Goyal and Rajib Prasad

Agriculture and Agri-Food Canada, Lethbridge Research Center,
Lethbridge, Canada
Abstract
Wheat, an important cereal crop, is cultivated worldwide and is second highest in production, just
after maize. Due to the increasing world population, there is need for a 40% increase in wheat produc-
tion to meet global food requirements. Wheat production is diminished mainly by biotic and abiotic
stresses all over the world. Of these, pathological diseases are the most important limiting factor of
wheat production as different pathogens infect wheat plants, causing severe losses in yield and qual-
ity. Wheat can be infected by biotrophic fungi, necrophytic species and nematodes, as well as viruses
and bacteria. Among these, different fungal diseases are the most prominent and pose a great challenge
to wheat production. Development of resistant varieties is the only solution to overcome this problem
and to attain the required wheat production. The development of resistant varieties has benefited
immensely from the use of molecular markers, genetic maps, physical maps, QTL analysis and marker-
assisted selection (MAS). However, we have to develop multidisease-resistant varieties to fulfil the
demand for wheat globally. This review highlights some major fungal diseases of wheat in different
parts of the world and the associated problems.
Introduction used as a fodder material and for ethanol

production for the past few years. Unlike
Common wheat (Triticum aestivum L. em. rice and maize, which prefer tropical envi-
Thell) is an important staple food crop and ronments, wheat is best adapted to temper-
ranks first among the three major crops ate regions, occupying 17% (one-sixth) of
(wheat, maize and rice), which together the total crop acreage worldwide (Gupta
constitute about half of the total world food et al., 2008). According to the FAO (2007),
production. Wheat feeds about 40% of the wheat occupies 20% of the cultivated crop
world population and provides 20% of the area (in 2007, 213m ha versus 150m for rice
total food calories and protein in human and 143m for maize) and its annual produc-
nutrition (Varshney et al., 2006). It is not tion is 619 Mt of grain. Over the past 20
only used for bread making but is also used years, there has only been a small increase
for making biscuits, cakes, breakfast cereals, in the area of land on which wheat is culti-
pasta and fermented products like beer, vated worldwide, but the tonnage of wheat
alcohol, vodka, etc. It is also becoming pop- grain produced on this land has tripled as a
ular as a forage crop. Wheat straw has been result of improved farming practices and

Some Important Fungal Diseases and Wheat Production 363
the development of better wheat varieties 2006). Recently, available molecular markers
(Marshall et al., 2001). A significant increase and functional genomics tools have helped
in wheat production has been observed in the breeder to manipulate the wheat genome
the past four decades; however, a slowing to develop disease-resistant cultivars and
down has been witnessed during the past achieve the target of wheat production.
few years (Gupta et al., 2008). Due to a con-
sistent increase in world population, there
is need for a 40% increase in wheat produc- Fungal Diseases of Wheat
tion to meet this requirement. Despite the
enormous progress that has taken place
Most of the important diseases of wheat are
around the world, there is less hope in
caused by fungal pathogens, while only a
achieving this goal. Resistance to both biotic
few are caused by viruses and bacteria
and abiotic stresses will be critical for reach-
(McIntosh et al., 1995; Rajaram and van
ing this target. Abiotic stresses include
Ginkel, 1996). Infection of fungal diseases
drought, untimely or excess heat, untimely
in wheat depends on the availability of free
or excess rain, water logging of soils, wind,
water on the host plant surface, susceptibil-
extreme cold, frost, acid soils and salinity,
ity of the host, the density of inoculum, tem-
nutrient imbalances and/or shortages, as
perature and other environmental factors.
well as micronutrient deficiencies.
Moreover, host–parasite interaction plays a
The impact of biotic stress on wheat
significant role in the development of dis-
production and quality is highly devastat-
ease and subsequent symptoms on the wheat
ing. Diseases in wheat, most caused by
plant. In this chapter, some of the com-
fungal pathogens and a few by viruses and
monly reported fungal diseases of wheat are
bacteria, are important production con-
described. Table 27.1 lists major fungal dis-
straints in almost all wheat-growing envi-
eases of wheat reported by different pathol-
ronments (Rajaram and van Ginkel, 1996).
ogists around the world.
Wiese (1987) identified over 40 fungal, 32
viral and 81 bacterial diseases that attack
wheat plants at different growth stages.
Although it is difficult to obtain accurate Fusarium head blight (FHB)
estimates of crop losses to different fungal
diseases, the British Agrochemicals Associa- Several species of Fusarium can cause Fusar-
tion (1993) suggests that, under farm condi- ium head blight (FHB), also known as scab of
tions where crop rotations, good husbandry cereal crops. Among these, F. graminearum,
and the application of pesticides are prac- found mainly in the USA, Canada, China
tised, losses to diseases can still be around and the EU, is accountable for severe losses
13%, while under conditions where crop in yield and quality of wheat production
protection measures are not taken, losses (Parry et al., 1995). An epidemic of FHB in
can be as high as 50%. It is the goal of wheat the USA and Canada in 1993 was a result of
breeders to introduce genetic resistance into changes in crop management practices (min-
their varieties to minimize chemical protec- imum or reduced tillage), changes in rainfall
tion measures and losses due to diseases. patterns and a low resistance in the culti-
Under different environments, breeders face vars against FHB (Dill-Macky and Jones,
problems of different spectra of locally prev- 1997). In the case of wheat, Fusarium spp.
alent diseases caused by specific biotypes, attacks different plant organs but mainly
serotypes and strains. For many diseases, targets the ear, which leads to great loss in
genes for resistance segregating in a simple seed quality. On the ear, Fusarium enters
‘Mendelian’ fashion have been identified; through the stomata to the palea and lemma
while for other diseases, resistance genes and destroys these tissues completely. The
still remain to be detected, due to either a first symptom of FHB is a tan or brown dis-
complex mode of inheritance or imprecise coloration at the base of a floret within the
disease-screening procedures (Gowda et al., spikelets of the head. The infection may be
364 A. Goyal and R. Prasad
Table 27.1. The major fungal diseases of wheat reported across the world.
Name of the Pathogenic fungal Tolerant varieties/

disease species genotypes References
Black point/kernel Alternaria alternata Sunco, Cascades Lehmensiek et al., 2004

smudge
Common bunt Tilletia caries, T. foetida AC Domain Fofana et al., 2008
Common root rot Cochliobolus sativus ND 652 Mergoum et al., 2005
Ergot Claviceps purpurea Carleton, Kenya farmer Platford and Bernier, 1970
Fusarium head Fusarium graminearum Bizel, Sumai 3 Bourdoncle and Ohm, 2003
blight (scab)
Leaf rust Puccinia recondita Tangmai 4, ND 652 Li et al., 2004;
(P. triticina) Mergoum et al., 2005;
Kolmer et al., 2007
Loose smut Ustilago tritici DT676 Knox et al., 2008
Powdery mildew Erysiphe graminis Tangmai 4 Li et al., 2004
Speckled leaf Septoria avenae Arina and Riband; Chartrain et al., 2009
blotch f. sp. tritici Courtot and Tonic
Glume blotch Stagonospora nodurum Red Chief Laubscger et al., 2008
Spot blotch Cochliobolus sativus Ning 8201, K8027 Sharma et al., 2007
Stem rust P. graminis f. sp. tritici ND 652, Tangmai 4 Li et al., 2004;
Mergoum et al., 2005
Stripe rust P. striiformis Tangmai 4 Li et al., 2004
Take-all Gaeumannomyces Xinong 1376, Xiaoning et al., 2004
graminis var. tritici Xinong 918, R859
limited to one spikelet, but if the fungus of 2 ppm and 1 ppm DON in soft wheat in
invades the rachis, the entire head may non-stable and baby foods, respectively.
develop symptoms of the disease. Discolor- Control of this disease has been diffi-
ation of the head starts due to the production cult, because of the complex nature of the
of mycotoxins [zearalenones and deoxyniva- host/pathogen interaction. Cultural prac-
lenol (DON)] by the Fusarium. The mycotox- tices, such as rotation with non-host crops
ins affect seed quality adversely, producing and management of crop residues, in com-
toxic dust and thus making the seeds unsuit- bination reduce primary infection. A mixed
able for human and livestock consumption fungicide composed of carbendazim and tri-
(Eudes and Laroche, 2003). The mycotoxin adimefon was reported to have a significant
DON, even in low doses of 1–3 ppm, can synergistic action (Wang, 1997). Under high
cause reduced feed intake and less weight disease pressure, Bravo or Folicur were
gain in animals, while a high dose up to reported to reduce levels of FHB, though
10 ppm can cause vomiting and refusal to these are not cost-effective under low dis-
feed. DON is also very harmful to humans; ease pressure (Agrios, 1997). Host resistance
therefore, different countries have estab- is a promising and effective management
lished laws to protect consumers. For exam- solution, but resistance has not been easy to
ple, the EU Member States allow a maximum achieve in the adapted cultivars.
of 1.25 ppm DON in unprocessed bread,
0.5 ppm in bread and bakery products and
only below 0.2 ppm in baby foods (Buerst- Wheat rust
mayr et al., 2009). The USA Food and Drug
Administration recommend only 1 ppm Wheat rust pathogens belong to genus Puc-
DON in finished wheat products, while cinia, family Pucciniaceae, order Uredina-
Health Canada have established guidelines les and class Basidiomycetes. Rust disease
is capable of causing considerable economic Cultural control provides at least par-

loss throughout the world (FAO, 2008). Rust tial control of wheat rust epidemics. Plant-
in cereals, found back in the late 17th cen- ing early maturing varieties is an efficient
tury, was caused by a fungal parasite which way to avoid losses due to stem rust infec-
was named later as Persoon’s P. graminis tion. Propiconazole (Tilt) and triadimefon
(Chester, 1946). In the beginning of the 20th (Bayleton) are found to be effective against
century, different fungal species were identi- stem rust (Agrios, 1997), though these chem-
fied for different rusts with contrasting host icals are cost-prohibitive. To save the world
ranges. In wheat, rust diseases are so impor- from the wheat epidemic, CIMMYT and
tant that in 2007, the CSIRO, Australia, pub- ICARDA started the Global Rust Initiative
lished a special issue on wheat rust in the (GRI) to coordinate efforts to track and study
Australian Journal of Agricultural Research. Ug99 and develop resistant varieties of wheat
(Stokstad, 2007). Later in 2008, it was taken
over by the Borlaug Global Rust Initiative
Stem rust (BGRI), chaired by Dr N.E. Borlaug, who said
he was optimistic that the fungus would be
Stem or black rust of wheat is a major disease beaten again (Stokstad, 2007). Efforts were
problem, caused by the fungus, P. graminis also taken to understand the rust’s epidemi-
Pers. f. sp. tritici. It has been a major disease ology and evolution, which led to the
on wheat since the rise of agriculture and the barberry eradication programme in North
Romans even prayed to a stem rust god, America and Europe (Singh et al., 2006).
‘Robigus’. The Italians, Fontana and Tozzetti,
independently provided the first report on
stem rust in wheat in 1767. In the early to
mid 1950s, stem rust epidemics caused Leaf rust
approximately 50% yield losses of wheat in
North America (Leonard, 2001). During the Wheat leaf rust, also known as brown rust,
1950s, Norman Borlaug and other scientists is caused by the rust fungus, P. triticina Rob.
started developing high-yielding wheat vari- Ex Desm. f. sp. tritici Eriks (syn. P. recond-
eties that were resistant to stem rust and ita). De Candole (1815) reported for the first
other diseases in North America and through- time that leaf rust was caused by fungus and
out the world. The rust-resistant, high- named it Uredo rubigovera. Later in the
yielding wheat variety banished chronic 19th century, the name was changed to P.
hunger in much of the world, ended stem recondita (Cummins and Caldwell, 1956).
rust outbreaks and won Borlaug the Nobel However, the present name, P. triticina, was
peace prize in 1970 (Singh et al., 2006). In suggested by Savile (1984) and Anikster
most areas of the world, the life cycle of P. et al. (1997). Up to 2007, more than 50 races
graminis consists of continual uredinial of leaf rust were detected all over the world
generations. The disease spreads either via (Kolmer et al., 2007; Mebrate et al., 2008).
airborne spores or occasionally locally from Leaf rust is the most prevalent of all the
wild susceptible barberry (Berberis sp.) wheat rust diseases, occurring in nearly all
plants (Eversmeyer, 2000). areas where wheat is grown. Depending on
Ug99, so called as it was first seen in the severity and duration of infection, losses
Uganda in 1999, is a new devastating race of in wheat can vary by up to 50% (Nagarajan
‘stem rust’ which has already travelled from and Joshi, 1975; McIntosh et al., 1995). The
Africa to Iran and can proceed to India, Pak- disease has caused serious epidemics in
istan and Bangladesh (Pretorius et al., 2000). North America, Mexico, South America and
It is particularly dismaying because of its some other countries. This fungus can infect
ability to infect crops in just a few hours wheat plants with a 3 h dew period at tem-
and its vast cloud of invisible spores can be peratures near 20°C. However, more infec-
carried by the wind for hundreds of miles tions occur with longer dew periods. The
(Singh et al., 2006). fungus initially starts covering leaves with
orange pustules of urediniospores (uredinia). reported in 1915 (Carleton, 1915) and seri-
The urediniospores are reddish-brown, ellip- ous outbreaks were reported in the western
tical to egg-shaped, echinulate structures. In states in the 1960s (Line, 2002; Boyd, 2005).
the later stage, the postules eventually darken For this disease, generally no cultural
due to the formation of black teliospores control measures are applicable, but in the
(Roberson and Luttrell, 1987). Infections can USA, where the disease occurs commonly,
result in a 1–20% yield loss since infected the removal of the alternate host is an estab-
leaves die earlier and all the nutrients are lished method of cultural control. Identifi-
directed to the growing fungi. Infection can cation and use of the resistant gene in
also cause grains to shrivel. The loss in yield resistant varities is the only way to reduce
depends on several factors that include time the impact of the disease on wheat produc-
of initial infection, crop development stages, tion. Many yellow rust resistance genes have
relative resistance or susceptibility of the been identified in wheat by different wheat
wheat cultivars. Higher yield losses result workers and to date, 41 of these (Yr1 to Yr41)
when the initial infection occurs early in have been designated (McIntosh et al., 2008).
the growing season before tillering. Infec- Most of the identified yellow rust resistance
tion occurring after heading when grain fill- genes have proven to be race-specific, with
ing is in progress will cause lesser crop loss resistance being effective only against iso-
(Agrios, 1997). lates of P. striiformis f. sp. tritici carrying
Chemical control with trizole fungi- the corresponding avirulence gene. Different
cides has been reported as useful in control- wild wheat varieties were also used to trans-
ling infections up to ear emergence, but is fer the resistance gene to hexaploid wheat
difficult to justify economically in attacks for stripe rust resistance (Kuraparthy et al.,
after this stage. Varietal control is again the 2007a,b; Singh et al., 2007; Chhuneja et al.,
best control for leaf rust. Resistant varieties 2008). More recently, a highly resistant gene
possess one or more special leaf rust resis- with broad spectrum on strip rust races,
tance genes called Lr genes. Currently, there namely Yr36, from wild emmer wheat was
are more than 58 different Lr genes avail- used for positional cloning (Fu et al., 2009).
able in wheat (Bansal et al., 2008; Chhuneja
et al., 2008; McIntosh et al., 2008), but most
varieties have only a few Lr genes. So, there
is a need to develop multi Lr gene-carrying Karnal bunt
varieties to defeat leaf rust disease.
Karnal bunt (partial bunt) of wheat has
become a disease of serious concern in some
parts of the world as it causes direct yield
Yellow rust or stripe rust losses and also has significance as an export
problem because many believe the patho-
Another rust of wheat, stripe or yellow rust gen to be a quarantine pest. Consequently,
which is caused by P. striiformis f. sp. tritici, stringent quarantine measures have been
can be as damaging as other rusts. Due to a adopted in several countries, which may
requirement for a very low optimum tem- affect not only the wheat grain trade but also
perature for its development, stripe rust is germplasm exchange (Royer and Rytter,
not found in many areas of the world. How- 1988). Karnal bunt caused by the smut fun-
ever, a total area of 9.4m ha (> 35%) under gus Tilletia indica Mitra Neovossia indica
wheat cultivation is affected by stripe rust (Mitra, 1931), a Basidiomycetes fungus, is a
(Singh et al., 2004). On the world level, serious floral-infecting disease of wheat in
stripe rust is found predominantly in north- the major wheat-growing areas of India
ern Europe, the Middle East, East Africa, (Gill, 1990) and some other wheat-growing
China, India and the continents of South countries of the world (Nath et al., 1981).
America, Australia and New Zealand (Saari The pathogen is known to infect bread
and Prescott, 1985). In the USA, it was first wheat, durum wheat and triticale (Agarwal
et al., 1977). The disease was first reported (Mitra, 1931) or a vestige of attached myce-
in 1931 in experimental wheat crop at the lium (Durán and Fischer, 1961). The disease
Botanical Station at Karnal, India (Mitra, cycle (Fig. 27.1) starts with the introduction
1931), and was for many years known only of teliospores on to a field. Contaminated
in the plains of India and Pakistan (Ahmad seeds are considered to be the major source
and Attaudin, 1991). Currently, it occurs in of teliospores, while other sources include
Afghanistan, India, Iran, Iraq, Mexico, Nepal wind, animals, contaminated equipment or
and Pakistan and in limited areas of the USA contaminated vehicles. Teliospores may
(Durán, 1972; Munjal, 1975; Singh et al., remain dormant but viable for several years
1989; Ykema et al., 1996). Recognition of (Ottman, 2002). Although planting infected
fungal structures (teliospores) on grain sam- seed is the primary means of getting the
ples from Lebanon and Syria suggest that spores into the soil, this may or may not
the disease is established in these countries produce infected plants directly in the first
as well (Locke and Watson, 1955). year. The greater threat of disease occurs the
Karnal bunt requires free water in the following year as the soil is turned over,
soil for teliospores, the overwintering life bringing these teliospores back to the sur-
stage of Karnal bunt, to germinate. Telio- face. At the flowering stage of host plants,
spores are brown to dark brown, spherical the teliospores produce sporidia that infect
or subspherical, or oval, 22–42 × 25–40 µm the plant florets and fungal hyphae enter the
in diameter, occasionally having an apicu- ovary (Aujla et al., 1977; Singh and Prasad,
lus (Roberson and Luttrell, 1987), papilla 1978; Khetarpal et al., 1980; Krishna and
Infected
grains
Primary
(partial systematic spread)
infection
Combining
and threshing
Germination
of soilborne
Subsequent teliospores
spread to
late tillers
Multiplication
on wheat and
Germination of primary
other plant leaves
spordia on whorl
Filliform
Allantoid secondary
secondary sporidia sporidia
Fig. 27.1. The life cycle of Karnal bunt caused by Tilletia indica Mitra.
Singh, 1982). Subsequent disease develop- Powdery mildew

ment in the embryo end of the kernel results
in the formation of new teliospores, which Powdery mildew of wheat, a wind-dispersed
are deposited back in the soil at harvest, disease, is an important and most common
adding further to soil inoculum. Cool, disease worldwide, particularly in humid
cloudy and very humid conditions or rain- regions (Oerke et al., 1994). It is of special
fall between awn emergence and the end of interest in epidemiology because it results
flowering are required for sporidia produc- in reduced kernel size and seed weight, and
tion, infection and for the disease to flourish ultimately lower yield. The fungal pathogen,
(Dhaliwal et al., 1983; Goates, 1988). The Blumeria graminis f. sp. tritici (an Ascomy-
incidence of Karnal bunt is usually very low cete), causing powdery mildew on wheat,
and rarely seen if the environmental require- is a biotrophic obligate parasite (Cooke
ments are not met. Karnal bunt affects the et al., 2006), which is highly sensitive to the
heads of wheat plants. The disease is not environment and its presence can vary from
easily detected in the field because few flo- season to season (Jenkyn and Bainbridge,
rets are typically infected and the area of the 1978; Jorgensen, 1988; Wolfe and McDer-
kernel affected might be small and facing mott, 1994). The powdery mildew fungus is
inwards. A mass of black teliospores is made up of different races and forms that
found at the embryo end of the kernel and, are highly specialized. Wheat cultivars
at higher levels of infection, along the crease might be resistant to a certain race of the
or in the entire kernel (Goel et al., 1977; mildew fungus, but susceptible to another
Dhaliwal et al., 1983). A fishy odour is emit- race. Some of the special features of pow-
ted from infected seeds due to the presence dery mildew, such as wide distribution,
of trimethylamine (Mehdi et al., 1973). rapid development within or on host tissue,
Conventional approaches to control massive production of spores, the ability to
this disease consist of the adoption of vari- remain viable after long-distance dispersal
ous cultural practices such as crop rotation and a high capacity to become virulent on
for longer periods, sowing of disease-free previously resistant cultivars, make it a dev-
seeds, adjustment of the nitrogen balance in astating disease of wheat (Boshoff et al.,
the soil and adjustment of the time of irriga- 2002).
tion to minimize disease incidence (Mitra, Powdery mildew oversummers on vol-
1937; Munjal, 1974; Goel et al., 1977; Singh unteer crops in the asexual stage, infects
and Prasad, 1978; Aujla et al., 1981, Singh the autumn-sown crop and, eventually,
and Singh, 1985; Gill et al., 1993). Control overwinters on the volunteers to infect the
through fungicides is not completely effec- crops in spring (Zadoks, 1961). In mild
tive as the disease is seed- and soilborne areas, volunteer wheat plants are abundant
(Singh et al., 1985). However, application of because of the relatively frequent rainfall
Tilt at heading and 1 week later can reduce in summer, while in dry regions, oversum-
disease incidence by 90% when environ- mering can depend on grass species
mental conditions are conducive to disease (Boshoff et al., 2002). For mildew, the asex-
development (Ottman, 2002). Hence, the ual cycle is the production of haploid
most economical, eco-friendly and effective conidia, while occasionally the ascospores,
approach to control the disease is the culti- which are the result of sexual cycle, can
vation of resistant varieties. The main initiate epidemics (Cooke et al., 2006). Mil-
sources of resistance against Karnal bunt dew also differentiates a sexual stage,
have been the Indian, Chinese and Brazilian which contributes to oversummering. In
wheats (Fuentes-Davila and Rajaram, 1994). early summer, B. graminis f. sp. tritici initi-
A new range of genetic variability for resis- ates the formation of generative mycelium
tance to Karnal bunt has been observed in and cleistothecia starting on the lower
synthetic hexaploid wheat derived from T. leaves. In the cleistothecia, 15–20 asci
turgidum × T. tauschii crosses (Villareal develop, each containing eight haploid
et al., 1996).
ascospores which are dispersed by wind, plant can use its full potential to fill the
even under high humidity after rain (Gotz grain. Fungicides can be applied based on
et al., 1996). Ascospores can develop at any the level of disease in the field, the known
time during the last half of the year; there- susceptibility of the variety and the selling
fore, sexual reproduction is more important price of the grain (Agrios, 1997). Growing
for powdery mildew on wheat. Apart from mildew-resistant cultivars is the most eco-
ascospores, conidia from the summer crop nomical way to control powdery mildew,
can also infect volunteer plants; thus, a though wheat varieties vary in their resis-
mixture of ascospores and conidia forms tance to powdery mildew and new races of
the inoculum for the winter crop. However, the fungus can attack previously resistant
the mildew population grown during varieties.
autumn on the winter crop can survive the
cold period in vegetative stage on overwin-
tering green plants (Cooke et al., 2006).
Mildew is more severe in dense stands of Conclusions
heavily fertilized wheat. Plants are most
susceptible during periods of rapid growth, Wheat is a most important cereal crop and
especially from stem elongation through is becoming more in demand due to the
heading growth stages. significant increase in the world popula-
Powdery mildew on wheat is recog- tion. To protect the world from the upcom-
nized by small, effuse patches (colonies) of ing threat of hunger, food and nutrition,
cottony mycelia on the upper and lower sur- wheat production must be doubled in time.
faces of the leaves. As these patches sporu- Major concern about wheat quality and
late and age, they become a mass of dull tan production is related to biotic stress. Differ-
colour. Chlorotic (yellow) patches may later ent methods, such as chemical control, cul-
surround the mildew colonies (Purdy, 1967; tural methods and eradication of alternate
Kingsland, 1982). Powdery mildew attacks hosts, are used to prevent the disease, but
the leaves, but stems and heads are also the most important and effective one is the
affected. The fungus grows primarily on the development of resistant varieties. For
surface of the host and feeds on the living durability of resistance against fungal
green cells of the plant. Damage occurs from diseases, breeders should focus on new
reduced photosynthetic ability when green sources of race-specific resistance genes
surfaces are shaded and the host is robbed from either adapted cultivars or wild vari-
of moisture and food by fungal growth. eties. However, extensive knowledge of the
Yields may be reduced by 20% or more. pathogen population is a vital criterion in
Spring wheat, other than soft white wheat, assessing resistance and guidelines for
are seldom affected at economic levels on breeders to incorporate useful resistance
the prairies, while winter wheat is affected genes into the desired background. Recent
to a greater degree. The disease will reduce studies have proved the usefulness of dif-
yields seriously if the flag and second leaves ferent marker systems and association map-
are affected (Gotz et al., 1996; Boshoff et al., ping of genes/QTLs controlling resistance
2002). against different fungal diseases (Crossa
Incorporating wheat residues into the et al., 2007). Similarly, MAS was also
soil, destroying volunteer wheat and crop employed successfully to improve quality
rotation can lessen the amount of overwin- and resistance against disease (see Dub-
tering inoculum in the field. Powdery mil- covsky, 2004; Anderson, 2007; Sorrells,
dew thrives where high rates of nitrogen 2007). In future, new molecular marker sys-
have been used. Therefore, use of a correct tems (e.g. ESTs, SNPs and DArTs) and func-
and balanced fertilization programme with tional genomics approaches (e.g. TILLING,
proper levels of N, P and K is advised. It is RNAi and epigenetics) can be used to facil-
important to keep the top two leaves of the itate the development of resistant varieties
plant as disease free as possible so that the in bread wheat.
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Index
Note: Page numbers in italic refer to tables and figures in the text
Abies spp., root and butt rot 347, 349, 353, 354 ecological functions 345
abiotic stresses management 350
endophyte protection of plants 189–190 root and butt rot
fenugreek 246–247 biological and chemical control
wheat 363 122–123, 354–356
Acaulosporaceae 166 diagnosis 350–351, 351
accelerated ageing germination (AAG) 332–333 effects of forest management 350
acetaldehyde 8 general control strategies 351–354
acetic acid 43, 341 infection biology 346–349, 347
Achyranthes japonica 40 integrated management 356–358
acibenzolar-s-methyle (ASM) 9 wood decay 348
Acremonium terricola 284 tree species 345–346
Actinomycetes, antibiotics 123 Alternaria spp. 232
Adgen Phytodiagnostic Septoria ELISA kit 299 fruit crops 5
aflatoxins 15, 29, 31, 39 inhibition by plant extracts 40–41
production inhibited by plant extracts 39, leaf blight
60, 61 castor 271–272
recommended limits 34 safflower 267–268
agglutinins 127 wheat 232–233, 241
Agrobacterium radiobacter 125, 125 leaf blight/black point 234–236
agronomic characters, and disease resistance 73, leaf spot
81–82 sesame 270
ajoene 20, 22 sunflower 265–266
aldehydes 42 Alternaria alternata 364
alfalfa 173, 207 Alternaria carthami 271
alimentary toxic aleukia (ATA) 31 Alternaria helianthi 266
alkaloids 33, 150, 190 Alternaria infectoria 234–236
allicin 20, 22 Alternaria padwickii 40–41, 55
Allium sativum extract 40 Alternaria sesame 270
allyl-isothiocyanate (AITC) 43 Alternaria solani 173
Aloe vera gel 9 Alternaria triticina 232–233
alpine European forests 345–346 aluminium tolerance 189
375
376 Index
Amaranthus spp. 311–312 control 133, 351–358

smut diagnosis 351, 351
incidence in cultivars 315, 316 aroeira extract 61
wild species 313, 315–316 aromatic compounds, fruit and vegetables 7–8,
Amaranthus hybridus 315–316 42–43
Amaranthus retroflexus 315–316 asarone 20, 22
AMF, see arbuscular mycorrhizal fungi Ascochyta hordei var. europaea 236–237
amino acids 177 Ascochyta leaf spot 252
anise 56, 60 Aspergillus spp.
antagonists 122 plant extract treatment 59–61
criteria for commercial production 111 seed spoilage 330–331
endophytes 185–186 Aspergillus flavus 29, 31, 32, 330
fruit storage pathogens 6–7, 110–111 Aspergillus niger 265, 330
intergration with other control measures Aspergillus ruber 330–331
115–116 AspireTM 110, 114
mechanisms of action 111–113 Avr gene products 129
root and butt rot fungi 355 AVR-Pita avirulence gen family 98
tan spot 283–284
antibiosis 9, 111, 123
KB-8A 270 Bacillus spp. 124–125, 252
Trichoderma 123, 127 bacteria
antiseptics, stored produce 34 biocontrol of soil diseases 124–125, 125
Aosta Valley forests 357, 357 endophytes 150
Aphanoderma album 212 bacterial disease
apoplast, protease activity 304 endophyte plant protection 186–187
apples 6, 43, 174 fenugreek 247, 248
hot air treatment 116 bags, seed storage 336, 338, 340, 341
potassium iodide wraps 6 bajra, rusts 209
arbuscular mycorrhizal fungi (AMF) 124 banana, mycorrhizae 176
effects of agricultural practices 163–165 bark beetle 357
importance in agriculture 162–163 bark extracts 40
interaction with fungal pathogens 122–123, barley, Fusarium head blight 79, 81–83, 87–88
172–175 barley yellow dwarf virus 187
mechanisms of disease control 175–177 basil extracts 40, 60–61
role in plant nutrition and growth 172 bavistin 332, 332, 333
signalling pathway 175 bayletan 332
soil propagule bank 164 bean, rust fungi 209–210, 214, 216
effects of tillage 164–167 beet, leaf endophytes 151–152, 153
taxonomy 172 benlate 332
Argentina benodanyl 214
fungicide use 292 benomyl 226
wheat pathogens 231 benomyl thiabendazole 34
Alternaria leaf blight 232–233 benzaldehyde 42
Alternaria leaf blight/black point benzanilide 214
234–236 benzimidazole 214
Ascochyta hordei leaf spot 236–237 Beta vulgaris var. esculenta, see beet
Cephalosporium gramineum stripe biological control
238–239 airborne disease 124
Cladosporium herbarum leaf spot commercial products and systems 125, 125
239–241 components 131–134
monitoring changes 241–242 constraints in development 113–115
Phoma soghina leaf spots 237–238 Trichoderma spp. 131, 133
Pyricularia grisea spot blight 241 definition 121–122
tan spot 276–284 efficacy, consistency of 114
wheat production 276, 291–292 endophytes 150–151, 185–190
arginine 177 mechanisms 122–123
Armillaria mellea sensu lato 347, 348, 349 Phytophthora sojae 323
Index 377
postharvest diseases 17–18, 110 α-cadinol 20, 22

rusts 212 calcium chloride 115
Septoria tritici blotch 303–305 camphor 20, 21
soilborne diseases 124–125, 125 captafol 214
tan spot of wheat 159, 283–284 captan, seed dressings 332, 332, 333
see also botanicals; essential oils; caraway oil 38
Trichoderma spp.; yeasts carbendazim 99, 226
bioprotection, AM fungi 171, 172–175 carboxin, seed treatment 334
BioSaveTM 7, 110 Carthamus oxycantha (Pohli weed) 269
Bipolaris spp., seed pathogens 55–56 Carthamus tinctorius, see safflower
Bipolaris sacchari 223–224 carvacrol 20, 22
Bipolaris sorokiniana 55 carvone 20, 21, 40, 53, 57
bitter leaf 40 caryophyllene 20, 21
‘black mars’ 29 cash crops, mycorrhizae 174
black pepper 174, 176 castor 271–273
black point, wheat (Alternaria infectoria) 234 cedarwood oil 38
black stem rust 203, 214 Cephalosporium gramineum stripe 238–239
bleaching powder, seed treatment 333 Ceratocystis paradoxa 225
blue mould decay 44, 116 Cercospora arachidicola 263–264
Blumeria graminis f.sp. tritici 368–369 Cercospora leaf spot
borax 355–356 fenugreek 249–252, 250
bordeaux mixture 214 sesame 271
botanicals 7–9, 17–18 Cercospora sesami 271
chemical structures 21–22 Cercospora traversiana 250–252, 250
effective against toxin producing fungi 39 cereal crops
effects on seed fungi 55–61 mycorrhizae 172–173
efficacy in fungal control 19–20 rust diseases 206
essential oils 8–9, 18 seedborne fungi, treatment with plant
seed treatments 38–39, 52–53 extracts 57
fruit crop disease control 7–9 see also individual cereal crops
potential advantages 45 cerebrosides 187
potential risks 45–46 charcoal rot, fenugreek 250
seed treatments 41 charcoal stump rot 174, 177
seedborne fungi chemical fungicides 36, 183
Alternaria 55 drawbacks of 36, 62
Aspergillus 59–61 Phytophthora sojae control 323
Bipolaris 55–56 regulation 110
Colletotrichum 56 resistance 99–100
Curvularia 56–57 rice blast disease 99–100
Fusarium 57–59 root and butt rot of alpine forests
Macrophomina 59 355–356
Penicillium 59–60 rust diseases 213–214
Botryosphaeria spp. 133 seed treatments 331–333, 332
Botrytis spp. storage diseases 16–17, 34
botanical control 42, 43–44 sugarcane diseases
grey mould of fruit 4 pineapple disease 225–226
grey rot of castor 272 rusts 221
Trichoderma control products 133 smut 219
Botrytis cinerea 4, 42, 43–44 tan spot 283
Botrytis ricini 272 wheat 283, 292
brassicol 332 Chenopodium procerum 40
brinjal, seed treatments 334, 335, 335 chestnut blight fungus 123, 186
Bromus spp., head smut 139–140, 140, 141 chickpea
brown rot 4 mycorrhizae and fungal diseases 176
brown rust, wheat 364, 365–366 seed treatments 333, 334
Bt genes 342 chilli seeds 335, 335
Burkholderia ambifaria 188 chitinases 125, 127, 177
378 Index
chitosan 9 crop residues, wheat 283

Cicer arietinum 209 crop rotation
1,8 cineole 20, 22 forests 353
cinnamaldehyde 20, 21, 53 Phytophthora sojae control 323
cinnamate derivatives 95 tan spot control 283
cinnamon oil 56, 58 cross-protection 123
citral 39, 53 Cryphonectria (Endothia) parasitica 123
citronellol 20, 21 cryptocin 186
citrus fruit 115 cucumber, mycorrhizae, effects on fungal
brown rot 4 diseases 176
essential oil treatments 44 cultural practices
green mould 115–116 alpine forests 350
root rot 174 effects on mycorrhizae 163–165
Cladosporium spp. 212 fruit production 5
Cladosporium herbarum, wheat leaves 239–241 wheat diseases 365, 368, 369
Claviceps 54 Septoria leaf blotch resistance 74
Clavicipitaceae 150 tan spot 283
Climacocystis borealis 347, 348 see also no-tillage systems
control 351–358 cumin 60
diagnosis 351, 351 Cuminum cyminum 174
climate curcumene 20, 22
India 15, 37 Curvularia spp. 56
and postharvest diseases 15 Curvularia protuberata 190
and powdery mildew of wheat 368–369 cyanide (HCN) precursors 177
Septoria tritici blotch development ρ-cymene 20, 21
298–299, 298 Cyprus rotundus extracts 40
climatic factors, seed storage fungi 340 cytomegalovirus, human (hCMV) 187
clove oil 38, 56, 60
clover, rusts 207
cocoa beans, postharvest damage 29 Dalbergia rust 206
coffee damping-off diseases
rust diseases 204–205, 206, 208 fenugreek 249, 252
fungicides 214 sunflower 267
cold storage 15 Trichoderma control products 133
collar rot defences, see plant defence responses
castor 272 deformed plants 30
fenugreek 250, 252 deoxynivalenol (DON) 80, 81, 83, 86, 364
groundnut 265 inhibition by essential oils 58
Colletotrichum spp. 56, 186 recommended levels 364
Colletotrichum falcatum 221–222 Dichanthelium lanuginosum 190
Colletotrichum gloeosporioides 7, 154 diene antifungal compounds 4
common bunt (Tilletia laevis) 143–145 discoloration of crops 29, 54
competition 112, 122–123 disease resistance
Trichoderma 127–128 agronomic characters 73, 81–82
‘compound interest diseases’ 205 FHB 80–81
containers, seed storage 336, 338, 340, 341 induced 9, 123–124, 128–129
copper-based fungicides 214, 335, 340 morphological 80, 81–82
corn smut 140–142, 143 non-specific (partial) 95–96, 322
cotton physiological, types of 80–81
mycorrhizae, effects on fungal diseases race-specific 321–322
176 RBD 95–99
rusts 207, 210 Septoria leaf blotch 70–73
coumestrol 177 see also endophytes, plant protection; plant
cowpea breeding; resistance genes
mycorrhizae 173, 176 DON, see deoxynivalenol
seed treatments 333, 334 downy mildew, sunflower 266–267
Crambus spp. 188 Drechslera tritici-repentis, see tan spot
Index 379
drought tolerance, and endophytes 189 ethanol 115–116

drying of produce 33, 341 eugenol 20, 53
dryland crops 246 Eutypa dieback 9
Dutch elm disease 188 expressed sequence tags (EST) 87
eye spot disease, sugarcane 223–224
early leaf spot, groundnut 263–264

egusi melon 61 fenchone 20, 21
endophytes fennel 60
bacteria 150 fenugreek
beet leaves 151–152, 153 abiotic disease 246–247
definitions 149–150, 183–184 bacterial disease 247, 248
ecological role and strategy 150–151 biology 245–246
as gene vectors 151 crop potential 246
groups 184 disease-resistant cultivars 257
non-grass plants 150 fungal disease 249–256, 257
plant protection 151, 159 collar rot 250, 252
abiotic stress 189–190 Fusarium wilt 252–253
bacterial disease 186–187 leaf spot 250, 252
fungal disease 159, 185–186 pod spot 250, 253
insects 188–189 powdery mildew 254–256, 257, 258
nematodes 188 spring black stem/leaf spot 253–254
viral disease 187 insect pests 247, 248, 249
potential of 190 nematodes 247, 248
research 151, 184–185, 190–192, 191 seed extracts, antifungal activity 249
soybean leaves 154–155, 154 seed treatments 334–335, 335
tomato leaves 152–154, 153 viral diseases 247
wheat 155–156, 157–158, 159 ferbam 213
environmental conditions fertilization 74, 369
and powdery mildew of wheat 368–369 fescue, tall 150, 187, 188
seed storage 337–340, 341 fescue toxicosis 150
Septoria tritici development 298–299 FHB, see Fusarium head blight
stored crops 31–33 fibre crops, rusts 207
enzyme-linked immunosorbent assays (ELISA) fig (Ficus spp.), rust fungi 210–211, 214
299 fir, silver, root and butt rot 347
enzymes flavour, damage by fungi 29
AM fungi 177 flavour compounds, fruit 7–8, 42–43
biological control agents 112, 125, 127 fluzilazol propiconazol 283
fungal pathogens 29–30, 32 Fomes annosum, see Heterobasidion annosum
epidemiology, definition 292 Fomitopsis pinicola 347, 348
ergot 31, 54, 364 control 351–358
ergovaline 150 diagnosis 351, 351
Erysiphe cichoracearum 270 food grains, loss in storage 329–330
Erysiphe polygoni 254–256, 257, 258 food security 14
Escherichia coli, recombinant 125 foot rot, black pepper 174
essential oils 8–9, 18, 37–38, 43–44 forage crops, rusts 207, 216
active components 37, 53 forest rotation 353
biocide formulation 42 forests
chemical structures 21–22 role of mycorrhizae 174, 175
efficacy 19, 20 see also alpine European forests
phytopathogenic fungi 38 free fatty acids (FFAs) 29–30, 331
seedborne fungi 38–39, 52–53, fruit
55–61 antifungal compounds in unripe 4
toxin producing fungi 39 aromatic and flavour compounds 7–8,
mode of action 44–45, 53 42–43
toxicity 45–46 cultural disease control 5
estrobirulinas 283 disease-resistant transgenic plants 10
380 Index
fruit continued Gliocladium spp. 130, 270

nutritional value 3 Gloeosporium rot 5
postharvest diseases 3, 4 Glomeromycota spp. 172
integrated management 7, 115–116 in no-tillage systems 163–164, 166–167
plant extract treatments 7–9, 42–44 Glomosporium amaranthi, see Thecaphora
prevention 5–7 amaranthi
production in India 4 Glomus coronatum 188
rust fungi 213 beta-1,3-glucanases 125, 177
frutiafol 283 glucosinolates 8, 43
fumigation, botanicals 5, 42, 43, 44, 60 glume blotch 364
fumonisins 39, 80 Glycine max, see soybean
fumonism 31 gram (Cicer arietinum), rusts 209
fungicides, see chemical fungicides grapes, postharvest technology 6
fusarenon-X (FUS-X) 80 grapevine, Eutypa dieback 10
Fusarium spp. grass species
mycotoxins 80 endophytes 150, 187, 188, 189
plant extract treatments 57–59 head smut 139–140, 140, 141
postharvest pathogens 79 tan spot hosts 279
toxin production 31, 364 green mould, citrus fruit 115–116
Trichoderma control products 133 green revolution, India 205–206
Fusarium avenaceum 79, 80 grey mould, strawberry 4
Fusarium culmorum 79, 80 grey rot, Botrytis 272
Fusarium graminearum 31, 32, 79, 80 groundnut 263
Fusarium head blight (scab/FHB) 78–79, collar rot 265
363–364, 364 early leaf spot 263–264
epidemics and crop losses 79 late leaf spot 264
host resistance 80–81 postharvest damage 29, 30, 33
barley 81–83, 87–88 role of mycorrhizae 173
wheat 83–87 rust 203, 206, 208
mycotoxins 364 fungicides 213, 214, 264
species isolated 79 resistant varieties 264
symptoms and effects 79–80, 363–364 seed and seedling diseases 264–265
Fusarium moniliformae 224–225 growth abnormalities, damaged seed 30
Fusarium oxysporum 252–253 guava 4
Fusarium oxysporum f.sp. ricini 272–273
Fusarium oxysporum f.sp. sesame 270
Fusarium poae 79, 80 hairpin-encoding genes 99
Fusarium wilt halogenation, seeds 342
and AM fungi 173 head blight, see Fusarium head blight (scab)
castor 272–273 head rot, Rhizopus 267
endophyte-induced resistance 185 head smut, Bromus spp. 139–140, 140
fenugreek 250, 252–253 heading date 73, 81–82
safflower 268 heat tolerance, and endophytes 190
sesame 270 heat treatments 15–16
Alternaria leaf blight 268
fenugreek seed 253
galactomannans, fenugreek 246 fruit crops 5, 116
garlic extracts 56, 59, 60, 61 red rot of sugarcane 223
gene silencing 9 heating of crops (deleterious) 30
geothermal soils 190 Helianthus annuus var. macrocarpus, see
geraniol 53 sunflower
germination Helicobacter pylori 187
and seed mycoflora 331, 331 Helminthosporium carbonum 10
and seed treatments 333–336, 333, 337 Helminthosporium oryzae 56
Gibberella moniliformis 224 Heterobasidion annosum sensu lato 347, 348,
Gigasporaceae 166 349, 350
ginger 44, 56 control 122–123, 351–358
Index 381
diagnosis 351, 351 larch (Larix), root and butt rot 347, 353, 354
species susceptbility 353–354, 354 late leaf spot, groundnut 264
Heterosporium medicaginis 253 latex 9
hevien 9 leaf blight
hexanal 42–43 Alternaria carthami 267–268
hexenal 42–43 Alternaria infectoria 234, 241
hinosan, seed dressings 332, 332, 333 Alternaria triticina 232–233, 241
honeybees 45 leaf blotch (Septoria) 70–74
horticultural crops leaf rust, wheat 364, 365–366
mycorrhizae 173–174, 176 leaf spot
rust diseases 207 Alternaria 265–266, 270
‘host shifts’ 94 Ascochyta 236–237, 252
host-specific toxins (HST) 223 Cercospora, sesame 271
hrf1 gene 99 groundnut 263–264
hydration-dehydration treatments 342 Phoma sorghina 237–238
hyperparasitism 123 Pyricularia grisea 241
hypersensitive response (HR), soybean 320 lectins 127
hypovirulence 123 legume crops
mycorrhizae 173
rust diseases 209–210
immunoassays, Septoria tritici 299 fungicide treatment 214
India seed treatments 333, 334
castor production 271 lemongrass oil 39, 58
climate 15, 37 leucine-rich repeats (LRRS) 212
endophyte research 190–192, 191 Leveillula taurica 270
fruit production 4 Lewia infectoria 235–236
green revolution 205–206 lime-sulphur 213
indole derivatives 186 limonene 20, 21, 53
induced resistance 9, 123–124, 128–129 Limonomyces roseipellis 284
insect pests linalool 20, 21, 53
fenugreek 247, 248, 249 lineage exclusion hypothesis 98
protective effects of endophytes 188–189 linseed 207, 209
stored products 28–29, 31 lipases 29–30, 127
integrated disease management 62, 124 logging, impact on root and butt rot fungi 350
fruit crop diseases 7 lolitrem B 150
Phytophthora sojae 323 Lr genes 366
postharvest fruit disease 7, 115–116 Luffa acutangula 340
postharvest fungi 115–116 Lycopersicon esculentum, see tomato
root and butt rot fungi of trees 356–358 lyso-phosphatidylcholine 175
rusts 215
Septoria leaf blotch 73–74
ionizing radiation treatments 5–6, 16 Macrophomina phaseoli 29
iron 130 Macrophomina phaseolina 272
isoleucine 246 Magnaporthe graminicola 293
Magnaporthe grisea 92–93
pathotypes 93–95, 97
jasmonates 8, 43 maize
javanicin 187 postharvest damage 30
jowar, rusts 206, 208–209, 216 rusts 206, 209, 216
jute bags, seed storage 336, 338 seed spoilage in storage 330
seedborne fungi, plant extract treatments
58
karnal bunt (partial bunt) 366–368, 367 smut 140–142, 143
mancozeb 213, 333, 333
maneb 213
Laetiporus sulphureus 347, 348 manganese 130
Laetisaria arvalis 284 mango 4
382 Index
marker-assisted selection (MAS) and endophytes 188

soybean 323 fenugreek 247, 248
wheat 86–87, 88, 369 and mycorrhizae 176–177
medicinal plants nitrogen fertilization 74, 369
fenugreek 246 nivalenol (NIV) 80
northern India 185, 190 no-tillage systems
rusts 207 and arbuscular mycorrhizae 163–164,
Meloidogyne incognita 188, 247 166–167
melon seeds, plant extract treatments 41, 61 problems of 163
menthol 20, 22 soil property changes 163
menthone 20, 21 and tan spot control 283
mercury fungicides 214 wheat crops 74, 276
methyl bromide 183 Norway spruce, root and butt rot 347, 352, 353,
methyl jasmonate 43 354, 354
methyl salicylate 46 notchi powder 41
MGR586 DNA repeat element 94 nucleotide-binding site plus leucine-rich repeat
microtubules 177 (NBS-LRR) genes 98
minerals, solubilization and sequestration by nutrients
Trichoderma 128–129, 130 competition for 112
moisture, stored crops 31–32 see also plant nutrients
molecular diagnostics, root and butt rot fungi nutritional requirements, fungi 32
351, 351
molecular markers
Phytophthora sojae 321 oat, rust 206
soybean 323 oats 283
wheat 86–87, 369 ochratoxin 31
moringa 41 odour changes 29
morphological disease resistance 80, 81–82 Oidium erysiphoides 270
mulberry 206 oils
mung bean 173, 333, 334 fruit skin coatings 6
muskmelon seed 334, 335 see also essential oils
mustard 334 oilseed crops
mycoparasitism 123, 127 mycorrhizae 173
mycoparasitism related genes (MRGs) 126 rust diseases 206
mycorrhizae 124 seed treatments 333, 334
soil infectivity 164 see also individual oilseed crops
see also arbuscular mycorrhizal fungi olive oil 38
(AMF) onion, mycorrhizae 176
Mycosphaerella arachidis 263–264 onion-pink rot 173
Mycosphaerella graminicola 69, 70, 231 Onnia tomentosa 347, 348
mycotoxins oocydin 186
aflatoxins 15, 29, 31, 39 oranges 44
control of production 39 orchard hygiene 5
DON 58, 80, 81, 83, 86, 364 oregano oil 38, 60
Fusarium head blight 80, 364 ornamental plants, rusts 207
mould species producing 32 orthodihydroxy (O-D) phenols 177
safe limits 34 oxanthiin-carboxin, rusts 214
myrcene 20, 21 oxycarboxin 214
Myrothecium roridum 284 oxygen 32
ozone treatments 5
nabam, rust diseases 213

neem extracts 6, 8, 34, 40, 41 paddy, seed treatments 334, 335, 335, 336
neem oil 42, 56, 60 palm oil 30, 33
neem seedlings, AMF 174, 175 palmarosa oil 58
neembicidine 333 papaya 5, 9, 41
nematodes pawpaw 40
Index 383
PCR-based diagnosis, root and butt rot fungi Septoria leaf blotch resistance 71–73
351, 351 wheat 86–87, 363, 369
pea, rust 209, 213 plant defence responses 95, 123–124
pearl millet effects of mycorrhizae 175–176
rust diseases 206, 209 provocation by Trichoderma spp. 304–305
seed spoilage/treatments 330, 335–336, plant extracts 39–40
336, 337, 338 effective against phytopathogenic fungi
Penicillium, plant extract treatments 59–61 39–40
pepper, mycorrhizae 176 effective against seed fungi 40–41
peppermint oil 38 fruit crop treatments 44
Peronospora trifoliorum 249 total number 37
peroxidase 129, 177 see also botanicals; essential oils
Pestalotiopsis microspora 186 plant growth
Phaeoisariopsis personata 264 and mycorrhizal associations 172
Phaeolus schweinitzii 347, 348 and Trichoderma spp. 129–130
phalsa 174, 213 plant height, and disease resistance 73
phenylalanine 177 plant nutrients
Phlebia gigantea 122–123 and fruit storage rot 5
Phoma spp., Trichoderma control products 133 solubilization/sequestration by
Phoma pinodella 249, 253–254 Trichoderma 128–129, 130
Phoma sorghina 237–238 uptake and mycorrhizal associations
Phomopsis oblonga 184 176–177
Phomopsis psidii 4 plantation crops
physiological specialization rust diseases 206–207
Phytophthora sojae 320–321 see also alpine European forests; forests
powdery mildew of wheat 368 plantavax w.p. 214
rice blast fungi 93–95, 97 plantibodies 9
tan spot on wheat 281–282 Plasmopara halstedii 266
Tilletia laevis 143–145 Plasmopara patens 266
Ustilago bullata 139–140, 141, 142 Plasmophara perennis 266
physiology of resistance, rice blast 95 Plebiopsis gigantea 355
phytoalexins 95, 129 pod spot, fenugreek 253
in AMF-containing plants 177 Pohli weed 269
induction by yeast antagonists 112 pokkah boeng disease 224–225
Phytophothora spp., Trichoderma control polyphenol oxidase 177
products 133 polythene bags, seed storage 336, 338
Phytophthora drechsleri 268 population genetics 292–293
Phytophthora infestans 185 postharvest diseases 14–15, 28, 29–30, 54–55
Phytophthora nicotianae var. parasitica biochemical effects 29–30
173–174 conditions favouring 31–33
Phytophthora parasitica var. sesame 269 crop losses 109
Phytophthora sojae 319 crop weight loss 30
life cycle 319–320, 319 discoloration of crops 29, 54
physiologic races 320–321 flavour and odour changes 29
Phytophthora spp., blight of sesame 269 fruit crops 4–5
Pi-ta gene 98 botanical as antifungal agents 7–9
Picea spp., root and butt rot 347, 349, 353, 354 integrated control 115–116
pigeon pea blight 173 management 5–7
pine oils 53 Fusarium spp. 79
pine, Scots, root and butt rot 347 growth abnormalities 30
pineapple disease, sugarcane 225–226 insects 28–29, 31
α-pinene 20, 21 management 33–34
Pinus spp., root and butt rot 347, 349, 353, 354 biocontrol products 110
plant breeding botanicals 17–18, 19–22
Fusarium head blight (FHB) resistance 80 preparation for attack by other agents
powdery mildew resistance 257 30–31
rust disease resistance 212 rotting and caking 30
384 Index
postharvest diseases continued RBD, see rice blast disease

see also seed storage; storage diseases reactive oxygen intermediates (ROI) 95
potassium metabisulphite 341 reactive oxygen species (ROS) 190
powdery mildew recombinant inbred lines (RILs) 85, 323
fenugreek 249, 250, 254–256, 257, 258 red rot, sugarcare 221–223
plant extracts 38 red smudge 278
sesame 270–271 relative humidity (RH)
wheat 368–369 seed storage 337–339, 339
predation 124 Septoria tritici blotch development 298, 298
preservatives, chemical 34 stored crops 31–32
prochloraz 283 repeat induced mutation (RIP) 10
propagative materials, prevention of storage repeated DNA sequences, Magnaporthe grisea
damage 34 93–95
propionic acid 341 resistance gene analogues (RGA) 99
proteases, apoplast 304 resistance (R) genes 10
Pseudonmonads 124 FHB 82–83
Puccinia arachidis 264 Hm1 10
Puccinia graminis f.sp. tritici 364, 365 race-specific 321–322
Puccinia helianthi 266 rice blast disease 96–99, 97
Puccinia kuehnii 220–221 rusts 212
Puccinia melanocephala 220–221 soybean 322–323
Puccinia striiformis f.sp. tritici 366 wheat 366
Puccinia triticina f.sp. tritici 365–366 resorcinols 4
pulegone 20, 21 Reynoutria spp. 38
pulses RFLP techniques 301
mycorrhizae 173 rhizobacteria-induced systemic resistance (RISR)
rust diseases 207, 209–210, 216 128
pungam 41, 42 Rhizoctonia solani 133, 249, 250, 252
Pyrenophora tritici-repentis, see tan spot rhizome rot, ginger 174
Pyricularia grisea 241 Rhizopus arrhizus 267
Pyricularia oryzae 186 Rhizopus nigricans 267
Pythium spp. Rhizopus oryzae 267
damping-off 249 Rhizopus stolonifer 5
Trichoderma control products 133 Rht-D1 locus 86
Qfhs.ifa-5A QTL 84 rice
QFhs.ndsu-3BS QTL 84–85 postharvest damage 30
Qrgz-2H-8 gene 83 seed fungi 55, 57
QTL, see quantitative trait loci sheath rot 42
quantitative trait loci (QTL) rice blast disease (RBD)
Fusarium head blight resistance 78, 82, control 99–100, 100, 186
84–87 crop losses 92
Phytophthora sojae resistance 323 epidemiology 93
Septoria leaf blotch resistance 71, 72 fungal agent (Magnaporthe grisea) 92–93
‘quelling’ 10 races 93–95, 97
fungicide resistant 99–100
host resistance
race typing, Ustilago scitaminea 218 genetics 95–99
radiation, stored food commodities 5–6, 16 non-specific 95–96
Radopholus similis 188 physiology 95
rainfall symptoms 93
and Septoria tritici blotch development Ricinus communis, see castor
298–299, 298 RISR, see rhizobacteria-induced systemic
and storage fungi 340 resistance
rainforests, mycorrhizae 175 ROI, see reactive oxygen intermediates
random amplification of polymorphic DNA root and butt rot fungi, biological and chemical
(RAPD) 95–96, 321 control 122–123, 354–356
raspberries 8 root knot nematodes 176–177
Index 385
root rot Scots pine, root and butt rot 347, 353, 354
Macrophomina 250, 272 seed abortion 54
Phytophthora 268–269 seed extracts, disease suppression 249
safflower 268–269 seed health 329
wheat (S. rolfsi) 172, 177 seed necrosis 54
ROS, see reactive oxygen species seed piece infection, pineapple disease 226
roses, rusts 210 seed storage
rubber plant 174, 177 containers 336, 338, 341, 342
Rumex crispus 40 postharvest strategies 341
rust fungi 204 seed treatments
bean 209–210 chemical fungicides 331–333, 332
coffee 208 fenugreek 251–252
cotton 210 persistence during storage 339–340
epidemics and losses from 204–205 plant extracts 39, 40–41
fenugreek 249, 250 essential oils 38–39, 52–53
fig 210–211, 214 Trichoderma spp. 129, 134
grain crops 206 and viability 333–336, 333, 337
gram 209 seed viability
groundnut 203, 206, 208, 209, 264 loss in storage 30, 330, 331–336, 331
key to species/varieties 216 and seed treatments 333–336, 333, 337
linseed (flax) 209 and storage environment 337–340, 341
maize 206, 209, 216 seedborne fungi
management strategies 211–215 Fusarium spp. 79
orange 220 management, plant extracts 40–41, 55–61
pea 209 symptoms of disease 54–55
pearl millet 206, 209 seeds
rose 210 artificial 342
safflower 269 drying 341
Sorghum spp. 206, 208–209, 216 free fatty acid content 29–30, 331
soybean 210 invigorating treatments 342
sugarcane 220–221 pelleting 342
sunflower 266 Septoria leaf blotch
systematics 201–204 causal agent 70
teleutospores 204 crop yield losses 70
urediniospores 203–204 integrated management 73–74
wheat 207–208, 364–366, 364 resistance 70–73
rye, rusts 206 Septoria tritici blotch (STB) 293
ryegrass, endophytes 187, 188, 189 ascendant movement of disease 299–301
‘ryegrass staggers’ 150 biological control 303–305
early detection 299
epidemiological studies 293–298
safflower 267–269 impact of climate 298–299
Alternaria leaf blight 267–268 population genetic studies 301–303
Fusarium wilt 268 serine 177
Phytophthora root rot 268–269 Serratia marcescens 186
rust 206, 213, 214 sesame 269–271
Salvia officinalis 38 Fusarium wilt 270
sambangi 41 Phytophthora blight 269–270
savoury oil 58 powdery mildew 270–271
sclerotia 33, 54 Sesamum indicum, see sesame
Sclerotinia spp., Trichoderma control sheath rot, rice 42
products 133 siderophores 130
Sclerotinia sclerotiorum, endophyte protection skin coatings, fruit 6
185 smut 138–139
Sclerotium rolfsii 172, 177, 265 amaranth 311–312
stem rot 265 characterization of pathogen 313–315,
Trichoderma control products 133 314
386 Index
smut continued seeds 337–340, 341

amaranth continued strawberries 4, 5, 8
incidence in amaranth cultivars 315, Streptomyces 124–125
316 stripe, Cephalosporium gramineum 238–239
wild hosts 313, 315–316 stromatization 54
head smut, Bromus spp. 139–140, 140, 141 stylar end rot 4
maize 140–142, 143 sugarcane
sugarcare 218–219 economic importance 217
‘smut whip’ 219 eye spot disease 223–224
sod webworms 188 pineapple disease 225–226
sodium bicarbonate 115–116 pokkah boeng disease 224–225
sodium bisulphate 5 red rot disease 221–223
sodium metabisulphite 341 rust diseases 220–221
soils smut diseases 218–219
acidity 189 sulphur 213
diseases control 124–125, 125 sulphur dioxide 5
mycorrhizae propagule banks 163, 164–167 sunflower 265–267
no-tillage systems 74, 163–164, 166–167, Alternaria leaf spot/blight 265–266
283 downy mildew 266–267
sorghum, seed treatments 335 head rot 267
Sorghum spp., rusts 208–209, 213 rusts 206, 213, 214, 266
sowing time 249 Suryanarayanan, Prof. T.S. 190, 191
soybean sustainable disease management
endophytic fungi 154–155, 154 rice blast 100, 100
mycorrhizae 173, 177 see also biological control; integrated
Phytophthora root/stem rot 318 management
crop losses 318
disease cycle 319–321
management 321–323 T-muurolol 20, 22
pathogen 319, 320–321 take-all 172, 364
symptoms 319 tamarind 41
rust fungi 206, 210 tan spot 231–232, 241, 276
seed treatments 333, 333 crop losses 276–277
Sphaerellopsis filum 212 disease cycle 278–280
Sphaerotheca fuliginea 270 management 159, 282–284
spike morphology 81–82 pathogen 277–278
spinach, seed treatments 334, 335 physiological specialization 281–282
sponge-gourd 41 prevalence and range in South America
spring black stem, fenugreek 253–254 276
spruce, Norway, root and butt rot 347, 352, 353, symptoms of infection 278
354, 354 tannins 40
star anise 56, 60 tea tree oil 53
stem rot, S. rolfsi 265 tebuconazol 283
stem rust (black rust), wheat 364, 365 teliospores 219
Stereum sanguinolentum 347, 349, 350 temperatures, seed and crop storage 32,
control 351–358 337–339, 339
diagnosis 351, 351 Terminalia ivorensis 175
steroidal sapogenins 246 terpenoids 44
storage diseases terpine-4-ol 20, 21
common fungal species 330 Thecaphora amaranthi 311–312
conditions favouring 31–33 Thecaphora amaranthicola 312
management 15–17, 33–34, 340–341 characterization 313–315, 314
botanicals and plant extracts 40–41 incidence in amaranth cultivars 315, 316
postharvest strategies 341 in wild amaranth species 313, 315–316
preharvest conditions 340 thermotherapy
storage environment see heat treatments
fruit crops 33, 34 red rot of sugarcane 223
Index 387
thiram urediniospores, Puccinia 221, 269

rust diseases 213 Ustilago bullata 139–140, 140, 141
seed dressings 332, 332, 333 Ustilago maydis 140–142, 143
thujone 20, 21 Ustilago scitaminea 218–219
thyme oil 9, 19, 38, 44–45, 56 Ustilago tritici 364
thymol 20, 21, 44–45, 53 UV illumination 5–6
tillage
effects on mycorrhizae 163, 164–166
see also no-tillage systems vegetable crops
Tilletia indica 366–368, 367 endophytes 185–186
Tilletia laevis (common bunt) 143–145 mycorrhizae 173–174, 176
time of sowing 249 rust diseases 207, 209–210, 216
tobacco 174, 177 seed treatments 334–335, 335
tomato verbenol 20, 22
endophytes 152–154, 153, 185 verbenone 20, 22
mycorrhizae 173–174, 176 Verticillium spp.
toxic metabolites 15, 29, 31, 39 antagonistic effects of endophytes 185–186
Alternaria 232 parasitic on rusts 212
Fusarium head blight 80 protective role of AMF 173
mould species producing 32 viral diseases
toxicosis, endophytes 150 endophyte plant protection 187–188
transgenic plants, disease-resistant 10 fenugreek 247
trenches, isolation 352 vitavax 214
triazoles, systemic 283 vomitoxin, see deoxynivalenol (DON)
Trichoderma spp. 123, 125–126 Vrs1 locus 82
chemicals produced 129
commercial use 131
compatibility testing 132–134 warehouse conditions 33, 34, 337–340, 341
delivery methods 134 water requirements, fungi 32
mass production and formulation 130–131, weed hosts, rice blast disease 94
132, 134 wheat
mechanisms of action 126–129 abiotic stresses 363
pesticide susceptibility 130 Alternaria leaf blight 232–233, 241
plant growth promotion 129–130 Ascochyta leaf spot 236–237
range of biocontrol uses 126, 126 black point/leaf blight (A. infectoria)
red rot of sugarcane 223 234–236
safflower wilt 268 breeding for disease resistance 363, 369
Septoria tritici blotch 303–305 MAS 86–87, 369
solubilization/sequestration of plant Cephalosporium gramineum 238–239
nutrients 128–129, 130 Cladosporium herbarum 239–241
tan spot control 284 common bunt (T. laevis) 143–145
Trichoderma harzianum 126, 223, 265, 270, endophytic fungi 155–159, 156–158
273, 284, 303–304 fungal diseases 69–70, 277, 363–369, 364
mass production and delivery 134 pathogen-specific thresholds 292
Trichoderma viride 126, 265, 270, 273 Fusarium head blight 363–364, 364
trichothecenes 80 crop losses 79
Type A 80 resistance 83–87
Type B 80 global demand and production 69, 275,
Triticum spp., see wheat 291–292, 362–363
Triticum dicoccoides 87 karnal bunt (T. indica) 366–368, 367
Triticum macha 87 Phoma sorghina leaf spots 237–238
γ tubulin 177 powdery mildew 368–369
‘tulsi’ 8 Pyricularia grisea 241
root rot 133, 172, 177, 265
rust diseases 204, 207–208, 364–366, 364
UG99 (stem rust) 365 fungicides 213, 214
urea treatments 355–356 leaf rust 364, 365–366
388 Index
wheat continued safflower 268

rust diseases continued sesame 270
stem rust 364, 365 wintergreen, oil of 46
yellow (stripe) rust 366 wood decay fungi 348
seed disease, plant extract treatments 57–58 wrappings, fruit 6
seed dressings 333–334
Septoria leaf blotch 70, 71–73
Septoria tritici blotch (STB) 293–305 xanthan gum 114
ascendant movement 299–301 Xanthomonas alfalfa 247, 248
biocontrol 303–305 Xylaria sp. 187
early detection 299
epidemiological studies 293–298
impact of climate 298–299 yams 30, 34, 40
population genetic studies 301–303 yeasts (biocontrol agents) 111
Sumai 3 cultivar 85–86, 87 constraints in commercial development
tan spot 231, 241, 276 113–115
crop losses 276–277 integration with other control measures
disease cycle 278–280 115–116
management strategies 159, 282–284 mechanisms of action 111–113
pathogen 277–278 yellow rice disease 31
physiological specialization yellow rust, wheat 364, 366
281–282 Yield-PlusTM 110, 114
prevalence and range in South
America 276
symptoms of infection 278 Zea, see maize
wilt, Fusarium zearalenone (ZEN/F2-toxin) 80
and AM fungi 173 zero-tillage, see no-tillage systems
castor 272–273 zineb, rust diseases 213
endophyte-induced resistance 185 zingiberene 20, 22
fenugreek 250, 252–253 ziram 213

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Management of Fungal Plant Pathogens

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Analía Edith Perelló

Assistant Professor and Research Scientist, CONICET - CIDEFI,

Management of fungal plant pathogens / edited by Arun Arya,

Typeset by AMA Dataset, Preston, UK.

PART I: BOTANICALS IN FUNGAL PEST MANAGEMENT

1 Recent Advances in the Management of Fungal Pathogens of Fruit Crops 3

2 Botanicals in Agricultural Pest Management 14

3 Deleterious Effects of Fungi on Postharvest Crops and Their

4 Exploitation of Botanicals in the Management of Phytopathogenic

5 Use of Plant Extracts as Natural Fungicides in the Management of

PART II: DISEASE CONTROL THROUGH RESISTANCE

6 Resistance to Septoria Leaf Blotch in Wheat 69

7 Barley and Wheat Resistance Genes for Fusarium Head Blight 78

8 Sustainable Management of Rice Blast (Magnaporthe grisea

PART III: BIOLOGICAL CONTROL MECHANISMS

9 Postharvest Technology – Yeast as Biocontrol Agents: Progress,

10 Biological Control of Plant Diseases: An Overview and

11 Physiological Specialization of Ustilaginales (Smut) of

PART IV: ENDOPHYTES IN PLANT DISEASE CONTROL

12 Status and Progress of Research in Endophytes from

13 Effect of Tillage Systems on the Arbuscular Mycorrhizal Fungi

14 Mechanism of Action in Arbuscular Mycorrhizal Symbionts

15 Role of Fungal Endophytes in Plant Protection 183

PART V: MANAGING FUNGAL PATHOGENS CAUSING LEAF DAMAGE

16 The Rust Fungi: Systematics, Diseases and Their Management 201

17 Etiology, Epidemiology and Management of Fungal Diseases of Sugarcane 217

18 New and Emerging Fungal Pathogens Associated with Leaf Blight

19 Diseases of Fenugreek (Trigonella foenum-graecum L.) and Their Control

20 Fungal Diseases of Oilseed Crops and Their Management 263

21 Occurrence of Pyrenophora tritici-repentis Causing Tan Spot in Argentina 275

22 Epidemiological Studies on Septoria Leaf Blotch of Wheat in Argentina 291

PART VI: ALTERNATIVE CONTROL STRATEGIES

23 Review of Thecaphora amaranthicola M. Piepenbr., Causal Agent

24 Population Biology and Management Strategies of Phytophthora sojae

25 Management of Fungal Pathogens – A Prerequisite for Maintenance of

The colour plate section can be found following page 50.

Gonthier, Paolo, Department of Exploitation and Protection of Agricultural and Forestry

Introduction Taken either as a dessert or processed, the

 CAB International 2010. Management of Fungal Plant Pathogens

Safe fumigating agents that disappear after

Chemically impregnated wrappers Various strains of antagonist must be com-

Ashok Kumar, Priyanka Singh and N.K. Dubey

Introduction the world is destroyed by various pests,

 CAB International 2010. Management of Fungal Plant Pathogens

Methods Fungi/mycotoxins References

management, botanical pesticides are well Conclusions

Plants Products Fungi/mycotoxins References

Hypericum linarioides EO/PEE/ 6 Fusarium spp. Cakir et al. (2005)

Ajoene Aspergillus niger, Candida albicans, Yoshida et al. (1987)

H3C CH3 CH2 H3C CH2 H3C CH3

H3C CH3 H3C CH3 H3C CH3

H3C CH3 H3C CH3 H2C CH3

Fig. 2.1. Chemical structures of some bioactive essential oil constituents.

CH3 H3C CH3 H3C CH3

OCH3 CH3 CH3

CH3 H3C CH3 H3C CH3

CH3 H3C CH3 H3C CH3