Natural Products in Plant Pest Management

NATURAL PRODUCTS IN PLANT PEST
MANAGEMENT
NATURAL PRODUCTS
IN PLANT PEST MANAGEMENT
Edited by
Nawal K. Dubey
Centre for Advanced Studies in Botany
Banaras Hindu University, Varanasi, India
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Library of Congress Cataloging-in-Publication Data
Natural products in plant pest management / edited by N.K. Dubey.
p. cm.
Includes bibliographical references and index.
ISBN 978-1-84593-671-6 (alk. paper)
1. Natural pesticides. 2. Agricultural pestsControl. 3. Plant products.
I. Dubey, N. K. II. Title.
SB951.145.N37N394 2011
632.96dc22
2010020068
ISBN-13: 978 1 84593 671 6
Commissioning editor: Sarah Mellor
Production editor: Fiona Chippendale
Typeset by AMA Dataset, Preston, UK.
Printed and bound in the UK by CPI Antony Rowe, Chippenham.
Contents
Contributors
Preface
1. Global Scenario on the Application of Natural Products
in Integrated Pest Management Programmes
N.K. Dubey, Ravindra Shukla, Ashok Kumar,
Priyanka Singh and Bhanu Prakash
2. Plant Products in the Control of Mycotoxins
and Mycotoxigenic Fungi on Food Commodities
Sonia Marn, Vicente Sanchis and Antonio J. Ramos
3. Natural Products from Plants: Commercial Prospects
in Terms of Antimicrobial, Herbicidal and Bio-stimulatory
Activities in an Integrated Pest Management System
J.C. Pretorius and E. van der Watt
4. Antimicrobials of Plant Origin to Prevent the Biodeterioration
of Grains
K.A. Raveesha
5. Some Natural Proteinaceous and Polyketide Compounds
in Plant Protection and their Potential in Green
Consumerization
L.A. Shcherbakova
6. Natural Products as Allelochemicals in Pest Management
Roman Pavela
vii
ix
21
42
91
109
134
vi
Contents
7. Potency of Plant Products in Control of Virus Diseases of Plants

H.N. Verma and V.K. Baranwal
149
8. Phytochemicals as Natural Fumigants and Contact Insecticides

Against Stored-product Insects
Moshe Kostyukovsky and Eli Shaaya
175
9. Prospects of Large-scale Use of Natural Products as Alternatives

to Synthetic Pesticides in Developing Countries
D.B. Olufolaji
191
10. Current Status of Natural Products in Pest Management

with Special Reference to Brassica carinata as a Biofumigant
Mara Porras
205
11. Fungal Endophytes: an Alternative Source of Bioactive

Compounds for Plant Protection
R.N. Kharwar and Gary Strobel
218
12. Suppressive Effects of Compost Tea on Phytopathogens

Mila Santos, Fernando Dinez and Francisco Carretero
242
13. Biotechnology: a Tool for Natural Product Synthesis

Sanath Hettiarachi
263
Index
280
Contributors
V.K. Baranwal, Advanced Centre of Plant Virology, I.A.R.I, New Delhi, India.
E-mail: vbaranwal2001@yahoo.com
Francisco Carretero, Plant Production Department. University of Almeria,
La Caada de San Urbano. 04120 Almera, Spain
N.K. Dubey, Department of Botany, Banaras Hindu University, Varanasi-221005,
India. E-mail: nkdubey2@rediffmail.com
Fernando Dinez, Plant Production Department. University of Almeria,
La Caada de San Urbano. 04120 Almera, Spain
Sanath Hettiarachi, Department of Botany, University of Ruhuna, Matara,
Sri Lanka. Email: sanath@bot.ruh.ac.lk
R.N. Kharwar, Mycopathology and Microbial Technology Lab, Department
of Botany, Banaras Hindu University, Varanasi-221005, India.
E-mail: rnkharwar@yahoo.com
Moshe Kostyukovsky, Agricultural Research Organization, the Volcani Center,
Israel, Bet Dagan, P.O. Box 6, 50250, Israel. E-mail: inspect@volcani.agri.gov.il
Ashok Kumar, Department of Botany, Banaras Hindu University, Varanasi221005, India
Sonia Marn, Food Technology Department, Lleida University, XaRTA-UTPV,
Lleida, Spain. E-mail: smarin@tecal.udl.cat
D.B. Olufolaji, Department of Crop, Soil and Pest Management, The
Federal University of Technology, P.M.B. 704, Akure. Nigeria.
E-mail: tundeolufolaji@yahoo.co.uk
Roman Pavela, Crop Research Institute, Drnovska 507, Prague 6 Ruzyne, Czech
Republic. E-mail: pavela@vurv.cz
Mara Porras, Department of Crop Protection, IFAPA Centro Las
Torres Tomejil, Aptdo. 41200-Alcal del Ro, Sevilla, Spain.
E-mail: mariaa.porras@juntadeandalucia.es
Bhanu Prakash, Department of Botany, Banaras Hindu University,
Varanasi-221005, India
vii
viii
Contributors
J.C. Pretorius, Department of Soil, Crop and Climate Sciences, University

of the Free State, P.O. Box 339, Bloemfontein 9300, South Africa.
E-mail: pretorjc.sci@ufs.ac.za
Antonio J. Ramos, Food Technology Department, Lleida University, XaRTAUTPV, Lleida, Spain
K.A. Raveesha, Herbal Drug Technology Laboratory, Department of Studies in
Botany, University of Mysore, Manasagangotri, Mysore-570 006, India.
E-mail: karaveesha@gmail.com
Vicente Sanchis, Food Technology Department, Lleida University, XaRTA-UTPV,
Lleida, Spain
Mila Santos, Plant Production Department. University of Almeria, La Caada de
San Urbano, 04120 Almera, Spain. E-mail: msantos@ual.es
Eli Shaaya, Agricultural Research Organization, the Volcani Center, Israel,
Bet Dagan, P.O. Box 6, 50250, Israel
L.A. Shcherbakova, Russian Research Institute of Phytopathology, VNIIF, B.,
Vyazyomy, Moscow reg., 143050, Russia. E-mail: larisa@vniif.rosmail.com
Ravindra Shukla, Department of Botany, Banaras Hindu University, Varanasi221005, India
Priyanka Singh, Department of Botany, Banaras Hindu University, Varanasi221005, India
Gary Strobel, Department of Plant Sciences, Montana State University, Bozeman,
MT 59717, USA
E. van der Watt, Department of Soil, Crop and Climate Sciences, University of the
Free State, P.O. Box 339, Bloemfontein 9300, South Africa
H.N.
Verma,
Jaipur
National
University,
Jaipur,
India.
E-mail: vermalko@yahoo.co.uk
Preface
The ever increasing global population needs substantial resources for food
production. However, food production as well as its protection is imperative.
The situation gets particularly critical in developing countries where the net
food production rate is slowing down relative to the population rise. The
world food situation is aggravated by the fact that, in spite of all the available
means of plant protection, a major fraction of the yearly output of food commodities gets destroyed by various pests including bacteria, fungi, viruses,
insects, rodents and nematodes. The production of mycotoxins by fungi has
added new dimensions to the gravity of the problem. Losses at times are
severe enough to lead to famine in large areas of the world that are densely
populated and dependent on agriculture.
The use of synthetic pesticides has undoubtedly contributed to a green
revolution in different countries through increased crop protection. However, recent years witnessed considerable pressure on consumers and farmers to reduce or even eliminate the deployment of synthetic pesticides in
agriculture owing to environmental risks emerging from their indiscriminate
use. Thus, there has been renewed interest in botanical pesticides as the alternative and eco-chemical option in pest management. It is also imperative for
sustainable agriculture to reduce the incidence of pests and crop diseases to
a degree that does not seriously damage the farmers products and also to
develop cost-effective strategies with minimal ecological side effects.
The use of locally available plants in the control of pests is an age-old
technology in many parts of the world. Some plants, namely Derris, Nicotiana
and Ryania, were used to combat agricultural pests during the prehistoric
era. Used widely until the 1940s, such botanical pesticides have been partially replaced by synthetic pesticides that are easier to procure and longer
lasting.
Higher plants, in this respect, harbour numerous compounds that may
offer resistance to pathogens. There has been a renewed interest in botanical
ix
Preface
antimicrobials owing to various distinct advantages. Botanicals, being the

natural derivatives, are biodegradable, and do not leave toxic residues or
byproducts to contaminate the environment. The main thrust of recent
research in the area has been to evolve alternative control strategies that
eventually reduce dependency on synthetic fungicides. Recently, in different
parts of the world, emphasis has been placed on exploitation of higher plant
products as the novel chemotherapeutants for plants because of their nontoxicity, systemicity and biodegradability. To date, different plant products
have been formulated as botanical pesticides for large-scale applications for
the eco-friendly management of plant pests and as an alternative to synthetic
pesticides in crop management. These products are cost-effective and have
low toxicity to humans and livestock. Therefore, such products from higher
plants may be exploited as the eco-chemical and biorational approach in
integrated plant protection programmes. The interests and trends in the
modern society for green consumerism involving fewer synthetic ingredients in the agriculture industry may favour the approval of plant-based
products as the safe alternative strategy in managing plant pests. Natural
plant chemicals will certainly play a pivotal role in the near future for pest
control in industrialized and developing countries as well. Because of their
biodegradable nature, systemicity following application, ability to alter the
behaviour of target pests and their favourable safety prole, the botanical
pesticides are considered the vital tools in achieving the evergreen revolution
and in the ecological strategy of controlling agricultural pests.
To date, natural plant products for pest management form the frontier
areas of research on a global scale. The present book deals with the current
state and future prospects of botanical pesticides in the eco-friendly management of plant pests. Looking at the growing interest of professionals and the
general public alike, it seemed imperative to produce a book that encompasses
as much information as possible, with an emphasis on the exploitation of
plant products in the possible management of agricultural pests. The book
may be useful to many, including plant pathologists, microbiologists, entomologists, plant scientists and natural product chemists. It is a compilation
of invited chapters from eminent scientists and professors from educational
institutions and research organizations of different countries. The book
addresses different topics under the domain of natural compounds in
agricultural pest management and is an attempt to include notable and
diversied scientic works in the eld carried out by leading scientists in
the world.
The book harbours a total of 13 chapters contributed by eminent scientists working in the eld so as to offer relevant and practical information on
each topic. Different issues, including the global scenario on the application
of botanical pesticides, plant products in the control of mycotoxins, the commercial application of botanical pesticides and their prospects in green consumerism, natural products as allelochemicals, their efcacy against viral
diseases and storage pests, and bioactive products from fungal endophytes,
have been covered in the book. I am very grateful to the contributors for their
generous and timely responses in spite of their busy academic schedules.
Preface
xi
I wish to extend my appreciation to all the contributors for their cooperation

and encouragement. Without their enthusiasm and the timely submission of
their excellent chapters, this work would not have been possible.
The book focuses on newer developments in agricultural pest management using natural products and provides up-to-date information from
world experts in different elds. It is expected that the book will be a source
of inspiration to many for future developments in the eld. It is also hoped
that the book will become useful for those engaged in such an extraordinary
and attractive area. The book would serve as the key reference for recent
developments in frontier research on natural products in the management of
agricultural pests and also for the scientists working in this area.
I convey the strength of my feelings to my wife Dr Nirmala Kishore,
daughter Dr Vatsala Kishore and son Navneet Kishore who have always
been the excellent intellectual companions without whose constant loving
support, patience, and unmatched help and sacrices it would have been
difcult to bring out the book on time. I also bow my head to my parents
(Sri G.N. Dubey and Smt Shanti Devi) for their blessings. My sincere thanks
are also due to my current research students, especially Ravindra Shukla, for
help and cooperation.
Thanks are due to CABI Publishers for publishing the book with utmost
interest; I am thankful to the staff members of CABI for sparing no pains to
ensure a high standard of publication. Special thanks goes to Sarah Mellor,
the Commissioning Editor, who initially motivated me to bring out this book
during the ICPP 2008 held in Turin, Italy.
N.K. Dubey
Global Scenario on the

Application of Natural Products
in Integrated Pest Management
Programmes
N.K. DUBEY, RAVINDRA SHUKLA, ASHOK KUMAR,
PRIYANKA SINGH AND BHANU PRAKASH
Department of Botany, Banaras Hindu University, Varanasi, India
Abstract
In recent years there has been considerable pressure in agriculture to reduce chemical
pesticides and to look for their better alternatives. The plant kingdom is recognized
as the most efcient producer of different biologically active compounds, which provide them with resistance against different pests. Some higher plant products have
been currently formulated as botanical pesticides and are used on a large scale as ecofriendly and biodegradable measures in managing agricultural pests. Botanicals used
in agricultural pest management are safer to the user and the environment. The interest in the possible use of natural compounds to control agricultural pests has notably
increased in response to consumer pressure to reduce or eliminate chemically synthesized additives in foods. There is a wide scope of use of plant-based pesticides in the
integrated management of different agricultural pests. A consolidated and continuous search of natural products may yield safer alternative control measures comparable to azadirachtin and pyrethryoids, which are being used in different part of the
world as ideal natural fungicides. The products from higher plants are safe and economical and would be in high demand in the global pesticide market because of their
diverse mode of application.
1.1 Introduction
Agriculture plays an important role in the survival of humans and animals.
It is the driving force for broad-based economic growth, particularly in developing countries. Tropical and subtropical regions have a greater potential for
food production and can grow multiple crops annually. Agricultural crops
suffer a colossal loss due to the ravages of insects and diseases thus causing
a serious threat to our agricultural production. In some years, losses are much
greater, producing catastrophic results for those who depend on the crop for
food. Major disease outbreaks among food crops have led to famines and
CAB International 2011. Natural Products in Plant Pest Management
(ed. N.K. Dubey)
N.K. Dubey et al.
mass migrations throughout history. Loss of crops from plant diseases may
result in hunger and starvation, especially in less developed countries where
access to disease-control methods is limited and annual losses of 3050 % are
common for major crops. Owing to the congenial climatic conditions and
particular environment, the agriculture in tropical and subtropical countries
suffers severe losses due to pests (Varma and Dubey, 2001; Roy, 2003).
Even during storage, foods are severely destroyed by fungi, insects and
other pests. The deterioration in the stored food commodities is mainly caused
by three agents, fungi, insects and rodents, under different conditions of storage. Insect pests cause heavy losses to stored grains, especially in humid and
warm areas of the world. The production of mycotoxins by several fungi has
added a new dimension to the gravity of the problem. Fungi are signicant
destroyers of foodstuffs during storage, rendering them unt for human consumption by retarding their nutritive value and sometimes by the production
of mycotoxins. According to FAO estimates, 25% of the world food crops are
affected by mycotoxins each year (Dubey et al., 2008). Generally, tropical conditions such as high temperature and moisture, unseasonal rains during harvest, and ash oods lead to mycotoxins. Poor harvesting practices and
improper storage during transport and marketing can also contribute to the
proliferation of mycotoxins. Among the mycotoxins, aatoxins raise the most
concern posing a great threat to human and livestock health as well as international trade. Aatoxins are the most dangerous and about 4.5 billion people in
developing countries are exposed to aatoxicoses (Williams et al., 2004;
Srivastava et al., 2008). Aatoxins are potent toxic, carcinogenic, mutagenic,
immunosuppressive agents, produced as secondary metabolites by the fungi
Aspergillus flavus and A. parasiticus on a variety of food products.
The entire effort of growing a crop will be lost in the absence of crop
protection, resulting in nancial loss to the grower. Therefore crop protection
against various pests is a must in agriculture. There is a need to reduce if not
eliminate these losses by protecting the crops from different pests through
appropriate techniques. Currently, the role of crop protection in agriculture
is of great importance and is a more challenging process than before. Hence,
there is an urgent need to pay proper attention to control quantitative losses
due to pest infestations of crops and their produce as well as qualitative
losses due to mycotoxin contamination.
1.2 The Need to Search for Eco-friendly Control Measures

The most conventional and common method of pest and disease control is
through the use of pesticides. Pesticides are the substances or mixture of substances used to prevent, destroy, repel, attract, sterilize or mitigate the pests.
Generally pesticides are used in three sectors, viz. agriculture, public health
and consumer use. These pesticides are largely synthetic compounds that kill
or deter the destructive activity of the target organism.
Many farmers and crop growers use insecticides to kill infesting
insects. The consumption of pesticide in some of the developed countries is
Global Scenario and Natural Products
almost 3000 g/hectare. Unfortunately, there are reports that these compounds
possess inherent toxicities that endanger the health of the farm operators,
consumers and the environment (Cutler and Cutler, 1999). Pesticides are
generally persistent in nature. Upon entering the food chain they destroy the
microbial diversity and cause ecological imbalance. Their indiscriminate use
has resulted in the development of a resistance problem among insects to
insecticides, pesticide residue hazards, upsetting the balance of nature and a
resurgence of treated populations. Insect resistance to phosphine is a matter
of serious concern (Rajendran, 2001; Benhalima et al., 2004; Thie and Mills,
2005; Dubey et al., 2008). The repeated use of certain chemical fungicides in
packing houses has led to the appearance of fungicide-resistant populations
of storage pathogens (Brent and Hollomon, 1998). The ability of some of
these pests to develop resistance curbs the effectiveness of many commercial
chemicals. Resistance has accelerated in many insect species and it was
reported by the World Resources Institute that more than 500 insect and mite
species are immune to one or more insecticides (WRI, 1994). Similarly about
150 plant pathogens such as fungi and bacteria are now shielded against
fungicides (Shetty and Sabitha, 2009). Moreover, the control of such pests has
become increasingly difcult because of reduced effectiveness of pesticides
caused by the emergence of pesticidal resistance in arthropod pests.
Reliance on synthetic chemicals to control pests has also given rise to the
destruction of benecial non-target organisms (parasitoids and predators),
thereby affecting the food chain and impacting on biological diversity. There
have also been cases of pests becoming tolerant to insecticides, resulting in
the use of double and triple application rates (Stoll, 2000).
Furthermore, the use of synthetic chemicals has also been restricted
because of their carcinogenicity, teratogenicity, high and acute residual
toxicity, ability to create hormonal imbalance, spermatotoxicity, long degradation period, environmental pollution and their adverse effects on food
and side effects on humans (Omura, et al., 1995; Unnikrishnan and Nath,
2002; Xavier, et al., 2004; Konstantinou, et al., 2006; Feng and Zheng, 2007).
Pesticides can be remarkably persistent in biological systems. They have
been reported to be accumulated in ecosystems. Osprey eggs in the Queen
Charlotte Islands, polar bear fat in the high Arctic, and the blubber of whales
in all the oceans of the world are contaminated with pesticide residues, even
though all these creatures live far from point sources of pesticide application
(CAPE, 2009). Water and wind, as well as the bodies of animals that serve
as prey for others (including humans) higher on the food chain, are the
universal vectors for pesticide dispersal. Highest on the food chain, human
breast milk contamination is of great concern because of high levels of
bio-accumulated pesticides. The World Health Organization (WHO) estimates that 200,000 people are killed worldwide, every year, as a direct result
of pesticide poisoning (CAPE, 2009). Pest control strategies, therefore, need
proper regulation in the interest of human health and environment. In recent
years there has been considerable pressure on consumers to reduce or eliminate chemical fungicides in foods. There is increasing public concern over the
level of pesticide residues in food. This concern has encouraged researchers
N.K. Dubey et al.
to look for alternative solutions to synthetic pesticides (Sharma and

Meshram, 2006).
Effort is therefore needed to nd alternatives or formulations for
currently used pesticides. There is, therefore, still a need for new methods of
reducing or eliminating food-borne pathogens, possibly in combination with
existing methods (Leistner, 1978). At the same time, Western society appears
to be experiencing a trend of green consumerism (Tuley de Silva, 1996; Smid
and Gorris, 1999), desiring fewer synthetic food additives and products with
a smaller impact on the environment.
Considering all these drawbacks, many components of the integrated
pest management (IPM) concept were developed in the late 19th and early
20th centuries, and biopesticides and bioagents became important tools
in pest management strategies that are practical, economical, eco-friendly
and protective for both public health and the environment. Sustainable
agriculture aims to reduce the incidence of pests and diseases to such a
degree that they do not seriously damage the farmers crops but without
upsetting the balance of nature. One of the aims of sustainable agriculture
is to rediscover and develop methods whose cost and ecological side-effects
are minimal.
1.3 Historical Use of Botanical Products in Pest Management

Plant diseases are known from times preceding the earliest writings.
The Bible and other early writings mention diseases, such as rusts, mildews,
blights and blast. Disease control measures are as old as modern civilizations
and were rst recorded long before the Renaissance in both the Western and
the Oriental worlds. The use of locally available plants in the control of pests
is an ancient technology in many parts of the world. Rotenone was originally
employed in South America to paralyse sh, causing them to come towards
the surface and be easily captured (Fig. 1.1a; Ware, 2002). It has been used in
fruit gardens to combat such pests as Japanese beetles, mites and stinkbugs.
Preparations of roots from the genera Derris elliptica, Lonchocarpus nicou and
Tephrosia sp. containing rotenone were commercial insecticides in the 1930s.
Rotenone is a avonoid derivative that strongly inhibits mitochondrial
function by inhibiting NADH-dependent coenzyme Q oxidoreductase
(Singer and Ramsay, 1994). Rotenone persists 35 days on the foliage after
application and is easily biodegradable. Nicotine obtained from several
members of the genus Nicotiana has been used commercially as an insecticide
(Fig. 1.1b). However, presently it is not accepted by most organic certication
programmes because of its residue and the potential harm it can cause to
humans and the agroecosystem (MMVR, 2003). Ryanodine, an alkaloid from
the tropical shrub Ryania speciosa, has been used as a commercial insecticide
against the European corn borer (Fig 1.1c). Ryania controls many insects,
particularly those with chewing mouthparts (Coats, 1994; Regnault-Roger
and Philogene, 2008).

(a)
5
(b)
OH
C
(c)
CH3
CH2
HO
HO
HO
HO
HO
O
O
HO
H
H
O
O
CH3
Fig. 1.1.
HN
OH
CH3
(a) Rotenone (b) Nicotine (c) Ryanodine.
Used widely until the 1940s, such botanical pesticides were partly displaced by synthetic pesticides that at the time seemed easier to handle and
longer lasting. With the knowledge of the adverse effects of synthetic pesticides, worldwide attention is currently being given to shifting to nonsynthetic safer pesticides. There is renewed interest in the application of
botanical pesticides for crop protection, and scientists are now experimenting and working to protect crops from pest infestations using indigenous
plant materials (Roy et al., 2005).
1.4 Plant Products in Current Worldwide Use as Pesticides

The plant kingdom is recognized as the most efcient producer of chemical
compounds, synthesizing many products that are used in defence against
different pests (Prakash and Rao, 1986; 1997; Charleston et al., 2004; Isman
and Akhtar, 2007). Higher plants contain a wide spectrum of secondary
metabolites such as phenolics, avonoids, quinones, tannins, essential oils,
alkaloids, saponins and sterols. Tens of thousands of secondary products of
plants have been identied and there are estimates that hundreds of thousands of such compounds exist. These secondary compounds represent a
large reservoir of chemical structures with biological activity. Therefore,
higher plants can be exploited for the discovery of new bioactive products
that could serve as lead compounds in pesticide development because of
their novel modes-of-action (Philogene et al., 2005). The rainforest plants
are particularly thought to have developed a complete array of defenceproviding chemicals. This resource is largely untapped for use as pesticides
(Tripathi et al., 2004).
Extracts prepared from different plants have been reported from time to
time to have a variety of properties including insecticidal activity, repellence
to pests, antifeedant effects, insect growth regulation, toxicity to nematodes,
mites and other agricultural pests, and also antifungal, antiviral and
antibacterial properties against pathogens (Prakash and Rao, 1986, 1997).
N.K. Dubey et al.
Natural pest controls using botanicals are safer to the user and the
environment because they break down into harmless compounds within
hours or days in the presence of sunlight. Botanical pesticides are biodegradable (Devlin and Zettel, 1999) and their use in crop protection is a practical
sustainable alternative. Pesticidal plants have been in nature for millions
of years without any ill or adverse effects on the ecosystem. Botanical pesticides are also very close chemically to those plants from which they are
derived, so they are easily decomposed by a variety of microbes common in
most soils. Their use maintains the biological diversity of predators (Grange
and Ahmed, 1988), and reduces environmental contamination and human
health hazards. Botanical pesticides tend to have broad-spectrum activity
and are sometimes stimulatory to the host metabolism (Mishra and Dubey,
1994). Botanical insecticides can often be easily produced by farmers and
small-scale industries. Recently, attention has been paid towards the exploitation of higher plant products as novel chemotherapeutics in plant protection. Such plant products have also been formulated for their large-scale
application in crop protection, and are regarded as pro-poor and cost-effective
(Dubey et al., 2009).
Neem
Neem (Azadirachta indica) is regarded as the Wonder Tree, Botanical
Marvel, Gift of Nature and Village Pharmacy in India. From prehistoric
times, neem has been used primarily against household and storage pests,
and to some extent against pests related to eld crops in the Indian subcontinent. Neem oil and seeds are known to have inherent germicidal properties
and have been in use for Ayurvedic (herbal) medicines in India for a long
time. Burning neem leaves in the evening is a common practice in rural India
to repel mosquitoes. Neem is widely grown in other Asian countries and
tropical and subtropical areas of Africa, America and Australia. It grows well
in poor, shallow, degraded and saline soil. Neem can be considered as the
most important among all biopesticides for controlling pests. Neem pesticides do not leave any residue on the crop and therefore are preferred over
chemical pesticides. In the past decade, neem has become a source of natural
pesticide due to its non-toxicity, environmental safety and so on, thereby
replacing synthetic pesticides. Neem derivatives have been applied against
several species of storage pests and crop pests as leaves, oil, cake, extracts
and as formulations in neem oil (Gahukar, 2000; Dhaliwal et al., 2004). Neem
pesticides are thus a potential alternative to chemical-based pesticides and
their use can avoid the dumping of thousands of tonnes of agrochemicals on
Earth every year.
Neem-based pesticides are sold under trade names such as Margosan-O,
Azatin Rose Defense, Shield-All, Triact and Bio-neem. They have been shown
to control gypsy moths, leaf miners, sweet potato whiteies, western ower
thrips, loopers, caterpillars and mealybugs as well as some of the plant
diseases, including certain mildews and rusts.
Neem products also function as insect growth regulators (IGRs). The

treated insects are usually prevented from moulting to develop into the next
life stage and they die. The treatment may also deter egg laying. Generally,
chewing insects are affected more than sucking insects. Insects that undergo
complete metamorphosis are also generally affected more than those that do
not undergo metamorphosis.
Neem seeds are a rich storehouse of over 100 tetranortriterpenoids and
diverse non-isoprenoids (Devkumar and Sukhdev, 1993). The neem tree contains more than 100 different limonoids in its different tissues (Isman et al., 1996).
Many of these are biologically active against insects as antifeedants. The
most touted biologically active constituent of neem has been highly
oxygenated azadirachtin and some of its natural analogues and derivatives.
Azadirachtin (molecular formula: C35H44O16, chemical structure shown in
Fig. 1.2), a highly oxidized triterpenoid, is the most widely publicized bioactive molecule in neem. It is systemic in nature, absorbed into the plant and
carried throughout the tissues, being ingested by insects when they feed on
the plant. This may make it effective against certain foliage-feeders that
cannot be reached with spray applications. Azadirachtin is more effective
when formulated in a neem oil medium together with the other natural
products of neem. Hence, it is preferable to use neem oil enriched with
azadirachtin as a stable feed stock for making pesticide formulations.
Neem as a harmless and safe pesticide ts into integrated pest management and organic farming. In toxicological studies carried out in the USA
and Germany, different neem products were neither mutagenic nor carcinogenic, and they did not produce any skin irritations or organic alterations in
mice and rats, even at high concentrations. Azadirachtin is considered
non-toxic to mammals, having a low mammalian toxicity with an LD50 of
>5000 mg/kg for rat (oral acute) (Raizada et al., 2001), sh (Wan et al., 1996)
and pollinators (Naumann and Isman, 1996). Hence it is classied by the
CH3
H
C
C
H3C
O
C
OH
O
O
H
CH3
OH
H3C O
CH3
H3C
H
C
O
O
O
OH
C
O
O
CH3
Fig. 1.2.
Azadirachtin.
O
H
N.K. Dubey et al.
Environmemtal Protection Agency (EPA) in class IV. Regarding its environmental impact, neem is sensitive to light and the half-life of azadirachtin is
one day (Kleeberg, 2006).
Pyrethrum
Pyrethrum is one of the oldest and safest insecticides, and is extracted from
the dried ower buds of Chrysanthemum sp. The ground, dried owers were
used in the early 19th century to control body lice during the Napoleonic
Wars. Even today, powders of the dried owers of these plants are sold as
insecticides. Pyrethrum is a mixture of four compounds: pyrethrins I and II
and cinerins I and II (chemical structures shown in Fig. 1.3; Ware, 2002).
Chrysanthemum plants, Chrysanthemum cinerariaefolium, are grown primarily in Kenya, Uganda, Tanzania and Ecuador. Pyrethrins affect the insect on
contact, creating disturbances in the nervous system which eventually result
in convulsions and death. Low doses, however, often cause temporary paralysis from which the insect may recover. For this reason, pyrethrums are
mixed with a synergist such as piperonyl butoxide (PBO) derived from sassafras or n-octyl bicycloheptane dicarboximide to increase insect mortality
and to extend their shelf life (Ware, 2002).
Pyrethrum products represent 80% of the total market of botanical insecticides and are favoured by organic growers because of their low mammalian
toxicity and environmental non-persistence (Isman, 1994). Pyrethrum is nontoxic to most mammals, making it among the safest insecticides in use.
Pyrethroids versus pyrethrin
Pyrethroids are synthetic materials designed to imitate natural pyrethrum.
They have been developed based on pyrethrins, but are much more toxic and
long lasting (Singh and Srivasava, 1999). They are marketed under various
trade names, for example Ambush or Decis. Pyrethroids can be useful insecticides, but some pyrethroids are extremely toxic to natural enemies. Pyrethroids are also toxic to honey bees and sh. Sunlight does not break them
down and they stick to leaf surfaces for weeks, killing any insect that touches
the leaves. This makes them less specic in action and more harmful to the
environment than pyrethrin. In addition they irritate the human skin.
Sabadilla
Sabadilla, also known as cevadilla, is derived from the seeds of the sabadilla
lily (Schoenocaulon officinale), a tropical lily that grows in Central and South
America (Soloway, 1976). The active ingredient is an alkaloid known as
veratrine which is commonly sold under the trade names Red Devil or
Natural Guard (for the structure, see Fig. 1.4). This compound was rst
used in the 16th century, and grew in popularity during the Second World
War, when other botanicals such as pyrethrum and rotenone were in short
supply. The dust is made from the seeds and the active components are
H3C
CH3
H
O
H 3C
H
H3C
CH3
CH2 Pyrethrin I
H
O
H3C
CH3
H
O
H 3C
H
H3C
CH3
CH3
Cinerin I
O
H3C
O
CH3
H
O
H3C
H
H 3C
CH3
CH2
Pyrethrin II
(Z)
H3C
O
CH3
H
O
H3C
H
H 3C
CH3
CH3
Cinerin II
Fig. 1.3.
Pyrethrum.
N
H
OH
OH
H
O
OH O
Fig. 1.4. Veratrine.
OH
OH
HO
10
N.K. Dubey et al.
lacking in the other plant parts (roots, bulbs, stems and leaves). It is interesting that the toxic constituents actually become more powerful after storage.
Sabadilla is considered among the least toxic of botanical insecticides,
with an oral LD50 of 40005000 mg/kg (in mice) (Dayan et al., 2009). It slows
down the shutting of Na+ channels and disturbs membrane depolarization,
causing paralysis before death (Bloomquist, 1996). No residue is left after the
application of sabadilla because it breaks down rapidly in sunlight.
Carvone
Carvone is a monoterpene of the essential oil of Carum carvi (see Fig. 1.5 for
the structure). It is a non-toxic botanical pesticide under the trade name
TALENT. It inhibits the sprouting of potato tubers during storage and protects them from bacterial rotting without exhibiting mammalian toxicity.
Thus, it enhances the shelf life of stored fruits and vegetables and inhibits
microbial deterioration without altering the taste and odour of the fruits after
treatment (Varma and Dubey, 1999). The LD50 value of carvone (in mice) is
reported to be 1640 mg/kg (Isman, 2006).
Allyl isothiocyanate
Allyl isothiocyanate (Fig. 1.6) is an organosulfur compound that serves the
plant as a defence against herbivores. Because it is harmful to the plant itself,
it is stored in the harmless form of the glucosinolate, separate from the
myrosinase enzyme present in plants. When an animal chews the plant, the
allyl isothiocyanate is released due to action of myrosinase enzyme, thus
repelling the animal.
Members of the plant family Brassicaceae are chemically linked by the
almost universal presence of glucosinolates, a class of sulfur-containing
glycosides, also called mustard oil glycosides or thioglucosides. These
compounds are considered the rst line of defence of crucifers against insects
and other organisms (Renwick, 1996).
O
CH3
H3C
C
CH2
Fig. 1.5.
Carvone.
N
Fig. 1.6.
Allyl isothiocyanate.
11
1.5 Other Plant Products

Other environmentally safe botanical pesticides are in use from plants,
namely from Annona squamosa (seeds), Pongamia pinnata (seeds) and Vitex
negundo (leaf) (Hiremath et al., 1997). Some plant species are known to
be highly resistant to nematodes. The best documented of these include
marigolds (Tagetes spp.), rattlebox (Crotalaria spectabilis), chrysanthemums
(Chrysanthemum spp.), and castor bean (Ricinus communis) (Duke, 1990).
These plants may be recommended as intercropping plants to control the
nematode population in soil through their exudates and leachates. In addition to protecting crops from infestation, many rainforest plants can be used
as insect repellents. Bright orange berries of Bixa orellana are effective in
deterring biting insects, in addition to being used as a body paint and dye
(Butler, 2009). In some of the members of Asteraceae, the photodynamic
compound alpha-terthienyl has been shown to account for the strong nematicidal activity of the roots (Fig. 1.7a). However, no plant-derived products
are sold commercially for the control of nematodes. Strychnine formulations
are used in commercial rodenticides (Fig. 1.7b). Quassia amara (Surinam
Wood), belonging to the family Simaroubaceae, is a tree species naturally
distributed in Suriname and several tropical countries. Traditionally, the bark
and leaves are used in herbal remedies and as medicine because the major
secondary metabolites of this tree, quassin and neo-quassin, exhibit pharmacological properties such as antimalarial, antifungal, anti-ulcerative, antiedimogenic and anticancer activity. The male reproductive system, particularly
spermatogenesis, sperm maturation and androgen biosynthesis, is highly
sensitive to the metabolites of Q. amara, which would be useful for insect pest
control but may also affect male reproduction in non-target organisms. Therefore, their pharmacological effects on mammals should be determined before
recommendation to avoid any handling problems with such chemicals. However, quassin has been recently assessed in trials in Australia to control pests
of Brassicaceae (Thacker, 2002).
Some plants have been reported to contain insect growth regulatory
chemicals (IGRs), which disrupt insect maturation and emergence as
adults. Juvabione (Fig. 1.8a), found in the wood of balsam r, was discovered
by accident when paper towels made from this source were used to line
insect-rearing containers resulting in a suppression of insect development
(a)
(b)
N
S
H
H
H
S
S
N H
O
Fig. 1.7.
(a) -terthienyl (b) Strychnine.
O
H
12
N.K. Dubey et al.

(a)
O
C
(b)
CH3
CH3
O
CH3
Fig. 1.8.
(a) Juvabione (b) Precocenes.
(Varma and Dubey, 1999). Analogues of insect juvenile hormones such as

juvocimenes in Ocimum basilicum have also been reported (Balandrin et al.,
1985). Precocenes isolated from essential oils of Matricaria recutita interfere
with the normal function of insect glands that produce juvenile hormones
resulting in the suppression of insect growth while moulting (Fig. 1.8b).
Many plant chemicals deter insects from feeding, thereby showing an
antifeedant effect. Azadirachtin and limonoids such as limonin and nomilin
from different plant species in Meliaceae and Rutaceae (e.g. from Citrus
fruits) have long been used successfully for insect control, especially in India.
Azadirachtin protects newly grown leaves of crop plants from feeding damage, thereby showing systemic antifeedant properties (Varma and Dubey,
1999).
1.6 Essential Oils

Since the middle ages, essential oils have been widely used for bactericidal,
virucidal, fungicidal, antiparasite, insecticidal, medicinal and cosmetic applications, especially nowadays in the pharmaceutical, sanitary, cosmetic, and
agricultural and food industries. In nature, essential oils play an important
role in the protection of the plants as antibacterials, antivirals, antifungals,
insecticides and also against herbivores by reducing their appetite for such
plants. They also may attract some insects to favour the dispersion of pollens
and seeds, or repel undesirable others. Some essential oils have been recognized as an important natural source of pesticides. Aromatic plants produce
many compounds that are insect repellents or act to alter insect feeding
behaviour, growth and development, ecdysis (moulting), and behaviour during mating and oviposition. Recently researchers have demonstrated such
compounds showing larvicidal and antifeedant activity (Adebayo et al., 1999;
Larocque et al., 1999; Gbolade, 2001), capacity to delay development, adult
emergence and fertility (Marimuthu et al., 1997), deterrent effects on oviposition (Naumann and Isman 1995; Oyedele et al., 2000), and arrestant and repellent action (Landolt et al., 1999). Plants with strong smells, such as French
marigold and coriander, act as repellents and can protect the crops nearby.
Most insect repellents are volatile terpenoids such as terpenen-4-ol. Other
terpenoids can act as attractants. In some cases, the same terpenoid can repel
certain undesirable insects while attracting more benecial insects. For instance,
geraniol will repel houseies while attracting honey bees (Duke, 1990).
13
Repellents and attractants modify the behavioural response of insects.

This is the basis for the principle of behavioural insect control, whereby a
given species is either attracted to a bait or pheromone, or repelled from a
host plant by a repulsive agent (Fagoonee, 1981). Some of the plants contain
chemicals which alter the behaviour and life cycle of insect pests without
killing them. Such chemicals are termed as semio-chemicals by the Organisation for Economic Cooperation and Development (Jones, 1998). The most
attractive aspect of using essential oils and/or their constituents as crop protectants is their favourable mammalian toxicity because many essential oils
and their constituents are commonly used as culinary herbs and spices. Such
products are generally exempted from toxicity data requirements by the
Environmental Protection Agency, USA. Some American companies have
recently taken advantage of this situation and have been able to bring essential-oil-based pesticides to market. Mycotech Corporation produces
CinnamiteTM, an aphidicide/miticide/fungicide for glasshouse and horticultural crops, and ValeroTM, a fungicide for use in grapes, berry crops, citrus
and nuts. Both products are based on cinnamon oil, with cinnamaldehyde as
the active ingredient. EcoSMART Technologies are aiming to become a world
leader in essential-oil-based pesticides (Shaaya and Kostjukovsky, 1998).
Several essential oil constituents are already in use as an alternative to conventional insecticides. For example, d-limonene is an active ingredient of
commercially available ea shampoos, pulegone and citronellal are used as
mosquito repellents, and 1,8-cineole is the structural base of the herbicide
cinmethylin (Duke et al., 2000). Many commercial products such as Buzz
Away (containing oils of citronella, cedarwood, eucalyptus and lemongrass),
Green Ban (containing oils of citronella, cajuput, lavender, safrole from
sassafrass, peppermint and bergaptene from bergamot oil) and Sin-So-Soft
(containing various oils) are in use as insect repellents (Chou et al., 1997).
A PushPull or stimulodeterrent diversionary strategy has been developed in South Africa for minimizing damage due to maize stem borer insects
(Cook et al., 2006). This strategy involves the selection of plant species
employed as trap crops to attract stem borer insects away from maize crops,
or some plant species are used as intercrops to repel insects. The trap and
repellent plants contain some semio-chemicals which attract or repel the
insect. Pennisetum purpureum and Sorghum vulgare attract the stem borer
insect, while Milinis minutiflra, Desmodium uncinatum and D. intorium are the
repellent plants. The PushPull strategy is also employed in the control of
Heliothis sp. in cotton elds (Pyke et al., 1987). The PushPull strategy exploiting the chemical ecology of plants would prove an interesting, indigenous
and readily available concept in the management of insect population in eld
crops. Plant owers such as marigolds and certain types of vegetables can
help to control pests in or around the main crop, which is sometimes called
companion planting (Kuepper and Dodson, 2001).
Some of the essential oils and their components show chemosterilant
activity making the insect pests sterile. The compound -asarone extracted
from rhizomes of Acorus calamus, possesses antigonadial activity causing the
complete inhibition of ovarian development of different insects (Varma and
14
N.K. Dubey et al.
Dubey, 1999) (Fig. 9.1). The products showing chemosterilant activity are
highly required in integrated pest management programmes to limit the
chances of physiological (resistant) race development by insects.
Some of the essential oils have been found useful against those species of
pests that are resistant towards synthetic pesticides. These essential oils are a
complex mixture of components including minor constituents, in contrast to
synthetic pesticides based on single products, and they act synergistically
within the plant as a defence strategy. Hence, it is likely that they are more
durable towards pests evolving resistance (Feng and Isman, 1995). Due to
their largely environmentally friendly nature, they can be efficiently used for
pest management in urban areas, homes and other sensitive areas such as
schools, restaurants and hospitals (Isman, 2006).
Octopamine (a biogenic amine found in insects) has a broad spectrum of
biological roles in insects, acting as a neurotransmitter, neurohormone and
circulating neurohormoneneuromodulator (Evans, 1980; Hollingworth et al.,
1984). Octopamine exerts its effects through interacting with at least two
classes of receptors which, on the basis of pharmacological criteria, have been
designated octopamine-1 and octopamine-2 (Evans, 1980). Interrupting the
function of octopamine results in a total breakdown of the nervous system in
insects. Therefore, the octopaminergic system of insects represents a biorational target for insect control. The lack of octopamine receptors in vertebrates probably accounts for the profound selectivity of certain essential oils
as insecticides. A number of essential oil compounds have been demonstrated to act on the octopaminergic system of insects (Enan et al., 1998).
Mode-of-action studies on monoterpenoids also indicate the inhibition of
acetylcholinesterase enzyme activity as the major site of action in insects
(Rajendran and Sriranjini, 2008).
Encapsulation is the suitable technology for the formulation of essentialoil-based pesticides. The method reduces the loss of the active agents and
offers the possibility of a controlled release of oil vapours (Moretti et al., 1998).
Essential oils can also be incorporated with polymers into sheets. Attractant
adhesive films with essential oils have been prepared to control insects in
agriculture and horticulture (Klerks Plastic Industries B.V., 1990).
Many of the commercial products that include essential oils are on the
Generally Recognised as Safe (GRAS) list fully approved by the Food and
Drug Administration (FDA) and Environmental Protection Agency (EPA) in
USA for food and beverage consumption (Burt, 2004).
CH3
O
H3C
CH3
O
CH3
Fig. 1.9.
-Asarone.
15
1.7 Higher Plant Products as Inhibitors of Aflatoxin Secretions

The post-harvest colonization of various moulds on food commodities
reduces their shelf life and market value, as well as rendering them unt for
human consumption because of the secretion of different types of mycotoxins, including aatoxin, that cause undesirable effects on human health.
Hence, both qualitative as well as quantitative losses of food commodities have been reported due to fungal infestations. For the complete protection of stored food commodities from fungal biodeterioration, a fungitoxicant
should be inhibitory to fungal growth as well as aatoxin secretion. Reducing aatoxin residue levels in food or feed can confer international trade
advantages in developing countries and there may also be long-term benets
for the local population through health improvement (Dichter, 1987). Some
natural products from plants such as allicin from garlic and onion extracts,
clove oil, and black and white pepper have been reported to control fungal
toxins (Ankri and Mirelman, 1999). Leaves of Garcinia indica (Selvi et al.,
2003), Morinda lucida and Azadirachta indica (Bankole, 1997) have been found
effective in controlling aatoxin production in food commodities. Some of
the natural products, such as cinnamon and clove oil (Sinha et al., 1993), phenols
(Singh, 1983), some spices (Hasan and Mahmoud, 1993) and many essential
oils (Razzaghi-Abyaneh et al., 2008) have been reported as effective inhibitors
of fungal growth and aatoxin production. The extracts of several wild and
medicinal plants have also been tested against aatoxin-producing fungi
(Bilgrami et al., 1980). Essential oils from Cymbopogon citratus, Monodora myristica, Ocimum gratissimum, Thymus vulgaris and Zingiber officinale have been
reported for their inhibitory effect on food spoilage and mycotoxin-producing
fungi. Recently, the essential oils of Cinnamomum camphora (Singh et al., 2008a),
Thymus vulgaris (Kumar et al., 2008) and Pelargonium graveolens (Singh et al.,
2008b) have been reported to suppress aatoxin B1 secretion by different toxigenic strains of A. flavus. However, there is little in the literature on the ability
to monitor aatoxin secretions by toxigenic fungal strains on food commodities.
1.8 Conclusion
Sustainable agriculture aims to reduce the incidence of pests and diseases to
such a degree that they do not seriously damage the farmers crop without
upsetting the balance of nature. One of the aims of sustainable agriculture is
to rediscover and develop strategies of which the cost and ecological sideeffects are minimal. The secondary compounds of plants are a vast repository
of compounds with a wide range of biological activities. Unlike compounds
synthesized in the laboratory, secondary compounds from plants are virtually guaranteed to have biological activity and that activity is likely to function in protecting the producing plant from a pathogen, herbivore, or
competitor. Among the variety of natures ecosystem services, natural pest
control is an important aspect. Hence, it is pertinent to explore the pesticidal
activity of plant products.
16
N.K. Dubey et al.
Natural pest controls using botanicals are safer to the user and the
environment because they break down into harmless compounds within
hours or days in the presence of sunlight. They are also very close chemically
to those plants from which they are derived, so they are easily decomposed
by a variety of microbes common in most soils. Because of greater consumer
awareness and negative concerns towards synthetic chemicals, crop protection using botanical pesticides is becoming more popular. There is a wide
scope for the use of plant-based pesticides in the integrated management of
different agricultural pests.
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Plant Products in the Control of

Mycotoxins and Mycotoxigenic
Fungi on Food Commodities
SONIA MARN, VICENTE SANCHIS AND ANTONIO J. RAMOS
Food Technology Department, Lleida University, Lleida, Spain
Abstract
Mycotoxins are naturally occurring secondary metabolites of several toxigenic fungi
that contaminate the whole food chain, from agricultural products, through to human
consumption. Restrictions imposed by the food industries and regulatory agencies on
the use of some synthetic food additives have led to a renewed interest in searching
for alternatives, such as natural antimicrobial compounds, particularly those derived
from plants. This chapter summarizes recent work on the antifungal activity of plant
products and their potential for use as food additives. During the past two decades
many publications have dealt with the inhibition of mycotoxigenic species by natural
plant products. Most of them showed the high efcacy of such products as antifungals. Their nal application to food products is, however, still in its infancy. The
reasons for this are: (i) different origin, varieties and extraction methods of plant
products result in essential oils and oleoresins that are widely varied in composition,
preventing a direct extrapolation of results, unless experiments are carried out using
pure components of these essential oils and oleoresins; (ii) plant products should be
applied in such a way and at a concentration that does not affect sensorial quality of
food products. Most in vitro studies used high concentrations of plant extracts and
direct contact as the screening technique, so the application of these extracts to foods
has not always been successful; and (iii) safety issues should be addressed prior to the
widespread application of such extracts.
2.1 Mycotoxins in Foods

Mycotoxins are naturally occurring secondary metabolites of several toxigenic fungi that contaminate the whole food chain, from agricultural
products such as peanuts and other nuts, fruits and dried fruits, and ultimately are consumed by man. These toxins can be produced in the eld during the growth of the fungus on the crop or later, as a result of substandard
handling or storage. Animal-derived foods such as milk, cheese and meat
can be other sources of mycotoxins, if animals have been given contaminated
(ed. N.K. Dubey)
21
22
S. Marn et al.
feed. The occurrence of mycotoxins may differ from year to year. The FAO
estimates that mycotoxins contaminate 25% of agricultural crops worldwide.
They also have a signicant impact on economics, by causing losses in farm
animals or giving rise to difculties in their management, or by rendering
commodities unacceptable for national or international trade because they
do not conform to existing regulations (EU, Commission Regulation
1881/2006 and 1126/2007, 105/2010 and 165/2010).
The generic term mycotoxicosis covers a variety of toxicities that target
exposed organs of animals and humans. In this regard, several mycotoxins
are potent animal carcinogens and have been classied by the International
Agency for Research in Cancer as potential human carcinogens. The toxic
effects of mycotoxins on human health are acute with a rapid onset and an
obvious toxic or chronic response as characterized by low-dose exposure to
mycotoxins over a long-time period. So, the real impact of the mycotoxins
in human health depends on the type and the amount of the mycotoxin
ingested.
2.2 Natural Preservatives for Control of Mycotoxigenic Fungi

and Mycotoxins in Foods
Synthetic preservatives are nowadays the more effective and widespread way
of chemical control of harmful microorganisms and diseases caused by them.
Both eld fungicides or antifungal food additives are efcient ways to control
fungal growth in foods and raw materials. Antifungal agents are chemicals
that prevent or delay mould growth. However, the presence of chemical residues in foods and labelling of preservatives in food packages are major concerns nowadays; furthermore, increasing fungal resistance has also become a
problem. The interest in the search for natural preservatives and their application in food products appear to be stimulated by modern consumer trends
and concerns about the safety of current food preservatives.
Restrictions imposed by the food industries and regulatory agencies on
the use of some synthetic food additives have led to renewed interest in
searching for alternatives, such as natural antimicrobial compounds, particularly those derived from plants.
2.3 Plant Products as Alternatives for Control of Mycotoxigenic

Fungi and Mycotoxins
Plants are continuously exposed to a wide range of pathogens and, even
though they do not possess an immune system, they have evolved a variety
of defence mechanisms against these pathogens. After the perception of the
primary wound, a signal is transducted locally and systemically through
the activation of different pathways which are integrated in the regulation
of the stress response (Doares et al., 1995). Several plant pathogenesisrelated proteins such as -glucanases, chitinases, chitin-binding proteins,
Control of Mycotoxins and Mycotoxigenic Fungi
23
polygalacturonase-inhibiting proteins and -amylases have been shown to

possess antifungal activity against toxigenic fungi. When attacked by insects
and pathogens, plants produce, together with defence proteins, a wide variety of volatile and non-volatile secondary metabolites, such as phytoalexins,
alkaloids, terpenes, aldehydes, etc.
Much research has been published on the antimicrobial activity of
plant products, including ours, plant extracts, oleoresins and essential
oils. A very interesting option is the use of essential oils as antimicrobial
additives, because they are rich sources of biologically active compounds.
Essential oils are mainly obtained by steam distillation from various plant
sources.
The antimicrobial activity of plant products has been extensively studied
and demonstrated against a number of microorganisms, mostly in vitro rather
than in tests with foods, and usually using a direct-contact antimicrobial
assay. In the direct-contact method, active compounds are brought into contact with the selected microorganisms, and their inhibition is monitored by
means of direct inspection or by measuring a physical property that is directly
related to microorganism growth, such as optical density, impedance or conductance. The solution of active compound may be added to a test tube or to
agar medium, and then microorganisms are inoculated. The disc diffusion
test consists of spiking a sterile disc with the active compound; spiked discs
are then added after inoculation of the medium, after which the inhibition
zones are measured, giving an indication of the antimicrobial strength. For
volatile compounds such as essential oils, the antimicrobial effectiveness in
the vapour phase is of particular interest. This variety of testing techniques
makes it difcult to compare results obtained by different researchers, mainly
because of the difculty in knowing the concentrations of active compounds
applied.
In vitro assays against mycotoxigenic fungi

Many plant extracts have been tested in vitro for antifungal activity against a
wide range of fungi associated with deterioration of food commodities and
herbal drugs.
Studies carried out by Karthikeyan et al. (2007) revealed that aqueous
leaf extract of zimmu (an interspecic hybrid of Allium cepa L. and Allium
sativum L.) exhibited strong antifungal activity against Aspergillus flavus,
Fusarium moniliforme and Alternaria alternata and caused in vitro fungal
growth inhibition of 73.3%, 71.1% and 74.4%, respectively. These moulds
cause mouldy sorghum grains. Essential oil and methanol extract of Satureja
hortensis had strong activity against A. flavus isolated from lemon fruit
(Dikbas et al., 2008).
Extracts of Cynara cardunculus were effective in the control of A. flavus,
A. niger, A. ochraceus, Fusarium tricinctum, Penicillium funiculosum, P. ochrochloron, Trichoderma viride and Alt. alternata (Kukic et al., 2008). Similar results
were obtained with aqueous extract of Adenocalymma sativum in order to
24
S. Marn et al.
control A. flavus, A. niger, A. terreus, F. oxysporum, F. roseum, P. italicum, Cladosporium cladosporioides and Alt. alternata. The minimum inhibitory concentration of extracts against A. flavus and A. niger were superior to those of two
commonly used synthetic fungicides (Shukla et al., 2008).
The essential oil of Amomum subulatum exhibited a fungitoxic spectrum
against a wide range of moulds such as A. niger, A. flavus, A. terreus,
A. fumigatus, Alt. alternata, Cladosporium herbarum, Curvularia lunata, F. oxysporum, Helminthosporium oryzae and Trichoderma viride. Their mycelial growth
was signicantly inhibited at 750 g/ml (Singh et al., 2008a).
An in vitro initial screening of a range of 37 essential oils (including
cinnamon leaf, Cynnamomum zeylanicum; clove, Syzygium aromaticum; lemongrass, Cymbopogon citrates; oregano, Origanum vulgare; and palma rose,
Cymbopogon martinii) on inhibition of mycelial growth of toxigenic strains of
F. verticillioides, F. proliferatum and F. graminearum under different temperatures (2030C) and water activities (aw) (0.950.995) was made. The basic
medium was a 3% maize meal extract agar. The agar medium was modied
with glycerol in order to get the assayed water activity and the essential oils
were incorporated at different concentrations (0, 500, 1000 g/ml). Although
water activity was determinant for the growth of the isolates, in general, the
preservative effects of the oils were not linked to aw. However, a trend to a
higher inhibition by the oils when aw was low was observed. Temperature
had a minor importance in the inhibitory effect of the essential oils (Velluti
et al., 2004a).
Essential oils of both cinnamon and clove were shown to effectively
inhibit growth of both A. flavus and P. islandicum by direct contact, and in the
vapour phase. In the atmosphere generated by these two essential oils eugenol was the major compound, while other essential oils with low levels of
eugenol proved to be ineffective, suggesting that eugenol was the antimicrobial agent (Lpez et al., 2005).
Guynot et al. (2003) proved that the volatile fractions of cinnamon leaf,
clove, bay (Laurus nobilis), lemongrass and thyme (Thymus vulgaris) showed
potential antifungal activity against A. flavus and A. niger.
In vitro assays for the inhibition of mycotoxin production

Species in Aspergillus section Flavi, mainly A. flavus and A. parasiticus are
among the most commonly occurring spoilage fungi. Some strains of these
species have the ability to produce aatoxins. These fungi invade agricultural commodities such as corn, peanuts and cottonseed, and herbal drugs,
the resulting contamination with aatoxin often making these products unt
for consumption. Moreover, these mycotoxins are considered carcinogenic
substances and their presence is a health concern, so their control is one of
the aims in order to obtain safe products. Little attention has been paid, however, to the efcacy of essential oils in inhibiting aatoxin production.
Recently, work has been carried out to study the effect of these products as
anti-aatoxigenic agents.
25
Bluma et al. (2008) carried out a study with several plant extracts. A total
of 96 extracts from 41 Argentinian plant species were screened against four
strains of Aspergillus section Flavi. Studies on the percentage of germination,
germ-tube elongation rate, growth rate, and aatoxin B1 (AFB1) accumulation were carried out. Clove, mountain thyme (Hedeoma multiflora) and
poleo (penny royal; Lippia turbinate var. integrifolia) essential oils showed the
most antifungal effect in all growth parameters analysed as well as AFB1
accumulation.
Essential oils from Pelargonium graveolens (Singh et al., 2008b) and
Artabotrys odoratissimus (Srivastava et al., 2009) exhibited fungitoxicity
against toxigenic strains of A. flavus at 0.75 g/l and 750 l/l, respectively. The
oils of P. graveolens and Aelagonium odoratissimus showed excellent antiaatoxigenic efcacy as they completely inhibited AFB1 production even at
0.50 g/l and 750 l/l, respectively.
In addition, an in vitro study with aqueous extracts of neem (Azadirachta
indica) leaves on A. flavus and A. parasiticus has shown that the extracts fail to
inhibit the vegetative growth of these moulds, while aatoxin biosynthesis
was essentially blocked in vitro (A. flavus 100% and A. parasiticus more than
95%, using extract concentrations at 10% v/v). AFB1 synthesis was also
inhibited at low extract concentrations of Allium sativum in a semi-synthetic
medium (Shukla et al., 2008).
The essential oil of Cinnamomum camphora was effective against other
toxigenic A. flavus isolates detected in medicinal plants. Srivastava et al.
(2008) evaluated other essential oils. In this work, the growth of a toxigenic
strain of A. flavus decreased progressively with increasing concentrations
of essential oils from leaves of C. camphora and the rhizome of Alpinia
galanga incorporated into SMKY medium. Both oils showed complete inhibition of growth of the toxigenic strain of A. flavus at 1000 mg/l. The oils
signicantly arrested AFB1 production by A. flavus. The oil of C. camphora
completely blocked AFB1 production at 750 mg/l, whereas that of Alpina
galanga showed complete inhibition at 500 mg/l only. The combination of
C. camphora and Alpina galanga oils showed more efcacy than the individual oils, showing complete inhibition of AFB1 production even at 250 mg/l.
The major components of C. camphora oil, as determined using GCMS,
were fenchone (34.82%), camphene (23.77%), -thujene (17.45%), l-limolene
(7.54%) and cis-p-menthane (5.81%). In case of Alpina galanga oil, bicyclo[4.2.0]oct-1-ene, 7-exo-ethenyl (58.46%), trans-caryophyllene (7.05%),
-pinene (14.94%) with camphene (2.15%), germacrene (1.78%) and citronellyl acetate (1.41%) were recorded as major components. In this study, the
oils showed anti-aatoxigenic properties at concentrations lower than their
fungitoxic concentration. Thus, the inhibition of fungal mycelia by these
oils may be through a mode other than the aatoxin inhibition. The difference in antifungal and aatoxin inhibition efcacy of essential oils may be
attributed to the oil composition. The components of the oils may be acting
by different modes of action for antifungal activity and aatoxin inhibition.
The interesting nding of this study is the better efcacy of the oil combination of C. camphora and Alpina galanga in controlling the mycelial growth as
26
S. Marn et al.
well as aatoxin production at a concentration lower than with the individual oils (Srivastava et al., 2008).
The results obtained by Sandosskumar et al. (2007) in in vitro experiments
conrmed the antifungal activity of the zimmu extract against toxigenic
strains of A. flavus. In addition, when the aatoxigenic strains were grown in
medium containing zimmu extract the production of AFB1 was completely
inhibited, even at a concentration of 0.5%. In addition, when AFB1 was incubated with this extract a complete degradation of the toxin was observed
5 days after incubation. It is possible that the reduction in AFB1 content may
be due to detoxication or catabolism of AFB1 by root exudates of zimmu.
Molyneux et al. (2007) suggested the hypothesis that aatoxin biosynthesis
is stimulated by oxidative stress on the fungus and the compounds capable
of relieving oxidative stress should therefore suppress or eliminate aatoxin
biosynthesis.
Srivastava et al. (2008) concluded that, in general, the inhibitory action of
natural products on fungal cells involves cytoplasm granulation, cytoplasmic
membrane rupture and inactivation and/or inhibition of synthesis of intracellular enzymes. These actions can occur in an isolated or in a concomitant
manner and culminate with mycelium germination inhibition. Phenolic compounds in the essential oils have been mostly reported to be responsible for
their biological properties; however, some non-phenolic constituents of oils
are more effective. The aldehyde group is also believed to be responsible for
antimicrobial activity. Among the alcohols, longer chain (C6C10) molecules
in the oils have been reported to be more effective. Such compounds present
in the oils may be held responsible for such biological activities.
In addition, the therapeutic use of essential oils and their combinations
comprising more than one fungitoxic ingredient may also provide a solution
for the rapid development of fungal resistance which is currently noticed in
cases of different prevalent antifungal therapeutics. Results obtained by other
researchers (Sidhu et al., 2009) conrmed the synergistic effect of plant
extracts. So, the combination of botanicals can be used for control of fungal
growth and aatoxin production.
Another mycotoxigenic mould is P. expansum, which is mainly responsible for decay in apples and pears kept in cold storage rooms. In addition, it is
regarded as the major producer of the mycotoxin patulin. When apples
invaded by P. expansum are used in making apple products (e.g. fruit juices),
these products will probably be contaminated with patulin. Its presence is a
health concern.
Neem leaf extracts inhibited patulin production at concentrations higher
than 12.5 mg/ml, reaching a 96% inhibition at 50 mg/ml of neem extract.
Patulin concentrations were reduced by neem extracts in cultures whose growth
was not inhibited. So, the inhibition of patulin production does not appear to
be simply a function of mycelial weight reduction (Mossini et al., 2004).
The aldehydes hexanal, trans-2-hexenal, citral, trans-cinnamaldehyde
and p-anisaldehide, the phenols carvacrol and eugenol, and the ketones
2-nonanone and ()-carvone were screened for their ability to control
P. expansum conidia germination and mycelial growth (Neri et al., 2006).
27
The in vitro spore germination and mycelial growth assay showed a consistent
fungicidal activity by trans-2-hexenal, whereas ()-carvone, p-anisaldehyde,
eugenol and 2-nonanone exhibited a progressively lower inhibition. The
aldehyde trans-2-hexenal was the best inhibitor of conidial germination with
a minimum inhibitory concentration (MIC) of 24.6 l/l, while carvacrol was
the best inhibitor of mycelial growth with a MIC of 24.6 l/l. Other in vitro
experiments carried out with this mould by Venturini et al. (2002) determined
that thymol and citral were the essential oil components that showed the
greatest inhibitory effects.
2.4 Research in Antimicrobial Plant Products Applied to Foods

Biologicals, because of their natural origin, are biodegradable and they do
not leave toxic residues or by-products to contaminate the environment.
Spices and herbs have been used for thousands of centuries by many cultures
to enhance the avour and aroma of foods. However, an increasing number
of researchers have demonstrated that commonly used herbs and spices,
such as garlic, clove, cinnamon, thyme, oregano, allspice, bay leaves, mustard
and rosemary, possess antimicrobial properties. The activity was mainly due
to the presence of essential oils (Table 2.1). Essential oils and their constituents
have been used extensively as avour ingredients in a wide variety of foods,
beverages and confectionery products. Many such products are classied as
Generally Recognized As Safe.
Cereals
Cereals are among food commodities that are mostly prone to bear mycotoxins.
Fusarium mycotoxins (including fumonisins, zearalenone and trichothecenes,
among others) and ochratoxin A are mainly found in cereals. Maize, in particular, is a suitable substrate for aatoxin accumulation. Therefore, plant
extracts, essential oils, or their puried components have been assayed for
their efcacy controlling fungi when applied directly to cereals. Together
with the effect of these compounds on mycotoxigenic fungi development
and on mycotoxin production, the phytotoxicity of the oils to the seeds has
been evaluated, mainly calculated as the percentage of germination of the
seeds, their viability or seedling growth. Maize has been the most investigated cereal, followed by wheat, sorghum, rice and others.
Maize
Lemongrass as powder or as essential oil has been one of the most frequently
assayed for its efcacy against mycotoxigenic fungi on maize. Thus, Adegoke
and Odesola (1996) found that the application of the essential oil of lemongrass
to maize could avoid the development of inoculated A. flavus and P. chrysogenum. Authors suggested that the presence of phytochemical components
such as tannins, alkaloids and glycosides, as well as the terpenes found in the
essential oil, could play an important role in this effect.
28
S. Marn et al.
Table 2.1. Most common essential oils investigated in relation to mycotoxigenic moulds
prevention in foodstuffs.
Essential oil
Plant species
Major compounds
References
Thyme
Thymus
vulgaris
Thymol, carvacrol,
linalool
Oregano
Origanum
vulgare
Carvacrol,
thymol
Cinnamon
Cinnamomum
zeylanicum
Eugenol,
cinnamaldehyde,
caryophyllene
Mustard
Allyl
Brassica hirta,
Brassica juncea, isothiocyanate
Brassica nigra,
Brassica rapa
Syzygium
Eugenol,
aromaticum
cariophyllene
Montes-Belmont and Carvajal,

1998; Soliman and Badeaa,
2002
Marn et al., 2003; Velluti et al.,
2003; Lpez et al., 2004;
Marn et al., 2004;
Souza et al., 2007
1998; Soliman and Badeaa, 2002;
Marn et al., 2003; Velluti et al.,
2003; Marn et al., 2004
Nielsen and Rios, 2000;
Mari et al., 2002;
Dhingra et al., 2009
Clove
Lemongrass
Cymbopongon
citratus
Geranial,
neral
Basil
Ocimum
basilicum
Thymol
Neem
Azadirachta
indica
Hexadecanoic acid,
oleic acid

1998; Nielsen and Rios, 2000;
Awuah and Ellis, 2002; Marn
et al., 2003; Velluti et al., 2003;
Marn et al., 2004; Matan et al.,
2006; Bluma and Etcheverry,
2008; Reddy et al., 2009
Adegoke and Odesola, 1996;
Dubey et al., 2000; Marn et al.,
2003; Velluti et al., 2003;
Fandohan et al., 2004; Velluti
et al., 2004b; Marn et al., 2004;
Somda et al., 2007;
Souza et al., 2007
1998; Soliman and Badeaa,
2002; Fandohan et al., 2004;
Atanda et al., 2007
1998; Owolade et al., 2000;
Fandohan et al., 2004;
Somda et al., 2007;
Reddy et al., 2009
Lemongrass essential oil has also been assayed to control the growth and
fumonisin B1 (FB1) production by F. proliferatum (Velluti et al., 2003; Souza et al.,
2007). For example, Marn et al. (2003) found that the essential oil (500 or 1000
g/g) inhibited growth of this fungus on articially inoculated irradiated
grains at 0.995 aw and 20 or 30C, but results where not satisfactory at 0.95 aw.
Inhibition of FB1 production was only effective when it was applied to grains
29
at 0.995 aw and 30C. Results were similar when assayed on naturally contaminated maize inoculated with F. verticillioides and F. proliferatum, showing
that antimycotoxigenic ability of lemongrass only took place at the higher
water activities (Marn et al., 2003). From this study it was suggested that competing mycobiota play an important role in FB1 accumulation and that the
efcacy of essential oils in cereals may be much lower than that observed in
the in vitro experiments using synthetic media. The main components found
in the essential oil used in these studies were geranial and neral, and in much
lower quantities limonene, geranyl acetate, geraniol and methyl heptenone.
Fandohan et al. (2004) also assayed lemongrass essential oil against
F. verticillioides, nding a total inhibition of F. verticillioides growth at a concentration of 8 l/g over 21 days. At 4.8 l/g fumonisin production was not
affected under open storage conditions, but a marked reduction was observed
in closed conditions. Unfortunately, at this dose the oil adversely affected
kernel germination.
When assayed on irradiated maize against a mycotoxigenic strain of
F. graminearum, lemongrass essential oil (500 or 1000 g/g) had an inhibitory
effect on growth rate of grains at 0.995 aw and at 20 or 30C, but at 0.95 aw
only the higher dose was effective. Deoxynivalenol (DON) production was
inhibited at 0.995 aw/30C at both doses, but no signicant effect was
observed for zearalenone (ZEA) control (Velluti et al., 2004b) (DON and ZEA
both being toxins produced by F. graminearum, amongst other Fusarium species). Similarly, several F. graminearum strains assayed on non-sterilized
maize with lemongrass essential oil showed a limited efcacy for DON control (total prevention at 0.995 aw/30C, but no effect at 20C) and a limited
effect for ZEA control (prevention at 0.95 aw /30C) (Marn et al., 2004).
Besides lemongrass, a huge number of essential oils have been tested for
fungal control or mycotoxin prevention on maize. Thus it has been conrmed
that essential oils from seeds of Azadirachta indica (neem tree; 500 and 1000
g/g) and leaves of Morinda lucida (500 g/g), two Nigerian medicinal plants,
completely inhibited A. flavus aatoxin synthesis in inoculated maize grains.
Similarly, essential oils of cinnamon, peppermint (Mentha piperita), basil (Ocimum basilicum), oregano, Teloxys ambrosioides (the avoring herb epazote),
clove and thyme caused a total inhibition of A. flavus development on maize
kernels (Montes-Belmont and Carvajal, 1998). Only around a 50% reduction
was observed with the oils of Eucalyptus globulus (eucalyptus) and Piper
nigrum (black pepper). Some of their constituents, when used at 2% concentration, such as thymol or o-methoxycinnamaldehyde signicantly reduced
A. flavus maize grain contamination. No effect was observed with the essential oils of Allium cepa (onion) and Allium sativum (garlic). However, other
authors have found that Allium extracts at 2.5% have a signicant effect on the
reduction of the incidence of F. proliferatum in maize grains (Souza et al., 2007).
Essential oils of Pimpinella anisum (anise), Pumus boldus (boldus), mountain thyme, clove and poleo were assayed against Aspergillus section Flavi
(A. flavus and A. parasiticus) in sterile maize grain under different water activities (0.9820.90 aw range) (Bluma and Etcheverry, 2008). Five essential oils
were shown to inuence lag phase, growth rate and AFB1 accumulation, in a
30
S. Marn et al.
way that depends on their concentration, substrate water activity and time of
incubation. Only the highest concentration assayed (3000 g/g) showed the
ability to maintain antifungal activity during a 35-day incubation period.
Ethanolic, methanolic and aqueous extracts of leaves, roots, scape and
owers of Agave asperrima (maguey cenizo) and Agave striate (espadin) have
been tested for their capacity to inhibit growth and aatoxin production by
A. flavus and A. parasiticus on maize. Leaves and roots showed no inhibitory
effect, and methanolic extracts from owers were the most effective. It was
found that 50% of the minimal inhibitory concentration of extracts (approximately 20 mg/ml) produced aatoxin reductions higher than 99% in maize
in storage conditions (Snchez et al., 2005).
Natural maize phenolic acids such as trans-cinnamic acid (CA) and
ferulic acid (FA), alone or in combination, have been tested to control A. flavus
and A. parasiticus growth and aatoxin production on maize (Nesci et al.,
2007). A combination of 25 mM CA + 30 mM FA was very effective in the
control of fungal growth and completely inhibited AFB1 production at all
aw assayed (0.990.93 aw range). However, some treatments, as a CAFA
mixture at 10 + 10 mM, could lead to stimulation of an A. parasiticus population,
and stimulation of AFB1 could be possible in some treatments.
Addition of whole or ground dry basil leaves at 50100 mg/g to maize
has shown to be effective in the reduction of aatoxin contamination in maize
stored for 32 days, with reductions in the range of 7594% (Atanda et al.,
2007). The authors suggested that aatoxins could be controlled by co-storing
whole dry sweet basil leaves with aatoxin-susceptible foods in a very
simple manner.
Aqueous extracts of leaves of some indigenous plants from Nigeria, such
as Ocimum gratissimum, Acalypha ciliata, Vernonia amyygdalina, Mangifera
indica and Azadirachta indica, had a signicant inhibitory effect on F. verticillioides development on maize, with Acalypha ciliata being the most effective
(Owolade et al., 2000).
The effect of cinnamon, clove, oregano and palma rose oils (5001000 g/g)
on growth and FB1 accumulation by F. verticillioides and F. proliferatum in
maize grain, at 0.995 and 0.950 aw and at 20 and 30C, have also been evaluated, resulting in different efcacies depending on the treatment conditions
(Marn et al., 2003; Velluti et al., 2003). These essential oils have also been used
to study their effects on ZEA and DON production by F. graminearum in nonsterilized maize grain in the same initial conditions. In this case the efcacy
of essentials oils was found to be poor, clove essential oil being that with a
broader applicability (Marn et al., 2004).
Ocimum vulgare (30 g/g) and Aloysia triphylla (lemon verbena; 45 g/g)
essential oils were evaluated on F. verticillioides FB1 production on maize
grain. The oregano essential oil decreased the production level of FB1, probably because of its content of monoterpenes (such as thymol, menthol and
cinnamaldehyde) that act as antioxidants and inhibitors of toxicogenesis and
sclerotial development. On the other hand, Aloysia triphylla increased the
production of FB1, probably because of the presence of myrcenone, alpha
thujone and isomers of myrcenone in the oil, compounds that showed
31
oxidant properties that increase lipid peroxidation and, consequently, fumonisin production (Lpez et al., 2004).
Essential oils of O. basilicum and O. gratissimum have been demonstrated
to reduce the incidence of F. verticillioides in maize, and totally inhibited fungal growth at concentrations of 6.4 and 4.8 l/g, respectively, although at the
4.8 l/g dose they did not affect fumonisin production. On the other hand,
oil of neem seeds has been shown to accelerate the growth of F. verticillioides
on maize grains (Fandohan et al., 2004).
Wheat
To control A. flavus development on wheat, the essential oils of C. citratus,
O. gratissimum, Zingiber cassumunar and Caesulia axillaris have been assayed.
The oils of Caesulia citratus and Caesulia axillaris showed fungistatic activity,
indicating their in vivo applicability as herbal fumigants (Dubey et al., 2000).
In the same way, vapours of the essential oil of Caesulia axillaris (1300
g/g, v/v) have been shown to control A. flavus, A. niger, A. fumigatus, A.
sulphureus, Rhizopus spp., Mucor spp., Curvularia spp., Penicillium oxalicum
and Absidia spp. during 12 months of storage. The essential oil of Mentha
arvensis (600 g/g, v/v) showed similar results. These oils were also effective
in controlling the insect pests Sitophilus oryzae (Varma and Dubey, 2001). Similarly, the practical applicability of essential oil from leaves of wormseed
(Chenopodium ambrosioides) as a fumigant for protecting stored wheat has
been assayed. A concentration of 100 g/ml applied as a fumigant was able
to control A. flavus, A. niger, A. parasiticus, A. terreus, A. candidus and P.
citrinum during 12 months of storage (Kumar et al., 2007).
The essential oils of thyme and cinnamon (500 g/g), Calendula officinalis
(marigold) (2000 g/g), Mentha viridis (spearmint), basil and Achillea
fragantissima (quyssum) (3000 g/g) completely inhibited A. flavus, A. parasiticus, A. ochraceus and F. moniliforme on wheat grains. Caraway oil (Carum
carvi) was inhibitory at 2000 g/g against A. flavus and A. parasiticus and at
3000 g/g against A. ochraceus and F. moniliforme. Anise oil completely inhibited the four fungi at 500 g/g. Worse results were obtained with essential
oils of chamomile (Matricaria chamomilla) and hazanbul (Achillea millefalium)
(Soliman and Badeaa, 2002).
Resveratrol, an extract of grape skin, has been demonstrated to be effective in controlling the ochratoxigenic fungi P. verrucosum and A. westerdijkiae
on naturally contaminated wheat (Aldred et al., 2008). Total populations of
fungi were signicantly reduced by the presence of this compound, often by
about 13 log colony forming units (CFU), but CFUs of the mycotoxigenic
inoculated fungi were reduced only about 12 logs. In experiments developed
at different water activities (0.9950.80 aw range) and temperatures (1525C)
during a 28-day period of storage, grain treated with resveratrol (200 g/g)
had signicantly less ochratoxin A (OTA) than the untreated controls. OTA
contamination was reduced by >60% in most of the treatment conditions.
Extracts of Argyreia speciosa and Oenothera biennis have also been
assayed. Hexadecanyl p-hydroxycinnamate and scopoletin isolated from
32
S. Marn et al.
roots of A. speciosa have demonstrated effective antifungal activity against

the mycotoxigenic species A. alternata on wheat grains (more than 80%
inhibition at 500 g/g, and 100% at 1000 g/g). Gallic acid from O. biennis
showed activity against F. semitectum at 1000 g/g (96.9% inhibition)
(Shukla et al., 1999).
Sorghum
Essential oils of sweet basil, cassia (Cinnamomum cassia), coriander (Coriandrum
sativum) and bay leaf (Laurus nobilis) were tested for their efcacy to control an
aatoxigenic strain of A. parasiticus on non-sterilized sorghum grains stored at
28C for 7 days (Atanda et al., 2007). Sweet basil seemed to be the best option,
as 5% dosage gave an optimal sorghum protective effect. The use of this oil is
adequate in terms of the possibility of practical application, and is within the
acceptable sensory levels of 15% for food consumption. Good results on fungal growth control were also obtained with the essential oils of cassia, and with
the combination of cassia + sweet basil essential oils. It was also suggested that
aatoxin production could be controlled in sorghum grains by co-storing
whole dry sweet basil leaves with the grains; 100 mg/g of basil leaves have
been shown to reduce the total aatoxin contamination by 90.6%.
The effect of essential oils of lemongrass, eucalyptus and crude oil of
neem has been tested against F. moniliforme, as well as against Colletotrichum
graminicola and Phoma sorgina, on sorghum grains (Somda et al., 2007). Of the
three plant extracts evaluated, lemongrass essential oil was the most potent.
Concentrations of 6 and 8% caused the greatest reduction of seed infection at
levels comparable to synthetic fungicides such as Dithane M-45. More than
50% of F. moniliforme growth was reduced by lemongrass essential oil.
An emulsiable concentrate (EC) of zimmu has been tested under
eld conditions to check its efcacy controlling A. flavus, F. moniliforme and
A. alternata on sorghum grains (Karthikeyan et al., 2007). A leaf extract of zimmu,
formulated to 50 EC, contaning 50% (v/v) zimmu extract, using an organic
solvent (cyclohexanone), an emulsier (Tween-80) and a stabilizer (epichlorohydrin), was prepared. Foliar application of zimmu formulation 50 EC at
3 ml/l (v/v) concentration 60, 75 and 90 days after sowing signicantly
reduced the incidence of grain mould (about 70% reduction) and increased
the grain weight and grain hardness. The plant extract compared favourably
with foliar applications of the fungicide mancozeb (2.5 g/l). A signicant
reduction in the AFB1 content in sorghum was observed when plants were
sprayed with a 50 EC formulation at 0.3% concentration.
Rice
Plant extracts of different parts of Allium cepa, Allium sativum, Azadirachta
indica, Eucalyptus terticolis, Ocimum sanctum, Annona squamosa, Pongamia glaberrima, Curcuma longa and Syzgium aromaticum have been assayed for their
activities against aatoxigenic A. flavus growth and AFB1 production on rice
(Reddy et al., 2009). Among the plant extracts tested, Syzgium aromaticum
(5 g/kg) showed complete inhibition of A. flavus growth and AFB1 production.
33
Curcuma longa, Azadirachta sativum and O. sanctum also effectively inhibited

the A. flavus growth (6578%) and AFB1 production (72.285.7%) at 5 g/kg.
Fruits
Penicillium expansum (blue mould) is the causative agent of the mycotoxin
patulin in apples and pears. Some researchers in this eld have focused on
the use of certain compounds of the essential oils of plants (Table 2.1), instead
of applying the essential oil directly. For example, marked fungicidal action
was obtained with carvacrol against Mucor piriformis at 125 g/ml, while
p-anisaldehyde stopped mycelial growth of P. expansum at 1000 g/ml
(Caccioni and Guizzardi, 1994). Allyl-isothiocyanate (AITC) can be employed
successfully in modied atmosphere packaging or as a gaseous treatment
before storage. Blue mould was controlled by exposing inoculated Conference and Kaiser pears for 24 h to an AITC-enriched atmosphere. At 5 mg/l,
AITC showed a high fungicidal activity on pears inoculated with P. expansum
at 104 conidia/ml, without causing phytotoxic effects. Disease incidence
appeared to be correlated with inoculum concentration and AITC treatment
concentration. Increasing the P. expansum inoculum concentration at a constant AITC concentration resulted in increasing disease incidence. In fruits
inoculated with the higher P. expansum concentration (106 conidia/ml) and
treated with 5 mg/l AITC, the infected wounds were reduced by only 20%
with respect to the control. At such a high inoculum concentration the efcacy of the most active molecules is usually signicantly reduced. The good
results obtained with AITC treatments delayed up to 2448 h after inoculation suggest that this compound has potential for postharvest disease control. The efcacy of AITC in controlling P. expansum in pears could allow it to
be used to control blue mould infection during packing-house fruit handling.
The ability of AITC to control thiabendazole-resistant strains of P. expansum
is particularly useful in this context because the proportion of thiabendazoleresistant strains is usually high. The results of analysis on the skin and pulp
of pears treated with AITC conrmed the extremely low concentration of
AITC residue in fruit, which is unlikely to have any implications for human
health. The use of AITC, produced from puried sinigrin or from Brassica
juncea defatted meal, against P. expansum appears very promising as an
economically viable alternative with a moderately low impact on the
environment (Mari et al., 2002). The potential use of volatile fungicides to
control postharvest diseases requires a detailed examination of their
biological activity and dispersion in fruit tissues, and the development of a
formulation that inhibits growth of pathogens without producing phytotoxic
effects on fruits.
Neri et al. (2006) showed that volatile eugenol at concentrations of
74 and 984 l/l was necessary to inhibit mycelial growth and conidial germination, respectively, in P. expansum. Apples harvested at commercial
maturity were subjected to water treatment, lecithin at 50 mg/ml, eugenolethoxylate (2 mg/ml) and pure eugenol (2 mg/ml) combined with
34
S. Marn et al.
50 mg/ml lecithin. All treatments were tested at 18 and 50C. Fruits were
dipped in the treatment solutions. After 2 min of treatment, fruits were
stored at 2C at normal atmosphere. Signicant disease incidence reductions were observed in fruits treated with eugenol mixed with lecithin at
50C; this combination reduced the incidence of P. expansum by 6090%. The
lecithineugenol formulation did not induce immediate or delayed phytotoxicity at room temperature. Investigation of its vapour phase properties
and its applicability in the storage room will surely facilitate the application of eugenol as a control agent for long periods and may avoid the problems of phytotoxicity induced by some liquid formulations (Amiri et al.,
2008).
Essential oils of Caesulia axillaris and Mentha arvensis were applied at
1500 and 1000 l/l to the storage atmosphere of P. italicum inoculated oranges.
The Caesulia-oil-treated oranges showed an increased storage life of 3 days,
and the Mentha oil-treated oranges showed an increase of 7 days. No visual
symptoms of possible injury caused by the oils were observed on the peel of
the fruits (Varma and Dubey, 2001).
Aspergillus section Nigri (formerly A. niger) is an ubiquitous fungal
contaminant of foodstuffs, such as fruits, vegetables, nuts and spices. Some
species in this section have been recently shown to produce ochratoxin A.
Pepperfruit (Dennetia tripetala) extracts have been tested in tomato puree
against common spoilage fungi, including A. niger. Extracts, as a single hurdle,
failed to inhibit fungal growth when compared to counts before treatment.
Moreover, the use of extracts alone would entail the use of concentrations
that may affect the sensorial properties of the tomato; however, combining
with heat treatment (80C for 1 min) or NaCl addition (10 mg/g) resulted in
effective treatments (Ejechi et al., 1999). Cinnamon essential oil has been
tested against a range of fungi isolated from tomato, including A. niger.
Although in vitro experiments showed promising results in terms of colony
development and fungal sporulation inhibition, when cinnamon oil was
tested as a volatile in the atmosphere of stored tomatoes and peppers no
major effects were observed (Tzortzakis, 2009). Tomatoes and strawberries
exposed to an enriched oil vapour showed improved fruit-quality-related
attributes, conrming the benets observed after exposure to cinnamon oil
vapour at different concentrations. These ndings may have considerable
commercial signicance.
Extracts of Zingiber officinale and Xylopia aetiopica were added (13%) to
A. niger, A. flavus or Rhizopus stolonifer inoculated orange and apple juices.
Although growth was reduced, the extracts either alone or in combination
did not impose enough stress to stop the growth of the fungi (Akpomeyade
and Ejechi, 1999).
The advantage of essential oils is their bioactivity in the vapour phase and
the limitation of aqueous sanitation for several commodities (e.g. strawberries
and grapes) make the essential oils useful as possible fumigants for stored
commodity protection. One limitation of the essential oils is the strong
avour they impart, thus restricting their applicability only to products with
a compatible avour.
35
Nuts
Salicylic acid, thymol, vanillyl acetate, vanillin and cinnamic acid completely
inhibited the germination of fungi contaminating walnut kernels. All ve
compounds showed somewhat similar activity in inhibiting the growth of
the potentially mycotoxigenic A. niger, A. flavus and P. expansum at the concentrations tested (up to 25 mM), with thymol showing the highest activity
(i.e. complete inhibition of growth at 5 mM) (Kim et al., 2006).
Powders from the leaves of O. gratissimum and cloves of Syzgium aromaticum were used as protectants at 3% in combination with various packaging methods to store 3.5 kg groundnut kernel samples (9.3% moisture)
articially inoculated with A. parasiticus. Selected treatments were repeated
with naturally infected kernels. A high level of protection was obtained with
Syzygium powder at 3% concentration using 12% moisture kernels (Awuah
and Ellis, 2002).
Calori-Domingues and Fonseca (1995) found that treatment of unshelled
peanuts with grapefruit seed extract was not efcient in controlling aatoxin
production during storage. Peanuts treated with grapefruit seed extract at
5,000 and 10,000 mg/kg had mean aatoxin contamination in the range
2,75756,334 g/kg and 6885,092 g/kg, respectively, while the control had
3,362108,333 g/kg. Of all of the chemicals tested only propionic acid was
effective in controlling aatoxin production. Treatments were considered
efcient when the aatoxin content (B1 + G1) remained less than 30 g/kg.
Shelled groundnut samples with moisture contents between 7.5 and
10.5% and inoculated with conidia of A. glaucus and A. parasiticus were stored
for 1590 days at 25C, and fumigated with synthetic food grade essential oil
of mustard (Brassica rapa) (100 l/l space). Deterioration of the samples was
assessed by estimating the percentage of kernels colonized by fungi, the
number of CFUs/kernel, and the accumulation of ergosterol and free fatty
acids. The values of these variables increased with the moisture content and
storage period, independent of the fumigation treatment; however, the rate of
increase was signicantly lower in fumigated samples (Dhingra et al., 2009).
2.5 Use of Plant Products in Active Packaging

Active packaging (AP) is an innovative food packaging concept that combines advances in food technology, food safety, and packaging and material
sciences in an effort to better meet consumer demands for fresh-like, safe
products. One specic application is to incorporate essential oil(s) into the
packaging to prevent the growth of microorganisms.
The use of preservative food-packaging lms offers several advantages
compared to the direct addition of preservatives to food products, since the
preservative agents are applied to the packaging material in such a way that
only low levels of the preservatives come into contact with the food. Either
of two approaches can be used to give packaging materials antimicrobial
activities. In preservative-releasing or migrating approaches, preservatives
36
S. Marn et al.
are introduced into their bulk mass, or applied to their surfaces, which subsequently migrate into the food or the headspace surrounding the food. In
non-migrating approaches, compounds are applied to the packaging surfaces
that inhibit target microorganisms when they come into contact with them.
The most widely used materials in food and drink packaging are various
kinds of papers and boards, which are usually wax-coated to improve their
water-resistance and increase the shelf-life of the packaged products. Adding
an active compound to the wax formulation before coating creates an AP
material. The shelf-life of the packaging manufactured using cinnamaldehyde-fortied-cinnamon essential oil was evaluated against A. flavus, and it
was found to retain its total activity over the whole 71-day test period. Finally,
the efcacy of the coatings was tested in trials with two varieties of strawberries. Complete protection was obtained during 7 days storage at 4C, during
which no visible fungal contamination developed and there were no apparent
visible or organoleptic changes in the strawberries (Rodriguez et al., 2007).
Lpez et al. (2007) demonstrated the potential utility of polypropylene and
polyethylene/ethylene vinyl alcohol copolymer lms with incorporated oregano or cinnamaldehyde-fortied-cinnamon essential oils at concentrations of
4% (w/w) as antifungal packaging materials, also against A. flavus. They
maintain their antimicrobial properties for more than 2 months, and their use
in contact with foodstuffs has been demonstrated not to be harmful to consumers health. The main drawback could be the organoleptical alteration of
the packaged food due to the chemicals released by the active package.
AP may be an alternative to modied atmosphere packaging (MAP) or
could complement it. For example, the volatile gas phase of combinations of
cinnamon oil and clove oil showed good potential to inhibit growth of spoilage fungi normally found in intermediate moisture foods when combined
with a modied atmosphere comprising a high concentration of CO2 (40%)
and low concentration of O2 (<0.05%). To prevent the growth of A. flavus,
4000 l of mixed oils was required to be added to the active MAP system and
higher ratios of cinnamon to clove oil were more effective (Matan et al., 2006).
A. flavus was found to be the most resistant microorganism; this is in accordance with other authors who found A. flavus to be the least inhibited by
essential oils in different antimicrobial tests.
The effect of MAP active packaging using volatile essential oils and
oleoresins from spices and herbs were tested against a range of fungi commonly found on bread (P. commune, P. roqueforti, A. flavus and Endomyces fibuliger). Mustard essential oil showed the strongest effect. Cinnamon, garlic
and clove also had high activity, while oregano oleoresin only inhibited
growth weakly. Vanilla (Vanilla planifolia) showed no inhibitory effect towards
the tested microorganisms at the applied concentrations. A. flavus was more
resistant than the other microorganisms. Mustard essential oil was investigated in greater detail. MIC for the active component, AITC, was determined
for the same species and an additional three moulds and one yeast. The minimum inhibitory concentration (MIC) values ranged from 1.8 to 3.5 g/ml
gas phase. Results showed that whether AITC was fungistatic or fungicidal
depended on its concentration, and the concentration of spores. When the
37
gas phase contained at least 3.5 g/ml, AITC was fungicidal to all tested
fungi. Results of sensory evaluation showed that hot-dog bread was more
sensitive to AITC than rye bread. The minimal recognizable concentration of
AITC was 2.4 g/ml gas phase for rye bread and between 1.8 and 3.5 g/ml
gas phase for hot-dog bread. These ndings showed that the required shelflife of rye bread could be achieved by AP with AITC. AP of hot-dog bread,
may nevertheless require the additional effect of other preserving factors to
avoid off-avour formation (Nielsen and Rios, 2000).
Comparisons between the effectiveness of the volatile gas phases and the
liquid phases of essential oils have shown that oil in the liquid phase is more
effective in preventing spoilage than when added via the gas phase. Higher
volumes are required if the essential oils contact only the contaminating
microorganisms in the gas phase. However, advantages of using a volatile
gas phase of essential oil for food products are that it may have a lesser
inuence of the nal taste and aroma of the product and its release may be
regulated more easily.
2.6 Conclusions
During the past decade many publications have dealt with the inhibition of
mycotoxigenic species by natural plant products. Most of them showed a
high efcacy of such products as antifungals. Their nal application to food
products is, however, still in its infancy. Several reasons are involved:
Different origin, varieties and extraction methods of plant products

result in essential oils and oleoresins that are widely varied in their composition. This heterogeneity prevents a direct extrapolation of results,
unless experiments are carried out using pure components of these
essential oils and oleoresins.
Plant products should be applied in such a way and at concentrations
that do not affect grain viability or the sensorial quality of food products.
Most in vitro studies used high concentrations of plant extracts and used
direct contact as the screening technique, so the application of these
extracts to foods was not always successful.
Finally, safety issues should be fully addressed prior to the widespread
application of such plant products.
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Natural Products from Plants:

Commercial Prospects
in Terms of Antimicrobial,
Herbicidal and Bio-stimulatory
Activities in an Integrated Pest
Management System
J.C. PRETORIUS AND E. VAN DER WATT
Department of Soil, Crop and Climate Sciences, University of the Free
State, Bloemfontein, South Africa
Abstract
The use of natural products developed from wild plants is gaining interest and
momentum throughout the world in both developed and developing countries. In
developing countries the use of natural plant extracts is simply the result of the inability of subsistence farmers to afford commercial synthetic pesticides. However, in
developed countries this is largely due to consumer resistance towards synthetic
chemicals, including antimicrobial, herbicidal and bio-stimulatory agents, believed to
be potentially hazardous to the environment and human health. In this chapter an
overview of the latter three pesticide groups is supplied in terms of its current natural product status from an integrated pest management perspective as it is applied in
the agricultural industry. First, some background is provided in order to cover the
history of natural product development in these three pesticide categories. Second, a
short synopsis of screening programmes that identied wild plants containing natural compounds which have the potential to be considered in natural product developing programmes is supplied. Lastly, the outcome of these development programmes
that realized commercialized natural products is covered.
3.1 Introduction
Plant diseases cause large yield losses throughout the world and all important food crops are attacked with disastrous consequences for food security.
In many cases, plant diseases may be successfully controlled with synthetic
fungicides, but this is costly to African peasantry and often has disadvantages and side effects on the ecosystem (De Neergaard, 2001). It is, however,
42

(ed. N.K. Dubey)
Natural Products from Plants
43
an established fact that the use of synthetic chemical pesticides provides

many benets to crop producers. These benets include higher crop yields,
improved crop quality and increased food production for an ever increasing
world population. Despite this, synthetic pesticides may pose some hazards
to the environment, especially when improperly used by farmers in developing countries who lack the technical skill of handling them, and who fail to
adapt to this technology easily. This may result in undesirable residues left in
food, water and the environment, toxicity to humans and animals, contamination of soils and groundwater and may lead to the development of crop
pest populations that are resistant to treatment with agrochemicals.
Moreover, in Africa and the Near East obsolete pesticides have become a
source of great environmental concern. Some stocks are more than 30 years
old and are kept in poor conditions because of inadequate storage facilities
and lack of staff trained in storage management. The Food Agricultural Organization (FAO) estimated that developing countries are holding stocks of
more than 100,000 tonnes of obsolete pesticides, of which 20,000 tonnes are in
Africa. Many of these chemicals are so toxic that a few grams could poison
thousands of people or contaminate a large area. Most of these pesticides
were left over from pesticide donations provided by foreign aid programmes.
In the absence of environmentally sound disposal facilities, stocks are constantly increasing (Alemayehu, 1996). Obsolete pesticide stocks are, therefore, potential time bombs. Leakage, seepage and various accidents related
to pesticides are quite common and widespread. Storage conditions rarely
meet internationally accepted standards. Many pesticide containers deteriorate and leak their contents into the soil, contaminating groundwater and the
environment. Most stores are in the centres of urban areas or close to public
dwellings. According to the World Health Organization (WHO) there are 25
million cases of acute occupational pesticide poisoning in developing
countries each year (Alemayehu, 1996).
As a result of the problems outlined above, farmers in developing countries
and researchers alike are seeking less hazardous and cheaper alternatives to
conventional synthetic pesticides. One such alternative is the use of natural
products from plants to control plant diseases in crops as part of an organic
approach to Integrated Pest Management (IPM) programmes. Justication for
pursuing this alternative can be found in the following statement published a
decade ago by the Environmental Protection Agency (EPA) regarding the
advantages of natural products from plants in the control of plant diseases:
Natural products from plants have a narrow target range and highly-specic
mode of action; show limited eld persistence; have a shorter shelf life and
present no residual threats. They are often used as part of Integrated Pest
Management (IPM) programmes; are generally safer to humans and the
environment than conventional synthetic chemical pesticides and can easily be
adopted by farmers in developing countries who traditionally use plant extracts
for the treatment of human diseases.
(Deer, 1999)
A further reason for exploring the use of plant extracts or natural products as biological pesticides more extensively can be found in the plant
44
itself. Plants have evolved highly specic chemical compounds that

provide defence mechanisms against attack by disease-causing organisms,
including fungal attack, microbial invasion and viral infection (Cowan,
1999). These bioactive substances occur in plants as secondary metabolites, and have provided a rich source of biologically active compounds
that may be used as novel crop-protecting agents (Cox, 1990). In nature
some wild plants have the potential to survive both harsh biotic and abiotic environmental conditions. This has initiated the postulate that such
plants might be utilized as sources for the development of natural products to be applied in agriculture by man as natural herbicides, bactericides, fungicides or products with bio-stimulatory properties in crude or
semi-puried form.
It is estimated that there are more than 250,000 higher plant species on
earth (Cowan, 1999) offering a vast, virtually untapped, reservoir of bioactive chemical compounds with many potential uses, including their application as pharmaceuticals and agrochemicals. It is generally assumed that
natural compounds from plants pose less risk to animals and humans and
are more environmentally friendly than their synthetic counterparts
(Johnson, 2001). As in pharmacology, biochemicals isolated from higher
plants may contribute to the development of natural products for the agricultural industry in three different ways (Cox, 1990): (i) by acting as natural
pesticides in an unmodied state (crude extracts); (ii) by providing the
chemical building blocks necessary to synthesize more complex compounds; and (iii) by introducing new modes of pesticidal action that may
allow the complete synthesis of novel analogues in order to counter the
problem of resistance to currently used synthetic products by bacterial and
fungal pathogens.
However, it is quite common that a natural compound isolated from a
plant may be of great biological interest but may not be sufciently robust for
use (Steglich et al., 1990). Subsequently, a need for the modication of natural
products into synthetic analogues that will give the desired effect may still
exist. One can, for example, isolate a natural compound with promising antimicrobial activity and, by introducing a stable chemical structure with higher
activity synthetically, develop a commercial product (Steglich et al., 1990).
The alternative that is now more vigorously being pursued in organic farming systems is the application of the plant material itself in a natural form.
However, to date and despite statements such as these made almost two
decades ago, the use of natural plant extracts as pesticides to control pathogens in crops, for example, are not widespread while synthetic chemical pesticides still remain the major tool in pest management systems. Nevertheless,
some natural products have been commercialized recently and the use of a
combination of synthetic chemicals and natural products in IPM programmes
will probably become more popular in future.
Another related area of organic farming systems is the potential to apply
natural plant extracts as either plant growth regulators or natural herbicides.
A plant growth regulator is an organic compound, either natural or synthetic,
that modies or controls one or more specic physiological processes within
45
a plant (Salisbury and Ross, 1992). If the compound is produced within the
plant it is called a plant hormone e.g. auxins, gibberellins, cytokines, abscissic
acid and ethylene. Two decades ago, Roberts and Hooley (1988) stated that
the potential exists to apply a plant extract as a foliar spray in order to stimulate growth in crop plants and hence increase yields. According to the authors,
a principal objective of the agricultural and horticultural industries is to
manipulate plant growth and development in such a way that the quantity
or quality of a crop is increased. After the late-1980s an elevated interest
developed in terms of identifying natural plant compounds that possess the
potential to manipulate plant growth and development over a short period,
e.g. a growing season.
From a crop production perspective, the term integrated pest management, in the broad sense of the word, means to apply more than one method
or product in order to control pests that farmers have to deal with on a
regular basis. The term pests, in the broad sense of the word, includes,
among other things, insect and weed pests as well as viral, bacterial and
fungal diseases. However, the term integrated pest management has a different meaning when applied in either conventional cropping systems or
organic agriculture. Pest control in conventional cropping systems is mostly
driven by the large-scale use of many different synthetic chemicals and it
cannot be denied that great success has been achieved in this way. Due to
consumer resistance and pressure generated by the green revolution
towards the use of synthetic chemicals, a shift towards organic agriculture
was inevitable. Restrictions in terms of synthetic chemical application in
organic agriculture have, in turn, forced a shift towards the use of natural or
organic products.
In conventional cropping systems integrated pest management is rather
straightforward and entails the use of any registered product on the market
that a farmer might choose, and this applies for any of the pests mentioned
earlier. The methods of application of these products are in many cases similar, e.g. foliar application, but might include integrated seed, soil and foliar
treatments. IPM in organic agriculture is much more complex and controlled
and may include sowing multiple crops, extended rotation cycles, mulching,
specic soil cultivation methods (Dayan et al., 2009) and the use of organically certied natural products.
Be that as it may, the reduction in the number of synthetic products as a
result of more stringent pesticide registration procedures (Dayan et al., 2009),
such as the Food Quality Protection Act of 1996 in the United States, has
opened the door for the vigorous pursuit of natural products from plants
over the past two decades. In the meantime, many natural compounds from
wild plants have been isolated, puried, identied and patented but, only a
few products are commercially available.
In this chapter attention will be given to the integrated pest management
concept, the rationale for considering natural compounds from plants and
their potential to be applied as agrochemicals in an IPM system in the agricultural industry as well as selected areas where some progress have been
made over the past three decades. In terms of the latter these will include
46
natural compounds with application potential as antimicrobial, herbicidal

and bio-stimulatory agents.
3.2 Rationale for Considering Natural Compounds from Wild

Plants to be Developed as Commercial Products
Wild plants are a valuable source for the development of new natural
products with the potential to be used for disease management in organic
crop production systems (Duke et al., 1995). Widespread public concern for
long-term health and environmental effects of synthetic pesticides, especially
in developed countries, has prompted a renewed effort to search for natural
compounds of both plant and microbial origin that can be applied as alternatives to existing synthetic pesticides (Ushiki et al., 1996). However, only a
small proportion has been investigated for possible use in plant disease
control in agriculture.
World agriculture still encounters huge crop losses annually due to plant
diseases and the situation is more serious for most subsistence farmers in
developing countries that depend on non-conventional disease management
practices, often with doubtful results. In contrast, crop producers in developed countries rely heavily on synthetic fungicides and bactericides to control plant diseases. Despite their efcacy against plant diseases, synthetic
chemicals are considered to cause environmental pollution and are potentially harmful to human health. According to the National Academy of
Sciences (Wilson, 1997), the carcinogenic risk of fungicide residues in food is
more than insecticides and herbicides put together.
The long-term effect of pesticides in contaminating the environment is of
particular concern, together with the fact that frequent application of fungicides has resulted in fungal mutation and, subsequently, new resistant strains
(Khun, 1989). Moreover, consumer resistance towards the use of synthetic
chemicals has escalated, especially in developed countries, supplying a rationale for the application of natural product alternatives in the agricultural
industry (Duke et al., 1995). Currently, in many developed countries, the tendency to shift to organic farming systems has evolved under consumer pressure in an attempt to reduce the risk of pesticide application. As a result,
research on the possible utilization of biological resources and its application
potential in agriculture has become very relevant. A promising approach in
this regard is the use of natural plant products as an alternative to synthetic
chemicals due to their apparently less negative impact on the environment
(Ganesan and Krishnaraju, 1995).
Conceptually and practically the use of natural plant products in
agriculture is not new, but dates back to the time of Democratus (470 bc),
where sprinkling of amurca, an olive residue, was recommended to control
late blight disease (David, 1992). In short, plant extracts have been recognized to solve agricultural problems ever since man took to farming (Pillmoor,
1993). For example, the control of insect pests through the use of natural
plant products, such as Pyrethrum extracted from the Pyrethrum plant,
47
Chrysanthemum cinerariefolium, and neem seed from Azadirachta indica

(Richard, 2000), has a long history in plant protection.
Despite the fact that reports on the practical application of natural plant
extracts towards the control of plant pathogens in modern agriculture are
found less frequently in the literature (Menzies and Blanger, 1996), a
considerable number of reports on natural chemicals that are biologically
active against various plant diseases is currently available. These natural
compounds are usually secondary metabolites and are synthesized in plants
as a result of biotic and abiotic interactions (Waterman and Mole, 1989;
Helmut et al., 1994). By means of bioassay guided screening, a number of
these natural plant compounds, with antimicrobial activity, have been
isolated and progress has also been made towards the identication and
structural elucidation of these bioactive compounds (Grayer and Harborne,
1994). Although extractable secondary metabolites have long been considered
as an important source of pharmaceuticals, the evaluation of their application potential in crop production systems has been largely neglected. A wide
range of activities with both positive and negative effects, including the control of microorganism, plant growth regulation (Adam and Marquardt, 1986),
the induction of plant resistance to various diseases (Daayf et al., 1995;
Schmitt et al., 1996) and promotion of benecial microorganisms in the soil
rhizosphere (Williams, 1992) have been reported. Despite these efforts, the
isolation of plant secondary metabolites has led to very few commercial
successes in the agricultural industry and more specically in crop management practices.
An accelerated search for alternative options to synthetic fungicides,
based on natural products from plants, therefore, seems to be an important
consideration in light of the current restrictions in pesticide use in both developed and developing countries. This especially applies to the search for
environmentally friendly bioactive components with broad-spectrum antimicrobial activity (Benner, 1993). This probably needs serious consideration
in developing countries where yield losses are high as a result of low-input
production systems due to the unaffordability of synthetic fungicides to local
farmers.
According to the Natural Antifungal Crop Protectants Research Agency
(Hall, 2002) spoilage and plant pathogenic fungi are responsible for some
20% loss of the potential global plant production for food and non-food use.
The very large amount of chemical crop protectants used to control these
losses can be detrimental to both the environment and human health.
Therefore research has been initiated to develop and implement non-synthetic
crop protectants using natural antifungal agents (green chemicals) or
antifungal metabolites from plants. These natural crop protectants will be
designed for use on food or non-food crops vulnerable to fungal deterioration (Hall, 2002). From an agronomic perspective, a secondary aim of research
on natural plant products is to cultivate bioactive plants, as alternative
agricultural crops, to serve as sources for the bioactive compounds.
Despite the inherent potential of compounds from plants to be applied as
natural products in agriculture, cognizance has to be taken of problems
48
associated with its use in plant disease control systems that have been
encountered in the past, and that have probably contributed to it not being
regarded as a viable strategy in the agrochemical industry. First, claims have
been made that the efcacy of plant extract compounds are not comparable
to that of synthetic fungicides and lack consistency (Benner, 1993). Second,
natural products have, in some cases, been reported to be phytotoxic to crop
plants (Benner, 1993). Third, some natural compounds are unstable and can
be broken down by UV-light or oxidation before the desired biological effects
have been produced (Seddon and Schmidt, 1999). However, these arguments
cannot be generally accepted as less than 10% of natural plants have been
screened for their application potential and in most cases only towards one
target pathogen (Hamburger and Hostettman, 1991). In light of the vast number of plant species known in the world today, the subsequent chemical
diversity should allow for the identication of desired biologically active
compounds with sufcient stability.
In contrast to the arguments against the use of natural plant compounds,
other arguments towards its potential benecial attributes should be considered. These include the possible reduction in the risk of fungicidal resistance,
and the fact that they are potentially less toxic to humans and animals
(Ganesan and Krishnaraju, 1995). Most importantly, it is envisaged that crude
plant extracts might be more affordable to subsistence farmers as they are
readily available and are probably cheaper to produce. Hence, attempts to
develop plant-derived natural products and the consideration of its application potential in disease management systems in both developed and
developing countries does not seem to be out of line.
In developing countries in particular, the consideration of applying natural plant products in their crude form should be high on the agenda. The
approach has long been used in most traditional farming systems in many
developing countries. Most African farmers possess substantial indigenous
knowledge of insect and pathogen control. Although this knowledge is probably not scientically based, some examples of natural insecticides applied
in Ethiopian subsistence farming systems are worth mentioning. Extracts of
chinaberry (Melia azedarach), pepper tree (Schiunus molle) and endod (Phytolacca dodecandra) are used to control insects in both organized agriculture
and home gardens (Gebre-Amlak and Azerefegne, 1998). Crude extracts of
all three plant species were reported by the authors to be effective against
Busseola fusca (maize stalk borer) larvae. With regard to disease management,
a crude extract of Dolichos kilimandscharicus L. (Bosha) has been used as a
slurry to treat sorghum seed in the control of covered (Sporisorium sorghi;
Ehrenberg) and loose (Sphacelotheca cruenta, Kuhn) kernel smuts in Ethiopia
(Tegegne and Pretorius, 2007). Experimentally, treatment of sorghum seed
with D. kilimandscharicus, in a powder form, provided excellent control of
both pathogens and was as effective as the standard chemical, Thiram.
However, this has been practised on a small and isolated scale. It seems necessary to obtain a more scientic base through additional research in order to
consider an expansion of these practices as well as to consider the economic
potential of this approach.
49
3.3 Secondary Metabolites: Characteristics, Functions and

Possible Applications in Agriculture
Except for primary metabolites such as carbohydrates, proteins, lipids and
nucleic acids, plants also contain a large variety of secondary metabolites.
Secondary metabolites are classied by different authors in different ways
but for the purpose of this prcis, a simple classication into three groups is
supplied: (i) isoprenoid; (ii) aromatic; and (iii) alkaloid components (Stumpf
and Conn, 1981; Dey and Harborne, 1989; Salisbury and Ross, 1992).
Isoprenoid components
This group of compounds is relatively diverse and three terms have been
allocated to it, namely isoprenoids, terpenoids and terpenes. The term
isoprenoid is relatively descriptive in the sense that the common factor that
relates these compounds is the 5C units, called isoprene units (Fig. 3.1).
However, the term terpene is probably used more often.
Under this group, known hormones such as gibberillic acid (GA), abscisic acid (ABA) and brassinosteroids as well as other components such as sterols, carotenoids, rubber and the phytol tail of chlorophyll are classied. All
of these consist of repeating isoprene units. Isoprene units join in different
ways to form components with chain, ring or combined chainring structures (Fig. 3.2).
Terpenes are found abundantly in nature and are, as far as chemical
structure is concerned, a diverse group of secondary metabolites. On the
basis of the number of isoprene units, terpenes are divided into seven classes.
Hemiterpenes consist of one isoprene unit (C5); they are not found in this
form (freely) in nature, but can be found in the form of alcohols or acids
(examples shown in Fig. 3.3).
Fig. 3.1.
An isoprene unit.
Repeating isoprene-units (chain)
Fig. 3.2.
Ring
Ring and chain combined
Different ways in which isoprene units can combine.
50
Monoterpenes, consisting of two isoprene units (C10), form the main

component of essential oils and are therefore economically important due to
their aroma (perfume), e.g. menthol, camphor, geraniol and pinene (Fig. 3.4).
Their application potential in agriculture lies in the fact that specic monoterpenes are growth inhibitors of higher plants (e.g. menthol and pinene) and
therefore have the potential to be developed as natural herbicides.
Sesquiterpenes consist of three isoprene units (C15) (Fig. 3.5). One of the
best known sesquiterpenes is absissic acid (ABA), which has a hormonal
function. Plants showing natural resistance towards drought stress synthesize more ABA than drought-sensitive crops. Theoretically, the external
application of ABA to crops has the potential to manipulate drought resistance in crops. Other sesquiterpenes have medicinal value e.g. as diuretics
and as antimicrobials (e.g. kadinene from juniper berry).
Diterpenes consist of four isoprene units (C20) and are the rarest types of
terpenes in plants. Of the most important in this group is the hormone gibberellic acid (GA; Fig. 3.6). Although natural hormones are used in the horticultural and agricultural industries on a small scale as growth stimulants, it
is expensive and not economically justiable on a large scale.
OH
COOH
Iso-amyl alcohol
Fig. 3.3.
Angelic acid
Hemiterpenes found in the form of alcohols or acids.
OH
O
OH
Menthol
Fig. 3.4.
Camphor
Geraniol
Examples of monoterpenes.
H3C
CH3
CH3
H
OH
O
H
CH3
ABA
Fig. 3.5.
Examples of sesquiterpenes.
COOH
Kadinene
Pinene
51
Triterpenes consist of six isoprene units (C30) and are divided into four
groups, namely common triterpenes, steroids, saponins and cardiac glycosides. Common triterpenes are commonly found in plants as a wax layer on
leaves and on some fruits e.g. limonene (Fig. 3.7).
Most steroids are hydrolysed on carbon 3 and are in fact all sterols. They
play important roles either as hormones in plants (e.g. brassinosteroids) or
vitamin precursors (e.g. 1--25-dihydroxy-vitamin D3 glycoside; Fig. 3.8).
Brassinosteroids (e.g. brassinolide; Fig. 3.9) have growth-stimulating and
yield-increasing properties and are regarded as the secondary metabolites
with great application potential in both the agricultural and horticultural
industries (see below).
OH
O
O
HO
COOH
Fig. 3.6.
Gibberellic acid.
O
O
O
Fig. 3.7.
Limonene.
OH
CH3
OH
HO
O
HO
H
A brassinosteroid (brassinolide)
Fig. 3.8.
Examples of triterpenes.
HO
OH
1--25-dihydroxy-vitamin D3 glycoside
OH
52
Other known sterols are cholesterol that acts as a precursor of other

steroids in plants, and ergosterol that is rare in plants but abundant in fungi
(Fig. 3.10). Usually, if the ergosterol concentration increases in plants, it is an
indication of fungal infection.
Saponins are so-called triterpene-glycosides coupled to a sugar group.
The molecule consists of two parts, namely the glycone (sugar) and aglycone
(triterpene) moieties (Fig. 3.11). Saponins have soap properties, foam in water
and have a bitter taste. They are also known for their medicinal properties in
Asiatic communities.
OH
CH3
OH
HO
O
HO
H
Fig. 3.9.
A typical brassinosteroid (brassinolide).
HO
HO
Cholesterol
Ergosterol
Fig. 3.10. Two known sterols, cholesterol and ergosterol.
Aglycone
OH
H
H
Sugar
HO
O
HO
Fig. 3.11.
A typical saponin.
H
CH2OH
53
Tetraterpenes consist of eight isoprene units (C40). The best known in this
group are the carotenoids, e.g. -carotene (Fig. 3.12). It is known as a colour
pigment supplying colour to owers, but also for its role in protecting
chlorophyll against over exposure to light.
Aromatic components
All compounds in the aromatic group of secondary metabolites contain at
least one aromatic ring (benzene ring; Fig. 3.13) in their structure, of which
one or more hydroxyl groups are substituted. Thousands of aromatic compounds have already been identied in plants. The group can be divided into
two sections, namely non-phenolic and phenolic aromatic compounds.
Non-phenolic aromatic compounds
Although most aromatic compounds contain one or more OH groups in the
benzene ring, the OH groups are absent in non-phenolic aromatic compounds. For the sake of convenience, this group is further divided into two
groups, namely non-phenolic amino acids and hormones, as well as tetrapyrroles. In the former group, the two amino acids phenyl alanine and tryptophan, also precursors of many phenolic compounds, and the hormone auxin
(indole acetic acid or IAA) are best known (Fig. 3.14). As is the case for GA,
auxin is also used as a growth stimulant in the horticultural industry, but on
a small scale.
In the tetrapyrrole group of non-phenolic aromatic compounds, chlorophyll (Fig. 3.15) is best known and consists of four pyrrole rings forming a
porphyrine head attached to a phytol tail. Another example is phytochrome that is a light-sensitive component, allowing plants to distinguish
between different day lengths.
H3C
H
CH3
H
CH3
Fig. 3.12.
CH3
CH3
-carotene.
OH
Fig. 3.13.
A typical aromatic ring (benzene ring).
CH3
H H3C
CH3
H
H3C
CH3
54
COO
COOH
NH3+
COO
NH3+
Phenyl alanine
Tryptophan
Auxin (IAA)
(indole acetic acid)
Fig. 3.14. Non-phenolic aromatic compounds.
H2C=CH
CH3 (H-C=O for chlorophyll b)
H3C
CH2CH3
N
N
Mg
H
N
H3C
H
N
H
CCH2CH2
H
C
HC
O
O =COCH3
H
H
H
CH3
O-CH2-C=C-CH2-CH2-CH2-C-CH2-CH2-CH2-C-CH2-CH2-CH2-C-H
CH3
CH3
CH3
CH3
Chlorophyll a and b
R
OO
N
N
H H
H
OOC
COO
Phytochrome (Pf-form)
Fig. 3.15.
Non-phenolic chlorophyll and phytochrome.
S-Protein
Phytol tail
(C20H39)
55
Phenolic aromatic compounds

Phenols are the most abundant aromatic and naturally occurring components of plants. The phenols are classied in different ways, depending on
authors. We will only briey discuss the simple phenols, phenyl propanoids,
avonoids, tannins and kinones.
Simple phenols are all monomeric (consisting of only one aromatic
ring; Fig. 3.16). However, the most general simple phenols with growthinhibiting allelopathic properties that have the potential to be developed as
natural herbicides are vanillin, vanillic acid (a benzoic acid) and hydrokinone (Fig. 3.17). p-Hydroxy-benzoic acid and vanillic acid are the most common growth-inhibiting benzoic acids involved with allelopathy and are
found in maize, wheat, sorghum and barley.
Phenol is probably the precursor of all other phenolic compounds found
in plants. Hydrokinone, resorsinol and catechol are found in low concentrations in plants and their functions are not fully understood. They are mostly
secreted by insects as a defence mechanism against other insects and animals. Salicylic acid, on the other hand, possesses anaesthetic properties and
is the active ingredient of Aspirin. In plants a number of functions have
been identied for salicylic acid, namely induction of owering as well as the
induction of pathogenesis related (PR) proteins (peroxidase, chitinase and
-1,3-glucanase) that increases a plants resistance to fungal infections. When
secreted by plants, it also has an allelopathic growth inhibiting effect on other
plants in the environment.
Phenyl propanoids are synthesized from the aromatic amino acid phenyl
alanine and contained in their structure is a 3-carbon side chain coupled to a
phenol, e.g. coumaric acid and caffeic acid (Fig. 3.18).
OH
Fig. 3.16.
OH
Hydrokinone
OH
OH
HO
Phenol
OH
OH
OH
Resorsinol
Catechol
COOH
Salicylic acid
Simple phenols.
OH
OCH3
HO
Hydrokinone
Fig. 3.17.
R
R = -COH=vanillin
R = -COOH=vanillic acid
Simple phenols with great potential to be developed as natural products.
56
p-Coumaric acid is the direct precursor of other coumarins such as

umbelliferone and scopoletin but coumarin itself is synthesized from
cinnamic acid (Fig. 3.19).
The potential of phenyl propanoids to be developed into natural products for the agricultural industry is probably underestimated at present. For
example, coumarin, isolated from Mellilotus alba (White Clover; a legume), is
a strong inhibitor of seed germination and has the potential to be developed
as a pre-emergence herbicide. However, depending on the concentration, it
is also known to stimulate IAA activity and therefore growth. Its potential as
bio-stimulant has not been researched to date. Coumarin can also combine
with monosaccharide sugars such as glucose to form coumarin glycosides,
e.g. umbelliferone glycoside (Fig. 3.20).
These glycosides inhibit the activity of the enzyme 6-phosphogluconate
dehydrogenase, the regulatory enzyme of the oxidative pentose phosphate
(OPP)-pathway, and therefore possess excellent potential to be developed as
natural herbicides. Umbelliferone also has an inhibiting effect on the growth
OH
R
COOH
p-Coumaric acid (R=H)
Caffeic acid (R=OH)
Fig. 3.18. Two well-known phenyl propanoids.
OH
CH3O
HO
HO
Umbelliferone
Fig. 3.19.
O
Scopoletin
Coumarin
O
Cinnamic acid
Phenyl propanoids synthesized from the precursor, p-coumaric acid.
glycosyl
Fig. 3.20.
Umbelliferone glycoside.
57
of certain seedlings and shows potential as an herbicide. Scopoletin has a

stimulating effect on seed germination but an inhibiting effect on seedling
growth, indicating a potential as pre-emergence herbicide.
Flavonoids are the largest group of natural phenolic compounds found
in plants. More than 5000 different avonoids have been described. All avonoids have one thing in common, namely a 15-carbon skeleton structured
in three phenyl rings (A, B and C; Fig. 3.21). Other examples are shown
in Fig. 3.22.
A large variety of biological activities have been associated with avonoids, including antiviral, anti-inammatory, antioxidant, antidiabetic,
anticancer, insect-repelling and free-radical-scavenging activities. What
makes this group extraordinary from an industrial perspective are the excessive quantities found in both edible and non-edible plants as well as their
potential to be developed into natural products. It is estimated that about 2%
of all carbon photosynthesized by plants is converted to avonoids and this
amounts to approximately 1 109 tons per annum. With this enormous
amount of avonoids available, and in light of the numerous bioactivities
identied to date, the time is now ripe to consolidate our knowledge of the
B
A
Fig. 3.21. Typical flavonoid structure.
OH
OH
HO
HO
OH
OH
OH
HO
OH
O
OH
OH
OH
OH
OH
OH
Epicatechin
Gallocatechin
(+)-Catechin
OH
OH
OH
OH
HO
HO
OH O
Hyperoside
Flavonoid examples.
H
O
Fig. 3.22.
OH
galactose
OH
Quercitrin
rhamnose
58
group and to evaluate the application potential of these compounds in the

agricultural industry (Pretorius, 2003 and references therein).
Tannins are generally found in the xylem and bark of vascular plants.
Two types of tannins have been distinguished namely hydrolysable and condensed tannins. When hydrolysable tannins are broken down (hydrolysed),
the well known gallic and digallic acids are formed (Fig. 3.23). Tannins combine with proteins to form insoluble co-polymers and this technique is used
to tan animal skins. However, tannins are toxic to humans and animals and
not suitable for natural product development (Borris, 1996).
Kinones are divided in three groups, benzokinones, naphtakinones and
antrakinones (Fig. 3.24). Most kinones are coloured but do not contribute to
colour in plants as they are found in the inside tissue layers. The best known
example of benzokinone is ubikinone (Fig. 3.25), also known as Co-enzyme
Q, which acts as an electron carrier during the light reaction of
photosynthesis. Juglone, the well known naphtakinone, has a strong
HO
C O
HO
HO
HO
COOH
HO
COOH
HO
HO
HO
Gallic acid
Digallic acid
Fig. 3.23. Two hydrolysable tannins.

O
Benzokinone
Juglone
(a naphtakinone)
Antrakinone
Fig. 3.24. Three groups of kinones.

O
H3C-O
H3C-O
H
O
Ubikinone
Fig. 3.25.
Ubikinone, the best known example of a benzokinone.
59
inhibiting effect on seed germination and seedling growth. Antrakinone is

known as an insect repellent.
Alkaloids
More than 3000 alkaloids have been isolated from plants. The best known
alkaloids are morphine (isolated from the poppy ower), nicotine (tobacco)
and caffeine (coffee; Fig. 3.26).
Alkaloids are best known for their medicinal properties but also for their
toxicity. Their functions in plants are not well known but are possibly linked
to the natural resistance of plants against insects and pathogens. Alkaloids
are also known to inhibit seed germination and seedling growth of certain
plants. Because of their toxicity, chances are slim that alkaloids will be
considered in natural product development programmes.
3.4 Antimicrobial Properties of Plant Extracts

Plants are under constant attack from various microorganisms and fungi.
Their survival is testamentary to their ability to defend themselves chemically against these attacks by producing a myriad of secondary metabolites
with antimicrobial properties (Cowan, 1999; Richard, 2001). Therefore, in
addition to other functions, one of the most important functions of secondary
metabolites in plants is antimicrobial activity against bacteria, fungi and
viruses as well as acting as deterrents towards insects and predators
(Lazarides, 1998; Minorsky, 2001). Failure of microorganisms to colonize wild
plants has often been attributed to the presence of these inhibitory compounds within challenged tissues. However, monoculture crops have lost
their ability to defend themselves against biotic stressors to a large extent.
Callow (1983) classied antimicrobial compounds (secondary metabolites) isolated from plants into two categories: (i) constitutive compounds,
which are present in healthy plants; and (ii) induced compounds synthesized
from remote precursors following infection. The term constitutive includes
compounds that are released from inactive precursors following tissue
O
HO
H3C
NCH3
O
CH3
N
CH3
HO
Fig. 3.26.
N
N
Morphine
Examples of alkaloids.
CH3
Nicotine
Caffeine
60
damage, for example the release of toxic hydrogen cyanide from cyanogenic
glycosides. Induced compounds include the types of secondary metabolites
that are low molecular weight antimicrobial compounds and are synthesized
by and accumulate in plants that have been exposed to microorganisms
(e.g. phytoalexins).
Since plants have evolved highly elaborate chemical defences against
attack, these have provided a rich source of biologically active compounds
that may be used as novel crop-protecting agents. It follows, therefore, that
secondary metabolites with antimicrobial activity puried and isolated from
plant extracts, possess the potential to be developed into natural products.
As a result, many organizations and institutions in different countries of the
world are currently concentrating on natural product research. For example,
the aim of the Agrochemical Discovery and Development Program of the
National Centre for Natural Products Research in the USA is to identify lead
compounds for the development of environmentally benign and toxicologically safe pest management agents (Borris, 1996). This programme is done in
collaboration with scientists in the Natural Products Utilization Research
Unit of the United States Development Agency (USDA) agricultural research
service. Emphasis is on the discovery and development of compounds that
are useful in the control of diseases affecting small niche crops. The research
centre is devoted to improving agricultural productivity through the discovery, development and commercialization of agrochemicals derived from
natural plant compounds (Johnson, 2001).
Moreover, an increasing number of scientists have become involved in
intensive plant screening programmes for bioactivity and have contributed
to the identication of plant species with the potential to be included in
programmes for the development of natural products for the agricultural
industry. It is therefore not surprising that a signicant amount of evidence
over the past three decades has demonstrated that plant extracts are active
against plant pathogenic fungi (Lawson et al., 1998), soil-borne fungi (Awuah,
1994; Bianchi et al., 1997), bacteria (Leksomboon et al., 2001) and nematodes
(Oka et al., 2000). In recent years research interest has turned towards isolating, purifying and identifying these active compounds that have application
potential in plant disease control.
According to Grayer and Harborne (1994), secondary metabolites with
antimicrobial properties include terpenoids (e.g. iridoids, sesquiterpenoids
and saponins), nitrogen- and/or sulphur-containing compounds (e.g. alkaloids, amines and amides), aliphatics (especially long-chain alkanes and fatty
acids) and aromatics (e.g. phenolics, avonoids, bi-benzyls, xanthones and
benzo-quinones). Most of these compounds have been studied for their antifungal and antibacterial activities and potential usefulness against plant
pathogens in vitro (Verpoorte, 1998; Baldwin, 1999; Paul et al., 2000) and some
also in vivo. Many aromatic compounds, including simple and alkylated
phenols, phenolic acids, phenylpropanoids, coumarins, avonoids, isoavonoids, quinones and xanthones, have been reported to show notable
antifungal activities (Grayer and Harborne, 1994). For example, Bae et al.
(1997) isolated avonol diglycoside from leaves of Phytolacca americana L.
61
which exhibited signicant antifungal activity against Botrytis cinerea, Botryosphaeria dothidea and Colletotrichum gloeosporioides (Glomerella cingulata).
Flavonoid classes most often associated with antifungal activity are avanones, avonols, certain biavones, chalcones and dihydrochalcones. For
example, three glycosides isolated from Terminalia alata (T. elliptica) roots
showed antifungal activity against the plant pathogen Aspergillus niger and the
human pathogen Candida albicans at extremely low concentrations of between
25 and 32 ppm (Srivastava et al., 2001). The three new glycosides identied
were: compound 1, 3,3-di-O-methylellagic acid 4-O--d-glucopyranosyl-(1,4)-d-glucopyranosyl-(1,2)--l-arabinopyranoside; compound 2, 5,7,2-tri-Omethyl-avanone 4-O--l-rhamnopyranosyl-(1,4)--d-glucopyranoside and
compound 3, 2-,3-,19-,23-tetrahydroxyolean-12-en-28-oic acid 3-O--d-galacto-pyranosyl-(1,3)--d-glucopyrano-side-28-O--d-glucopyrano-side (Fig.
3.27). Compound 1 was a glycoside of an ellagic acid, whereas compounds 2
and 3 were a avanone glycoside and a triterpene saponin, respectively.
Saponins are one of several groups of compounds that originate from triterpenoids and show a wide range of biological activities. For instance, fungicidal triterpenoid saponins, which also have molluscidal activity, have been
isolated from roots of Dolichos kilimandscharicus (Marston et al., 1988). The
saponins from Dolichos were identied as the 3-O--d-glucopyranosides of
hederagenin, bayogenin and medicagenic acid (Marston et al., 1988). Saponins
from Mimusops elengi and M. littoralis seeds were also reported to be active
against Phytophthora palmivora and Colletotrichum capsici (Johri et al., 1994).
The anti-infective potency of extracts from plants emphasizes the vast
potential for natural compounds to be developed into commercial products
(Duke, 1990). Although many of these plant compounds have been applied
OCH3
O
O
HO
OCH3
H3CO
OR
OR
H3CO
H3CO
O
Compound 1
O
Compound 2
HO
COOR
HO
RO
HOCH2
Compound 3
Fig. 3.27. Three new glycosides with antifungal properties isolated and identified from
Terminalia alata (redrawn after Srivastava et al., 2001). See text for names of compounds.
62
in the pharmaceutical industry (Naseby et al., 2001) and folk medicine

(Duncan et al., 1999), their potential for plant disease management has not
yet been fully realized. Crop production still depends heavily on syntheticbased products (Philip et al., 1995) despite consumer resistance towards their
possible residual effects. For this reason the switch to organic farming,
including the use of natural products in disease management systems, has
become a priority (Benner, 1993; Michael, 1999). The next section provides an
overview of selected plants containing compounds with antimicrobial properties, identied during the past two decades, as well as the current status of
natural products that have recently been commercialized.
Plants with antifungal properties

Two decades ago Naidu (1988) assayed young and mature leaf extracts of
Codiaeam variegatum for antifungal activity. All extracts inhibited Alternaria
alternata and Fusarium oxysporum in vitro, with the young leaves being more
active against A. alternata and the old leaves more active against F. oxysporum.
The active secondary metabolites from the leaves extracts were identied as
phenolic compounds by chromatographic analysis. Phytochemical screening
of the leaves also revealed other metabolites that may be responsible for
antifungal activity.
In another study in the same year Bandara et al. (1988) reported that
steam distillates of the leaves of Croton aromaticus and C. lacciferus and root
extracts of C. officinalis inhibited mycelial growth of Cladosporium cladosporioides in vitro. Root extracts of C. lacciferus were moderately active while those
of C. aromaticus were inactive. Of the six compounds isolated from root
extracts showing antifungal activity, only 2,6-dimethoxybenzoquinone
obtained from the chloroform extract of C. lacciferus was signicantly active.
Comparatively small quantities of this compound were required to inhibit
growth of the pathogens Botryodiplodia theobromae and Colletotrichum
gloeosporioides (Glomerella cingulata).
Also in the same year Gonzalez et al. (1988) extracted Alnus acuminata
nodules with either 5% NaOH or water while constituents were separated by
silica gel column chromatography. The following compounds were isolated:
xylose, ribose, an aromatic carboxylic acid, a fatty acid, a phenolic biarylheptanoid and a avonoid glycoside. The avonoid glycoside was found to
inhibit the growth of Fusarium oxysporum and Pythium species.
In the 1990s a number of contributions by natural product researchers
were published. Only a few are mentioned here. Ajoene, a secondary metabolite derived from garlic (Allium sativum), was shown to inhibit spore germination of some fungi including Alternaria solani and other Alternaria spp.,
Collectotrichum spp., Fusarium oxysporum and other Fusarium spp. that cause
serious diseases in some important crop plants in India (Singh et al., 1990). In
a study conducted by Hoffmann et al. (1992) a methanol extract of Castela emoryi was active as both a preventative and curative agent against grape downy
mildew caused by Plasmopara viticola. An active secondary metabolite was
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identied as a glycoside, 15-glucopyranosyl-glaucarubolone. Saponins from

Mimusops elengi and M. littoralis seeds and crude extract of Ammi majus were
86100% effective against Phytophthora palmivora in vitro and the saponins
were 100% effective against Colletotrichum capsici (Johri et al., 1994). Encouraging results were obtained in 2 years of eld trials using these products for the
control of pathogens on Piper betle. No phytotoxicity was observed. Bae
et al. (1997) isolated an antifungal secondary metabolite, avonol diglycoside,
from the leaves of Phytolacca Americana L. and identied the compound as
kaempferol-3-O--d-apiofwanosyl-(1,2)--d-lucopyranoside by spectral analyses. The compound exhibited signicant antifungal activity against Botrytis
cinerea, Botryosphaeria dothidea and Colletotrichum gloeosporioides (Glomerella
cingulata).
Earlier reports on the potential of compounds from plants to be considered in natural product research most probably played a role in the elevation
of large-scale screening programmes that followed during the past decade.
For example, Pretorius et al. (2002a) performed a wide search for South
African plant species with fungitoxic properties against plant pathogens of
economic importance in agriculture. For this study, 39 plant species, representing 20 families from the subclasses Rosidae, Asteridae, Commelinidae and
Liliidae, were collected from the Blyde River Canyon Nature Reserve,
Mpumalanga, South Africa. Crude extracts were prepared and bio-assayed,
at equal concentrations, for their antifungal potential by determining the
inhibitory effects on the mycelial growth of seven economically important
plant pathogenic fungi. Statistically, signicant differences between plants
and plant parts were observed as well as the resistance of different fungi to
treatment with different plant extracts. The most signicant broad spectrum
mycelial growth inhibition was obtained with extracts from two species of
the subclass Liliidae, namely Aristea ecklonii and Agapanthus inapertus. The
crude extract of A. ecklonii performed best of all extracts as it totally inhibited
the mycelial growth of all seven of the plant pathogenic test organisms and
outperformed the inhibition by a broad-spectrum synthetic fungicide
(carbendazim/difenoconazole). Crude extracts of A. inapertus showed complete inhibition of four, and strong inhibition of the remaining three, plant
pathogenic fungi.
In the same year Pretorius et al. (2002b) also performed an in vivo study
on the control of black spot (Ascochyta blight) in pea leaves, caused by
Mycosphaerella pinodes, by a crude bulb extract of Eucomis autumnalis. The
fourth internode leaves were removed from 4-week-old pea (cv. Mohanderfer) plants, placed on moist lter paper in Petri dishes and inoculated with an
M. pinodes spore suspension before and after treatment with the extract. The
crude extract prevented M. pinodes spore infection of the leaves when the
leaves were inoculated with spores both before and after treatment with
the extract, conrming complete inhibition of spore germination. The crude
E. autumnalis extract showed no phytotoxic effect on the leaves even at the
highest concentration applied.
Equally promising was the results of a comprehensive study conducted
by Chen et al. (2002) to determine the inhibitory effect of 58 plant extracts on
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spore germination and the effective control of grape downy mildew

(Plasmopara viticola). Among the plant extracts, those of Chloris virgata, Dalbergia hupeana, Pinus massoniana, Paeonia suffruticosa and Robinia pseudoacacia
inhibited spore germination of the pathogen signicantly. An in vivo leaf disc
test showed that the infected leaf discs, treated with these ve plant extracts,
exhibited no disease symptoms. Their effects were the same or better than
that of the traditional fungicide, liquid Bordeaux.
Pandey et al. (2002), similarly, compared the antifungal potential of leaf
extracts from 49 angiosperms, collected in Uttar Pradesh, India, with commercial fungicides, by screening them against Helminthosoporium sativum
(Cochliobolus sativus). The leaf extract of Mangifera indica completely inhibited
the mycelial growth of the test fungus while four plant species, A. sativum,
Azadirachta indica, Lawsonia inermis and Matricaria chamomila (Chamomilla
recutita) showed more than 90% inhibition. On assaying different parts of
Mangifera indica, the leaf and seed extracts were found to possess the highest
activity and, together with a leaf extract of Matricaria chamomile, performed
better than the commercial fungicides.
An approach to screen for plants with antimicrobial activity against plant
pathogens that often results in success is to exploit indigenous knowledge on
medicinal plants and to screen these known plants for likely candidates. The
rationale behind this approach is to screen traditional medicinal plants
known in a specic area for their antimicrobial properties instead of randomly choosing potential candidates from the long list of currently known
owering plants, conifers, ferns or bryophytes. Rajiv et al. (2002) conducted a
study to screen for the most effective extracts out of 15 medicinal plants
against Helminthosporium nodulosum (Cochliobolus nodulosus) causing blight in
nger millet. These included Impatiens balsamina, Solanum nigrum, Tagetes
erecta, A. sativum, A. indica, Datura metel, Emblica officinalis (Phyllanthus
emblica), Eucalyptus citriodora, Euphorbia pulcherrima, Lantana camara, Mentha
arvensis, Mimosa pudica, Nerium indicum (N. oleander), Ocimum sanctum
(O. tenuiflorum) and Ricinus communis. Extracts were sprayed on the potted
nger millet plants at 15, 30, 45, 60 and 75 days after sowing. Crude extracts
of S. nigrum and I. balsamina showed the highest mycelial growth inhibition,
followed by T. erecta. Overall, the crude extract of S. nigrum recorded the best
result in vitro but was found inferior to the I. balsamina extract in in vivo tests
(Rajiv et al., 2002).
Besides large-scale screening programmes, data from quite a number of
smaller projects that included the screening of one or more plants were published during the past decade. In most cases these smaller projects included
the isolation and identication of active compounds involved, even if only
the chemical group level. Although large-scale screening programmes are
important to at least identify plant orders, families, genera or species with
promising potential, the more concerted approach is probably preferable.
Only a few approaches are mentioned here.
In their study of antimicrobial properties of plant extracts, Orlikowski
(2001a) used grapefruit extract (GE; Biosept 33 SL) to control Phytophthora
spp. Amendment of peat with GE at a concentration of 165 g cm3 resulted
65
in a drastic decrease in colony-forming units of the pathogen Phytophthora

cryptogea and suppression of its development in potted gerbera (Gerbera jamesonii) and cypress (Chamaecyparis lawsoniana). About 40 g GE ml1 inhibited
approximately 50% of the mycelial growth, whereas the pathogen did not
develop at all in the presence of GE at a concentration of 1000 g ml1. The
antifungal property of GE against P. cryptogea was attributed to the presence
of the active ingredient 7-geranoxycoumarin in GE (Orlikowski et al.,
2001b).
Kishore et al. (2002) reported on the antimicrobial activity of aqueous leaf
extracts from Lawsonia inermis and Datura metel against Mycosphaerella berkeleyi causing late leaf spot in groundnuts (Arachis hypogaea). Field experiments
were conducted at the International Crops Research Institute for the SemiArid Tropics (ICRISAT), Patancheru, India during 1999 and 2000 using a susceptible groundnut cultivar (TMV2). The D. metel extract continuously
reduced disease progress up to 115 days after sowing while the severity of
late leaf spot at harvest was signicantly less than that of the controls. The
L. inermis extract was slightly less effective, containing disease progress up to
95 days after sowing, but disease severity was also considerably less than
that of the untreated and the positive chlorothalonil treated controls. Pod
yields in plots sprayed with L. inermis and D. metel extracts were 20 and 48%
higher, respectively, than in the control plots.
In the same year the aqueous extracts from bird cherry tree (Padus avium
[Prunus padus]), aspen (Populus tremula), and celandine (Chelidonium majus)
effectively suppressed the germination of Puccinia triticina (Puccinia recondite)
uredospores (Karavaev et al., 2002). Fungitoxic activity of the extracts was
attributed to the high phenolic compound content and high peroxidase
activity in the leaves of these plants.
Curir et al. (2003) investigated the phenol compositions of two cultivars
of carnation (Dianthus caryophyllus) namely Gloriana and Roland, which
were partially and highly resistant, respectively, to Fusarium oxysporum subsp.
dianthi. The aim was to determine if endogenous phenols could have an antifungal effect against the pathogen. Analyses were performed on healthy and
F. oxysporum-inoculated tissues in vitro as well as on plants in vivo. Two benzoic acid derivatives, protocatechuic acid (3,4-dihydroxybenzoic acid) and
vanillic acid (4-hydroxy-3-methoxybenzoic acid), were found within healthy
and inoculated tissues of both cultivars, together with the avonol glycoside
peltatoside (3-[6-O-(-l-arabinopyranosyl)--d-glucopyranosyl] quercetin).
These molecules proved to be only slightly inhibitory towards the pathogen.
2,6-Dimethoxybenzoic acid was detected in small amounts only in the inoculated cultivar Gloriana, while the highly resistant cultivar Roland showed
the presence of the avone datiscetin (3,5,7,2-tetrahydroxyavone). The
latter compound exhibited an appreciable fungitoxic activity against
F. oxysporum subsp. dianthi.
Natural plant extracts have in recent years also received attention as a
signicant and safe resource for the control of soil-borne pathogens due to
the phasing out of methyl bromide, which has played a major role as a soil
fumigant worldwide (Eshel et al., 2000). Methyl bromide, the major fumigant
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used, is scheduled to be phased out, because it was dened by the Montreal

Protocol of 1991 as a chemical that contributes to the depletion of the ozone
layer (Bowers and Locke, 2000). Presently, pre-plant soil fumigation and fungicide applications are used to control wilt diseases. Subsequently, extracts
from a number of plants have been identied as possible alternatives to
methyl bromide against soil-borne pathogens.
Due to environmental and safety concerns associated with pesticides, as
well as the need for a replacement of methyl bromide, Bowers and Locke
(2000) investigated the effect of several formulated plant extracts and essential oils on soil populations of F. oxysporum. Fusarium wilts are some of the
most widespread and destructive diseases of many major ornamental and
horticultural crops (Bowers and Locke, 2000). Treatment of the soil with 10%
aqueous emulsions of the formulated extracts of a chilli pepper extract and
essential oil of mustard mixture, a cassia tree extract and clove oil reduced
populations of Fusarium by 99.9, 96.1 and 97.5%, respectively, 3 days after soil
treatment. The same formulations also suppressed disease development in
the greenhouse and resulted in an 80100% plant stand after 6 weeks. The
observed reductions in the pathogen population in soil and increase in plant
stand in the greenhouse indicated that these natural plant products may play
important roles in future biologically based management strategies for the
control of Fusarium wilt diseases.
Similar results were observed with neem tree (Azadirachta indica) extracts
against soil-borne fungi (Ume et al., 2001). It was shown that extracts from
the leaves and seed kernels possess antifungal activity against Sclerotium rolfsii (Corticium rolfsii). Both leaf and seed extracts showed some effect against
different growth stages of the fungus, but the effects were fungistatic rather
than fungitoxic. The non-polar extracts of the seed kernels were reported to
be more effective than those rich in polar terpenoids such as azadirachtin
and an aqueous leaf extract was also more effective than the kernel-derived
material. Neem is perhaps the most useful traditional medicinal plant in
India and each part of the tree has some medicinal property that has made it
commercially exploitable (Kausik et al., 2002).
Amadioha (2002) evaluated the antifungal activities of different extracts
of neem, both in vitro and in vivo. The oil extract from seeds as well as water
and ethanol leaf extracts of the plant were effective in reducing the radial
mycelial growth of Cochliobolus miyabeanus in culture and in controlling the
spread of brown spot disease in rice. However, the oil extract was found to
be the most effective, followed by the ethanol leaf extract, in inhibiting the
growth of the pathogen in vitro and in controlling the development of the
disease in vivo. The oil and ethanol extracts compared favourably with carbendazim (Bavistin) at 0.1% active ingredient and had the potential to control the brown spot disease of rice in vivo. Additionally, Bohra and Purohit
(2002) studied the effects of the aqueous extracts of 17 plant species collected
from Rajasthan, India on a toxigenic strain of Aspergillus flavus. The neem
extract recorded the highest mycelial growth inhibition of the fungus.
The antimicrobial potential of plant extracts has also been reported
against the highly resistant fungi that cause soil-borne damping-off disease
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in plants. The efcacy of crude, boiled water and acetone extracts of 17 plant
species against Rhizoctonia solani, causing wet root rot disease in chickpeas,
was evaluated under laboratory conditions (Kane et al., 2002). The crude,
boiled water and acetone extracts of A. sativum, the crude and boiled water
extract of Eucalyptus sp., as well as the boiled water and acetone extracts of
Zingiber officinale contributed to 100% inhibition of the mycelial growth of the
pathogen. In the same year Prabha et al. (2002) reported on the antifungal
properties of extracts from Foeniculum vulgare, Coriandrum sativum, Trigonella
foenum-graecum, Anethum graveolens and Cuminum cyminum against three
fungi, including Fusarium oxysporum. All extracts showed a relatively greater
inhibitory effect on F. oxysporum, with stem extracts from A. graveolens exhibiting complete growth suppression, while the mycelial growth of the other
two fungi was also inhibited signicantly.
Om et al. (2001) also reported the effect of essential oils extracted from
Callistemon lanceolatus (Callistemon citrinus), Citrus medica, Eclipta alba, Hyptis
suaveolens and Ocimum canum (O. americanum) against Rhizoctonia solani, the
cause of damping-off disease of tomato and chilli (Capsicum annuum). The
essential oils of Citrus medica, E. alba and O. canum completely inhibited
the growth of the fungus within 24 h. The essential oils of C. lanceolatus and
O. canum controlled the damping-off disease of tomato by 57 and 71%
and that of chilli by 40 and 83%, respectively. The same effect was also
observed by applying crude, boiled water and acetone extracts from
A. sativum, and a boiled water extract of Eucalyptus sp., as well as boiled
water and acetone extracts of Zingiber officinale (Kane et al., 2002).
In some cases plant extracts not only inhibit fungal mycelial growth, but
also spore germination. Chen et al. (2002) reported that Chloris virgata, Dalbergia hupeana, Pinus massoniana, Paeonia suffruticosa and Robinia pseudoacacia
extracts inhibited spore germination of grape downy mildew (Plasmopara
viticola) and the effect was comparable to that of a traditional fungicide, liquid Bordeaux. Other work also demonstrated that leaf extracts of Lawsonia
inermis and Datura metel possess antifungal activity against Mycosphaerella
berkeleyi, causing late leaf spot in groundnuts (Kishore et al., 2002). Particularly, the D. metel extract continuously reduced disease progress up to 115
days after sowing while the severity of late leaf spot at harvest was
signicantly less than that of the controls.
Plant seeds also contain compounds with antimicrobial properties. Seed
extracts of 50 plant species, belonging to different families, were evaluated
for their ability to inhibit the growth of Trichoderma viride in vitro (Bharathimatha et al., 2002). Of the various seed extracts, that of Harpullia cupanioides
(Roxb.), belonging to the family Sapindaceae, displayed very high antifungal
activity. The seed extract of H. cupanioides strongly inhibited the growth of
Rhizoctonia solani, Curvularia lunata (Cochliobolus lunatus), Colletotrichum musae
and Alternaria alternata and retained its antifungal activity even after heating
at 100C for 10 min or autoclaving at 121C for 20 min. Rodriguez and Montilla (2002) reported on the in vitro and in vivo antimicrobial effect of a Citrus
paradisi seed extract (Citrex) on F. oxysporum lycopersici causing tomato wilt.
Five treatments were evaluated: (i) immersion of plant roots in a solution of
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Citrex before transplant; (ii) weekly application to the foliage; (iii) weekly
application to the soil; (iv) weekly application to the foliage and to the soil;
and (v) immersion of plant roots at transplant plus weekly application to the
soil. The control was infested soil without application of the product. Treatments (i) and (iii) reduced wilting by 85%, indicating that it is possible to
control soil-borne pathogens with the C. paradisi seed extract.
Extremely promising, from a natural product development perspective,
is that many plant extracts compare favourably with commercially synthesized fungicides. For example, leaf extracts of Azadirachta indica, Atropa belladonna, Calotropis procera, Ocimum basilicum, Eucalyptus amygdalina, Ailanturs
excelsa (Ailanthus excelsa) and Lantana camara, at different concentrations,
were compared to the fungicides Bavistin (carbendazim), Dithane M-45
(mancozeb), captan, thiram and Topsin M (thiophanate-methyl) at standard
concentrations against F. oxysporum inducing fenugreek wilt under greenhouse conditions. Seeds of fenugreek were separately soaked overnight in
each leaf extract as well as the fungicide solutions (Gupta and Bansal, 2003).
All the leaf extracts, except that of Ailanturs excelsa, significantly inhibited the
mycelial growth of Fusarium compared to the untreated control. Maximum
germination of fenugreek seeds was observed with Bavistin (93.33%), followed by Atropa belladonna (90.66%), Azadirachta indica (87.99%), L. camara
(87.99%), C. procera (85.99%), O. basilicum (85.33%) and E. amygdalina (82.40%).
Dithane M-45 (80.00%), Captan (86.66%), thiram (86.66%) and Topsin M
(86.66%) were on par with the leaf extracts.
An approach adopted by Solunke et al. (2001), following the increasing
availability of data in the literature on plants with antifungal activity, was to
evaluate the potential of plant extracts to be applied in IPM programmes.
The authors conducted a study to manage sclerotium rot (Sclerotium rolfsii
[Athelia rolfsii]) of potato using a commercial fungicide, carbendazim, separately and together with plant extracts. Sensitivity of fungal isolates (SRP-1,
SRP-2, SRP-3 and SRP-4) against carbendazim was determined beforehand.
Subsequently, aqueous extracts of Azadirachta indica, Allium cepa, Glossocardia
bosvallea and Vinca rosea (Catharanthus roseus) were mixed with carbendazim
in solution and used to treat potato slices. Based on the minimum inhibitory
concentration of carbendazim against the four isolates, SRP-4 appeared to be
tolerant while SRP-1 was sensitive. The percentage control efficacy (PCE) of
carbendazim alone and in mixture with plant extracts were tested against the
tolerant isolate, SRP-4. Using carbendazim in combination with plant extracts
increased its PCE. Application of carbendazim along with A. indica and G.
bosvallea recorded a PCE of 100%. Carbendazim with V. rosea and with A. cepa
recorded PCEs of 90.77 and 80.53%, respectively. The results showed that it is
possible to reduce the selection pressure of carbendazim when combined
with plant extracts.
Finally, an aspect that needs special reference is that the antimicrobial
efficacy of plant extracts compare favourably with that of commercial fungicides in controlling plant diseases. In fact, research into antimicrobial activities of plant extracts has offered some potential solution to disease control in
the absence of effective chemical control. For example, few commercial
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fungicides have been effective in inhibiting teliospore germination of Tilletia

indica, the causal agent of Karnal bunt of wheat. This disease is becoming
more widespread, in part, because of the lack of effective chemical control.
Various extracts of native plants from Sonora, Mexico were evaluated to
determine their antifungal activity against T. indica. Dichloromethane and
methanol extracts were incubated with the fungus to measure the inhibition
of mycelial growth. Dichloromethane extracts from Chenopodium ambrosiodes
and Encelia farinosa reduced radial mycelial growth signicantly, but total
inhibition occurred at a relatively high concentration of 500 mg ml1 of the
dichloromethane extract from Larrea tridentata. Teliospores subjected to treatment with the latter plant extract showed no viability when transferred to
fresh culture media, conrming the extracts potential to be applied as
controlling agent for T. indica (Rivera et al., 2001).
Plants with antibacterial properties

Already in the 1990s, the application of natural plant extracts under both
greenhouse (Blaeser and Steiner, 1999; Nikolov et al., 1999; Scholz et al., 1999)
and postharvest (El-Ghaouth et al., 1995; Bhaskara et al., 1998) conditions
against various pathogens conrmed the potential of natural compounds
from plants to be developed into commercial products. Although the number of natural products applied in biological control systems is currently a
relatively small percentage of the total world market, it is predicted to
increase substantially. This implicates the possible decrease of pesticide
application in agriculture in the future. In turn, this approach could create a
signicant market opportunity for alternative products to be applied on a
larger scale in organic crop production systems.
A vast number of reports on large-scale screening programmes to evaluate wild plants for antibacterial activity are available in the literature; we
refer to only a few. The antibacterial and antifungal activities of 38 plants
belonging to 17 families were tested by Ghosh et al. (2000). The solvent
extracts of different morphological parts of these plants were tested against
14 bacterial and 18 fungal strains. Out of these, Alpinia mutica, Cephalandra
indica, Croton bonplandianum, Curcuma amada, Holarrhena antidysenterica, Moringa oleifera and Zingiber spectabile were found to contain antimicrobial properties (Ghosh et al., 2000). Khan and Omoloso (2002) studied the antibacterial
and antifungal activities of methanol extracts of Harpullia petiolaris leaves,
stems, root barks and heartwoods (collected from Papua New Guinea). Antibacterial activities were found to be highest in fractions of root bark, petrol
and dichloromethane fractions of stem bark, the petrol fraction of heartwood
and the butanol fraction of leaves. Antifungal activity was only observed in
the petrol fractions of the root bark and stem heartwood.
Ethanol and water extracts of leaves, owers, shoots, bark and fruits of
30 herbal and woody plant species were tested for in vitro growth inhibition
of Erwinia amylovora by Krupinski and Sobiczewski (2001) using an agar diffusion method. Active extracts were found in 23 species, while in 13 of these
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the active substances were found for the first time. The highest growth
inhibition of this bacterium was recorded for extracts of Aloe arborescens,
Juglans regia, Rhus typhina (R. hirta), Salvia officinalis and Satureja hortensis. In
almost all cases ethanol appeared to be a better solvent of active plant
substances against E. amylovora than water.
A similar approach was followed by Morais et al. (2002) in screening
crude extracts of 45 known medicinal plants against bacterial pathogens of
tomato. The antibacterial activity of crude extracts was tested against Xanthomonas campestris pv. vesicatoria, Ralstonia solanacearum and Clavibacter michiganense subsp. michiganense. Some assays were also performed to verify the
capability of these plant extracts to show antibiosis. Five of the 45 extracts
showed significant activity against the test bacteria, confirming the potential
of using either these extracts or active substances contained in them as natural products under field conditions. In the same year, Devanath et al. (2002)
compared the antibacterial effect of extracts from three medicinal plants
(Psidium guajava, Aloe vera [A. barbadensis] and Datura stramonium) to two
standard fungicides (Streptocycline and Blitox or copper oxychloride) against
Rhizoctonia solanacearum in vitro. The aqueous extract of Aloe vera was most
effective in suppressing the growth of R. solanacearum followed by the extract
of Psidium guajava.
Zeller et al. (2002) reported on the effect of an extract from Hedera helix
against fire blight on pome fruits, caused by Erwinia amylovora. The highest
growth inhibition of this bacterium was recorded for extracts of Aloe arborescens, Juglans regia, Rhus typhina (R. hirta), Salvia officinalis and Satureja hortensis. In almost all cases ethanol appeared to be a better solvent of active plant
substances against E. amylovora than water. Also using the bacterium E. amylovora as a test organism, Jin and Sato (2003) searched for secondary metabolites in aqueous extracts from succulent young shoots of pear (Pyrus).
Aqueous extracts of the tissue of succulent young shoots exhibited strong
antibacterial activity against E. amylovora. The active compound was isolated
from the extract by steam distillation in vacuo, purified through charcoal
powder column chromatography and identified as benzoquinone (2,5-cyclohexadiene-1,4-dione) by NMR spectra, mass spectra and HPLC analysis. In the same year, Pretorius et al. (2002a) demonstrated the broad-spectrum
antibacterial activity of crude extracts from Acacia erioloba, Senna italica and
Buddleja saligna against the plant pathogens Agrobacterium tumefaciens,
Clavibacter michiganense pv. michiganense, Erwinia carotovora pv. carotovora,
Pseudomonas solanacearum and Xanthomonas campestris pv. phaseoli.
Additionally, plant extracts with antibacterial properties can provide an
alternative to certain antibiotics. Zeller et al. (2002) reported on such alternatives to the antibiotic streptomycin for the control of fire blight on pome
fruits, caused by E. amylovora, which is of great economic importance for
German and European fruit producers. An antagonistic preparation,
BIOPRO, showed a control efficacy of up to 60% and the plant extract from
Hedera helix revealed a high efficacy in the field in combination with a low
concentrated copper compound and a metal salt. The control of fire blight in
this manner was comparable to that by the antibiotic streptomycin under
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articial and natural infection conditions. The latter is important in light of

the fact that the use of copper-containing antimicrobials is phasing out in
Europe due to recent legislation, while the use of antibiotics, such as streptomycin, is highly opposed.
Current status of antimicrobial products from plant extracts

Extracts from various plant species appear to have promising potential for
their application as natural products in the agricultural industry against both
plant pathogenic bacteria and fungi and to be integrated in plant disease
management systems. However, to date, the number of commercially developed natural plant products that emerged from screening programmes is
alarmingly low. The natural plant product Milsana, extracted from the giant
knotweed (Reynoutria sacchalinensis), is probably best known (Daayf et al.,
1995). The product has been reported to control powdery mildew, caused by
Sphaerotheca fuliginea, in the long English cucumber under greenhouse conditions, and also showed broad spectrum activity against powdery mildew of
tomato, apple and begonia as well as downy mildew of grapevine and rust
of bean (Daayf et al., 1995). It was concluded by Petsikos et al. (2002) that
Milsana can substantially contribute to the management of cucumber
powdery mildew in organic or integrated farming systems.
A volatile natural product, Carvone, derived from dill and caraway
seed, has been developed to inhibit the growth of storage pathogens and to
suppress sprouting of potatoes in the warehouse (Moezelaar et al., 1999). Carvone is currently marketed as Talent in the Netherlands. Additionally,
Fungastop and Armorex II, two natural products developed in the USA
(Soil Technologies Corp.), are commercially available for the control of
various plant diseases in agriculture.
Dayan et al. (2009) supplied an excellent summary of new natural antimicrobial products that were recently commercialized for use in either conventional or organic agricultural or both industries. To name a few, these include
Bla-S (against rice blast disease in eastern Asia), Kasugamin (against rice
blast and other crop diseases in Japan), Mildiomycin (against powdery
mildews mainly in Japan), Delvolan (against fungal diseases of ornamentals) and Validacin (used for Rhizoctonia spp. control on a variety of crops).
All of these products are fermentation secondary products from Actinomycetes, mostly Streptomyces spp., commercialized for use as agricultural
fungicides in Japan, and to a lesser extent in other parts of the world.
Several plant essential oils are marketed as fungicides for organic farmers (Dayan et al., 2009). These include E-Rase from jojoba (Simmondsia californica) oil, Sporan from rosemary (Rosemarinus officianalis) oil, Promax
from thyme (T. vulgaris) oil, Trilogy from neem (A. indica) oil and GC-3
being a mixture of cottonseed (Gossypium hirsutum) oil and garlic (A. sativum)
extract. According to Dayan et al. (2009), few scientic papers deal with these
products or the actual active components, and their modes of action against
individual plant pathogens are largely unknown.
72
A fairly new approach to microbial disease control is via systemic

acquired resistance (SAR) or, in other words, the activation of natural defence
mechanisms within the plant via elicitors (Dayan et al., 2009). Harpin protein
that induces SAR in plants is sold as Messenger or ProAct. Brassinosteroids that induce the synthesis of PR proteins that resist fungal infection in
crops, are sold as ComCat (Roth et al., 2000). The latter product seems to be
used more often as a bio-stimulatory agent; an aspect that will be dealt with
later in this chapter.
These past successes and the publics current concern over the impact of
synthetic pesticides on the environment ensures a continued, if not an
increased, interest in searching nature for environmentally friendlier pest
management tools (Dayan et al., 2009).
3.5 Natural Bio-herbicides

The herbicide component of all pesticides sold increased from less than 20%
in the 1950s to almost 50% of the market in the 1980s (Jutsum, 1988) and this
has since risen to over 60%. Today, herbicides account for more than half of
the volume of all agricultural pesticides applied in the developed world and
their use has also contributed to the concern of consumers towards the potential health and environmental impact they may have (Dayan et al., 2009).
However, almost three decades ago, McWhorter and Chandler (1982) estimated that weeds contribute to a signicant 12% loss in worldwide crop
production, emphasizing the need to control weeds. These two perspectives,
namely the need to control weeds and the negative attitude of consumers,
have in part fuelled the current elevated interest in the discovery of natural
products to be applied as an environmentally friendly method of weed
control.
However, the complexity of the environment in terms of weed control
and especially the possible side effects of herbicides or biological control
agents on non-targeted plants, calls for a comprehensive study on
control strategies, whether biological, chemical, cultural or a combination of
these in an integrated weed management system. The biological approach to
weed control can be separated into three distinct strategies: conservation,
inundative and classical depending on the setting (e.g. range, row crops,
urban), the extent of the infestation and the biology of the system (McFadyen,
1998; Goeden and Andrs, 1999). According to the authors, conservation
biocontrol refers to situations where a natural biocontrol agent is already
present and is able to control the weed but requires assistance in the form of
cultural practices or integrated management decisions that enable the agent
to thrive. Although conservation strategies are less common in the biological
approach to weed control than in the biocontrol of insects, examples do exist
(Goeden and Andrs, 1999).
Inundative biocontrol involves the release of large numbers of a biological control agent at a time when weed populations are expected to escape
control or exceed a critical economic or competitive threshold. When the
73
agent is already present at a level that does not provide adequate, continuous
control of the target, this strategy is known as augmentation biocontrol
(Goeden and Andrs, 1999). In general, inundative biocontrol relies on the
released organisms themselves to control the target without any expectation
of continued control by future generations of the agent (Elzen and King,
1999). In an inundative strategy the agent may need to be released or applied
several times during a single crop cycle in the event of re-growth or
re-emergence of the target weed. Thus, inundative strategies typically apply
to relatively high-input systems.
Classical biocontrol, sometimes referred to as inoculative biocontrol,
involves the practice of identifying co-evolved natural enemies from the
native range of a target weed species and releasing them into the invaded
range to reduce the presence of the weed to acceptable levels (McFadyen,1998).
In classical biocontrol, the agents are expected to reproduce and proliferate
on the target weed and disseminate throughout its invaded range, reaching
an ecological equilibrium with the target weed and providing continuous,
perpetual control. Successful control depends almost wholly on damage
caused by the descendants of the released individuals rather than by the
released individuals themselves (Elzen and King, 1999). Classical biocontrol
is generally practiced in low-input systems. Biologically based weed control
can also take the form of weed-resistant properties in crop plants, akin to
host-plant resistance versus insect and pathogen pests.
Allelopathy, the production by a plant of secondary metabolites that
inhibit growth of nearby plants, is a phenomenon that has been studied for
its potential utility in weed control for many years (Rector, 2008). This implies
the use of natural compounds from plants, referred to as allelochemicals, in
natural product development programmes. The term allelopathy is derived
from two Greek words allelos and patos meaning to suffer on each other
(Delabays and Mermillod, 2002). According to the authors, allelopathy is
dened as a chemical process whereby certain plants release natural compounds into the environment that can either stimulate or inhibit the growth
and development of surrounding plants. In nature the latter is more likely, as
a form of competition for growing space, where allelochemicals are released
from the roots of one plant and absorbed by the roots of surrounding plants
preventing them from growing in the same area. There are several ways in
which an allelopathic plant can release its protective chemicals. These include:
(i) volatilization, where a chemical is released in the form of a gas through
the leaves and, on absorption, sensitive surrounding plants are stunted or
die; (ii) leaching of allelochemicals from decomposing abscised leaves preventing surrounding plants from establishing in the same area; and (iii) exudation of allelochemicals into the soil through the roots, preventing
surrounding plants from prospering (Delabays and Mermillod, 2002).
Despite its long history, little progress has been made in incorporating
allelopathy into mainstream weed management programmes due to a failure
to provide adequate weed control while maintaining other agronomic qualities of the crop (Belz, 2007). There is a strong possibility that formulation has
played a key role in the failure of bio-herbicides in the market and their
74
efcacy in the eld. This is understandable when the bottom line criterion for
any natural product developed from plant extracts, including natural
herbicides, is considered. This criterion is that the product must be effective,
safe and have consistent results and must have an adequate shelf life of at
least 1 year.
Despite the apparent failure to develop bio-herbicides from plants or
other biological sources at present (see current status later in this chapter),
the need to do so is increasing due to weeds becoming more resistant towards
the commercially available synthetic herbicides (Dudai et al., 1999; Duke et
al., 2000; Tworkoski, 2002). For example, in New Zealand the problems posed
by invasive weeds are among the most severe in the world with an estimated
cost of NZ$100 million each year (Juliena et al., 2007). Of all the weeds currently identied in New Zealand, only a third has been targeted by biological
control. The latter emphasizes the need to integrate either biological control
agents or chemical bio-herbicides or both into a broader management
programme for weeds.
Plants with herbicidal properties

The greatest potential for developing natural herbicides probably lies in the
plant kingdom, a largely untapped reservoir of natural compounds with
allelopathic herbicidal properties. Although little success in terms of natural
product development has been realized, extensive research has been done in
the past to screen for the growth inhibitory activity of extracts from many
plant species (Wu et al., 2002). Only a few recent examples are supplied to
emphasize the principle.
Nie et al. (2002) reported that extracts of Wedelia chinensis reduced seed
germination, inhibited seedling growth, resulted in yellowing leaves and
reduced resistance to disease in weeds such as Cyperus difformis, Paspalum
thunbergii, Alternanthera sessilis and Cynodon dactylon at relative low concentration of 0.4 g FW (fresh weight) ml1 water. This study conrmed the potential of W. chinensis extracts sprayed before crop emergence to control the
germination of weed seeds. Another study by Randhawa et al. (2002) showed
that a sorghum extract reduced seed germination and seedling growth of the
weed Trianthema portulacastrum substantially at high concentrations (75
100%) but promoted shoot length of the weed at low concentrations (25%).
Similar contrasting results were reported for the effects of extracts from
eight lucerne cultivars on seed germination as well as on root and hypocotyl development of lettuce seedlings (Tran and Tsuzuki, 2002). Extracts
from some lucerne cultivars had a stimulatory effect in terms of seed germination as well as root and hypocotyl growth, whereas others showed the
direct opposite effect, conrming that crop plants can also be affected by
plant extracts aimed at controlling weed growth. Singh et al. (2003) conrmed this phenomenon by showing that aqueous leaf leachates of Eucalyptus citriodora inhibited the germination and seedling growth of all test
crops (Vigna radiata, V. mungo and Arachis hypogaea) investigated. Further,
75
in their study of bioactivity of plant extracts, Deena et al. (2003) demonstrated

the inhibitory effect of leaf, stem and root leachates from Andrographis
paniculata on germination and seedling growth in rice. From this it became
clear that the bioactivities of plant extracts are unpredictable and may give
different and often contrasting results with regard to inhibition or promotion of growth and development in other plants. The reaction of crops to
treatment with plant extracts may depend on the interaction between different types of plant species or even on the concentration of the extracts
(Channal et al., 2002a).
Research into the allelopathic activity of plant extracts has resulted in the
identification of active ingredients responsible for both inhibition or stimulation of either seed germination or seedling growth. In this respect, Chung
et al. (2002) reported on the inhibitory effect of ferulic, p-hydroxybenzoic,
p-coumaric and m-coumaric acids isolated from three rice cultivars on the
growth of barnyard grass. This suggested that these compounds may be, at
least, a key factor in rice allelopathy on barnyard grass, and the information
presented may contribute to the development of natural herbicides.
Similar active allelochemicals were isolated by Sasikumar et al. (2002). In
their study on the allelopathic effects of Parthenium hysterophorus leachates
on cowpea, pigeonpea, greengram, blackgram and horsegram, the authors
reported significant seed germination inhibition for all test crops. Gas chromatographic analysis showed the presence of phenolic acids, namely caffeic,
p-coumaric, ferulic, p-hydroxybenzoic and vanillic acids in the leachates
from different plant parts (leaf, stem, flower and root) of P. hysterophorus. A
mixture of allelopathic compounds in bioassays significantly inhibited the
germination and vigour index of all test crops. However, leachates from
flowers had no inhibitory effect on the germination of blackgram and
greengram seeds.
Kato and Kawabata (2002) isolated a growth-inhibiting compound from
the acetone extract of 30-day-old lemon balm (Melissa offinalis) shoots by
means of silica gel column chromatography. This uncharacterized compound
inhibited the growth of cress seeds at concentrations higher than 0.3 g ml1.
Iqba et al. (2002) showed that living buckwheat reduced weed biomass compared to plots without buckwheat. A laboratory study revealed that root exudates from buckwheat (collected from Aomori, Japan) suppressed root and
shoot growth of the weeds Trifolium repens, Brassica juncea, Amaranthus palmeri, Echinochloa crus-galli and Digitaria ciliaris but also that of lettuce, and
reduced weed dry weight. Fagomine, 4-piperidone and 2-piperidinemethanol were isolated from a chloroform extract and identified as the active
ingredients.
Several allelochemicals have also been characterized from Helianthus
annuus that inhibit seed germination and seedling growth of Amaranthus
albus, Amaranthus viridis, Agropyron repens (Elymus repens), Ambrosia artemisiifolia, Avena fatua, Celosia cristata (C. argentea var. cristata), Chenopodium album,
Chloris barbara (Chloris barbata), Cynodon dactylon, Digitaria sanguinalis, Dactyloctenium aegyptium, D. ciliaris, E. crus-galli, Flaveria australasica, P. hysterophorus, Portulaca oleracea, Sida spinosa, Trianthema portulacastrum and Veronica
76
persica (Macias et al., 2002). The inhibitory effects of this crop may be utilized
for weed management to attain reduced herbicide usage in sustainable
agricultural systems (Azania et al., 2003).
Previous studies by Salamci et al. (2007) conrmed that the essential
oils of T. aucheranum and T. chiliophylllum, characterized by the relatively
high content of 1,8-cineole, camphor, borneol and -terpineol, exhibited
potent inhibitory effects on seed germination and seedling growth of
A. retroflexus. All commercialized essential oils act as non-selective, contact
herbicides that can provide good but transient weed control. The use of
essential oils for weed control in organic agriculture seems promising, but
these natural herbicides all act very rapidly and their efcacy is limited by
the fact that they probably volatize relatively quickly (Dayan et al., 2009).
Other oils such as pine and clover oils have also been used in organic
farming systems, but with limited success because the relatively high rate
of use required for control makes it expensive compared to only one
treatment of glyphosate with the same or even better control of weeds
(Dayan et al., 2009).
Current status of natural herbicides

Although several natural products from various sources are probably currently under development and targeted against specic weeds across the
globe, very little literature is available on the use and environmental impact
of natural products in organic agriculture and little success can be reported
at present. According to Babua et al. (2003), reasons why so many promising
bio-herbicides have failed to reach the commercial phase probably include:
(i) the economics of patenting and registration; (ii) inadequate or noncommercial market sizes; and (iii) technological constraints, especially relating to formulation chemistry. Discovery and development of a synthetic
chemical herbicide can easily cost US$30 million (Heiny and Templeton,
1993) or more. It is generally expected that the development of a natural
product herbicide should cost less. Indeed, two notable successes include
COLLEGO and BIOMAL, developed from exotic pathogens, where the
development costs were US$1.5 million and US$2.6 million respectively. The
costs involved with the development of these two examples of natural products can probably not be accepted as a rule and there are no guarantees that
prospective entrepreneurs will experience the same. However, from an ecological perspective natural products are much more favoured (Heiny and
Templeton, 1993) as a result of added advantages over classical synthetic
weed control agents, including their narrow host ranges that reduce their
impact on non-targeted plants in the environment (Rector, 2008), and this
alone serves as a driving force for natural product developers to continue
with this entrepreneurial business enterprise. Additionally, the quickly
expanding organic agriculture industry does not allow synthetic pesticides,
including herbicides (Anonymous, 2009), and this provides more momentum
for the natural product industry.
77
In an excellent recent review by Dayan et al. (2009), a number of natural compounds from which bio-herbicides have been commercialized for
weed control in organic agriculture have been listed. A few examples
include corn gluten meal (e.g. WeedBan, Corn Weed Blocker and Bioscape Bioweed), a mixture of essential oils and other organic compounds
(e.g. Burnout, Bioorganic, Weed Zap and GreenMatch) as well as
pelargonic acid mixed with related short-chain fatty acids and parafnic
petroleum oil (e.g. Scythe). However, despite the examples of natural
products mentioned above, a tripeptide obtained from the fermentation
culture of the actinomycete Streptomyces hygroscopis and registered as
Bialaphos is regarded by Dayan et al. (2009) to be the only true commercialized natural product herbicide to date. It is a pro-herbicide that is
metabolized into the active ingredient l-phosphinothricin (Fig. 3.28) in the
treated plant.
Bialaphos and phosphinothricin inhibit the enzyme glutamine synthetase which is necessary for the production of glutamine and for ammonia
detoxication. The elevated ammonia levels in tissues of treated plants stops
photosynthesis and results in plant death. Both Bialaphos and phosphinothricin are broad-spectrum post-emergence herbicides that can be used for
total vegetation control in many agricultural settings or in non-cultivated
areas and to desiccate crops before harvest (Dayan et al., 2009).
The list of commercialized bio-herbicides has expanded substantially
during the past two decades. However, their mere existence must be considered in view of the following very valid comments by Dayan et al. (2009):
(i) as opposed to traditional synthetic herbicides, none of the natural herbicidal compounds allowed for use in organic agriculture are very active and
they must, therefore, be applied in relatively large quantities that may lead
to undesirable effects on the environment and the soil fauna and microbes,
which is in direct opposition with the philosophical positions and purpose
of those who practice organic agriculture; (ii) the use of organic weed management tools may be enhanced in the context of an integrated pest management programme that includes sowing multiple crops, extended
rotation cycles, mulching, and soil cultivation and cover; (iii) existing natural herbicides show very little crop selectivity and still require laborious
application methods to ensure they do not come in contact with the desired
crop; and (iv) organic weed management methods may be possible in
small-scale farming and high-value crops but do not seem feasible in the
production of the agronomic crops such as grains grown in large-scale
farming enterprises.
HO
OH
NH2
Fig. 3.28.
L-phosphinothricin (redrawn from Dayan et al., 2009).
78
3.6 Natural Compounds from Plants with Bio-stimulatory

Potential
Allelochemicals found in plants are probably all secondary metabolites that
are distinctive from primary metabolites in that they are generally nonessential for the basic metabolic processes such as respiration and photosynthesis (Richard, 2001). They are numerous and widespread, especially in
higher plants (Pillmoor, 1993), and often present in small quantities (15%) as
compared to primary metabolites (carbohydrates, proteins and lipids).
Approximately 88,000100,000 secondary metabolites have been identied
in all plant forms, showing both structural and activity diversity (Verpoorte,
1998). Ecologically, these chemicals play essential roles in attracting pollinators, as adaptations to environmental stresses and serve as chemical defences
against insects and higher predators as well as microorganisms (Rechcigl
and Rechcigl, 2000). Although the purpose of the production of secondary
metabolites in plants has long been argued among researchers, it is now universally accepted that they are produced as a result of abiotic (Beart et al.,
1985) and biotic stresses (Bourgaud, et al., 2001), probably as part of a plant
defence arsenal. Besides the role secondary metabolites play in plant metabolism, the growth promotion or inhibitory properties of certain natural
compounds from plants have been extensively researched (Wu et al., 2002).
Current status of plant products with bio-stimulatory potential

Already, at the end of the millennium, two successful natural products developed in Moldavia (formerly part of the Soviet Union) are Moldstim and
Pavstim, extracted from hot peppers (Capsicum annum L.) and leaves of
Digitalis purpurea L., respectively (Waller, 1999). Both products have been
used on a large scale as plant-growth regulators and for disease control.
These developments are excellent examples of how natural plant resources
can be exploited and applied in agriculture.
However, from an agricultural perspective, plant extracts containing
growth-promoting substances have always been of interest to the research
community in terms of the role they could play in addressing future food
security issues. The ideal breakthrough would be to identify a plant or plants
that contain bio-stimulatory substances promoting growth and resistance to
pathogens, as well as yields in agricultural and horticultural crops. At this
point it seems appropriate to consider recent discoveries of bio-stimulatory
compounds from plants that have the potential to adhere to the ideal breakthrough criterion in terms of their application potential as natural products
in the agricultural industry.
Extracts from numerous plant species, with bio-stimulatory properties,
were identied and evaluated for their commercial potential. Channal et al.
(2002b) reported on seed germination as well as seedling growth enhancement of sunower and soybean by leaf extracts from three tree species
79
(Tectona grandis, Tamarindus indica and Samanea saman). Terefa (2002) reported
similar effects for P. hysterophorus extracts on tef (Eragrostis tef) while Neelam
et al. (2002) demonstrated similar effects for Leucaena leucocephala extracts on
wheat (Triticum aestivum). However, none of these studies revealed that treatment with the different plant extracts had any effect on the nal yields of the
crops under investigation.
In this regard, a report by Ferreira and Lourens (2002) demonstrating the
effect of a liquid seaweed extract (now trading as a natural product under the
name Kelpak) on improving the yield of canola must be regarded as signicant. Kelpak applied singly or in combination with the herbicide Clopyralid at various growth stages of canola (Brassica napus) was assessed in a
eld experiment conducted in South Africa during 19981999. Foliar application of 2 l Kelpak ha1, applied at the four-leaf growth stage, signicantly
increased the yield of the crop. The active compounds in Kelpak are auxins
and cytokinin.
In the same year, a study directed towards identifying bio-stimulatory
properties in plant extracts was performed by Cruz et al. (2002a). The authors
treated the roots of bean, maize and tomato with an aqueous leachate of Callicarpa acuminata and followed the in vitro effects on radicle growth, protein
expression, catalase activity, free radical production and membrane lipid
peroxidation in the roots. The aqueous extract of C. acuminata inhibited the
radicle growth of tomato but had no effect on root growth of maize or beans.
However, expression of various proteins in the roots of all treated plants was
observed. In treated bean roots the expression of an 11.3 kDa protein by the
leachate, showing a 99% similarity with subunits of an -amylase inhibitor
found in other beans, was induced. In treated tomato an induced 27.5 kDa
protein showed 95% similarity to glutathione-S-transferases of other Solanaceae species. Spectrophotometric analysis and native gels revealed that
catalase activity was increased twofold in tomato roots and slightly in bean
roots, while no signicant changes were observed in treated maize roots.
Luminol chemiluminescence levels, a measure of free radicals, increased
fourfold in treated tomato roots and twofold in treated bean roots. Oxidative
membrane damage in treated roots, measured by lipid peroxidation rates
revealed an almost threefold increase in peroxidation in tomato while no
effect was observed in maize or beans (Cruz et al., 2002a).
The signicance of this study lies in the fact that various metabolic
events can be manipulated in plants by treatment with certain plant extracts.
What has to be established by researchers is whether these altered metabolic
events contribute towards positive or negative physiological changes within
the treated plants. The rationale for this type of research lies in the search for
natural compounds to be applied in sustainable yield-improving, as well as
weed-, pest- and disease-controlling, management systems (Singh et al.,
2001). According to the authors, natural compounds isolated from some
plants show strong bio-herbicidal activity at high concentrations but at low
concentrations these extracts can promote crop seed germination and seedling growth, hence showing a potential to be applied as bio-stimulatory
agents or growth-promoting substances in agriculture. There is general
80
consensus amongst scientists that research in this regard should concentrate

on both the inhibitory and stimulatory effect of plant extracts on seed germination, seedling growth and the physiology of other test plants in order to
verify the action at hand (Khan et al., 2001; Ameena and George, 2002; Cruz
et al., 2002b; Duary, 2002; Obaid and Qasem, 2002).
Probably the most effective compounds to enhance crop yield, crop efciency and seed vigour have been identied as brassinosteroids (BRs;
Mandava, 1979; 1988), rst extracted from rape (Brassica napus L.) pollen
(Adam and Marquard, 1986). In a recent mini-report, Zullo and Adam (2002)
conrmed the prospective agricultural uses of BRs. The assumption of their
application potential was made from data collected over the past three
decades and only a few examples are presented here. Yield increases that
were in most instances signicant were reported, as cited by Zullo and Adam
(2002), in beans and lettuce (Meudt et al., 1983), rice (Lim, 1987), maize (Lim
and Han, 1988), wheat (Takematsu et al., 1988), chickpea (Ramos, 1995) and
tomato (Mori et al., 1986). Many other examples of BR use for increasing crop
yield can be found in the literature (Kamuro and Takatsuto, 1999; Khripach
et al., 1999; Khripach et al., 2000).
Besides their yield-improving effects, BRs have been shown to increase
plant growth in crops (Rao et al., 2002) and especially root growth (Mssig
et al., 2003), to increase resistance in crops towards low-temperature injury
(Kamuro and Takatsuto, 1999) and to increase resistance of potato to infections by Phytophthora infestans and Fusarium sulfureum (Kazakova et al., 1991).
Although many BRs, such as 24-epibrassinolide, are commercially available
and employed in some countries, more accurate studies on dosage, method
and time of application, its suitability for the plant or cultivar, and association with other phytohormones are needed, because many of the results were
obtained by experiments performed in greenhouses or small elds (Zullo
and Adam, 2002).
A report on a prototype bio-stimulatory natural product developed from
a BR-containing extract of Lychnis viscaria came from Roth et al. (2000). In
2003, after 12 years of intensive research under laboratory, greenhouse and
eld conditions at the University of the Free State, South Africa, a product
was listed in Germany as a plant-strengthening agent under the trade name
ComCat and commercialized by a German company, Agraforum AG.
Foliar applications of ComCat have been demonstrated to enhance consistently root development in seedlings and nal yields in a number of vegetable, fruit and row crops, as well as to induce resistance in crops towards
abiotic and biotic stress conditions, and the mechanisms of action were elucidated on both a metabolic and genetic level (unpublished results, Pretorius,
J.C. and van der Watt, E., University of the Free State, South Africa). Recently,
signicant yield increases in tomato, preharvest treated with ComCat,
were reported by Workneh et al. (2009). The authors also claimed more than
70% shelf life extension and higher marketability in tomato fruit harvested
from plants treated with ComCat during the vegetative growth phase compared to the untreated control under ambient storage conditions. Preharvest
ComCat treated tomatoes contained lower total soluble sugar levels at
81
OH
OH
OH
OH
CH3
OH
OH
HO
HO
O
24-epi-secasterone
O
24-epicastasterone
HO
O
HO
H
O
Brassinolide
Fig. 3.29. Brassinosteroid active compounds contained in the plant-strengthening

agent ComCat.
harvest and showed better keeping quality in terms of physiological weight

loss and juice content compared to untreated controls. In light of the diverse
positive effects of ComCat on agricultural and horticultural crops,
the product largely adheres to the ideal candidate criterion stated earlier.
Three BRs have been identied as the main active components of ComCat
and these include 24-epi-secasterone, 24-epicastasterone and brassinolide
(Fig. 3.29).
The future of commercialized bio-stimulants seems positive in light of
the elevated costs of fertilizer. In this regard, research in terms of the use of
bio-stimulants in combination with fertilizer levels lower than the recommended standard for different crops seems to be important in an attempt to
lower the input costs that have become a gloomy issue for farmers recently.
3.7 Conclusions
Discovery programmes by the agrochemical industry are mostly driven by
large-scale synthetic programmes followed by screening to identify potential
new bio-pesticides, including antimicrobials and herbicides. Most companies have a more modest effort to evaluate natural products from outside
sources and, to a lesser extent, from in-house isolation efforts. Although the
literature is replete with reports of the isolation and characterization of phytotoxins from many sources, and many of these compounds have been patented for potential use, the use of natural or natural-product-derived
herbicides in conventional agriculture is limited (Dayan et al., 2009).
Since the Second World War, traditional agricultural practices have
included the use of synthetic chemicals for the management of plant pathogens, pests and weeds. This has, without any doubt, increased crop production but with some deterioration of the environment and human health
(Cutler, 1999). Research indicates that even if one never uses pesticides, one
can still be exposed to them by being a consumer of commodities that others
have treated with pesticides, e.g. through food.
82
In addition to the target pathogen, pesticides may kill various benecial

organisms and their toxic forms can persist in the soil. The increasing incidence of resistance among pathogens towards synthetic chemicals is also a
cause for serious concern. The above is not only of major concern to the
developed countries, where consumer preferences are for organically produced foods, but also in the developing world, such as Africa, where synthetic pesticides are too expensive for subsistence farming. Because of these
problems there is a need to nd alternatives to synthetic pesticides.
Among the various alternatives, natural plant products that are biodegradable and eco-friendly are receiving the attention of scientists worldwide.
Such products derived from higher plants and microbes are relatively bioefcacious, economical and environmentally safe and can be ideal candidates
for use as agrochemicals (Macias et al., 2002). Additionally, the manufacturers
of natural bio-stimulants applied in agriculture claim increased production,
prot increases, cutting of operating costs and reduced fertilizer costs with
no detrimental effect to the environment (Chen et al., 2002). A number of
plants showing the potential to act as donor plants for these natural products
have been outlined in this chapter.
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Arras, G., Piga, A. and DHallewin, G. (1993)
The use of Thymus capitatus essential oil
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Antimicrobials of Plant Origin

to Prevent the Biodeterioration
of Grains
K.A. RAVEESHA
Department of Studies in Botany, University of Mysore,
Manasagangotri, India
Abstract
A signicant portion of stored food becomes unt for human consumption due to the
biodeterioration of grains. The incessant and indiscriminate use of synthetic chemicals in crop protection has been one of the major factors in polluting soil and water
bodies. Thus, there is a need to search for effective, efcient and eco-friendly alternative methods for preventing the biodeterioration of grains during storage. Antimicrobials of plant origin are an important alternative, which could be better exploited to
prevent grain biodeterioration. Fungi are signicant destroyers of food so the biodeterioration of grains can be prevented by inhibiting fungal growth. Of late, many
plant extracts have been screened for antifungal activity. Decalepis hamiltonii Wight &
Arn. (Asclepiadaceae) and Psoralea corylifolia L. (Leguminosae) have revealed highly
signicant antifungal activity. The antifungal active compounds from these plants
have been isolated by antifungal activity guided assays and characterized using
NMR, IR and mass spectral studies. The biomolecules responsible for the activity
were identied as 2H-Furo [2,3-H]-1-benzopyran-2-one in P. corylifolia and 2-hydroxy4-methoxybenzaldehyde in D. hamiltonii. In vitro and in vivo evaluations of these
biomolecules have shown promising inhibitory activity of important biodeterioration-causing fungi. Comparative evaluation of the bioactive compounds with those of
the routinely used chemical fungicides is highly encouraging. Results suggest the
potential of these biomolecules for commercial exploitation to develop eco-friendly
herbal remedies for preventing the biodeterioration of grains during storage.
4.1 Introduction
Storage of food is a necessity to ensure the availability of food throughout the
year. Stored foods are prone to postharvest loss in quality and quantity due
to infestation by different groups of organisms. Biodeterioration may be
dened as the quality and quantity loss of stored food caused by organisms.
The organisms broadly responsible for such deterioration are microbes,
insects and rodents. Among microbes, fungi are signicant destroyers of
(ed. N.K. Dubey)
91
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K.A. Raveesha
food, particularly species of Aspergillus and Penicillium. Grain produced is

not a grain until it is consumed without quality loss (Neergaard, 1977). In
spite of the developments in the methods of food preservation, nearly 30% of
the food produced is lost during storage, due to poor handling and storage
practices.
The incessant and indiscriminate use of chemical pesticides in agriculture has resulted in drug resistance, residual toxicity, and has caused soil and
water pollution. This has necessitated the search for natural eco-friendly
alternatives, which are biodegradable and non-toxic to non-target species.
Plants are a repository of novel biomolecules with various biological activities. Among the 250,000500,000 species of plants available on earth, the
number of plants screened phytochemically is a small percentage, among
these the number of plants screened for various biological activities is innitesimally small. In recent years there have been serious efforts to screen
plants for various biological activities and the scientic endeavour is focused
mainly on utilizing plants for human health and least towards plants for
plant health. Considering the fact that large amounts of synthetic pesticides
known to cause environmental pollution are used in present-day agriculture,
there is an urgent need to search for natural eco-friendly alternatives. Antimicrobials of plant origin appear to be one of the safest and eco-friendly
alternatives. Hence, there is a need to search for antimicrobials of plant origin
to manage eld crop loss in general and to prevent biodeterioration of grains
during storage in particular.
Clinical microbiologists have two reasons to be interested in antimicrobials of plant origin. First, it is very likely that these phytochemicals or
biomolecules will nd their way into the arsenal of prescribed antimicrobial
drugs. Second, the public is becoming increasingly aware of the problems
with and side effects from the over prescription and traditional use of
antibiotics (Cowan, 1999).
Agricultural microbiologists have many more reasons to be interested in
antimicrobials of plant origin: (i) it is very likely that these phytochemicals
may nd their way into the array of antimicrobial drugs prescribed; (ii) it is
known that the effective life span of any antimicrobial is limited, hence newer
antimicrobials are necessary; (iii) it may help to overcome the rising incidence of drug resistance amongst pathogenic microbes and the mechanism
of action could be different; (iv) the phytochemicals present in plant extracts
may alleviate the side effects that are often associated with synthetic antimicrobials; (v) phytomedicines usually have multiple effects on the plant
body and their actions are often growth promoting, more systemic and
beyond the symptomatic treatments of disease; and (vi) it may help in developing cost effective remedies that are affordable to the population (Cowan,
1999; Doughari, 2006).
Finding healing powers in plants for human health needs is an ancient
idea and practice. Searching herbal remedies for crop protection in general
and prevention of biodeterioration in particular is recently gaining importance. Agricultural microbiologists around the world are evaluating a number of plant extracts for antimicrobial activity against important plant
Antimicrobials to Prevent Biodeterioration of Grains
93
pathogens in vitro. Some attempts are also being made to identify the potential
of plant extracts to prevent biodeterioration of grains during storage.
The body of literature available in the area of antimicrobials of plant origin is voluminous and concerns different areas of study. In the present context, the literature reviewed concerns mainly the antifungal agents of plant
origin with specic reference to important biodeterioration-causing fungi.
The body of literature available may be broadly categorized under the following headings:
In vitro evaluation of plant extracts for antifungal activity against important biodeterioration-causing fungi.
In vitro evaluation of the active compounds for antifungal activity against
important biodeterioration-causing fungi.
Isolation and identication of the antifungal active compounds from
plants.
4.2 In Vitro Evaluation of Plant Extracts for Antifungal Activity

against Important Biodeterioration-causing Fungi
Screening plant extracts for antimicrobial activity is the rst step towards the
isolation of the active component. Water and/or different organic solvents,
singly or in combination, are generally used in the preparation extracts.
Aqueous extracts
Antifungal activity assays of leaf extract obtained from Ocimum sanctum,
Adhatoda vasica, Emblica officinalis, Saussurea lappa, Glycyrrhiza glabra, Zingiber
officinale, Piper longum, Piper nigrum, Onosma bracteatum, Tinospora cordifolia,
Fagonia cretica and Terminalia chebula were tested against Aspergillus flavus at
10% and 50% concentration. P. longum and Z. officinale showed signicant
antifungal activity (Farooq and Pathak, 1998). Crude extracts of 40 Iranian
and Canadian plants were tested for antifungal activity against several species of Aspergillus. Of these, 26 plants (65%) showed activity and the spectrum of activity was wide in case of Diplotaenia damavandica, Heracleum
pessicum, Sanguisorba minor and Zataria multiflora (Sardari et al.,1998).
Sinha and Saxena (1999) conducted an antifungal activity assay of leaf
extract of Allium cepa and Allium sativum (garlic) against Aspergillus niger and
reported that the garlic was more effective in inhibiting the germination of
spores and mycelial growth of the fungus. Mahmoud (1999) evaluated the
antifungal activity of ve different concentrations of aqueous extract of Lupinus albus, Ammivis naga and Xanthium pungens against A. flavus and production of aatoxins and reported the inhibition of mycelial growth of A. flavus
at 2, 4, 6, 8 and 10 mg/ml.
Aqueous extracts of 50 plants belonging to 27 families were screened for
antifungal activity against A. flavus and A. niger and it was found that only
94
K.A. Raveesha
four plants, namely Trachysper neumammi, Allium sativum, Syzygium aromaticum and Plectranthus rugosus were effective against both species of Aspergillus
(Singh and Singh, 2000). Aqueous extract of Terminalia australis has
been reported to be effective in inhibiting Aspergillus strains by Carpano
et al. (2003).
Aqueous extracts of Aloe barbadensis, Datura stramonium, Zingiber officinale, Murraya koenigii and Azadirachta indica were evaluated against species of
Aspergillus by employing the poisoned food technique and seed inoculation
method. All the plant extracts showed signicant inhibitory activity.
D. stramonium and A. indica showed higher activity (Sharma et al., 2003). The
extracts of Toona ciliata (stem bark) and Amoora rohituka (stem bark) exhibited
signicant in vitro antifungal activities against A. flavus at 20 and 30%
concentration (Chowdhury et al., 2003).
Solvent extracts
Welsh onion ethanol extract was tested against A. flavus and A. parasiticus
for mycelial growth inhibitory activity and aatoxins production (Fan
and Chen, 1999). Sharma et al. (2002) evaluated an alcoholic extract of Semecarpus anacardium L. against Aspergillus fumigatus at different concentrations
(20400 g/ml) and observed complete inhibition at 40 g/ml.
Petroleum ether and methanolic extract of Eupatorium ayapana were
tested for antifungal activity at 250, 500, 750 and 1000 g/ml against A. niger
and A. flavus. Petroleum ether extract showed higher antifungal activity than
the methanolic extract (Gupta et al., 2002). An ethyl acetate soluble fraction of
acidied aqueous mother liquor and buffer soluble fraction of neutral mother
liquor of stem bark of Alangium salvifolium were tested for antifungal activity
against species of Aspergillus by Katyayani et al., (2002) and reported
signicant activity at 10 mg/ml concentration.
The methanolic extract of stem bark of Ailanthus excelsa partitioned with
chloroform recorded signicant antifungal activity against A. niger, A. fumigatus, Penicillium flequentence, and Penicillium notatum at 300, 90, 70, and 140
g/ml concentrations, respectively (Joshi et al., 2003). Moderate antifungal
activity of ethanolic extracts of the trunk bark of Zanthoxylum fagara, Z. elephantiasis and Z. martinicense was observed against A. niger, and A. flavus at
500 and 1000 g/disc (Hurtado et al., 2003). Ethanolic extract of seeds of Piper
guineense and other solvent fractions obtained by column chromatography
tested against A. flavus, revealed highly signicant antifungal effect (Ngane
et al., 2003). Methanol and methyl chloride extracts of 20 Indonesian plants
were tested for antifungal activity against A. fumigatus at 10 mg/ml by the
poisoned food technique. Extracts of six plants, viz. Terminalia catappa, Swietenia mahagoni, Phyllanthus acuminatus, Ipomoea spp., Tylophora asthmatica and
Hyptis brevipes recorded signicant antifungal activity (Goun et al., 2003).
Extracts of aerial parts of Achillea clavennae, Achillea holosericea, Achillea
lingulata and Achillea millefolium (hexane:ether:methanol = 1:1:1) were tested
for antifungal activity by disc diffusion assay against A. niger. All four species
95
exhibited antifungal activity against tested strains (Stojanovic

et al., 2005).
Organic solvent leaf extracts of two Moroccan Cistus L. species Cistus
villosus L. and Cistus monspeliensis L. (Cistaceae) used in traditional medicine
were tested for their antifungal properties against A. fumigatus. The extracts
differed in their antifungal activities. C. villosus extracts exhibited higher
activity than C. monspeliensis (Bouamama et al., 2006).
Antifungal activity of Trapa natans L. fruit rind, extracted in different solvents with increasing polarity was observed and 1,4-dioxan, chloroform,
acetone, dimethylformamide, ethanol, and water did not reveal any activity
against A. niger (Parekh and Chanda, 2007).
4.3 In Vitro Evaluation of the Active Components for Antifungal

Activity against Important Biodeterioration-causing Fungi
Plants possess unlimited ability to produce secondary metabolites and more
than 12,000 of them have been isolated, which is probably less than 10% of
the total. Useful antimicrobial phytochemicals are phenolics and poly phenols, quinones, flavones, flavonoids and flavanols, tannins, coumarins, terpenoids and essential oils, alkaloids, and lectins and polypeptides. Cowan
(1999) has critically reviewed the antimicrobials of plant origin and has listed
the plants with antimicrobial activity.
Many secondary metabolites have been identified and many more are
yet to be discovered. There is growing evidence that most of these compounds are involved in the interaction of plants with other species, primarily
in the defence of the plant from plant pests. Secondary compounds represent
a large reservoir of chemical structures with biological activity (Duke, 1990).
This resource is largely untapped for use as pesticides.
Considering the advantages of organic pesticides over synthetic pesticides, nowadays, attempts are made by many workers to screen plants for
antimicrobial activity, isolate and characterize the bioactive compounds from
different parts. Such attempts to isolate antimicrobials of plant origin and
test their efficacy against different storage fungi to prevent biodeterioration
of grains are, however, very few.
The high terpene hydrocarbon content in the oils of chamomile, lavender,
eucalyptus, and geranium are responsible for their antifungal activity against
A. niger and Aspergillus ochraceous (Lis-Balchin et al., 1998). Antimicrobial
compounds, such as 3-0-methylveracevine, 3-0-[3-(2,2,2,-trifluoroethoxy)]-5(methoxybenzoyl) veracevine 3-0-(3,5-diiodobenzoyl) veracevine, 3-0-(3thienoyl) veracevine, isolated from the seeds of Schoenocaulon officinale
showed highly significant antifungal activity against A. flavus and A. niger
(Oros and Ujvary, 1999).
Mathekga et al. (2000) isolated an acylated form of a phloroglucinol from
Helichrysum caespititium. The structural elucidation revealed that the compound was 2-methyl-4-2-4(6-trihydroxy-3-(2-methylpropanoylphenyl)2-0-enylacetate). This compound completely inhibited the mycelial growth
96
K.A. Raveesha
of A. niger and A. flavus at 1.0 g/ml. Bioactive compounds

12-hydroxysandarocopimar-15-one and 2-propionoxyo--chesorcylic acid
isolated from methanolic extract of leaves of Trichilia heudelotti showed highly
signicant antifungal activity against A. niger at 100 g/ml (Aladesanmi and
Odediran, 2000).
Investigations on the antifungal active methanol fraction of the root of
Epinetrum villosum (Exell) Troupin (Menispermaceae) led to the isolation of
the bisbenzylisoquinoline alkaloid cocsoline, which displayed signicant
antifungal activity with a minimum inhibitory concentration (MIC) for
A. flavus and A. niger of 31.25 g/ml (Otshudi et al., 2005).
Phytochemical analysis of the leaves of Vernonia amygdalina yielded two
known sesquiterpene lactones, vernolide and vernodalol, that exhibited signicant antifungal activity. Vernolides exhibited high activity and the 50%
lethal concentration (LC50) values ranged from 0.2 to 0.4 mg/ml for P. notatum, A. flavus, A. niger and Mucor hiemalis. Vernodalol showed moderate
inhibitory activity against A. flavus, A. niger and P. notatum with LC50 values
of 0.3, 0.4 and 0.5 mg/ml, respectively (Erasto et al., 2006).
4.4 Isolation and Identification of the Antifungal Active

Component from Plants
The isolation and identication of antimicrobials of plant origin involve the
following stepwise approach:
Selection of the plant, followed by the selection of the plant part.

Selection of appropriate solvent for extraction.
Separation and purication of the active component.
Characterization and structural elucidation of the active component.
In vitro evaluation of the active component for antimicrobial activity
against test fungi.
In vivo evaluation of the active component for antifungal activity.
Employing bioactivity-directed fractionation and isolation (BDFI) for a

specic antimicrobial activity against biodeterioration-causing fungi is
advantageous. Two plants have been extensively studied using this method,
namely Psoralea corylifolia and Daecalepis hamiltonii. The active component
responsible for antifungal activity has been isolated, characterized and the
activity has been demonstrated in vitro. Further investigations have been
done to prove its efcacy to prevent biodeterioration in vivo.
Potential of 2H-furo[2,3-H]-1-benzopyran-2-one isolated from Psoralea
corylifolia L. to prevent the biodeterioration of maize
Psoralea corylifolia L. (Leguminosae) is an annual herb (Fig. 4.1) reported to
have medicinal properties. In traditional medicine, the seeds (Fig. 4.2) are
used in the treatment of psoriasis, leucoderma and inammatory disease of
Fig. 4.1.
Psoralea corylifolia L. (plant).
Fig. 4.2.
Psoralea corylifolia L. (seeds).
97
98
K.A. Raveesha
the skin. An antifungal activity guided assay of different solvent extracts

revealed that petroleum ether and methanol extracts were highly active.
Hence a combination of petroleum ether and methanol was employed for the
isolation of the antimicrobial active component from the seeds of P. corylifolia
(Kiran and Raveesha, 2004).
Isolation and characterization of the antifungal active component
Powdered seeds of P. corylifolia were reuxed in a petroleum ether and methanol mixture [9:1(v/v)] in a Soxhlet apparatus for 8 h at 60C. An excess of
solvent was removed by distillation under reduced pressure. The concentrated extract was cooled for 48 h at 5C to obtain the pure compound as
transparent, colourless, rectangular prism-shaped crystals (Fig. 4.3). The
average yield of the compound was 5 mg per 25 g of seed. The melting point
was 138C. The purity of the compound was conrmed using thin-layer
chromatography (TLC) and the Rf value was 0.47.
The bioactive compound was subjected to infra red (IR), 1H-NMR,
13C-NMR (Al-Fatimi et al., 2006) and gas chromatographymass spectral
(GCMS) analyses (Yanez et al., 2005) for structural elucidation.
The IR spectrum showed an absorption band in the region of 1652.9 cm1
for CO-stretching. Further absorption bands at 1550.7 cm1 and 1454 cm1
were due to the presence of a coumarin ring oxygen and a furan ring oxygen,
respectively.
In 1H-NMR spectra, the signal due to C3H and C4H of coumarin
appeared at 6.41 as a doublet and at 7.81 as a singlet. The aromatic protons
Fig. 4.3. Stereo micrograph of the active compound [2H-furo[2,3-H]-1-benzopyran-2one] isolated from seeds of P. corylifolia, showing transparent, colourless, rectangular,
prism-shaped crystals.
99
(C7 + C8) are mingled together and appeared at 7.7 as a multiplet. The
signal due to the C3 and C2 protons appeared at 6.84 and 7.49 as a doublet,
respectively.
13C-NMR data of the bioactive compound showed peaks at 146.85(C ),
2
143.8 (C4), 119.77(C3), 115.5(C7), 114.76(C8), 2 106.3(C3), and 99.86 (C2), which
are inconsistent with structure.
GCMS analysis showed a molecular ion peak at M/z 186.17 consistent
with the molecular formula C11H6O3. The peak at M/z 158 was due to the
formation of the coumarin cation. The recorded chromatogram of the plot
matched with the chromatogram of an already known compound, 2H-furo[2,3H]-1-benzopyran-2-one. Figure 4.4 presents the molecular structure of the
bioactive compound.
In vitro evaluation of the antifungal active component
Ten species of Aspergillus, viz. A. flavus, A. niger, A. terreus, A. tamarii, A. flavus
oryzae, A. fumigatus, A. candidus, A. ochraceous, A. flavipes and A. flavus columnaris, and two species of Penicillium, viz., P. chrysogenum and P. notatum, isolated
from maize seeds and known to cause biodeterioration of grains served as test
fungi for the antifungal activity assay using the poisoned food technique.
The poisoned food technique is as follows. Malt extract salt agar (MESA)
medium amended with different concentrations of the bioactive compound
were prepared and poured into sterile Petri plates and allowed to cool and
solidify. Mycelium discs (5 mm diameter) of 7-day-old cultures of species of
Aspergillus and Penicillium were placed at the centre of the plates and incubated
at 25 1C for 7 days. The MESA medium without the bioactive compound
served as a control. The colony diameter was measured. Similarly the fungicides Captan (C9H8Cl3NO2S) and Thiram (C6H12N2S4) were also tested against
all the test fungi at the recommended dose of 2000 ppm concentration for comparative evaluation. The percentage inhibition of mycelial growth if any was
determined by the formula PI = C T/C 100; where C is the diameter of control colony and T is the diameter of treated colony (Pinto et al., 1998). MICs for
each of the test fungi were determined on the basis of the concentration needed
to inhibit totally the test fungi (Fig. 4.5).
The total inhibition of A. flavus was observed at 100 ppm. A. niger and
A. fumigatus were totally inhibited at 500 and 600 ppm, respectively. A. flavus
oryzae and A. flavus columnaris were totally inhibited at 700 ppm. A. ochraceous
and A. flavipes were totally inhibited at 900 ppm. Total inhibition of
O
Fig. 4.4. Molecular structure of the bioactive compound, 2H-furo [2,3-H]-1-benzopyran-2one, isolated from seeds of P. corylifolia.
100
K.A. Raveesha
1000
Concentration of the bioactive
compound (ppm)
900
800
700
600
500
400
300
200
100
A.
fla
vi
pe
s
te
rre
fla
vu
us
s
or
yz
A
A.
ae
fla . ca
nd
vu
id
s
Pe
us
co
lu
ni
m
ci
lliu
na
ri
m
no s
P.
t
at
ch
um
ry
so
ge
nu
m
us
eo
A.
at
us
ig
oc
hr
ac
ar
ii
fu
m
r
ge
A.
ni
A.
ta
m
A.
A.
A.
As
pe
rg
i
llu
fla
vu
Fungi
Fig. 4.5. Minimal inhibitory concentration (MIC) of the bioactive compound [2H-furo[2,3-H]1-benzopyran-2-one] isolated from seeds of P. corylifolia L. against species of Aspergillus
and Penicillium.
A. tamarii, A. terreus and A. candidus was not observed even at 1000 ppm.
Penicillium notatum and P. chrysogenum were totally inhibited at 800 ppm and
900 ppm concentration, respectively.
The concentration of the bioactive compound needed for total inhibition
of Aspergillus and Penicillium species was much lower than the recommended
dose of the test fungicides.
In vivo evaluation of the active component to prevent the biodeterioration of
maize
Maize grains naturally infected with diverse species of Aspergillus and Penicillium were treated with 250, 500, 1000 and 1500 ppm concentration of the
bioactive compound. Untreated seeds served as a control. The treated and
untreated seeds were stored at room temperature (30 2C) for 4 months and
the moisture content of the seed sample was maintained at 13.5% (Janardhana
et al., 1999). Samples were drawn at regular intervals of 30, 60, 90 and 120
days from each treatment and subjected to seed mycoora analysis employing the standard method (ISTA, 1999), determination of protein content
(Lowry et al., 1951) and carbohydrate content (Dubios et al., 1956) to assess
the level of biodeterioration.
Results revealed a signicant reduction in the seed mycoora in all the
treatments with a total elimination of seed-borne fungi including species of
Aspergillus and Penicillium in the grains treated with 1000 ppm and 1500 ppm
concentration of the bioactive compound in all the storage periods tested. No
101
change in protein and carbohydrate content was observed in the grains

treated with 1000 and 1500 ppm even after 4 months of storage, suggesting
that the active compound, 2H-furo[2,3-H]-1-benzopyran-2-one isolated from
seeds of P. corylifolia L. could be exploited to prevent the biodeterioration of
grains.
Potential of 2-hydroxy-4-methoxybenzaldehyde isolated from Decalepis

hamiltonii Wight & Arn. to prevent biodeterioration of paddy
Decalepis hamiltonii Wight & Arn., a member of the family Asclepiadaceae, is
an important medicinal plant widely used in traditional medicine. The
rhizome (Fig. 4.6) of this plant is largely used in South India for pickling.
An antifungal activity assay of different solvent extracts of the fresh
rhizome of this plant revealed highly signicant activity in the petroleum
ether extract against seed-borne fungal pathogens (Mohana et al., 2006). Further experimentation conducted to isolate and characterize the antifungal
active principle from the petroleum phenolic fraction using chloroform as a
Fig. 4.6.
Rhizome of Decalepis hamiltonii Wight & Arn.
102
K.A. Raveesha
solvent for TLC revealed the presence of seven bands. An antifungal activity
assay of each of these bands revealed that band ve with an Rf value of 0.77
showed signicant antifungal activity, whereas the other bands did not show
any antifungal activity. Light blue uorescence at 365 nm was observed.
The active principle was isolated and subjected to 1H-NMR, 13C-NMR
and MS analysis to conrm the identity of the compound. The 1H-NMR
analysis of the compound showed NMR peaks at 3.85 (s,-0CH3), 6.52 (dd,
J = 2 HZ; 3-H), 6.55(d, J = 7 Hz, 5-H), 7.40 (d, J = 7 Hz; 6-H), 9.70 (s, CHO), 11.6
(s,-OH) functional groups. 13C-NMR analysis of the compounds showed
eight carbon signals 135.6 (1-CH), 108.7 (3-CH), 167.2 (C of carbonyl), 101.05
(5-CH), 164.8(2-C), 115.5(C), 194.7 (6-CH), and 56.09 (CH3) and its identity
was conrmed by MS analysis [m/z (% abundance): 57(48), 95(46), 108(24),
121(20), 151(100), 152(70)]. The strong molecular ion peak (m/z, 152) and
stronger M-1 ion peak (m/z, 151) observed were characteristic of an aromatic aldehyde. The melting point of the active compound is 46C. Results
revealed that the active compound responsible for the activity was
2-hydroxy-4-methoxybenzaldehyde (Fig. 4.7), reported in the literature by
Nagaraju et al. (2001).
The active component isolated was subjected to an in vitro antifungal
activity assay by the poisoned food technique and in vivo experiments to
evaluate the potential of this active compound to prevent the biodeterioration of paddy during storage.
An in vitro antifungal activity assay against important seed-borne fungi
associated with paddy known to cause biodeterioration during storage
revealed a highly signicant inhibitory activity against the test fungi. Complete inhibition of the test fungi was observed at 650 g/ml of the compound (Table 4.1). Further in vivo experimentation done to assess the
efcacy of the active principle to prevent biodeterioration of paddy up to
90 days of storage revealed a high potency of the compound to prevent
biodeterioration of grains. The percentage incidence of seed-borne fungal
species decreased signicantly in the samples treated with 1 g/kg of the
active compound. Comparative efcacy studies revealed that the
concentration of the active compound needed to prevent growth of fungi
responsible for biodeterioration of paddy was much less than that of thiram
(2 g/kg) (Table 4.2).
CHO
OH
O C H3
Fig. 4.7. Molecular structure of the bioactive compound, 2-hydroxy-4-methoxybenzaldehyde

isolated from the rhizome of Decalepis hamiltonii Wight & Arn.
3.25 0.3
8.09 0.5
11.33 0.4
16.71 0.5
21.82 0.7
34.34 0.7
43.07 0.6
59.94 0.7
64.78 0.7
76.14 0.5
91.49 1.1
100.0 0.0
100.0 0.0
100.0 0.0
100.0 0.0
100.0 0.0
100.0 0.0
Alternaria
alternata
3.34 0.2
6.6 0.6
19.72 0.4
24.20 0.6
34.60 0.5
45.89 0.6
67.10 0.3
86.69 0.4
91.35 0.5
100.0 0.0
100.0 0.0
100.0 0.0
100.0 0.0
100.0 0.0
100.0 0.0
100.0 0.0
100.0 0.0
Drechslera
tetramera
11.47 0.3
24.33 0.3
39.31 0.4
53.05 0.3
56.90 0.5
68.53 0.3
71.26 1.2
83.14 0.4
90.07 0.4
98.33 0.7
100.0 0.0
100.0 0.0
100.0 0.0
100.0 0.0
100.0 0.0
100.0 0.0
100.0 0.0
Fusarium
oxysporum
12.72 0.4
23.35 0.1
42.30 0.4
51.63 0.2
57.58 0.4
68.06 0.5
79.06 0.3
86.21 0.3
92.78 0.6
100.0 0.0
100.0 0.0
100.0 0.0
100.0 0.0
100.0 0.0
100.0 0.0
100.0 0.0
100.0 0.0
Fusarium
proliferatum
0.00 0.0
5.66 0.4
10.34 0.2
14.36 0.4
17.77 0.4
22.64 0.2
26.60 0.3
42.76 0.7
56.57 0.6
63.54 2.3
67.94 0.4
78.19 0.5
85.88 0.7
89.79 0.6
97.57 0.3
100.0 0.0
100.0 0.0
Pyricularia
oryzae
Mycelium growth inhibition (%) of seed-borne pathogenic fungi of paddy
Data given are mean of four replicates standard error.

Analysis of variance (ANOVA) d.f. = 15 at P < 0.0001.
40
60
80
10
100
150
200
250
300
350
400
450
500
550
600
650
Thiram
Concentration of
active compound
(g/ml)
Table 4.1. The antifungal activity of 2-hydroxy-4-methoxybenzaldehyde isolated from D. hamiltonii and that of thiram against
phytopathogenic fungi isolated from paddy.
2.10 0.5
3.61 0.5
8.84 0.6
11.88 0.4
12.64 0.5
24.67 0.6
26.55 0.5
31.41 0.7
37.18 0.5
41.75 1.0
49.56 0.5
65.00 0.6
76.19 0.6
90.08 0.8
100.0 0.0
100.0 0.0
100.0 0.0
Trichoconis
padwickii

103
26 0.5
51 1.2
27 0.5
40 0.8
42 0.7
34 0.8
26 0.8
25 0.5
16 0.8
42 0.8
28 0.6
41 0.8
36 0.3
21 0.4
22 0.5
23 0.7
13 0.4
30
22 0.5
21 0.5
21 0.8
27 0.8
42 0.5
49 0.9
44 0.4
31 0.5
64 0.5
28 0.3
60
26 0.5
19 0.5
25 0.4
58 0.9
54 0.8
48 0.7
39 0.8
72 0.4
27 0.8
90
30
6 0.3
5 0.5
8 0.8
0.0
0.0
0.0
0.0
0.0
6 0.5
4 0.3 11 0.5
0.0
0.0
0.0
9 0.3 14 0.3
4 0.5 12 0.6
5 0.3
4 0.5
8 0.8
19 0.4
10 0.5
9 0.5
13 0.3
26 0.4
14 0.6
60
90
10 0.8
8 0.7
12 0.5
28 0.5
15 0.8
14 0.8
18 0.8
35 0.5
19 0.6
0.5 g/kg active compound
Per cent incidence is based on four replicates with 100 seeds each, F = 321.64; P < 0.001.
Alternaria
spp.
Aspergillus
spp.
Curvularia
spp.
Drechslera
spp.
Fusarium
spp.
Penicillium
spp.
Pyricularia
spp.
Trichoconis
padwickii
Trichothecium
spp.
Storage
periods (days)
Untreated
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0
7 0.4
60
11 0.7
90
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
2 0.9
8 0.3
6 0.3
0.0
4 0.3
0.0
2 0.3
4 0.8
18 0.5
13 0.6
4 0.3
6 0.4
5 0.2 12 0.6 17 1.1
0.0
30
1 g/kg active compound
Incidence of seed-borne fungal species in paddy seeds (%)
30
8 0.6
60
3 0.4
0.0
0.0
0.0 0.0
0.0 0.0
0.0 0.0
0.0
0.0
0.0
0.0 8 0.6 15 0.6
0.0 0.0
0.0 0.0
0.0 0.0
0.0 5 0.6 23 0.5
0.0 0.0
Thiram (2 g/kg)
Table 4.2. Effect of 2-hydroxy-4-methoxy-benzaldehyde isolated from D. hamiltonii and thiram on seed-borne fungi of paddy stored
up to 90 days.
0.0
1 0.6
2 0.3
22 0.5
8 0.5
1 0.2
3 0.3
32 0.5
11 0.7
90
104
K.A. Raveesha
105
The evaluation of the bioactive compound to understand the impact on the

nutritional quality of the paddy grains stored up to 90 days revealed that
1 g/kg treatment is effective in preventing biodeterioration of paddy grains
without affecting the nutritional quality in terms of dry matter loss, lipid, carbohydrate and protein content (Fig. 4.8ad), suggesting that 2-hydroxy-4-methoxybenzaldehyde is an important antimicrobial of plant origin that could be exploited
to prevent biodeterioration of grains during storage (Mohana et al., 2009).
Even though a few antimicrobial active components have been isolated
and characterized by earlier workers, evaluations of these to assess their potency
to prevent biodeterioration of grains are lacking. The active compounds isolated from the seeds of P. corylifolia L. and from the rhizome of D. hamiltonii
Wight & Arn. have been successively evaluated to assess their potency to
Changes in total carbohydrates
4.3
0.70
0.68
0.66
0.64
0.62
0.60
0.58
0.56
0.54
0.52
4.2
4.1
4.0
3.9
3.8
3.7
60
30
Storage period (days)
90
30
60
90
Control
0.5 gm/kg
Control
0.5 gm/kg
1 gm/kg
Thiram
1.0 gm/kg
Thiram
Changes in lipids
(c)
Dry matter loss
(d)
6
40
35
30
25
20
15
10
5
0
Dry matter loss (%)
Lipid contents (mg/g)
Changes in proteins
(b)
Protein content (mg/g)
Carbohydrate content (g/g)
(a)
5
4
3
2
1
0
30
60
90
30
60
90
Control
0.5 g/kg
Control
0.5 g/kg
1 g/kg
Thiram
1 g/kg
Thiram
Fig. 4.8. (ad) Comparative efficacy of the bioactive compound 2-hydroxy-4-methoxybenzaldehyde isolated from D. hamiltonii (0.5 g/kg and 1 g/kg) and thiram (2 g/kg) on fungi
inducing nutritional losses in paddy grains stored up to 90 days.
106
K.A. Raveesha
prevent the biodeterioration of grains during storage. D. hamiltonii is an important edible plant and thus has an additional advantage as a herbal, eco-friendly
remedy for the prevention of grain biodeterioration. Further investigations on
the standardization of methods for high yield of the active compounds, toxicological aspects, development of formulations, treatment procedures and a package of practices to be adapted may pave the way for commercial exploitation.
4.5 Conclusion
Plants have been over exploited for human health needs while under
exploited for plant health needs. One of the possible reasons for this may be
the high-input cost that could be made good in human health care, while it
may be difcult in plant health care. Considering the cost of environmental
pollution and other related damages caused by the use of chemical pesticides
in crop protection, the development of eco-friendly natural pesticides is a
necessity. Screening plants for antimicrobial activity against phytopathogens
in general and against biodeterioration-causing fungi in particular is the rst
step towards this goal. Subsequent to demonstrating the science behind the
antimicrobial activity, research needs to be focused on developing an appropriate, cost-effective package of practices employing biotechnological
approaches. Some of the possible approaches to decreasing the cost of production are: (i) identify the gene responsible for the production of the active
component and considerably enhance the yield of the active component
through bioengineering; (ii) isolate and identify the endophytes, if any, associated with the production of the active compound and develop fermentative
methods for the large-scale production of the active component; (iii) develop
a package of practice, preferably utilizing the edible plant part pieces or
powder directly as an amendment in an appropriate quantity during grain
storage; and (iv) the chemical synthesis of the active compound.
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Some Natural Proteinaceous

and Polyketide Compounds
in Plant Protection and
their Potential in Green
Consumerization
L.A. SHCHERBAKOVA
Russian Research Institute of Phytopathology, Moscow, Russia
Abstract
Plant protection from diseases with the use of natural compounds, which are
indigenous and biodegradable in the environment, ts the purpose of green consumerization. Natural compounds controlling plant pathogens belong to different chemical classes and are produced by a wide range of organisms. The structural and
functional diversity of these compounds provides a great potential in crop protection
technologies in correspondence with green consumerization objectives. Disease prevention by natural compounds results from either direct or plant-mediated inuences
on targeted pathogens. Some natural substances affecting causative agents directly
do not produce a biocidal effect but specically attack pathways related to their
pathogenicity. Biogenic compounds inuencing plants induce resistance or enhance
tolerance to diseases. They elicit natural defence responses in plants and, unlike
chemical pesticides, do not promote the occurrence of resistant forms in targeted
pathogens. Two bacterial proteins, CspD and MF3, that have elicitor properties have
been well documented to provide resistance against various phytopathogens and
play a promising role in green consumerization. Fungal polyketides, statins, inhibiting a pathogenicity-related pathway in melanin-producing fungi are considered as
prospective candidates for developing biopesticides and reducing the impact of plant
diseases without killing pathogenic microorganisms.
5.1 Introduction
Plant diseases continue to cause considerable damage to global agriculture
resulting in yield losses and deterioration of agricultural products, including
their contamination with hazardous substances, e.g. mycotoxins, which
induce toxicological problems in people and animals. Despite amazing progress achieved in crop protection due to breeding for resistance to diseases
(ed. N.K. Dubey)
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L.A. Shcherbakova
and availability of effective agrochemicals, a number of problems are faced

in managing the diseases successfully. The resistant cultivars provide only
partial plant protection from a disease because of the occurrence of new
pathogenic races that are able to overcome the resistance. Sometimes, breeding for resistance is too complicated because of the diversity and high variability of plant pathogens and the variety of defence responses at different
stages of pathogenesis and plant development.
Chemical protection gives a powerful tool for effective control of many
disease-causing agents, but its implementation has a range of undesirable
side impacts. Thus, the use of chemical pesticides is associated with environmental risks, such as pollution of soil, water and crop plants with xenobiotic pesticide residues, killing benecial microorganisms or insects,
thereby interfering with the natural management of pests and pathogens,
a reduction in biological diversity, and so on. Sometimes, chemical pesticides are not always effective and many treatments with several fungicides are needed to control some fungal pathogens. As a result of continual
pesticide application on plants and stored agricultural products, toxic residues of chemical pesticides may appear and accumulate in food. Lastly,
perhaps the most disturbing consequence of chemical protection is the
development of pesticide resistance in pathogens that makes a protective
effect changeable and insufciently effective. The health and ecological
problems related to chemical pesticides are now receiving a great deal of
attention. Recognition of these problems has created the phenomenon of
green consumerization.
The biocontrol of phytopathogens as an additional or alternative plant
protection method looks more natural and environmentally compatible than
chemical treatments. The use of non-pathogenic or benecial microorganisms and/or natural compounds, which are native and biodegradable in the
environment, is the main objective of green consumerization. A few hundred
biocontrol products based on numerous benecial strains predominantly
of Trichoderma, Bacullis, Pseudomonas, Agrobacterium, Streptomyces and
Gliocladium species are produced, commercialized and used as biopesticides
and/or biofertilizers.
Natural compounds effectively controlling plant pathogens and pests
are represented by a diversity of substances belonging to different chemical
classes and produced by a wide range of living organisms (Varma and Dubey,
1999). Currently, the list of these natural compounds is growing longer from
year to year. Many natural products have already been used or recommended
as biopesticides (e.g. various secondary plant metabolites such pyrethrins,
glucosinolate-sinigrin-producing allyl isothiocyanate, carvone, azadirachtin,
rotenone, ryania, nicotine, some essential oils and other volatile plant substances). The plant-mediated mode of action of some natural compounds,
which does not establish contact with pathogens, decreases the risk of resistant pathogenic form selection. Because the search for natural substances tting phytosanitary regulations and environmentally compatible cropping
methods is a continuous active process, novel compounds are being discovered in plants, animals and microorganisms and are marketed in place of
Proteinaceous and Polyketide Compounds in Plant Protection
111
chemical pesticides in order to be adopted by so-called cottage industry or

governmentally regulated farming.
The disease-preventing effect of natural compounds that are used or can
be used for crop protection is a result of a direct or plant-mediated inuence
on targeted pathogens. Affecting phytopathogens directly, the compounds
interrupt pivotal metabolic pathways and/or disrupt vitally important structures that have as a consequence the death of the pathogens (biocidal effect).
Some natural substances or their analogues do not produce lethal effects to
plant pathogens but specically inuence pathways related to pathogenicity
or/and toxigenesis, e.g. mycotoxin production (Kumar et al., 2008; Shukla
et al., 2008). Such non-biocidal suppressors can impair the pathogenicity for
plants as well as the toxic risk for people and animals. In the case of a
plant-mediated mode of action, natural compounds elicit and activate
defence responses in plants that result in induced resistance to diseases (e.g.
Shcherbakova et al., 2007; 2008; Odintsova et al., 2009). Lastly, some of biogenic compounds inuencing plants do not induce resistance but enhance
tolerance to pathogens (sometimes by improving the physiological state of
plants). The structural and functional diversity of natural compounds as well
as their abundance provide a great potential for bringing plant protection
technologies in line with green consumerization.
The main goal of this chapter is to analyse properties of a few chemically
and functionally different natural compounds and demonstrate their prospective as promising candidates for new plant protection products with signicant potential in relation to green consumerization. The chapter will focus
on the characterization of two proteinaceous compounds of bacterial origin,
the elicitor properties of which provide disease resistance, and also on fungal
statins as natural compounds, which inhibit a pathogenicity-related pathway in melanin-producing fungi and can be used for developing biopesticides that reduce the harmful impact of crop diseases without killing
pathogenic organisms. In addition, microbial and plant proteinaceous compounds that have been previously well documented to be promising for
green consumerization will be briey surveyed.
5.2 Proteinaceous Compounds of Microbial and Plant Origin in

the Biocontrol of Plant Pathogens
Proteins are abundant and ubiquitous natural compounds strongly associated with all living organisms, hence it is possible to believe their involvement in plant protection will not conict with green consumerization
principles. Because proteinaceous compounds are important constituents of
a plant immune system and take part in the natural plant defence against
pathogens, scientists have emphasized their potential for plant protection for
a long time. Basically, two general approaches to the practical realization of
the protective potential of these compounds are the formulation of producers
(biocontrol or non-pathogenic microorganisms) or the engineering of transgenic plants. The compounds synthesized by formulated biocontrol agents
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L.A. Shcherbakova
interact with pathogenic microflora or induce resistance in plants. Constitutive

or inducible expression of protein-encoding genes transferred into plants
confer them resistance to many economically important diseases.
Several groups of microbial and plant proteinaceous substances that
have well-established potentialities in plant protection will be very briefly
reviewed below. The antipathogenic properties of two proteins, which were
isolated from biocontrol bacteria and relatively recently studied as putative
elicitors of disease resistance in plants, will be described in more detail.
The biocontrol potential of proteinaceous compounds will be considered
almost exclusively by using examples of fungal and bacterial pathogens.
Their antipest properties will be not covered within the scope of this chapter.
Nevertheless, it should be emphasized that many proteins and peptides discussed in this section (microbial peptides, defensins, proteinase inhibitors
and lectins) are well known as biological insecticides or nematicides of great
potentiality. Biopesticides based on microbial peptides or transgenic plants
carrying genes of defensins, lectins and proteinase inhibitors are good tools
for the fight against pests of plants.
Antimicrobial peptides produced by microorganisms and plants
The generation of antimicrobial peptides is one of the widespread natural
defence mechanisms of innate immunity of living organisms, and these compounds are interesting targets for green consumerization. Plants, including
agricultural crops, produce numerous defensive peptides. Microorganisms,
including those used in plant protection, synthesize and excrete antimicrobial peptides that are often responsible for the biocontrol effect. In several
authors opinions, peptides have advantages over more elementary organic
antimicrobial compounds when used for plant protection goals. Because the
peptide molecules contain from 10 to 50 (sometimes up to 85) amino acid
residues, they can more specifically interact with their protein targets in
causative agents (Park et al., 2009). Biopesticidal peptides can work against
pathogens by inhibiting nucleic-acid and protein biosyntheses and enzyme
activity or by interacting with the plasmalemma and destroying its integrity
(Huang, 2000).
Antimicrobial peptides from biocontrol microorganisms
Peptides possessing antimicrobial effects have been reported to be produced by bacteria, fungi, plants, invertebrates and vertebrates (GarciaOlmedo et al., 1998; Zasloff, 2002; Degenkolb et al., 2003; Bulet et al., 2004;
Nybroe and Sorensen, 2004; Carvalho and Gomes, 2009). According to the
recent review focused on antimicrobial peptides related to plant-disease
control (Montesinos, 2007), about 900 antimicrobial peptides are produced
by living organisms via ribosomal or non-ribosomal synthesis. Biocontrol
microorganisms have been reported to use both types of the peptide synthesis (Finking and Marahiel, 2004; Montesinos, 2007) and generate a wide
range of these compounds. Antimicrobial peptides of microorganisms
113
(AMPM) have been classied into several groups of linear and cyclic
peptides (fungal defensins, bacteriocins, peptaibols, cyclopeptides and
pseudopeptides) based on their most essential structural characteristics,
and have been inventoried according to name, composition and producer
microorganisms (Montesinos, 2007).
The efcacy of the natural antimicrobial peptides produced by biocontrol microorganisms towards various plant pathogenic bacteria, fungi and
oomycetes in vitro and in vivo has been well documented (Table 5.1).
Table 5.1. Instances of plant pathogens sensitive to antimicrobial peptides of microorganisms.

Targeted
plant pathogens
Fungi
Alternaria spp.
Botrytis cinerea
Group of effective AMPM
Reference
Pseudopeptides
Cyclopeptides
Fungal defensins
Peptaibols
Pseudopeptides
Stein, 2005
Lavermicocca et al., 1997
Ongena et al., 2005
Vila et al., 2001
Moreno et al., 2003
Moreno et al., 2005
Xiao-Yan et al., 2006
Stein, 2005
Selim et al., 2005
Selim et al., 2005
Ongena et al., 2005
Stein, 2005
Vila et al., 2001
Moreno et al., 2003
Moreno et al., 2005
Pedras et al., 2003
Moreno et al., 2003
Moreno et al., 2005
Stein, 2005
Gueldner et al., 1988
Romero et al., 2007
Stein, 2005
Asaka and Shoda, 1996
Bassarello et al., 2004
Nielsen and Sorensen, 2003
Ongena et al., 2005
Stein, 2005
Pedras et al., 2003
Gouland et al., 1995
Bipolaris sorokiniana
Colletotrichum spp.
Erysiphe sp.
Fusarium avenaceum
Fusarium oxysporum
Peptaibols
Peptaibols
Cyclopeptides
Cyclopeptides
Peptaibols
Fusarium solani
Fusarium spp.
Cyclopeptides
Fungal defensins
Leptosphaeria maculans
Magnaporthe grisea
Cyclopeptides
Fungal defensins
Pseudopeptides
Monilina fructicola
Podosphaera fuca
Podosphaera sp.
Rhizoctonia solani
Cyclopeptides
Cyclopeptides
Cyclopeptides
Cyclopeptides
Peptaibols
Pseudopeptides
Sclerotinia sclerotium
Sclerotium cepivarum
Cyclopeptides
Peptaibols
Continued
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L.A. Shcherbakova
Table 5.1. Continued.

Targeted
plant pathogens
Sphaerotheca sp.
Uncinula nector
Ventura inaequalis
Phytophthora infestans
Pythium intermedium
Pythium ultimum
Bacteria
Erwinia amylovora
Erwinia caratovora
Clavibacter michiganesis
Pseudomonas syringae
Rhodococcus fascians
Group of effective AMPM
Reference
Pseudopeptides
Pseudopeptides
Cyclopeptides
Cyclopeptides
Cyclopeptides
Cyclopeptides
Stein, 2005
Stein, 2005
Burr et al., 1996
De Bruijn et al., 2007
De Souza et al., 2003
Nielsen et al., 2002
Pseudopeptides
Brady et al., 1999

Jin et al., 2003
Selim, 2005
Bais et al., 2004
Bassarello et al., 2004
Cyclopeptides
Peptaibols
Cyclopeptides
Cyclopeptides
Antimicrobial peptides from plants

A broad family comprising antimicrobial peptides produced by plants is
called defensins. One of their functions in plants is the involvement in defence
mechanisms against pathogens, pests and abiotic stresses (Terras et al., 1995;
Lay and Anderson, 2005). These are basic peptides structurally and functionally related to the defensins of mammalia and insects. Molecular masses of
plant defensins that are characterized at present range from 5 to 7 kDa. Their
primary structure is formed with 4555 amino acid residues. Two structural
characteristics of the defensins, the presence of a pattern of conserved cysteine
residues and a domain with extremely variable amino acid sequence, are
typical components for mature peptide molecules of plant defensins. With
rare exceptions, defensins found in plants have a similar globular spatial
structure that is stabilized by a structural motif formed using disulde bonds
between eight cysteine residues. Possibly, defensins can aggregate in vivo
into dimers or oligomers (Terras et al., 1992). A certain correlation is observed
between the primary structure and antimicrobial activity of some plant
defensins. In general, the incorporation of basic amino acids (viz. arginine)
that add positive charge to a peptide molecule resulted in a considerable
increase in the antimicrobial activity (Terras et al., 1992; De Samblanx et al.,
1997; Landon et al., 2000).
In the overwhelming majority of cases, plant defensins possess inhibitory activity against fungi; however, the growth of Gram-negative bacteria is
also arrested after exposure to these peptides (Terras et al., 1993; Segura et al,
1998; Wong et al, 2006). An analysis of publications by Carvalho and Gomes
(2009) showed many damaging plant pathogens of economically important
crops can be inhibited in vitro with plant defensins. Various fungal species
(Alternaria brassicola, Alternaria solani, Botrytis cinerea, Cladosporium colocasiae,
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Cladosporium sphaerospermum, Colletotrichum lindemuthianum, Diploidia maydis, Magnaporthe grisea, Mycosphaerella arachidicola, Mycosphaerella fijinesis,
Nectria haematococca, Penicillium digitatum, Penicillium expansum, Phaeoisariopsis personata, Physalospora piricola, Rhizoctonia solani, Septoria tritici, Verticilium
albo-atrum, V. dahliae, and the toxigenic species Fusarium culmorum, F. decemcellulare, F. graminearum, F. oxysporum, F. verticillioides and Aspergillus niger);
two species of oomycetes (Phytophthora infestans and P. parasitica) and also
bacteria, viz. C. michiganensis and Ralstonia solanacearum, are among them.
Effective concentrations of plant defensins differ depending on the tested
peptides and the targeted pathogens (values of IC50, a protein concentration
that is required for 50% growth inhibition, vary from 1100 g/ml). The level
of antimicrobial activity may be regulated with bivalent ions (Terras et al.,
1992; 1993; Osborn et al., 1995; Segura et al., 1998; Wong and Ng, 2005). Along
with the growth inhibitory effect, some plant defensins cause morphological
changes in fungal mycelia (Carvalho and Gomes, 2009).
Studies on the mode of action of antimicrobial plant defensins are in
progress. There are conrmed hypotheses that an interaction with the cell
membrane of microorganisms resulting in ion efux and reactive oxygen
species (ROS) generation signicantly contributes to the mechanisms responsible for antifungal properties of plant defensins. Besides antimicrobial activity, plant defensins possess a range of biological functions (Lay and Anderson,
2005; Carvalho and Gomes, 2009).
The availability of antimicrobial peptides produced by plants or microorganisms through ribosomal synthesis for crop protection has been demonstrated by an increased disease resistance of transgenic plants expressing
fungal and plant defensin genes (Montesinos, 2007; Carvalho and Gomes,
2009). Several agricultural crops, e.g. tobacco, tomato, rice, aubergine, papaya
and canola, which are transformed with these genes and produce the corresponding peptides, have little or no disease development in laboratory,
greenhouse or eld experiments. A number of peptides produced by microorganisms are insecticidal or nematicidal. The ability of plant defensins to
inhibit -amylase and proteases can contribute to plant defence against pest
insects (Lin et al., 2007).
Enzymes, proteinase inhibitors, lectins and PR proteins
Lytic enzymes
To obtain nutrients, microorganisms synthesize various lytic enzymes that
can attack polymeric compounds of different origin. Biocontrol agents can
use these enzymatic activities on plant pathogens. Microbial chitinases, glucanases and proteases are lytic enzymes of most importance for the biocontrol of phytopathogens. These enzymes hydrolyse chitin, -glucans and
proteins, which can result in direct suppression of pathogen development or
generate products that function as resistance inducers. For instance, biocontrol isolates of Trichoderma harzianum and Trichoderma atroviride produce
endochitinase, -1,3-glucanase and alkaline proteinase, which degrade plant
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L.A. Shcherbakova
pathogenic fungi in vitro, halt their growth in planta and play a role in
mycoparasitism (Elad et al., 1982; Benitez et al., 1998; Lorito, 1998; Chernin
and Chet, 2002). Oligosaccharides or chitosan derived from fungal cell walls
exposed to microbial glucanases and chinases elicit a cascade of defence
responses in plants: generation of ROS, induction of pathogenesis-related
proteins (PR proteins) (including plant chitinases and glucanases),
phytoalexins and lignication (Dyakov and Ozeratskovskaya, 2007).
The potential of lytic enzymes for plant-disease management was well
demonstrated by studying chinolytic systems in the biocontrol bacteria Bacillus cereus, Pantoea agglomerans, Pantoea dispersa, and fungi, especially in the
widely used biocontrol fungus Trichoderma. Chitinases produced by Trichoderma are effective on virtually all chitinous pathogens, non-toxic for plants
and possess higher antifungal activity than such enzymes isolated from other
sources. The antifungal activity of chitinases from Trichoderma can reach the
level of some chemical pesticides (Lorito, 1998; Bonaterra et al., 2003; Chang
et al., 2003; Gohel et al., 2004).
Along with use of enzyme-producing biocontrol agents, there are several
other application strategies for chinolytic enzymes. The most conventional
approach consists of conferring resistance via engineering transgenic plants
containing heterologous chitinase and glucanase genes. Overexpression of
these genes in response to pathogen invasion can cause higher levels of the
enzymes in the plant cells followed by a faster and effective neutralization of
the pathogen. Indeed, transgenic broccoli, potato and tobacco plants expressing the T. harzianum endochitinase gene have been found to show resistance
against A. alternata, A. solani, B. cinerea and R. solani. Transgenic tobacco and
cabbage, carrying a bean chitinase gene were protected against R. solani.
Transgenic cucumber, rice, grapevine, strawberry and wheat transformed
with chitinase genes from rice (Oryza sativa) were resistant to B. cinerea,
R. solani, M. grisea, Sphaerotheca humuli and F. graminearum, respectively
(Gohel et al., 2006). Expression of exochitinase genes in transgenic apple trees
confers resistance to apple scab (Venturia inaequalis), a pathogen which is controlled by multiple applications of chemical fungicides during the growing
season (Bolar et al., 2000). These results show the broad potential for the
microbial chitinase transgenesis into plants for controlling fungal phytopathogens. The additional strategies are related to fermentation and different
ways of improving the enzyme producers (Gohel et al., 2006).
Other cases suggesting feasibility of crop protection with enzymes can
be illustrated by the examples of constructing transgenic potato carrying
glucose oxidase gene from A. niger or apple, potato and tobacco plants
expressing the bacteriophage T4 lysozyme gene (Wu et al., 1997). Glucose
oxidase is an enzyme involved in generating plant ROS. Expression of the
glucose oxidase gene led to accumulation of peroxide ions in plant tissues
that increased resistance to fungal diseases, e.g. to late blight (P. infestans),
wilt (Verticillium dahliae) and early blight (A. solani). Lysozymes are widespread enzymes that hydrolyse peptidoglycan of bacterial cell walls. Apple
plants with the T4L gene showed signicant resistance to the re blight agent
E. amylovora (Ko et al., 2000), while potato and tobacco was resistant to
117
E. carotovora subsp. carotovora (During et al., 1993). However, lysozyme excretion can have adverse effect on soil microbiota. Thus, the growth of B. subtilis
has been observed to be suppressed in rhizosphere transgenic T4 lysozymeproducing potato plants (Ahrenholtz et al., 2000).
Lectins
Plant lectins are a heterogeneous collective of proteins that specically bind
carbohydrates in a reversible way and take part in phytopathogen recognition.
Interacting with like components, lectins can attach to the cell surface. The
structure and functions of these compounds are discussed in detail (e.g.
Chrispeels and Raikhel, 1991). Lectins are well known naturally occurring
insecticides of widespread effect. Some free lectins strongly affect microbe
growth in plants and probably contribute to inhibiting pathogenesis. There are
chitin-specic lectins synthesized in the phloem and translocated via vessels.
These ndings suggest that lectins are potential antifungal agents.
Proteinase (protease) inhibitors of plant origin
Plant inhibitors of proteinases are a large group of peptides or small proteins
able to bind proteolytic enzymes of different organisms with competitive
inhibition of their activity. In plants, they are abundant in seeds and storage
organs, where their content can be up to 10% of water-soluble proteins. These
compounds are considered as reserve proteins and regulators of protein status or enzyme activity in plants. The proteinase inhibitors differ in substrate
specicity, have various isoforms, and their oligomers can combine or
dissociate with an inuence on the inhibitor properties.
A defensive function of proteinase inhibitors towards insects was initially
revealed when insects, after feeding, became inactive as a result of trypsin
inactivation. Protease inhibitors of plant origin were also shown to be active
against plant pathogenic nematodes. Since many phytopathogenic fungi and
bacteria secrete extracellular proteolytic enzymes, which play an important
role in pathogenesis (Valuyeva and Mosolov, 2004), plants use inhibition of
such enzymes as a defence strategy towards these microorganisms (Ryan,
1990; Habib and Khalid, 2007).
There are now ample data on protease inhibitors effective against phytopathogens in vitro and in vivo. For instance, inhibitors from potato inactivate
proteinases secreted by F. solani or F. sambucinum into cultural liquid. Inhibitors from buckwheat and pearl millet suppress spore germination and the
growth of many fungi including Aspergillus flavus, Aspergillus parasiticus,
F. moniliforme, F. oxysporum, A. alternata and Trichoderma reesei. Inhibitors of
proteinases are accumulated in response to pathogen invasion and prevent
disease development (e.g. in tomato inoculated with P. infestans). In some
cases, correlation between disease resistance and the constitutive inhibitors
is found (e.g. between wheat resistance to smut, or lupine and soybean to
fusarial wilt). Cells of potato tubers treated with elicitors, such as salicylic or
arachidonic acids, are able to excrete potatin and three chymotrypsin
inhibitors (Habib and Khalid, 2007).
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L.A. Shcherbakova
At least 14 genes encoding different protease inhibitors alone or in

combination with other heterologous genes have been reported to be transferred into cultured plants, which showed increased resistance predominantly to insects (Valuyeva and Mosolov, 2004). Plant protease inhibitors
have also been used to engineer resistance against viruses in transgenic
plants. For example, expression of the gene encoding the cysteine proteinase
inhibitor from rice, oryzacystatin, by transgenic tobacco was found to confer
resistance against tobacco etch virus and potato Y virus, replication of which
depends on cysteine proteinase activity (Valuyeva and Mosolov, 2004; Habib
and Khalid, 2007).
In the green consumerization context, it is important that some of the
available proteinase inhibitors are inactive for non-pathogenic microorganisms and do not inhibit activity of proteinases of animal origin. Thus, chestnut cystatin, which strongly inhibits the protease activity and the growth of
pathogenic B. cinerea, Colletotrichum graminicola, and Stagonospora nodorum,
has no effect on the protease activity and the growth of the saprophyte Trichoderma viride (Pernas et al., 1999), and a proteinase inhibitor extracted from
bean seeds specically suppresses serine proteinase of pathogenic C. lindemuthianum but does not inuence animal trypsin and chymotrypsin activity
(Valuyeva and Mosolov, 2004). Non-specic antipathogenic activity of protease inhibitors suggests that transgenic crops producing inhibitors of insecticidal or nematicidal proteinases may be incorporated into integrated
systems of plant protection against pests and pathogens. A further advantage of this approach is the possibility that inhibitory activity against proteinases could be combined with the insecticidal activity of lectins, resulting in a
synergistic antipathogenic effect.
PR proteins
Plants have numerous defence mechanisms that are activated in response to
pathogen attacks, abiotic stresses and chemicals that mimic pathogen
challenge. Production of proteins related to pathogenesis (PR proteins) is one
such inducible mechanism (Van Loon et al., 1994; Van Loon et al., 2006). These
proteins are not detectable at all or are present only at basal concentrations
in healthy plant tissues. Since the term PR proteins based on the above
characteristics was often used for all constitutive plant proteins for which
content or increasing activity was induced by microorganisms, the new term
inducible defence-related proteins was recently introduced (Van Loon et al.,
2006).
PR proteins consist of a large variety of families with members that differ
in occurrence, expression and biological activities; they are divided into 17
classes (Sels et al., 2008). A range of the above-mentioned plant proteins and
peptides such as chitinases, -1,3-glucanases, peroxidase, defensins and proteinase inhibitors are PR proteins and vice versa; some typical PR proteins
are antimicrobial and inhibit pathogen growth in vitro. For instance, tobacco
PR-1a is antifungal, and tomato proteins of the PR-1 family inhibit zoospore
germination and reduce pathogenicity of P. infestans. The ability to disrupt
119
fungal membranes has been shown for toumatin-like proteins (permatins) of

the PR-5 class. Overexpression of PR protein genes in plants renders disease
resistance. For example, the high level expression of PR-1 in transgenic
tobacco plants promotes control of Perenospora tabacina and Phytophthora
nicotiana (Dyakov and Ozeratskovskaya, 2007).
Biochemical functions of PR proteins, their role in defence mechanisms,
engineering of transgenic plants with enhanced resistance to plant pathogens and characteristics of individual PR proteins have been comprehensively surveyed by many researchers (Van Loon, 1985; Loon and Van Strien,
1999; Van Punja, 2001; De Lucca et al., 2005; Edreva, 2005; Van Loon et al.,
2006; Sels et al., 2008).
Proteinic inducers of plant resistance as a promising strategy for green

consumerization
The protective effect of some natural compounds against plant diseases
results from the induction of plant resistance to pathogens rather than from
biocidal activity towards the causative agents. This strategy is worthy of special consideration because it avoids a direct effect on a pathogen and involves
a plant-mediated mode of action. This activates the natural defence responses
of plants, minimizing the probability of the targeted pathogens developing
resistance.
Active defence mechanisms are initiated in plants upon recognition of
structural and chemical characteristics particular to a pathogen, collectively
referred to as pathogen-associated molecular pattern (PAMPs). The PAMP
components represented by compounds of different chemical origin were
named as general elicitors (or general inducers). These elicitors initiate a conserved set of plant defence responses such as ROS production, deposition of
callose, protein phosphorylation, and transcriptional activation of early
response genes, resulting in PAMP-triggered immunity or induced resistance. Natural compounds conferring disease resistance on plants in the
manner of general elicitors is of interest because of the non-specic character
of the induced resistance lots of pathogens can be controlled with one active
compound. The success of applying elicitor compounds is directly dependent on understanding their properties and mechanisms of action. As well as
all other biogenic or abiogenic, general or specic inducers, proteinaceous
elicitors produced by plant pathogens (e.g. glicoproteins, agellins, elongation factor Tu, elicitins and transglutaminases from Phytophthora spp., proteins and peptides from Cladosporium flavum, monolicollin, cold shock
proteins, harpins etc.) are the subject of genetic and biochemical research of
signalling pathways and molecular mechanisms underlying plant resistance
to diseases. Non-protein small compounds (plant signalling molecules and
hormones) participating in signalling as well as chemical compounds that
have been found to mimic elicitors are also being studied in this way. Transgenic plants with genes encoding microbial elicitors (e.g. elicitins) are created
for research purposes using biotechnological methods.
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Regarding the application, general elicitors to plant protection, chitosan,

arachidonic, salicylic and -aminobutyric acids may be referred to as examples of active ingredients that have been formulated and introduced into
agricultural practice as commercial biogenic non-protein inducers of plant
resistance. For instance, a supplement of tomato growth substratum with
chitosan suppressed the root rot caused by F. oxysporum f. sp. radicis-lycopersici (Lafontaine and Benhamou, 1996). Moreover, several chitosan formulations collectively known as chitozars, which are effective against root rots,
late blight and powdery mildew on cereals, potato and legumes, have been
included in biological protection systems of these crops in Russia. Among
protein elicitors, bacterial harpins are the most important. Harpins (hypersensitivity response and pathogenicity) are components of a transport system used by bacteria to transfer proteins through bacterial and plant
membranes. In the bacterial genome, harpins are products a hrp-gene cluster.
One of the rst harpins studied was HrpN, the product of hprN gene
expressed in E. amylovora. HrpN has been shown to be an acidic glycine-rich
thermostable protein with a molecular mass of 44 kDa that induces resistance
in Arabidopsis (Wei and Beer, 1996). Harpin has been commercialized and
marketed as a product that enhances resistance of some eld, ornamental
and vegetable crops to many diseases, and that stimulates plant growth and
owering.
In the coming years, we can expect the agricultural use of other elicitor
proteins for increasing plant resistance to diseases both by engineering of
plants containing elicitor protein genes and by exposure of plants to protein
elicitor-based products. Two bacterial proteins with plant-protecting activity
are considered below as examples of very promising compounds for developing both resistant transgenic crops and products for plant treatment.
Cold shock protein CspD as an elicitor of disease resistance in plants
Cold shock protein D (CspD) isolated from Bacillus thuringiensis culture broth
is a thermostable low molecular weight protein (molecular mass 7.2 kDa).
The B. thuringiensis gene encoding CspD was cloned and sequenced (CspD, #
AY272058 in GenBank). Nucleotide and amino acid sequences of CspD show
high homology with other bacterial CSPs (Dzhavakhiya et al., 2000, Kromina
and Dzhavakhiya, 2004).
CSPs are highly conserved proteins produced by various bacteria constitutively or in response to cold shock. They have also been studied in plants,
e.g. in Arabidopsis, tobacco and wheat. CSPs are involved in cell growth and
adaptation to low temperatures. They bind nucleonic acids and are considered to be putative translation anti-terminators. Bacterial CPSs contain a specic conserved cold shock domain (CSD), which occurs in CSPs from other
organisms. For instance, a fragment of high homology to bacterial CSD was
found in a family of plant CSPs referred to as glycine-rich proteins (GRPs).
Expression of grp-genes is dependent on the level of plant hormones (indolyl
acetic, salicylic, abscisic acids), illumination and the plant development
phase, and is upregulated by cold and wounding. The synthesis of GRPs can
121
also be enhanced upon pathogen attack both in compatible and incompatible

plantpathogen combinations. Alien CSPs are recognized by plants as components of PAMP and elicit resistance to some pathogens. Thus, CSPs from
Micrococcus lysodeikticus non-specically induced defence responses in
plants. It was found that a peptide consisting of 15 amino acid residues
(csp15) that represented a consensus sequence of RNA-binding PNP-1 and
RNP-2 motifs was responsible for eliciting activity towards some Solanaceae
plants (Felix and Boller, 2003). The peptide csp15 induced an oxidative burst
in tobacco (Nicotiana tabacum, cv. Havanna 425) and potato (Solanum
tuberosum), but was ineffective towards rice and cucumber cells.
CspD from B. thuringiensis has been reported to induce resistance both in
monocotyledonous and dicotyledonous crops against a wide range of pathogens: from lamentous microorganisms (fungi and oomycetes) to viruses
(Dzhavakhiya et al., 2000). For instance, CspD applied by dropping onto
wheat leaves or by spraying potato and rice seedlings with water- or bovine
serum albumin (BSA)-stabilized CspD solutions produced resistance against
S. nodorum, P. infestans and M. grisea, respectively. Effective crop protection
was observed in both greenhouse and eld experiments. Exposure of tobacco
plants to CspD induced resistance to tobacco mosaic virus (TMV) and potato
X-virus. Peptide csp15 of CspD protein (VKWFNAEKGFGFITP) also showed
resistance-inducing activity on plants and in model plantpathogen systems.
Treatments of tobacco leaf halves with 110 mol csp15 in 0.1% BSA a day
before inoculation with TMV resulted in a drastic reduction of lesion spot
number on the treated halves as compared to 0.1% BSA-treated (control)
halves or whole control leaves of the same plant (Kromina and Dzhavakhiya,
2004).
Several tests were carried out to identify which host defence responses to
the pathogen challenge are activated by CspD and csp15. Applying csp15 to
the surface of discs cut from potato tubers (cv. Istrinskiy, R1) increased the
hypersensitive response of potato cells to the incompatible P. infestans race r4
with a decreased number of dead cells and induced accumulation of salicylic
acid in the tuber tissues. Both CspD and csp15 have no fungitoxicity. Addition of the peptide to the suspension of cultured tobacco cells activated the
H+ pump and caused a reversible change in the extracellular pH. The resistance induced with csp15 or CspD in plants has a systemic character (Kromina
To transfer the CspD gene into tobacco plants, the pBilt7 plasmid, containing the CspD expression cassette (P35S/CspD/pACaMV), on pBin19 vector
background was constructed and transformed into A. tumefaciens. As a result,
seven lines of cv. Xanthi (NN) and ve lines of cv. Samsung (nn) were produced. Expression of CspD in these lines was conrmed by real-time PCR.
The transgenic tobacco plants had the same habitus as control plants and
produced fertile projeny. The transgenic lines showed increased resistance to
Alternaria longipes and TMV that coincided with the range of antipathogenic
activity observed for the elicitor protein per se. Importantly, the level of CspD
expression coincided with the resistance level to the both pathogens among
all tested lines as well as inside any one line. These ndings lead to the
122
L.A. Shcherbakova
assumption that disease resistance of transgenic tobacco is due to defence

responses that are elicited with CspD synthesized inside the plant (Kromina
The above results can be used in breeding programmes for production of
crop cultivars with resistance against a wide range of phytopathogens or
CspD-based biopesticides providing complex protective effects against fungi,
oomycetes and viruses.
MF3 protein, bacterial peptidyl-prolyl cis/trans isomerase, conferring plant
resistance to pathogens
The thermostable protein MF3 (16.9 kDa) is produced by biocontrol Pseudomonas fluorescens isolate 197. After cloning and sequencing the MF3-encoding
gene from P. fluorescens 197, a full primary structure of the protein was
determined, and high homology between amino acid sequences of MF3 and
peptidyl-prolyl cis/trans isomerases of the FK506-binding protein (FKBP)
type was found (Dzhavakhiya et al., 2005; Shumilina et al., 2006).
Peptidyl-prolyl cis/trans isomerases (PPIases) are enzymes that accelerate the slow cis/trans isomerization of the peptide bond between proline residues and the adjacent amino acid in protein. Initially, de novo synthesized
polypeptides have the trans conformation, which stabilizes the polypeptide
chain, but to form the native protein structure, about 7% of the peptide bonds
formed with proline residues need to be isomerized into the cis conformation. PPIases take part in the renaturation of denatured proteins, in protein
synthesis de novo and in the formation of biologically active conformations of
some polypeptides. PPIases are highly conservative enzymes produced by
prokaryotes, eukaryotes, viruses and phages. They have been classified into
three families, referred to as cyclophilins, FKBPs, which regulate signal
systems of eukaryotic cells, and parvulins.
In plants, FKBPs were initially detected in Vicia fava, Triticum aestivum
and Arabidopsis thaliana. It was found that A. thaliana contains 23 FKBPs and
FKBP-like low- or high-molecular weight proteins. Plant PPIases of the FKBP
type control plant fertility, cell division and differentiation as well as the perception of plant hormones. Plant FKBPs are expressed both constitutively
and in response to various stresses such as wounding, salinity or heat shock.
PPIases produced by phytopathogens can contribute significantly to the
pathogenic process. For instance, the plant pathogenic fungi M. grisea and B.
cinerea produce cyclophilins. Mutations in the cyclophylin genes impair virulence of these fungi. The M. grisea mutants form low-turgor appressoria
unable to penetrate through leaf cuticle. Deletion of the cyclophylin gene
does not influence M. grisea growth in vitro but affects formation of fungal
conidia (Viaud et al., 2002).
It is likely that PPIases are involved in plantpathogen relationships.
On the one hand, some pathogens are in need of host plant PPIases for establishing the infection process. On the other hand, interaction of plant PPIase
with pathogen proteins can result in induction of disease resistance. Thus
cysteine proteinase AvrRpt2, that is transported by Pseudomonas syringae to
123
Arabidopsis cells during pathogen invasion must be folded with the plant
cyclophylin ROC1 to form an active conformation able to cleave plant
proteins. Along with other proteins, the activated bacterial protease cleaves
protein RIN4, which is combined in Arabidopsis in an inactive complex with
a signal protein RPS2. Proteolysis of RIN4 releases RPS2 that triggers defence
responses (Mackey et al., 2003). PPIases of one organism have been reported
to inhibit the growth of another organism by means of competitive binding
with a receptor, and probably some PPIases can suppress growth of cultured
plant pathogens. For instance, cyclophylin C-CyP isolated from Chinese
cabbage (Brassica campestris L. ssp. pekinensis) inhibits in vitro growth of
several fungi including Rhizoctonia solani, B. cinerea, F. solani and F. oxysporum
(Lee et al., 2007).
Various experiments have shown that MF3 induced resistance in various
monocotyledonous and dicotyledonous plants against several fungal and
viral pathogens. MF3 was found to protect tobacco against TMV, potato Y
virus and A. longipes, and barley against Bipolaris sorokiniana. At the same
time, this protein did not influence TMV infectivity, was not fungitoxic and
had no phytotoxicity towards tobacco plants and cereals. Moreover, treatment of barley and wheat seeds, naturally infected by B. sorokiniana and
F. culmorum, with MF3 promoted formation of a well-developed root system
in the diseased barley and wheat seedlings, that conferred plant tolerance to
root rots and was consistent with growth-stimulatory functions for PPIases
in plants (Dzhavakhiya et al., 2005; Shumilina et al., 2006).
Comparative analysis of MF3 with 45 proteins of different homology levels revealed two conserved sequences in the MF3 polypeptide chain. Proteolysis with trypsin inside one of these conserved sequences produced
fragments that did not induce resistance to TMV in tobacco leaves. These
data allowed the assumption that the analysed sequence contained a motif
responsible for the resistance-inducing activity of MF3. Such a motif,
IIPGLEKALE GKAVGDDLEVAVEPEDAYG, was detected and named MF329 because it was found to consist of 29 amino acid residues. This fragment
was necessary and sufficient for induction of tobacco resistance to TMV. Biological tests on isolated tobacco leaves showed that treatments of tobacco
leaves with chemically synthesized oligopeptide MF3-29 at concentrations
0.5, 5 and 50 nM were as effective against the virus as the whole protein at the
same concentrations.
In order for products based on protein elicitors to effectively control
plant pathogens, they have to access plant receptors recognizing PAMPs. The
large size of the molecules or hydrophobic barriers on the plant surface (such
as cuticle) can impede physical contact or chemically mediated recognition
of elicitor proteins and the subsequent induction of defence responses in
plants. To solve this problem, special molecular carriers facilitating elicitor
transport to plant cells should be developed. Various polycationic molecules,
especially chitosan, which are used now for the delivery of large biological
molecules (DNA or proteins) to their outer or intracellular receptors, look
promising as putative carriers of proteinaceous elicitors. Experiments with
the wheat leaf spot agent S. nodorum and turnip mosaic virus (TuMV)
124
L.A. Shcherbakova
demonstrated that MF3 integration with chitosan, a non-toxic and

biodegradable natural polymer with elicitor properties, enhanced resistanceinducing activity of MF3, and enlarged the scope of the antipathogenic action
of this protein. No inhibitory effect on fungal growth in vitro was observed if
MF3, chitosan or the MF3chitosan complex was added to culture media.
Neither the protein nor chitosan applied separately on wheat leaves protected plants against S. nodorum. However, leaf treatment with the MF3
chitosan complex before plant inoculation with the pathogen signicantly
reduced disease severity. Spraying cabbage seedlings (cv. Krautman) with
the MF3 chitosan-complex resulted in at least a week's delay in TuMV accumulation, whereas virus spread was not delayed in seedlings sprayed with
MF3 alone or chitosan alone (Shumilina et al., 2005).
Rape lines (cv. Westar) with an mf3 gene insertion showed increased
resistance to fungal pathogens Plasmodiophora brassicae and TuMV. Most of
the transgenic plants carried more than one gene insertion. The bacterial protein concentrations in some lines amounted to 37 pg/mg of fresh weight. An
increased level of rape resistance to TuMV was kept independently of MF3
variation in plant tissues. Identically high resistance was observed at eight
different protein concentrations in transgenic lines. This may suggest that
tiny amounts of MF3 are sufcient for conferring plant resistance to the virus.
It was shown that ve transgenic lines of the six that were resistant to TuMV
also expressed an enhanced endurance to P. brassicae for 50 days after inoculation. The disease symptoms were signicantly less severe on roots of transgenic plants as compared to symptoms developing on non-transgenic rape
roots or roots of transgenic lines that had lost the mf3 insertion. It is believed
that the best lines obtained may be included in breeding programmes for
creating rape cultivars with resistance against both pathogens (Dzhavakhiya
et al., 2005; Shumilina et al., 2006).
Thus, the wide range of elicitor activity of CspD and MF3, as well as the
possibility to use general approaches of biocontrol, gene engineering technology and plant treatments in CspD- and MF3-based plant protection demonstrate the great potential of proteinaceous elicitors of natural defence
mechanisms for the environmentally safe control of phytopathogens. Toxicology studies and in-house laboratory tests demonstrated that the proteins
are non-toxic to animals and plants. Both of these compounds are patented
as new proteins with plant protecting properties (EP0868431A1 and PCT
WO2005/061533 A1). Due to the systemic character of the resistance induced
with CspD and MF3, the resistance spreads to the whole plant even if only
the seeds or leaves are treated. After a single treatment with the protein solutions, induced resistance remained effective for not less than 3 weeks. In
many cases this can cover the period of highest risk infection. The large-scale
introduction of transgenic cultivars resistant to a certain pathogen is sometimes limited because different pathogens dominate when the climatic and
soil conditions change. Moreover, there is the problem of unpredictable seasonal alteration of pathogens, which decreases the effect of pre-sowing treatments. Crop engineering or/and developing new products based on general
elicitors such as CpsD or MF3 inducing universal resistance would promote
125
overcoming these limitations. Searching for novel proteins and peptides

would promote progress in this eld. Finding elicitor activity in MF3 protein
belonging to PPIases that were never meant to be designated as disease resistance inducers shows desired properties may be discovered during screening of natural biologically active compounds which were previously studied
and characterized as non-hazardous for people and the environment. This
presents additional prospects for developing new biocontrol products
complying with green consumerization requests.
5.3 Statins as New Promising Candidates for Biopesticides

One of the effective plant disease management strategies is based on the
control of metabolic relationships in a plantpathogen system. This can be
reached by using compounds that alter or partially block specic pathways
of biosynthesis in plants or microorganisms, resulting in derangements in
the trophic relationships of pathogens with host plants, or in the production
of microbial metabolites related to pathogenicity. Such imbalances may not
cause the rapid death of pathogens but can lead to a reduction of their vital
capacity or/and impair their pathogenic properties. Similar metabolic effects
may be caused by plant inhibitors of fungal and bacterial proteases as
described above, as well as by the inhibitors of insect and nematode enzymes.
Statins, secondary metabolites of microbial origin belonging to the polyketide
group, represent another class of natural compounds that have recently
been discovered to modulate metabolic plantpathogen interactions. The
metabolic pathway targeted by statins is sterol biosynthesis.
Sterols are well known to be ubiquitous and important components of
outer membrane and intracellular membranes of eukaryotic organisms and
to play essential roles in their physiology. These compounds are required for
growth, development and reproduction of plant pathogens. A number of
organisms including insects, nematodes and oomycetes are not able to synthesize sterols or even their precursors. Sterol-dependent phytopathogens
obtain free sterols or intermediates from host plants and may be controlled
by compounds inducing alterations in content, availability or composition of
plant sterols.
Currently, several chemical fungicides are available that inhibit the synthesis of ergosterol in fungi. They prevent cellular membrane formation, stop
fungal growth and may suppress sporogenesis in established infection
agents. However, statins are natural inhibitors of sterol biosynthesis that presumably can cause similar effects. Statins were found to be produced by
different microorganisms, predominantly by lamentous fungi. They inhibit
the enzyme -hydroxy--methylglutaryl-CoA reductase (HMG-CoA reductase) and prevent sterol biosynthesis at the level of conversion of HMG-CoA
to mevalonic acid. Statins are widely applied in medicine as drugs against
atherosclerogenic diseases because they effectively decrease the blood cholesterol level. Thus, the biological compatibility and safety of statins are well
proven.
126
L.A. Shcherbakova
Recently, studies were made to investigate the potential of two statins,

lovastatin and compactin, in plant protection against diseases (Dzhavakhiya
and Petelina, 2008; Ukraintsteva, 2008). In these studies, both statins were
obtained by means of microbial synthesis using the superproducer strains
Aspergillus terreus 45-50 (lovastatin) and Penicillium citrinum 18-12 (compactin); these statins were examined in greenhouse and eld experiments with
plant treatments and in vitro tests on several pathogenic fungi. As statins are
insoluble in water, the authors used aqueous solutions of the statin sodium
salts that are referred to below as compactin and lovastatin.
The studied statins were found to possess fungicidal activity in vitro
against a number of plant pathogenic fungi. The addition of lovastatin at
concentrations from 0.001 to 0.1% to agar media inhibited growth of M. grisea,
S. nodorum and Coletotrichum atramentarium. Compactin arrested in vitro
growth of Cladosporium cucumerinum, S. nodorum and M. grisea at the same
concentration range. In addition, 0.001% compactin solution signicantly
reduced germination of S. nodorum spores while the 0.01% solution completely suppressed spore germination in this fungus. Among tested plant
pathogens, the fungus M. grisea, rice blast agent, showed the highest sensitivity to both statins (for lovastatin IC50 was about 0.003%), while S. nodorum
was most resistant to lovastatin (IC50 = 0.02%) and C. cucumerinum tended to
compactin resistance. Besides a growth-inhibitory effect, the presence of the
statins in nutrition media resulted in a discolouration of fungal mycelia in all
fungi, especially in M. grisea and C. atramentarium exposed to lovastatin. This
observation suggested suppression of fungal melaninogenesis. In all experiments, dicolouration-inducing concentrations of the statins were at least
tenfold lower than growth-inhibiting concentrations.
The studied statins were revealed to possess disease-preventing properties. They delay and decrease disease development on treated plants when
applied simultaneously or prior to inoculation with pathogens. Lovastatin
and compactin protected wheat against S. nodorum and tobacco from A. longipes. In addition, lovastatin showed protective activity against M. grisea on
rice, whereas compactin was effective against Puccinia graminis on wheat and
P. infestans on potato. Both fungicidal and protective effects were strongly
dose-rate dependent, but fungitoxic concentrations also made a toxic impact
on plants, whereas protection from diseases was provided by far lower concentrations that were non-phytotoxic. Thus, almost total or 2736% S. nodorum growth inhibition in vitro was observed at 0.1% and 0.01% lovastatin
concentrations in nutrition media, respectively. Both doses showed phytotoxicity on wheat leaves, but only the higher concentration caused retardant
effect on growing plants. When 0.1% lovastatin solution was used for wheat
seed soaking for 1.52 h, reduction of seedling length averaged 50%, and
0.01% concentration caused no inhibitory inuence on plant growth. A considerable reduction of the disease index was found after application of only
0.0005% lovastatin solution on isolated wheat leaves. The applied concentration did not suppress S. nodorum germination and in vitro growth but prevented disease development with 94 and 72% protection efcacy at 3 and
7 days after inoculation, respectively. This suggests that a mechanism other
127
than fungicidal activity contributes to the protective effect of statins

(Dzhavakhiya and Petelina, 2008).
The resistance of fungi to extreme environmental conditions and biotic
stresses is largely determined by their ability to produce protective highmolecular-weight pigments. One of the common fungal pigments in cell walls is
melanin. Melanin is a coloured polymer produced by many plant pathogenic
fungi, including M. grisea, S. nodorum, C. lagenarium and C. atramentarium,
through the pentaketide pathway and depends on the availability of acetylCoA. Melanin plays a signicant role in the infectivity of these fungi. For
instance, the ability of the rice pathogen M. grisea to penetrate into tissues of the
host plant is directly associated with the presence of melanin in the fungus.
Strains of M. grisea and C. lagenarium defective in melanin formation lose pathogenicity and are incapable of forming mycelial overgrowth in host plants. Revertants restoring wild-colour type regain pathogenicity (Dzhavakhiya et al., 1990).
Interestingly, antimelanogenic compounds, aminoalkylphosphinates, a
family of phospho-analogues of natural amino acids, suppress biosynthesis
of melanin and some toxic metabolites in the polyketide pathway of M. grisea,
and are also fungicidal. The aminoalkylphosphinates serve as analogues of
alanine, a precursor to pyruvic acid that is required for melanin biosynthesis.
Exposure of fungi to the phospho-analogues of amino acids inactivates pyruvate dehydrogenase, thus inhibiting synthesis of acetyl-CoA and melanin
(Zhukov et al., 2004) as well as aatoxins (Khomutov, Khurs, Shcherbakova,
Mikityuk, Dzavakhiya and Zhemchuzhina; unpublished data). The discovery that non-fungicidal lovastatin and compactin concentrations can induce
mycelium de-pigmentation and decrease disease severity on plants suggests
that their protective effect may be associated with impairing pathogenicity
due to an effect on melanin biosynthesis in causative agents. Statins and fungal melanin are both polyketides. Thus, the metabolism of the two compounds is interrelated, and it is not improbable that statins can negatively
mediate a stage of the polyketide pathway involved in the melanization of
plant pathogenic fungi.
Although the mode of protective action of statin is required for understanding and further research, rst small-plot eld trials suggest they may be
of certain interest from a practical point of view. For example, one preplanting treatment of potato tubers by soaking in 0.1% compactin solution for
half an hour resulted in a 1-month delay of late blight (P. infestans) emergence
on plants and a slower course of disease. Only extremely high statin concentrations of 0.5% undesirably inuenced plant physiological characteristics. By
the end of the growing season, an insignicant reduction of potato late blight
was observed on plants arising from the treated tubers, but these plants
produced fewer diseased tubers. There is reason to suppose that lovastatin
and compactin possess anti-phytoviral activity (Ukraintseva, 2008).
Further research of the mechanisms responsible for protective activity
could help statins take a tting place among the biopesticides of tomorrow.
Lovastatin and compactin per se might serve as base molecules for biochemical engineering of active analogues, and the approaches used to studying
their plant-protecting properties might be implicated in screening of other
128
L.A. Shcherbakova
pathogen-controlling compounds within the statin group and their derivatives. Such screening might lead to the detection of new natural polyketides
with high protective activity that would not have an adverse effect on plants.
Since the agricultural application of statin-based formulations does not
demand as high a level of statin purication as that in the pharmacological
industry, using a relatively simple isolation procedure (Dzhavakhiya, 2008)
and the superproducer strains suggests that statin-based plant-protection
technology would be a more economical and ecologically safe strategy than
any method using chemical pesticides.
5.4 Conclusion
The disease-preventing effect of natural compounds is a result of a direct or
plant-mediated inuence on targeted pathogens. The compounds can affect
phytopathogens directly, interrupting pivotal metabolic pathways and
results in the death of the pathogens (biocidal effect). Some natural substances or their analogues specically inuence pathways related to pathogenicity and toxigenesis. In the case of a plant-mediated mode of action,
natural compounds elicit and activate defence responses in plants that result
in induced resistance to diseases. In addition, some biogenic compounds
inuencing plants do not induce resistance but enhance tolerance to pathogens by improving the physiological state of plants. The structural and functional diversity of natural compounds as well as their abundance provide a
great potential for bringing plant-protection technologies in line with green
consumerization.
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Doklady Biochemistry and Biophysics 397,
210212.
Natural Products as
Allelochemicals in Pest
Management
ROMAN PAVELA
Crop Research Institute, Prague, Czech Republic
Abstract
Allelochemicals are one of the most plentiful groups of substances in the vegetable
kingdom. The signicance and use of these chemicals in nature are as varied as allelochemicals themselves. Allelochemicals also include a group of substances called allomones. These substances are created by plants as a defence against phytophagous
insects and comprise repellents, anti-ovipositants and antifeedants. This group of
plant metabolites can be of practical use in many areas of human activity. This chapter
deals with possibilities of using plant extracts containing allomone in the protection
of plants against pests. Substances with antifeedant effects, in particular, appear to be
highly promising for the development of new, environmentally safe insecticides. In this
chapter, we have therefore focused mostly on this group of substances and critically
considered the perspective of using antifeedant substances in plant protection.
6.1 Introduction
Plants have developed alongside insects since the very beginning of their
existence. Thanks to their common history, very fragile mutual relationships
have formed between individual plant and insect species, which we are
trying to clarify today using scientic approaches. On the one hand, plants
utilize insects, especially as their pollinators, and on the other, insects use
plants as the source of their food. Therefore, for balanced mutual interactions
between plants and insects, communication and mutual inuence must exist
between them to prevent uncontrolled excessive reproduction of any species
in order to maintain equilibrium in the ecosystem with maximum possible
biodiversity. Plants thus created many strategies in the course of their
co-evolution to protect themselves efciently against insect pests, and the
insects try to circumvent such strategies. Understanding their mutual relationships helps us not only to understand the world around us but also
provides information that may lead to a practical use.
134

(ed. N.K. Dubey)
Allelochemicals in Pest Management
135
As mentioned above, there are many types of plantinsect interactions;

however, the interaction between plants as a source of food and phytophagous insects as their pests is one of the most interesting and important ones
for practical applications in agriculture. In this case, it is a relationship between
food on one hand and consumer on the other; the plants were forced to
develop numerous defensive mechanisms to prevent uncontrolled destructive pest attacks. The most important defensive plant mechanisms include the
synthesis of biologically active compounds, the so-called secondary metabolites. First, such substances may provide direct insecticide effects, causing
mortality of phytophagous insects, and/or second, they may exert indirect
insecticide effects, only inuencing insect behaviour in some manner. Knowledge of such relationships leads not only to useful information necessary for
cultivating plants with resistance, but also to the direct utilization of extracts
in cultural plant protection using the so-called botanical insecticides.
Although the rst group of compounds, i.e. those with direct insecticidal
activity, has been used by humans as extracts for millennia, both in ghting
phytophagous pests as well as against parasites or storage pests, the other
group of compounds, generally called allelochemicals, has become the
subject of more profound interest only in recent decades (Pavela, 2007a).
The term allelochemicals (from Greek allelon: one another) is used to
describe the chemicals involved in interspecic interactions. It is dened as
a chemical signicant to organisms of a species different from its source.
Allelochemicals are divided into four subgroups, depending on whether the
emitter, the receiver, or both benet in the interaction.
An allomone (from Greek allos another; horman to stimulate) is

dened as a chemical substance, produced or acquired by an organism,
which evokes in the receiver a reaction adaptively favourable to the
emitter, e.g. a plant emits allomones to deter herbivores.
A kairomone (from Greek kairos opportunistic) is dened as a
chemical substance, produced or acquired by an organism, which evokes
in the receiver a reaction adaptively favourable to the receiver but not to
the emitter, e.g. secondary plant compounds help herbivores in nding
plants to feed on.
A synomone (from Greek syn with or jointly) is dened as a chemical
substance, produced or acquired by an organism, which evokes in the
receiver a reaction adaptively favourable to both the emitter and the
receiver. This group of allelochemicals includes oral scents and nectars
that attract insects and other pollinators and substances that play an
important role in symbiotic relationships.
An apneumone (from Greek a-pneum breathless or lifeless) is dened
as a substance, emitted by a non-living material, which evokes a reaction
adaptively favourable to the receiving organism, but detrimental to
another organism that may be found in or on the non-living material.
For example, parasites or predators are attracted to non-living substances in which they may nd another organism, their host or prey, by
apneumones released from the non-living substance.
136
R. Pavela
In many cases not a single semiochemical has an effect on its own but
different groups of chemicals in a precisely dened mixture act in an effectively combined manner. In general, it can be said that the whole group of
allelochemicals may nd its application in plant protection. Nevertheless,
metabolites falling in the allomone group represent the most studied group
with the most potential at present.
6.2 Allomones A Prospective Group of Substances for

Alternative Plant Protection
As explained above, this group of substances includes plant metabolites that
have some type of negative effect on insect behaviour providing benet to
the plants. The chemicals can be divided as follows according to their mode
of action:
1.
2.
3.
Repellents
Anti-ovipositants
Antifeedants
Repellents are substances that directly deter insects from settling on the
plant. They include an entire group of simple aromatic hydrocarbons, which
may be released into the environment, thus having a direct effect on insect
chemoreceptors (Koul, 2005). Repellency often tends to be connected with
anti-oviposition, as such substances deter females from settling on nutritive
plants and prevent oviposition at the same time. However, anti-oviposition
need not always be connected with repellency. It may be connected with antifeedancy very often when upon settling on the plant, the female nds that
the plant cannot provide food of good quality or acceptable for its descendants, and thus she ies off to seek a more suitable plant. The group of substances with an anti-oviposition effect includes a whole range of chemicals
from simple aromatic terpenes, phenols or alkaloids to molecules falling in
the group of polyphenols or limonoids (Koul, 2005).
The last group of substances antifeedants deter phytophagous insects
against food consumption. This group of substances has been studied on a
large scale in recent times and the use of such substances in plant protection
against pests is connected with their signicant potential. This group of
chemicals also range from simple aromatic terpenes, phenols or alkaloids to
molecules falling in the group of polyphenols or limonoids. At present, there
are high expectations for this group of substances due to their practical use in
plant protection (Isman, 1994; Koul, 2005; Pavela, 2007b).
The good prospects for the practical use of antifeedants are based on
several factors. Most of the repellent substances fall into the group of
aromatic hydrocarbons, which are volatile in the environment and their efciency time is therefore reduced depending on period of application and
dosage used. Numerous substances belonging to the polyphenols and
higher terpenes are included in the category of antifeedants, which may
have a much longer persistence time, thereby extending the efciency time
137
of products (Pavela and Herda, 2007a,b). Moreover, their antifeedant efciency is often connected with further biological activity such as growth inhibition and cumulative mortality.
Allomones with antifeedant activity include a high percentage of
substances that also meet the following requirements for development of
new botanical insecticides:
They are natural plant metabolites, safe for health and the environment;
Such substances provide specic effects on individual pest species they
thus show high selectivity;
Mixtures of substances with synergistic effects can be obtained, which
prevent the development of pest resistance;
Antifeedant substances usually show other biological effects such as
growth inhibition, mortality and reduced fertility of surviving individuals, which increase the practical efciency of the products (Isman, 2002;
Koul, 2005).
All these characteristics increase the prospects for the use of antifeedant
substances in plant protection. However, products based on allomones
should be applied as a preventative measure, for example, at the time of pest
invasion or its initial occurrence or at the time of larval incubation. Although
a large number of biologically active substances and extracts from plants
with antifeedant effects have been tested, only a limited number of products
have been subjected to a detailed eld application and in the development of
plant protection products (Isman, 1994).
6.3 Current State of Antifeedant Research

The history of the research of substances that show antifeedant effects dates
back to the 1930s. As early as 1932, Metzger and Grant tested about 500 plant
extracts against Popilla japonica, although results were not substantially
encouraging. Later, Pradhan et al. (1962) evaluated extracts of the Indian
neem tree, Azadirachta indica, that prevented feeding by the desert locusts.
Although terrestrial plants produce a diverse array of secondary metabolites,
probably more than 100,000 unique compounds (Isman, 2002), today, about
900 compounds have been identied to possess feeding deterrence against
insects (Koul, 2005).
In addition to various compounds isolated from plants or synthesized as
insect antifeedants, several studies demonstrate the antifeedant efcacy in
metabolite mixtures of plant essential oils or total extracts against a variety of
insect species.
In recent years studies have revealed the antifeedant potential of plant
essential oils against postharvest pests, aphids, thrips, lepidopterans, termites and mite pests (Hori, 1999; Hou-HouaMin et al., 2002; Koul, 2005;
Isman, 2006). Similarly, during the past few years much emphasis has been
placed on demonstrating the antifeedant efcacy in total plant extracts
(Mancebo et al., 2000a,b; Wang et al., 2000; Pavela, 2004a,b; Zhang et al., 2004;
138
R. Pavela
Debrowski and Seredynska, 2007) as they seem to exhibit the activity as

multicomponent systems. However, it is also well known that antifeedants
show interspecic variability (Isman, 1993). The existence of such interspecic differences, as shown for many insect species, is encouraging with a
view to searching selectively for specic feeding deterrents.
As already indicated, present research provides very important information on plant substances and their antifeedant efciency. Nevertheless, it
must be noted that the antifeedant efciency of a signicant number of
known antifeedant compounds is dependent on the exposure period and
their concentration. After a long exposure period, some of these compounds
lose their antifeedant efciency due to the development of resistance in the
insects consuming the contaminated food (Koul, 2005). Nevertheless, such
compounds may have other biological effects, at the same time, such as
cumulative mortality or perhaps larval growth inhibition.
However, it should be emphasized that most of the antifeedant research is
in the preliminary trial stage, although the activity of more than 900 compounds and several hundreds of plant extracts are known (Koul, 2005). However, it is expected that in the near future some novel efcacious plant-based
compounds will be formulated as antifeedants, from the huge biodiversity
provided by nature. Attention is currently being paid to promote the indigenous
antifeedant plants by extolling their practical application to the farmers.
6.4 Mechanism of Antifeedant Action

Food selection among insect herbivores is a highly specialized phenomenon.
While olfactory and physical aspects of plants or their organs can be important in insect host nding and acceptance (Miller and Strickler, 1984), the
choice of food is based primarily upon contact chemoreception of various
allelochemicals (Frazier, 1986; Stadler, 1992). In particular, dietary experience has inuenced the ability of insects to taste plant chemicals that may
have served as signals of suitability or unsuitability. Certain dietary constituents appeared to suppress the development of taste sensitivity to deterrents in an insect (Renwick, 2001). Avoidance of allelochemicals, when
looked at from a behavioural point of view, is the outcome of interactions
with chemoreceptors characterized by broad sensitivity to a spectrum of
deterrents (Mullin et al., 1994).
According to Schoonhoven et al. (1992), there are four basic reasons why
the chemosensory perception of feeding deterrents by phytophagous insects
warrants special attention:
Feeding deterrents are apparently more important in host-plant recognition than phagostimulants.
A huge number of feeding deterrents exist, with variable molecular
structures adding to their diversity.
There are fewer deterrent receptors.
Different deterrents may elicit different behavioural reactions, indicating
the presence of a differential sensory coding system.
139
Studies of the chemosensoric insect system are only at the initial point of
research, and knowledge of the mode of action of the substances is supercial
and should be studied in detail. Such a lack of knowledge is particularly
caused by the fact that suitable technologies for chemosensoric investigations
have been available only relatively recently, making it possible to perform
reliable measurements of response to substance at insect chemoreceptors.
Although research of chemoreceptors is important for a general understanding of efciency of individual antifeedant substances, from the practical point of view, experiments based on simple biological tests are those
mostly used in antifeedancy studies.
Bioassays against insects have been used for decades as a means of
elucidating the activity of many chemical components or extracts. The major
goals achieved by employing bioassay techniques are to determine the roles
of naturally occurring chemicals, identify the mechanism of resistance in
crop plants and to nd various insect control agents. The basic design to
study deterrents is to present to an insect a substrate with the candidate
chemical and to measure the response of the insect. Therefore, substrate
choice and presentation are important factors for a successful bioassay. Both
natural and articial substrates are used, depending upon the goal of the
experiment. On one hand one may emphasize that articial substrates offer
uniformity, but at the same time studies have shown that thresholds for the
same deterrent may vary as much as 1000 times between natural and articial substrates (Schoonhoven, 1982), perhaps due to differences in porosity or
uptake rates by the insect. For sucking insects, the principal articial substrate used has been a chemically dened liquid presented between natural
or articial membranes (Koul, 2005).
However, whatever the substrate may be, it is important that no textural
differences should occur between the control and test substrates. Colour differences may also inuence insects during testing. Care is needed to ensure
the least hindrance with the presentation to the insect chemoreceptors, which
should be in the usual way. Natural substrates could be whole plant, leaves,
leaf discs, or specialized substrates such as twigs, blocks of wood, board, and
paper towel discs. Articial substrates usually include agar-based articial
diets, simple liquid-based articial diets, styropors, or discs of foamed
polystyrene, or polyurethane, and glass bre discs (Koul, 2005).
Leaf discs are commonly used in preference or consumption bioassays
with chewing insects. These assays are important in estimating the biological
potential of the antifeedant effect of plant extracts in screening studies, and
they correspond as much as possible to the conditions of the practical application. However, it must be emphasized that these assays are short term. For
the purpose of practical use, further biological assays must be performed in
extracts or substances showing the best biological activity, which will be of a
long-term nature and will provide evidence on the practical applicability of
the substances or extracts in plant protection. Nevertheless, such assays are
irreplaceable as screening assays.
Individual types of biological assays used for evaluating antifeedant
efciency are discussed by Koul (2005). However, in general, such assays can
140
R. Pavela
be divided into two groups according to the mode of the experiment: a choice
assay or a no-choice assay.
The principle is that insects can choose either control or treated discs
(choice) or insects may be exposed to the test substance only (no choice). The
no-choice situation often is more representative of our agricultural system,
especially for monophagous species, but at the same time it is very sensitive
(Fig. 6.1).
The general procedure adopted in this test is that measured leaf discs are
punched out from substrates and treated either on one side or both sides
with a known quantity of test material in a carrier solvent. It is preferable to
use emulsified solutions in water in order to avoid interference with leaf disc
texture due to solvents (Isman, 2002). A method has been described by which
leaf surfaces can be covered with a uniform amount of a test chemical for bioassay with leaf-feeding insects. Chemicals are dissolved in gelatine solutions,
which can be sprayed evenly and which will adhere well to many leaf surfaces. Upon accurate application, the dosage per leaf area can be determined,
which is an important practical viewpoint for the application itself.
After application, the leaf discs are dried at room temperature and then
fed to candidate insects. Usually the arenas used are Petri dishes of variable
sizes in which one treated and one control disc is placed (choice), or both the
leaf discs are treated (no choice). In certain experiments five to ten treated
and untreated leaf discs are used and placed alternately in the Petri dishes in
a choice situation. The number of larvae introduced into each arena is variable depending upon the size and stage of the larvae used. There is also considerable variation in the duration of experiments, both long term and short
Fig. 6.1. No-choice test with extract obtained from Leuzea carthamoides against larvae
Leptinotarsa decemlineata, 48 h after application of 1% extract.
141
term. The consumption in each experiment is measured using various digitizing leaf area meters.
The bioassay is conducted for a very short duration (2 to 8 h) or until 50%
of either disc is consumed. Several formulas and ways of denomination are
presented in the literature for calculating the biological effect (Koul, 2005).
Nevertheless, the following formula to calculate feeding deterrence provides
the highest accuracy:
Feeding deterrence (%) = (C T)/(C + T) 100, where C and T are
the consumption of control and treated discs, respectively (Koul, 2005;
Pavela et al., 2008).
As mentioned above, no-choice assays are most important from the practical point of view, because they are closest in nature to practical application.
It is thus advisable to favour such assays.
When 90100% feeding deterrence is obtained in the assays, efcient
concentration (EC50 and EC95) can be determined in subsequent assays,
which is another important parameter for the mutual comparison of extract
or substance efciency. Moreover, it is also of equal importance to determine
the time during which the given insect does not consume food treated with
EC95, as insects may become habituated to antifeedant substances or the biologically active substances may be degraded due to the action of the environment (Koul, 2005). The insects can thus overcome the initial resistance against
food treated with antifeedant substances, which may cause the products to
lose their primary efciency. However, it must be emphasized that in this
respect, information on the period during which the insect does not consume
food treated with antifeedant substances is very sporadic, and therefore the
experiments must be completed with such information.
6.5 Current Practical Use of Antifeedant Substances

A long way still remains ahead in order to arrive at the full application potential offered by plant allomones in plant protection. No commercial products
based purely on antifeedant efciency are yet available. Nevertheless, some
products that primarily contain substances with an insecticide effect, and at
the same time also show an antifeedant and anti-oviposition effect, are used
at present.
Perhaps the most widely known application of antifeedant effects relates
to extracts made of the Indian plant Azadirachta indica A. Juss (syn. Melia azadirachta). A. indica has been well known in India and neighbouring countries
for more than 2000 years as one of the most versatile medicinal plants having
a wide spectrum of biological activity. A. indica A. Juss and M. azedarach are
two closely related species of Meliaceae.
Extracts made of seeds of this plant contain numerous biologically active
substances with insecticidal, fungicidal and bactericidal effects, used in many
industries, ranging from medicine to agriculture. Many works have been
published concerning the plant itself, its useful substances, and biological
142
R. Pavela
efciency including antifeedant effects (Jacobson, 1989; Schmutterer, 1990;

Ascher, 1993).
In the case of A. indica, substances falling in the group of limonoids
(azadirachtin, salanin, nimbin etc.) are responsible for antifeedant activity.
Both primary and secondary antifeedant effects have been observed in the
case of azadirachtin (Ascher, 1993). Primary effects include the process of
chemoreception by the organism (e.g. sensory organs on mouthparts which
stimulate the organism to begin feeding), whereas secondary processes
are effects such as gut motility disorders due to topical application only
(Schmutterer, 1990; Ascher, 1993). Inhibition of feeding behaviour by
azadirachtin results from the blockage of input receptors for phagostimulants or by the stimulation of deterrent receptor cells or both (Mordue and
Blackwell, 1993). In a recent study by Yoshida and Toscano (1994), the relative consumption rate of Heliothis virescens larvae treated with azadirachtin
was 25% of the control, equivalent to the lowest assimilation efciency of all
natural insecticides tested. In another study, larvae of Heliothis virescens consumed less food, gained less weight, and were less efcient at converting
ingested and digested food into biomass (Barnby and Klocke, 1987). Sensitivity between species to the antifeedant effects of azadirachtin is profound.
Order Lepidoptera appear most sensitive to azadirachtins antifeedant
effects, with Coleoptera, Hemiptera and Homoptera being less sensitive
(Mordue and Blackwell, 1993).
A whole range of commercial products based on azadirachtin are sold at
present; however, they utilize another signicant effect of azadirachtin type
tetranotriterpenoids, namely the growth inhibition effect (Schmutterer, 1990).
Extracts from the Indian tree of the Pongamia genus are another example of the commercial application of products based on antifeedant and
anti-oviposition effects. This genus has one species only, that is Pongamia
pinnata L. (syn. P. glabra Vent.; Derris indica Lamk.) which belongs to family
Leguminosae; subfamily Papilionaceae (Kumar and Kalindhar, 2003).
P. pinnata is a rich source of avonoids, the B-ring is either linked to a
furan or pyran ring. Some of these avonoids are known to have biological
activity. Antifeedant activities of various extracts of P. pinnata were observed
against many insect pests of different crops. Under laboratory conditions,
0.1% water emulsion of pongam oil (so-called karanj oil) showed antifeedant
activity against Amsacta moorei Butler (Verma and Singh, 1985) or Spodoptera
litura F. (Kumar and Kalindhar, 2003). The pongam oil is known to possess
strong repellent activity for egg-laying against many insect pests (Khaire et al.,
1993; Kumar and Kalindhar, 2003). The aqueous extracts of seeds and plants
of this species are known to possess ovicidal action against Phthorimaea
operculella Zell. and Helopeltis theivora Waterh. (Deka et al., 1998).
The repellent activities, including host deterrence and anti-oviposition,
of pongam oil against the adults of the common greenhouse whitey Trialeurodes vaporariorum Westwood were tested in greenhouses (Pavela and
Herda, 2007a,b). Chrysanthemum plants treated with different concentrations (0.52.0%) of water-suspended pongam oil showed relatively longlasting host deterrent and anti-oviposition effects on the adults of greenhouse
143
whitey. Although the repellent effect declined in time and concentration,

strong effects on the reduction of oviposition were found, while this effect
lasts at least 12 days after application, dependent on concentration.
Another example of commercially produced extracts with antifeedant
effects is represented by aromatic plants. For aromatic plants, substances
responsible for the smell and those that may be isolated using distillation
or supercritical extraction have been studied the most. This group of substances includes the mono- and di- sesquiterpenes, phenols and some other
hydrocarbons similar in structure (Pavela, 2008a).
Many monoterpenes from plant sources have been evaluated as feeding
deterrents against insects (Koul, 1982). However, capillin, capillarin, methyl
eugenol and ar-curcumene isolated from Artemisia capillaris show promise as
antifeedant compounds against cabbage buttery larvae, Pieris rapae crucivora. The relative strong antifeedant activity of capillin and capillarin suggest
that the C=O carbonyl group instead of CH2 methylene group, a CC in a
side chain and a lactone ring are some of the many factors that contribute to
the biological activity (Yano, 1987). Various derivatives of these base compounds such as methyl eugenol reveal that the 3,4-dimethyl group and
1-substituent of 3,4-dimethoxy-1-substituted benzenes contribute to the
antifeedant activity (Yano and Kamimura, 1993).
Aromatic hydrocarbons show a signicant direct insecticide activity,
so consequently they have been best studied (Pavela, 2006b, 2008a,b). Nevertheless, antifeedant efciency is connected not only with aromatic hydrocarbons in aromatic plants, but also with other polyphenolic substances
synthesized by such plants as part of their defence strategy against diseases
and pests.
These polyphenolic substances are currently being studied and because
of their huge biochemical variety, it is highly probable that an antifeedant
activity will be discovered in the near future that would lead to practical
application.
Recent results of biological activity studies of substances obtained from
Eurasian region plants, too, provide evidence that primary research is necessary and may lead to commercial application. Very strong, long-lasting antifeedant activity of seed extracts and root extracts of Leuzea carthamoides plants
against the Colorado potato beetle (Leptinotarsa decemlineata) was found
(Pavela, 2004a, 2006a) (Fig. 6.2. a,b). This antifeedant efciency against the
above mentioned pest led to the formulation of a new product based on antifeedant activity. The research on this aspect, which has been performed both
in our laboratory and in departments around the world, is thus providing its
rst results.
6.6 Prospects for Products Based on Antifeedant Substances

Many feeding inhibitors from plant sources have so far given excellent results
in laboratory conditions. In eld situations only a few of them are satisfactory
alternatives to traditional pest management. The chemical control is usually
144
R. Pavela
(a)
(b)
Fig. 6.2. Efficiency of extract obtained from L. carthamoides against larvae Leptinotarsa
decemlineata. (a) Control; (b) 15 days after application.
with broad-spectrum insecticides, and they have to be broad spectrum

by necessity. They have to sell in amounts large enough to accommodate
nancial development, research, and marketing.
Nevertheless, the use of antifeedants in pest-management programmes
has enormous appeal. They satisfy the need to protect specic crops while
145
avoiding damage to non-target organisms so the potential value is great. In

fact, insect damage to plants results from feeding or from transmission of
pathogens during feeding; therefore, the chemicals that reduce pest injury by
rendering plants unattractive or unpalatable can be considered as potential
substitutes for conventional insecticides. The host choice of generalists and
to some extent specialists may be modied when feeding inhibitors are used.
The range of insect species targeted may be chosen by either the chemical
structure of the inhibitor or the composition of a mixture of inhibitors, if different inhibitors are active against different species within the range. Therefore, a multi-component defence strategy of plants themselves could be used,
as shown in number of recent studies with non-azadirachtin types of limonoid
inhibitors (Koul, 2005) where non-azadirachtin limonoids have two different
modes of action, such as feeding deterrence and physiological toxicity, which
play a signicant role in the potentiation effect. Moreover some other plant
extracts have high potential in commercial application (Pavela, 2007b).
For the research to be successful, further assays must be performed and
suitable, new substances with high antifeedant efciency must be sought.
Important research will also concern determining synergism of the effects of
biologically active substances as such synergism seems to be very signicant
in increasing the biological activity of substances that have insecticide effects
(Pavela, 2008b).
Most feeding inhibitors are less stable chemicals than traditional insecticides and act with lower residual activity and environmental impact. Natural
predators and parasitoids remain unharmed by feeding deterrents targeting
the herbaceous host insects. As the target sites of antifeedants are different,
pesticide-resistant insect populations will still be affected by feeding inhibitors. Multi-component tactics will also slow down the resistance development to these new compounds. In fact, lack of resistance is very useful for the
practical application of antifeedants as it is unlikely that oligophagous insects
could develop general resistance to such deterrents, because this would
result in a rapid change of their host-plant range, which is determined mainly
by the occurrence of such chemicals in the non-host plants. Different molecular structures of possible antifeedant compounds could be another advantage. The blend of active constituents might diffuse the selection process,
mitigating the development of resistance compared to that expected with a
single active ingredient. This also supports the earlier mentioned contention
that combination mixtures of antifeedants could be more effective than
individual compounds.
The huge variety of defensive mechanisms of plants, including the
synthesis of allelochemicals, thus provides a research focus in seeking new
environmentally safe products to provide plant protection against phytophagous insects. However, such research should be intensied and needs
full cooperation between basic or applied research, manufacturers and the
users. Such cooperation may lead subsequently to an important reduction in
the dependence of agriculture on chemical industries synthesizing toxic
insecticides, as well as increasing the quantity of safe foodstuffs and improving
the health of this planets inhabitants.
146
R. Pavela
Acknowledgement
This study was supported by grants of the Czech Republic Ministry of
Education, Youth and Sports (ME09079).
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Potency of Plant Products

in Control of Virus Diseases
of Plants
H.N. VERMA1 AND V.K. BARANWAL2
1Jaipur
National University, Jaipur, India; 2Advanced Centre of Plant

Virology, New Delhi, India
Abstract
The exploitation of the inherent resistance phenomenon and manipulation of inducible
defence in plants is currently receiving much attention by researchers to control virus
infection. Recent advances in the molecular biology of resistance to virus infection
have presented new approaches for making susceptible crops resistant against virus
infection. These approaches include pathogen-derived resistance to viruses (coatprotein-mediated resistance, movement-protein-mediated resistance, replicase- and
protease-mediated resistance) and virus resistance through transgenic expression of
antiviral proteins of non-viral origin. Endogenously occurring substances in a few
higher plants have also been reported to induce systemic resistance in susceptible
hosts against virus infections. Ribosome-inactivating proteins (RIPs) may also play an
important role in the prevention of virus infection.
7.1 Introduction
Viruses prove to be a menace to humans and the environment because of
their disease-causing nature. They harm crops and cause economic losses.
The recent outbreak of cotton leaf curl virus disease in cotton in the northern
cotton-growing region of India has led to a huge yield loss of cotton bre.
Approximately 12,000 hectares of cotton were affected by leaf curl virus
disease during 1996 in Rajasthan alone. An annual loss of US$300 million is
caused by mungbean yellow mosaic virus (MYMV) by reducing the yield of
black gram, mungbean and soyabean (Varma et al., 1992). The worldwide
losses caused by viral diseases are estimated at about US$60 billion
per year.
To reduce losses, scientists have explored several strategies to control
virus infection. However, it is the exploitation of the inherent resistance
phenomenon and manipulation of inducible defence in plants that are
receiving much attention from researchers. The common approach for
(ed. N.K. Dubey)
149
150
introducing resistance against a virus in crops has been achieved through

conventional plant breeding. Limited success has been achieved through
this method. Recent advances in the molecular biology of resistance to
virus infection have provided new approaches to making susceptible
crops resistant against virus infection. These approaches include
pathogen-derived resistance to viruses (coat-protein-mediated resistance,
movement-protein-mediated resistance, replicase- and protease-mediated
resistance) and virus resistance through the transgenic expression of
antiviral proteins of non-viral origin (Baulcombe, 1994).
Resistance of plants to virus diseases may be broadly categorized into
two groups: (i) constitutive; and (ii) induced. Constitutive resistance is heritable and occurs in cultivars, which have gene(s) conferring resistance to
viral infection, whereas induced resistance has to be conferred afresh upon a
susceptible plant and is normally not heritable. Induced resistance operates
through the activation of natural defence mechanisms of the host plant. The
two forms of induced resistance are systemic acquired resistance (SAR) and
systemic induced resistance (SIR). In both SAR and SIR, plant defences are
preconditioned by prior infection or treatment that results in resistance (or
tolerance) against subsequent challenge by a pathogen or parasite. Great
strides have been made over the past 20 years in understanding the physiological and biochemical basis of SAR and SIR. Much of this knowledge is due
to the identication of a number of chemical and biological elicitors, some of
which are commercially available for use in conventional agriculture. However, the effectiveness of these elicitors to induce SAR and SIR as a practical
means to control various plant diseases is just being realized.
The infection of plants by necrotizing pathogens, including fungi, bacteria and viruses, induces systemic resistance to subsequent attack by the
pathogens. This resistance is called SAR (Kessman et al., 1994; Ryals et al.,
1994). It can also be activated in numerous plants by pre-inoculation with
biotic inducers including pathogens (Sticher et al., 1997). Endogenously
occurring substances in a few higher plants have been reported to induce
systemic resistance in susceptible hosts against virus infections. Such plant
extracts have been used for protecting economically important crops against
virus infections (Verma and Baranwal, 1989). Endogenously occurring virus
inhibitors may also be ribosome-inactivating proteins (RIPs) (Barbieri and
Stirpe, 1982; Mansouri et al., 2006; Zhang et al., 2007). Virus infection is prevented if a mixture of RIP and virus is exogenously applied on the leaf surface of a susceptible host. RIPs presumably inhibit virus infection by entering
the cytoplasm along with the virus particle and inhibiting protein synthesis
on host ribosomes, thus preventing early virus replication (Reddy et al.,
1986).
On the basis of their antiviral activity, virus inhibitors from plants can be
grouped into two categories:
1. Plant products that inhibit virus infection by inducing an antiviral state
either at the site of application (local resistance) and/or at a remote site
(systemic resistance) when applied a few minutes or hours prior to virus
Control of Virus Diseases of Plants
151
challenge (Verma and Mukerji, 1975; Verma and Awasthi, 1979, 1980; Verma
et al., 1984; Verma et al., 1995a; Prasad et al., 1995; Verma et al., 1996). Such
plant products have been called systemic resistance inducers (SRIs).
2. Basic proteins from plants that function by inactivating ribosomes of the
host and have been called RIPs (Barbieri et al., 1993, 2003; Van Damme et al.,
2001).
7.2 Systemic Resistance Inducers (SRIs)

Induction of systemic resistance by plant extracts has been reviewed from
time to time (Verma, 1985; Verma and Prasad, 1992; Verma et al., 1995a; Verma
et al., 1998). Physico-chemical characteristics of systemic resistance inducers
from plants such as Boerhaavia diffusa (Verma and Awasthi, 1979, 1980; Srivastava, 1995); Mirabilis jalapa (Verma and Kumar, 1980); Cuscuta reflexa
(Awasthi, 1981); Clerodendrum aculeatum (Verma et al., 1984; Verma et al., 1996;
Kumar et al., 1997; Srivastava et al., 2008); Bougainvillea spectabilis (Verma
et al., 1985; Verma and Dwivedi, 1984; Srivastava, 1995); Pseuderanthemum
atropurpureum (Verma et al., 1985) have been studied in some detail (Table
7.1).
Induced resistance operates through the activation of natural defence
mechanisms of the host plant. Extracts from brinjal (Verma and Mukherjee,
1975) and a few other higher plants such as Boerhaavia diffusa (Verma et al.,
1979), Bougainvillea (Verma and Dwivedi, 1984), Clerodendrum (Verma
et al., 1996; Kumar et al., 1997) and Datura (Verma et al., 1982) induce systemic
resistance to viral multiplication in plants. The active products present in these
Table 7.1. Characteristics of systemic resistance inducers obtained from some higher plants.
SRI-yielding plants
Characteristics
BD
CA/CI
BS
PA
CR
MJ
Source
Thermal inactivation
points (C )
Nature
Molecular weight
(kDa)
Active against
Root
80
Leaf
90
Leaf
80
Leaf
0
Leaf
70
Leaf
90
Glyco-protein
30
Basic Protein
34/29 and 34
Protein
28
0
0
Protein
1418
Protein
24
TMV
SHRV
GMV
TMV
SHRV
GMV
TMV
TMV
TMV
SHRV
SHRV
SHRV
CGMMV CGMMV GMV
PLRV
TMV
SHRV
PVY
TmYMV
BD = Boerhaavia diffusa; CA = Clerodendrum aculeatum; CI = C. inerme; BS = Bougainvillea spectabilis;

PA = Pseuderanthemum atropurpureum; CR = Cuscuta reflexa; MJ = Mirabilis jalapa. TMV = Tobacco
mosaic virus; SHRV = Sunnhemp rosette virus; GMV = Gomphrena mosaic virus; TRSV = Tobacco
Ringspot virus; TmYMV = Tomato Yellow Mosaic virus; CGMMV = Cucumber green mottle mosaic virus;
PVY = Potato virus Y; PLRV = Papaya leaf reduction virus.
152
extracts have no direct effect on viruses; their antiviral activity is mediated by

host cells in which they induce the antiviral state. All groups of plants respond
to treatment with extracts of these plants. The active products in plant extracts
inducing resistance are mostly small molecular weight proteins which sometimes may be glycosylated. These proteins are highly thermostable and can
withstand prolonged treatment at alkaline/acidic pH. Plant products from
different species vary in molecular weight and may differ in other characteristics too. Incubation of viruses with these antiviral agents has no effect on viral
infectivity, but they exert their antiviral effect by rendering host cells incapable
of supporting viral replication. Resistance development is inhibited by actinomycin D and cyclohexamide. This implies that resistance is dependent on
DNA-coded information of the cell and the antiviral activity of the antiviral
agent is indirect. The antiviral agents showed no host specicity and were
active against a wide range of viruses (Faccioli and Capponi, 1983).
Systemic resistance inducers obtained from plants have been shown to
be effective against a wide range of viruses (Verma and Awasthi, 1979). Leaf
extract of Clerodendrum aculeatum is active against TMV, SHRV, GMV, TmYMV,
(Verma et al., 1984), and an extract of Mirabilis jalapa is effective against
TmYMV, PLRV, CMV, CGMMV (Verma and Kumar, 1980). The extracttreated plants become resistant to attack by diverse pathogens such as fungi,
bacteria and viruses (McIntyre et al., 1981).
Mechanism of systemic induced resistance by botanicals

Systemic induced resistance against virus infection by botanicals is not yet
fully understood. The botanical resistance inducers themselves do not act on
the virus directly. Verma and Awasthi (1980) demonstrated the de novo synthesis of a virus inhibitory agent (VIA) in untreated leaves of Nicotinia glutinosa, whose basal leaves were treated with root extract of B. diffusa. These
induced substances inhibited almost completely tobacco mosaic virus in N.
glutinosa, Datura stramonium and D. metel but inhibition of tobacco ring spot
virus or Gomphrena mosaic virus in Chenopodium amaranticolor was less
pronounced. In another study Verma and Dwivedi (1984) found that VIA
induced in C. tetragonoloba inhibited completely the infection of tobamoviruses in all the seven hypersenstive hosts tested. Yet in another study by
Khan and Verma (1990), it was observed that VIA produced in C. tetragonoloba
following treatment with extract of Pseuderanthemum bicolor inhibited completely SHRV, TMV, cucumber green mottle mosaic virus and PVX in their
respective hypersensitive hosts, namely C. tetragonoloba, D. stramonium,
C. amaranticolor and G. globosa. However, the VIA produced by B. spectabilis in
N. tabacum cv. Samsun NN, N. glutinosa, and D. stramonium was less effective
against TMV (Verma and Dwivedi, 1984). Thus, it appears that induction of
systemic resistance by botanicals is non-specic and is effective against a
broad spectrum of viruses.
The production of VIA is maximum after 24 h of application of B. diffusa
root extract in N. glutinosa. However, B. spectabilis induced maximum VIA
153
activity after 48 h of its application in N. glutinosa. Thus, VIA production in a

host is time specic but a general phenomenon. Properties of VIA produced
in C. tetragonoloba after application of B. spectabilis leaf extract has been studied by Verma and Dwivedi (1984). The VIA could be precipitated by ammonium sulfate and hydrolysed by trypsin but not by ribonulease indicating
that it was a protein rather than a nucleic acid. The VIA induced by Pseuderanthemum bicolor in C. tetragonoloba was also proteinaceous with a molecular
weight of 15 kDa (Khan and Verma et al., 1990). The production of VIA was
sensitive to actinomycin D (Verma and Dwivedi, 1984). The mobile signal
produced VIA in the entire plant system. It appears that the putative messenger becomes active soon after induction and starts producing VIA, reaches
a maximal concentration and then starts to decline.
Commonly associated with systemic acquired resistance induced by
pathogens is the systemic synthesis of several families of serologically distinct low molecular weight pathogenesis-related (PR) proteins. The localization and timing of some PR proteins suggested their possible involvement in
acquired resistance against viruses. However, denite proof that the induction of PR proteins causes the acquired resistance has not been given. Plobner
and Leiser (1990) did not nd the production of PR proteins during systemic
resistance induced by carnation extract in Xanthi-nc tobacco plants. Absence
of PR proteins during induction of SAR by botanicals suggests that another
biochemical chain of reaction, other than one operating in pathogen / chemical induced SAR, might be operating during botanical induced systemic
resistance against virus infection in plants.
Durrant et al. (2004) reported that SAR is a mechanism of induced defence
that confers long-lasting protection against a broad spectrum of microorganisms. SAR requires the signal molecule salicylic acid (SA) and is associated
with accumulation of PR proteins, which are thought to contribute to
resistance.
Hansen (1989), while reviewing antiviral chemicals for plant disease
control, gave the following characteristics of an ideal antiviral compound
that will serve all purposes for virus disease management in crops:
Soluble in water or non-phytotoxic solvents.

Effective against at least some agriculturally important viruses at
non-phytotoxic concentrations.
Easily taken up by plants and distributed throughout the system.
Non-toxic by itself and in its catabolic forms to humans, plants and
wildlife.
Botanical resistance inducers can be classied as ideal virussuppressing agents, as they have all the characteristics of an ideal antiviral
compound. The resistance-inducing proteins from Boerhaavia diffusa and
Clerodendrum aculeatum can be applied directly by spraying on systemic
hosts, for management of some commonly occurring virus diseases under
greenhouse conditions or eld conditions. The agricultural role of endogenous antiviral substances of plant origin has been reviewed by Verma
et al. (1995).
154
The induced antiviral state in N. glutinosa by SRI from C. aculeatum

decreased considerably after 3 days (Verma and Varsha, 1994). However, the
resistance inducing ability of C. aculeatum SRI could be enhanced up to
6 days by priming it with certain proteinaceous additives (Verma and Versha,
1994). The activity is probably enhanced by modication or enhanced stability of proteinaceous inducers by these additives. Thus, one unexploited
approach to engineering virus resistance is the manipulation of inducible
defences in plants. The production of systemic resistance by the use of botanicals will be effective against a broad spectrum of viruses and will not break
down when plants are exposed to high temperatures.
Suppression of disease symptoms by true inhibitors may be accomplished either by acting on the rst stage of the infection process, which is the
adsorption of the virus into the host cell, or by blocking or competing with
the virus receptor sites on the leaf surface (Ragetli, 1957; Ragetli and
Weintraub, 1962) or by affecting the susceptibility of the host by altering host
cell metabolism (Verma and Awasthi, 1979). Bozarth and Ross (1964) suggested the phenomenon of SAR as a result of the initial infection by which a
signal was generated at the site of application and transported throughout
the plant to respond more effectively to the subsequent infection.
Induction of systemic resistance by resistance inducers obtained from
plant extracts was rst detailed by Verma and Mukerjee (1975). Extracts from
a few plants induce an antiviral state by acting through an actinomycin D
(AMD) sensitive mechanism (Verma and Awasthi, 1979; Verma et al., 1984).
AMD is an inhibitor of protein synthesis at the transcription level. Concomitant application of AMD with SRI reversed the induction of resistance in
susceptible plants. However, induction of resistance remains unaffected
when AMD is applied 12 h post-treatment. This gives an indication that
plant-extract-induced resistance is a host-mediated response (Verma et al.,
1979).
The activity at a distance from the point of application might be explained
by the supposition that the SRI present in the plant extract selectively attaches
at the surface, and a type of chain reaction starts that elicits the transcription
of defence-related genes, leading to the production of a new VIA (Verma and
Awasthi, 1979).
Ribosome inactivating proteins (RIPs)

The basic proteins that function by inactivating the ribosome of the host have
been called RIPs. The RIPs have been shown to be N-glycosidases, which
remove a specic adenine base in a conserved loop of the 28s rRNA of
eukaryotic organisms (Endo and Tsurugi, 1987; Endo et al., 1987) or the 23s
rRNA of prokaryotes (Hartely et al., 1991). Such damaged ribosomes can no
longer bind the elongation factor-2 (Gessner and Irvin, 1980; Rodes and Irvin,
1981). The RIPs damage the ribosome, arrest protein synthesis and cause cell
death. RIPs show antiviral activity against both animal and plant viruses
(Barbieri and Stirpe, 1982) and have been classied into two types (Stirpe
155
et al., 1992; Barbieri et al., 1993). Type-I RIPs consist of a single polypeptide
chain that is enzymatically active. These are scarcely toxic to animals and
inhibit protein synthesis in cell-free systems, but have little or no effect on
whole cells. The three well known antiviral proteins PAPs, dianthins and
MAP belong to this category. Type-II RIPs contain two types of polypeptide
chains. Chain A is linked to chain B through a disulfide bond. Chain B binds
the toxin to the cell surface and chain A enzymatically inactivates the ribosomes (Olsnes and Pihl, 1982). These are toxic to cells and inhibit protein
synthesis in intact cells and in cell-free systems. Several similarities exist
among the type-I RIPs. They are all basic proteins with a molecular weight in
the range 2632 kDa. They have an alkaline isoelectric point and are usually
stable, being resistant to denaturing agents and protease. A majority of the
RIPs are glycoproteins. Type-I RIPs are strongly immunogenic. Strocchi et al.
(1992) established that cross reactivity between RIPs obtained from unrelated
plants was either very weak or absent.
Recently cloning and expression of antiviral/ribosome-inactivating
protein from Bougainvillea xbuttiana was reported by Choudhary et al. (2008).
They reported that full-length cDNA encoding ribosome inactivating / antiviral protein (RIP/AVP) consisted of 1364 nucleotides with an open
reading frame (ORF) of 960 nucleotides encoding a 35.49 kDa protein of 319
amino acids.
The three well known antiviral proteins, namely PAPs (from Phytolacca
americana), dianthins (from Dianthus caryophyllus) and MAPs (from Mirabilis
jalapa) belong to the category of type-I RIPs.
Pokeweed antiviral proteins (PAPs)

The antiviral protein present in the leaves of Phytolacca americana was purified
to homogeneity and its molecular weight determined as 29 kDa (Irvin, 1975).
This protein, called pokeweed antiviral protein (PAP), had a PI of 8.1 (Irvin,
1983). The antiviral effect of PAP was most pronounced when it was coinoculated with the virus. PAP also inhibited virus infection when applied
prior to virus challenge. Local lesion formation by TMV on N. tabacum cv.
Xanthi-nc was inhibited by nearly 70% even after 48 h of treatment. The virus
inhibitory effect of PAP increased with the decrease in the time lapse between
treatment and challenge inoculation. PAP was less effective in preventing
virus infection when applied a short time after virus inoculation. No inhibition was observed when PAP was applied 50 min after virus infection (Chen
et al., 1991). PAP reduced infectivity of several mechanically transmitted RNA
and DNA viruses when the purified virus or sap from virus-infected plants
was mixed with an equal volume of PAP solution and the mixture rubbed on
the leaves of the local-lesion hosts (N. glutinosa / TMV; Chenopodium quinoa /
CMV; C. amaranticolor / TMV, CMV, alfalfa mosaic virus, PVY; Gomphrena globosa / PVX) or systemic hosts (Brassica campestris / cauliflower mosaic virus;
N. benthamiana / African cassava mosaic virus). PAP, thus, appears to be a
general inhibitor of virus infection (Tomlinson et al., 1974; Stevens et al., 1981;
156
Chen et al., 1991; Picard et al., 2005). PAP also shows antiviral activity against
several animal viruses. It is toxic to cells infected with poliovirus (Ussery
et al., 1977) and inuenza virus (Tomlinson et al., 1974). It inhibits multiplication of herpes simplex virus type 1 (Arnon and Irvin, 1980) and human
immunodeciency virus (HIV; Zarling et al., 1990). P. americana is now known
to contain three proteins (PAP I, II and III) with similar biological properties.
A new insight into the antiviral mechanism of PAP is that PAP depurination
of Brome mosaic virus RNA impedes both RNA replication and subgenomic
RNA transcription (Picard et al., 2005).
Rajmohan et al. (1999) reported that PAP isoforms PAP-I, PAP-II and
PAP-III depurinate RNA of HIV-I. A non-toxic PAP mutant inhibiting pathogen infection via a novel SA-independent pathway was reported by
Zoubenko et al. (2000). PAP inhibits translation by depurinating the conserved sarcin/ricin loop of the large ribosomal RNA. Depurinating ribosomes are unable to bind elongation factor 2, and, thus, the translocation step
of the elongation cycle is inhibited. Ribosomal conformation is required for
depurination that leads to subsequent translation inhibition (Mansouri et al.,
2006).
Carnation antiviral proteins (Dianthins)

Sap from carnation leaves shows virus inhibitory activity (Van Kammen
et al., 1961; Ragetli et al., 1962). Dianthin 30 and 32 were isolated from the
leaves of Dianthus caryophyllus (Ragetli et al., 1962). Local lesion production
by TMV on N. glutinosa was inhibited by 100% when the inhibitor was coinoculated with the virus (Stevens et al., 1981). The molecular weights as
determined by SDSPAGE are 29.5 and 31.7 kDa, respectively (Stirpe et al.,
1981). Immunoelectrophoresis revealed that dianthin 32 is distributed in the
growing shoots and in the young and old leaves of D. caryophyllus and dianthin 30 is distributed throughout the plant (Reisbig and Bruland, 1983). The
two are glycoproteins containing mannose and show a weak cross reaction.
The nucleotide sequence of cDNA encoding dianthin 30 has been determined
(Legname et al., 1991). The carnation proteins are also inducers of systemic
resistance (Plobner and Leiser, 1990). Cho et al. (2000) performed isolation
and characterization of cDNA encoding ribosome inactivating protein from
Dianthus sinensis L.
Mirabilis antiviral protein (MAP)

The roots, leaves and stem of Mirabilis jalapa show high inhibitory activity
against plant viruses. The Mirabilis jalapa leaf extract, when used as a foliar
spray 24 h prior to virus inoculation, suppressed disease symptoms on a few
systemic hosts (tomato/tomato yellow mottle virus; Cucumis melo var.
momordica/CMV; Cucumis sativa/cucumber green mottle mosaic virus;
tomato/tomato yellow mosaic virus; urd/yellow mosaic of urd) (Verma and
157
Kumar, 1980). A 5060% reduction of the virus content in the treated plants
was observed in the infectivity assays. M. jalapa extract was able to check the
population of aphids and whiteies and, thereby, control the natural spread
of a few viruses on the systemic hosts (Verma and Kumar, 1980).
MAP isolated from root inhibits mechanical transmission of TMV, PVY,
cucumber green mottle mosaic virus, and turnip mosaic virus on local-lesion
and systemic hosts and can induce systemic resistance of a low order when
applied to basal leaves (Kubo et al., 1990). The puried protein consists of a
single polypeptide without a sugar moiety and has a molecular weight of
24.2 kDa. It is a basic protein rich in lysine content, with a PI of 9.8 (Kubo et
al., 1990). The complete amino acid sequence of MAP has been determined.
It consists of 250 amino acids and its molecular weight as determined from
the sequence is 27,833 kDa. The native MAP was resistant to protease
digestion (Habuka et al., 1989).
MAP produced by M. jalapa cells in suspension culture showed comparable biological activity with that of the roots and leaves and also reacted
positively with anti-MAP serum (Ikeda et al., 1987). Several nutritional and
hormonal factors also affect the formation of MAP by M. jalapa cells in suspension culture (Ikeda et al., 1987). Bolognesi et al. (2002) reported ribosomeinactivating and adenine polynucleotide glycosylase activities in M. jalapa L.
tissues.
Ricinus (RICIN A) antiviral protein

The ricinus antiviral protein is a type II RIP isolated from Ricinus communis
that has a molecular weight of 65 kDa. Its is a heterodimer consisting of chain
A similar to the type-I RIP and chain B linked together by a disulde bond.
Chain B has great similarity to mammalian lectins A. Once inside the cell
chain A acts as a type-I RIP and inactivates protein translation machinery.
The cDNA for ricin has been cloned (Lamb et al., 1985). Both A and B chains
are encoded by a single gene which has no intron. Detection of ricin and
other ribosome-inactivating proteins by an immuno-polymerase chain
reaction assay was reported by Lubelli et al. (2006).
Role of RIPs
The above examples of RIPs show their important role against both plant
viruses and also RNA- and DNA-containing animal viruses (Barbieri et al.
1993). The mechanism of antiviral activity suggested for both plant and
animal system involves increased permeability of and easier entry of RIPs
into virus-infected cells, blocking of protein synthesis and reduced virus
multiplication (Barbieri et al., 1993).
Virus resistance previously observed in transgenic plants expressing coat
protein genes, and so on, has been specic for the virus from which the genes
are derived or for closely related viruses (Beachy et al., 1990) but transgenic
158
tobacco and tomato plants expressing the pokeweed antiviral gene are found
to be resistant to a broad spectrum of plant viruses (Lodge et al., 1993).
A problem often encountered in using type-I RIPs is the fact that they
cannot inhibit protein synthesis in intact cells. However, when coupled to
type-II RIP, they can be used effectively. The toxicity of plant materials
containing type-II RIPs have long been known and has great medicinal
potential. Recently, apoptosis was described in both lymphoid tissue and in
the intestine of abrin- and ricin-poisoned rats. Apoptosis was also observed
in tissue culture of cancer cells treated with ricin.
The antiviral activity of the RIP could be due to inactivation of ribosome
of the infected plant cell. As compared to the usefulness in the plants, the role
of RIPs in human and animal systems has been much more widely documented. In spite of all these efforts, the biological signicance of RIPs in
nature is not yet known and moreover, their antiviral action does not seem to
depend upon the inhibition of host ribosomes (Chen et al., 1993).
Great potential exists today in elucidating the possible signicance of
RIPs and their exact antiviral role which may not always depend upon inhibition of host ribosomes. Their co-action with another antiviral mechanism
also needs to be explored, especially their place in the cascade of events following viral infection up to establishment of resistance, both systemic or
localized.
7.3 Pathogen-induced Systemic Acquired Resistance (SAR)

The infection of plants by necrotizing pathogens, including fungi, bacteria
and viruses, induces systemic resistance to subsequent attack by the pathogens. This resistance is called systemic acquired resistance (SAR) (Kessman
et al., 1994; Ryals et al., 1994; Prasad et al., 2001). Also, it can be activated in
numerous plants by pre-inoculation with biotic inducers including pathogens (Sticher et al., 1997). One of the prominent features of SAR is that resistance is expressed against pathogen which can be widely different from the
initial infecting pathogens.
In a range of plant species, the development of necrotic lesions in response
to pathogen infection leads to induction of generalized disease resistance in
uninfected tissue. Thus, TMV inoculated hypersensitive tobacco cultivar
develops systemic resistance against TMV (virus), Phytophthora parasitica var.
nicotinae (fungi) and Pseudomonas tabaci (bacteria). TMV also induced resistance against Peronospora tabacina and reduced reproduction of the aphid
Myzus persicae (McIntyre et al., 1981). Thus a single viral agent induced resistance in tobacco against diverse challenges.
The rst report of virus induced SAR came in 1952. Primary inoculation
of the lower leaves of D. barbatus with carnation mosaic virus (CarMV)
resulted in the development of fewer lesions on the upper leaves upon challenge inoculation with CarMV (Gilpatrick and Weintraub, 1952). Virusinduced resistance was further substantiated by Ross (1961a, 1961b) and
Loebenstein (1963). Cucumber plants infected with tobacco necrosis virus
159
(TNV) protected the plant systemically against disease caused by the fungus
Colletotrichum lagenarium (Jenns and Kuc, 1977). SAR expessed in Vigna plants
following inoculation with TNV against challenge by TNV was not expressed
against challenge by a CMV which infects the host systemically (Pennazzio
and Roggero, 1991). Associated with SAR was the stimulation of ethyleneforming enzyme activity. Infection of ecotype Dijon of Arabidopsis thaliana
with turnip crinkle virus (TCV) leads to the resistance against further infection by TCV or Pseudomonas syringae (Uknes et al., 1993). SAR in cucumber
plants against powdery mildew disease, caused by Sphaerotheca fuliginea
(Schlechtend Fr.) Pollacci, was induced by localized infection in cucumber
cotyledons with TNV (Farrag et al., 2007).
The SAR phenomenon is observed both in dicotyledonous and monocotyledonous plants; it provides the third and nal line of defence against
pathogens. The rst line of defence consists of genetically inherited resistance mechanisms that make plants constitutively resistant to the majority of
pathogens present in the environment. The second line of defence is activated in the immediate vicinity of the infected or wounded site in an attempt
to prevent the spread of pathogens throughout the plant. The local resistance
response develops more rapidly than SAR and involves cell-wall and cuticle
strengthening, synthesis of toxins, antifeedants and the production of
defence-related proteins including the PR proteins. In addition to long-distance signal molecules, local resistance may be partially mediated through
relatively immobile endogenous elicitors, which include oligogalacturonide
fragments of the plant cell wall (Lamb and Dixon, 1990). Several lines of evidence suggest that endogenous SA is a signal molecule in SAR. Involvement
of SA in SAR came from the discovery that endogenous SA increases by at
least 20-fold in the virus-inoculated leaves of tobacco (Malamy et al., 1990).
The increase coincides with the appearance of hypersensitive response (HR)
lesions on the inoculated leaves. Accumulation of SA increased with the
intensity of HR and was proportional to the dose of virus inoculum (Yalpani
et al., 1991). Tissue accumulation of SA in TMV inoculated xanthi-nc tobacco
paralleled or preceded detectable increase in the levels of PR-1 mRNA in
both inoculated and uninoculated leaves (Malamy et al., 1990).
7.4 SAR and the Role of PR Proteins

SAR strongly correlates with the coordinate expression of at least nine families of genes (sar genes), of which several encode the PR proteins (Ward
et al., 1991). Generally, tissues in which a signicant amount of PR protein has
been induced are more resistant to infection by pathogen than those that lack
PR proteins. PR proteins were rst discovered in 1970 in tobacco plants reacting hypersensitively to TMV infection (Gianinazzi et al., 1970; Van Loon and
Kammen, 1970). PR proteins are produced in plants in response to infection
by viruses, bacteria (Metraux and Boller, 1986), fungi (Gianinazi et al., 1980)
and viroids (Conejero et al., 1979) and are also synthesized in response to
chemical treatments and specic physiological stresses (Stintzi et al., 1993).
160
Several classes of the PR proteins either possess direct antimicrobial activity

or are closely related to classes of antimicrobial proteins. These include -1,3glucanase, chitinase, cysteine-rich proteins related to thaumatin and PR-1
proteins. An in vivo role in disease resistance has not been demonstrated for
any of the PR proteins. Alexander et al. (1993) demonstrated that constitutive
high level expression of PR 1-a in transgenic tobacco results in tolerance to
infection by Peronospora tabacina and Phytophthora parasitica var. nicotinae. On
the other hand, transgenic tobacco plants expressing PR 1-b gene exhibited
no reduction in the severity of TMV symptoms (Cutt et al., 1989). Quite often
SAR was not correlated with the induction of PR proteins (Kopp et al., 1989; Ye
et al., 1989; Cohen et al., 1993; Kessman et al., 1994). Fraser found a poor
correlation between the levels of PR 1-a protein and also gave evidence for
the occurrence of PR proteins in leaves of healthy tobacco plants during
flowering (Fraser, 1981; 1982). In conclusion, most of the evidence shows that
the PR proteins are closely associated with, and not necessarily responsible
for, induced resistance.
7.5 Induced Resistance and the Role of Induced Antiviral

Proteins
Virus-induced new antiviral protein components
Antiviral substances are formed in plants responding hypersensitively to
virus infection (Verma and Prasad, 1992) and have been recognized as phosphorylated glycoproteins (Faccioli and Capponi, 1983), glycoproteins (Wieringa and Dekker, 1987), RNA (Kimmins, 1969), traumatic acid (Kato and
Misawa, 1976), and protein-like substances (Nienhaus and Babovic, 1978).
Chadha and MacNeill (1969) found the formation of an antiviral principle in
tomato plants systemically infected with TMV. These antiviral compounds
are not generally specific to the plants. Thus, antiviral substances produced
in capsicum plants could reduce PVX infection on Gomphrena globosa as well
as Solanum tuberosum (Nagaich and Singh, 1970).
The presence of an antiviral factor (AVF) was established in virus-infected
plants which could decrease the number of local lesions produced by TMV
and PVY (Sela and Applebaum, 1962). Partially purified AVF from TMV
infected N. glutinosa plants was found to contain both protein and RNA (Sela
et al., 1964). It was sensitive to ribonuclease and was resistant to proteolytic
enzymes (Sela et al., 1966).
Mozes et al. (1978) established that the purified AVF is a phosphorylated
glycoprotein of molecular weight 22 kDa on SDS gels. It is sensitive to pronase under conditions suitable for proteolysis of glycoproteins. It remains
active after treatments with SDS and is stable at pH 2.0. It resembles interferon in many of its properties (Mozes et al., 1978).
Previously it was believed that TMV infection was necessary for AVF
production. But Edelbaum et al. (1983) found that TMV infection could be
substituted by treatment with a mixture of Poly (I), Poly (C), cAMP
161
and cGMP for the induction of active AVF in leaves and callus cultures of
N. glutinosa.
Inhibitor of virus replication

Loebenstein and Gera (1981) reported, for the rst time, an inhibitor of virus
replication (IVR). IVR is released into the medium from TMV-infected cells of
Samsun NN plants and inhibits replication in protoplasts from local-lesionresponding Samsun plants. IVR is detected as soon as 24 h after inoculation
of protoplasts and it is effective when applied up to 18 h after inoculation.
IVR is neither host nor virus specic. It inhibits TMV, CMV and also PVX
(Gera and Loebenstein, 1983). Gera and Loebenstein (1983) reported that IVR
also inhibited TMV replication in intact leaves when applied to cut stems or
when used as a spray.
Plant-extract-induced virus inhibitory agent

It would be interesting to study the entire cascade of events and properties of
the virus inhibitory agent (VIA) induced by antiviral agents. Treatment of
lower / upper leaves of hypersensitive or systemic hosts of virus with
antiviral agents results in the development of resistance throughout the plant
a few hours later. This is detectable by challenge inoculation with viruses
producing local lesions or systemic symptoms. The lesions are either reduced
or totally absent and systemic symptoms are either milder or totally suppressed. Plant extracts or the semi-puried proteins from these plants stimulate the hosts to produce VIAs that spread to surrounding tissues and other
plant parts (Verma et al., 1996). The VIAs have been isolated from leaves of
plants treated with phytoproteins and they have been shown to inactivate
the viruses in vitro (Verma and Awasthi, 1980; Verma and Dwivedi, 1984;
Verma et al., 1996). The production of VIA leading to resistance seems to be
an activation of a pre-existing system and hence is easily stimulated. VIA is
able to move from one leaf to another through the vascular system of the
plant.
Therefore, the antiviral agents from plants can be broadly grouped into
two categories based on their mode of action: (i) those affecting virus in vitro;
and (ii) those affecting via host plants.
Amongst the latter, we can distinguish (1) those that act by affecting host
susceptibility and which need to be applied in leaf tissue before or at the time
of virus inoculation, e.g. protein inhibitors from Phytolacca spp., Dianthus
spp., Chenopodium spp. and so on, and (2) those acting by inducing the host
resistance mechanism, which can truly be called antiviral agents, and act by
obstructing the establishment of virus. They exert their effect when applied a
few hours before virus inoculation. Their effect is visible even on non-treated
parts of susceptible plants (Verma and Mukherjee, 1975; Verma and Awasthi,
1979; Verma et al., 1979, 1980, 1982, 1985, 1995, 1996; Kumar et al., 1997).
162
Verma and Awasthi (1980) reported that the synthesis of VIA is inhibited
if AMD is applied soon after extract treatment. The VIA synthesized is neither
virus specific nor host specific. Extracts containing VIA when incubated with
the virus reduced their infectivity. VIAs from a few hosts have been characterized. The VIA synthesized in the leaves of N. glutinosa, treated with B.
diffusa root extract reduced infectivity of TMV on N. glutinosa, Datura stramonium and D. metel (Verma and Awasthi, 1980). It was, however, less effective
in inhibiting TRSV and GMV on C. amaranticolor. The VIA production was
maximum after 24 h treatment with the extract. The VIA has a proteinaceous
nature (Verma and Awasthi, 1980). VIA synthesized in C. tetragonoloba plants
following treatment with B. spectabilis leaf extract prevented infection of
tobamoviruses in seven hypersensitive hosts (Verma and Dwivedi, 1984) and
its production was maximum after 48 h treatment. The VIA showed characteristics of a protein (Verma and Dwivedi, 1984). VIA produced in the same
host upon treatment with Pseuderanthemum bicolor extract completely prevented infection of SHRV, TMV, CGMMV, and PVX on C. tetragonoloba, D.
stramonium, C. amaranticolor and G. globosa, respectively (Khan and Verma,
1990).
VIA and the antiviral state are induced concomitantly with the systemic
resistance induction in host plants, following treatment with certain plant
extracts. VIA is an inducible gene product like AVF, IVR and PR proteins. The
phytoproteins occurring in the root / leaf extracts of Boerhaavia diffusa, Clerodendrum aculeatum, Clerodendrum inerme and B. spectabilis etc. possess strong
systemic-resistance-inducing properties (Verma and Prasad, 1992).
Clerodendrum aculeatum and Boerhaavia diffusa, the two potent

antiviral plants
Clerodendrum and Boerhaavia are economically important plants because
many of their species possess medicinal properties. Almost every part of
these plants is credited with some medicinal properties and is employed in
traditional Indian system of natural therapy.
Virus inhibitors from Boerhaavia diffusa and Clerodendrum aculeatum
inhibit several plant viruses. These inhibitors modify the susceptibility of
host plants towards virus infection. The roots of B. diffusa are a rich source of
a basic protein, which has been used for inducing systemic resistance in
many susceptible crops against commonly occurring viruses (Verma and
Awasthi, 1979, 1980; Verma et al., 1979; Awasthi et al., 1984, 1985, 1989; Verma
et al., 1995, 1999). This protein or antiviral agent was active against spherical
and tubular viruses in hypersensitive hosts such as Datura metel, Nicotiana
tabacum var Ky-58, N. glutinosa / TMV, Cyamopsis tetragonoloba / SHRV, Vigna
sinensis / SHRV, and systemic hosts such as Nicotiana tabacum c.v NP-31 / TMV,
Crotolaria juncea / SHRV, N.glutinosa / TRSV, when applied a few hours (1724
h) before virus inoculation or when tested after mixing with virus inoculum
(Verma and Awasthi, 1979; Awasthi et al., 1984). The inhibitor is a basic glycoprotein (7080% protein, 813% carbohydrate) with a molecular weight of
163
1620 kDa as determined by gel-filtration chromatography (Verma et al.,

1979). The protein has a PI of around 9 and has a molecular weight of 30 kDa
(Srivastava, 1995). The RIP was found to be extremely thermostable (Verma
and Awasthi, 1979). Following treatment with the systemic RIP, the host produces a VIA. The VIA shows characteristics of a protein and reduces infectivity of the viruses both in vitro and in vivo (Verma and Awasthi, 1980). Upon
gel filteration on Sephadex G-25, two active fractions exhibiting protein
characteristics were recovered (Verma and Awasthi, 1980).
The protein occurring in B. diffusa functions as a signal molecule and is of
great interest as it has a role in stimulating the defence system of plants
against viruses (Singh, 2006; Verma et al., 2006). The VIA is present in both
treated and untreated leaves (Verma and Awasthi, 1980). Micropropagation
of B. diffusa has been carried out for producing the systemic RIP for viral
disease management (Gupta, 1999; Gupta et al., 2004).
Susceptible healthy hosts upon treatment with C. aculeatum extract
develop complete resistance within 46 h. The SIR is reversed by the simultaneous application of AMD (Verma et al., 1984). Treatment with SRI from C.
aculeatum leaves, triggers accumulation of a new defensive VIA in treated
and non-treated leaves of healthy host plants (Verma et al., 1984; Verma et al.,
1996, 1999; Srivastava 1999). The SRI in crude sap is resistant to denaturation
by organic solvents and extremely thermostable (Verma et al., 1984). The
resistance-inducing activity of C. aculeatum SRI (CA-SRI) is not affected by
exogenous application of proteases (Verma et al., 1996). Leaf extract of C. aculeatum is most effective for controlling virus diseases in crop plants (Verma
et al., 1995a; Srivastava 1999; Srivastava et al., 2004). Leaf extract, when
sprayed on susceptible host plants, prevents infection of mechanically and
white fly transmitted viruses in several hosts, e.g. tomato yellow mosaic
virus, tobacco mosaic virus, sunnhemp rosette virus and tobacco leaf curl
virus (Verma et al., 1984; Verma et al., 1995a) (Table 7.2).
Table 7.2. Plants containing virus inhibitory activity.

Name
Family
Reference
Amaranthus albus
Alternanthera brasiliana
Acacia arabica
Amaranthaceae
Amaranthaceae
Leguminosae
(Fabaceae)
Aceraceae
Agavaceae
Simaroubaceae
Amaranthaceae
Amaranthaceae
Papaveraceae
Chenopodiaceae
Smookler, 1971
Noronha et al., 1983
Gupta and Raychauduri, 1971a
Acer insulera
Agava americana
Ailanthes excelsa
Alternanthere ficoidea
Amaranthus caudatus
Argemone mexicana
Beta vulgaris
Yoshi et al., 1954

Simon, 1963
Patel and Patel, 1979
Noronha et al., 1983
Smooker, 1971
Patel and Patel, 1979
Paliwal and Narinai, 1965
Continued
164
Table 7.2. Continued.

Name
Family
Reference
Boerhaavia diffusa
Nyctaginaceae
Bougainvillea spectabilis
Brassica oleracea
Callistemon lanceolatus
Carissa edulis
Celosia plumose
Chenopodium amaranticolor
Chenopodium ambrisioides
Cinchona ledgeriana
Citrus medicatimonum
Clerodendrum aculeatum
Nyctaginaceae
Brassicaceae
Myrtaceae
Apocynaceae
Amaranthaceae
Chenopodiaceae
Chenopodiaceae
Rubiaceae
Rutaceae
Verbenaceae
Clerodendrum fragrans
Cocos nucifera
Cucurbita maxima
Cuscuta reflexa
Datura metel
Datura stramonium
Dianthus caryophyllus
Eucalyptus tereticornis
Eugenia jambolana
Euphorbia hitra
Gomphrena globosa
Gyandropsis pentaphylla
Helianthus annus
Hordeum vulgare
Verbenaeceae
Arecaceae
Cucurbitaceae
Cucurbitaceae
Solanaceae
Solanaceae
Caryophylaceae
Myrtaceae
Myrtaceae
Euphorbiaceae
Amaranthaceae
Asteraceae
Asteraceae
Gramineae
(Poaceae)
Nyctaginaceae
Cactaceae
Solanaceae
Euphorbiaceae
Phytolaccaceae
Acanthaceae
Verma and Baranwal, 1988; Mehrotra

et al., 2002; Sukhdev, 2006; Singh,
2006
Verma and Dwivedi, 1983
Verma, 1973
Gupta and Raychaudhuri, 1971a,b;
Tolo et al., 2006
Patil, 1973
Smookler, 1971 Alberghina, 1976
Verma and Barranwal, 1983
Gupta and Raychauduri, 1971b
Ray, et al., 1979
Verma et al., 1984; Srivastava 1999;
Srivastava et al., 2004; Singh, 2006
Verma et al., 1984
Gendron, 1950
Weintrab and Willison, 1983
Awasthi, 1982
Verma and Awasthi, 1975
Paliwal and Narinai, 1965
Van Kammen et al., 1961
Ray et al., 1979
Verma et al., 1969
Weeraratne, 1961
Grasso and Shephard, 1978
Paliwal and Nariani, 1965
Johari, et al., 1983
Leah, et al., 1991
Mirabilis jalapa
Opuntia robusta
Petunia hybrida
Phyllanthus frafernus
Phytolacca dodecandra
Pseudoranthemum
atropurpureum
Rumex hastatus
Solanum nigrum
Terminala chebula
Tetragonia expansa
Zingiber officinale
Polygonaceae
Solanaceae
Combretaceae
Tetragoniaceae
Zingiberaceae
Kataoka et al., 1991

Simons et al., 1963
Singh, 1972
Saigopal et al., 1986
Reddy et al., 1984
Verma et al., 1985
Singh, et al., 1977
Vasudeva and Nariani, 1952
Gupta and Raychauduri, 1971b
Benda, 1956
Ray et al., 1979
The systemic resistance inducer from Clerodendrum aculeatum and

Clerodendrum inerme have been puried and characterized. A 34-kDa basic
protein was isolated from the leaves of C. aculeatum: 64 g/ml of protein
provided complete protection of untreated leaves against TMV infection in
165
N. tabacum Samsun NN (Verma et al., 1996). Two basic proteins of 29 and

34 kDa (CIP-29 and CIP-34) were isolated from the leaves of C. inerme. A
resistance-inducing protein of 31 kDa was isolated from the same plant
(Parveen et al., 2001). The minimum amount of puried proteins required to
induce systemic resistance varied from 16 g/ml for CIP-29 to 800 g/ml
for CIP-34 (Prasad et al., 1995). CAP-34, a protein from C. aculeatum, when
applied to lower leaves of Carica papaya, suppressed the disease caused by
papaya ring spot virus (PRSV). In the control papaya plants the typical disease symptoms of PRSV, that is, mosaic to liformy, appeared in 95% of the
plants between 30 and 60 days after virus inoculation. In the CAP-34-treated
papaya plants the symptoms appeared in only 10% of the plants during the
same period. The presence of PRSV was determined by ELISA and RTPCR
(Srivastava et al., 2009). It appears that protein from C. aculeatum is a good
candidate for utilization in management of virus diseases. The systemic
virus inhibitory activity of these proteins is due to the host-mediated phenomenon of formation / accumulation of a new virus inhibitory protein in
the treated plants showing systemic resistance. When TMV is mixed with
the induced virus inhibitory protein, the virus is completely inhibited. The
fact that CA-SRI also behaves as an RIP, as it inhibited in vitro protein synthesis in rabbit reticulocyte lysate and wheat germ lysate (Kumar et al.,
1997), strongly conrms this belief that antiviral proteins generally have the
properties of RIPs. The sequence of the CA-SRI protein showed varying
homology (1154%) with the RIPs from other plant species (Kumar et al.,
1997). However, the absence of hybridization between the CA-SRI gene and
the DNA / RNA of Mirabilis, Bougainvillea, rice, pea and tobacco shows that
the virus inhibitory genes do not react with leaf proteins from Mirabilis,
Bougainvillea, Boerhaavia, rice, pea and tobacco. These only indicate that
antiviral proteins from different plants may behave differently and can be
used specically in particular hostvirus systems.
The pre-inoculation spray of SRIs from C. aculeatum, C. inerme, Boerhaavia
diffusa, B. spectabilis and Pseuderanthemum bicolor modied the susceptibility
of several host plants such as tomato, tobacco, mungbean, urdbean, bhendi
and sunnhemp against subsequent infection by viruses. The treatment helps
to protect the susceptible hosts during the vulnerable early stages of development. Induced resistance appears to be a universal process in all susceptible hosts and can be used to advantage in plant protection. Pre-inoculation
sprays (four sprays) of B. spectabilis leaf extract protected plants of Cucumis
melo against cucumber green mottle mosaic virus (CGMMV), Crotalaria juncea against sunnhemp rosette virus (SRV) and Lycopersicon esculentum against
TMV for 6 days (Verma and Dwivedi, 1983). Since the duration of resistance
conferred by SRIs was up to only 6 days, it was realized that the durability
of resistance needed to be prolonged under eld conditions to achieve better
protection against virus infection. It has been shown that proteinaceous
modiers, such as papain, enhanced the activity of CA-SRI and also prolonged the durability of induced resistance against sunnhemp rosette
tobamovirus in Crotalaria juncea up to 12 days (Verma and Varsha, 1995). In
another study, ve weekly sprays of CA-SRI in potted plants of tomato in
166
open elds protected the plants from natural virus infection for more than 2
months (unpublished results). However, in another study it was shown that
weekly sprays of leaf extract of C. aculeatum delayed the symptom appearance of leaf curl virus in tomato and promoted the growth of the plants
(Baranwal and Ahmad, 1997). A natural plant compound called NS-83 was
shown to reduce and delay the disease symptoms by TMV, PVX and PVY in
tobacco and tomato plants under eld conditions and the fruit yield in
tomato was increased by 23.4% (Xin-Yun et al., 1988). Plant extracts from C.
inerme and Ocimum sanctum provided a high degree of resistance against
tobacco chlorotic mottle virus in cowpea, possibly by induction of systemic
induced resistance (Mistry et al., 2003). Sumia et al. (2005) demonstrated
inhibition of local lesions and systemic infection induced by Tobacco streak
virus in cowpea and French bean by pre-treatment of a proteinaceous substance from seeds of Celosia cristata. Foliar sprays with aqueous leaf extract
of C. aculeatum plant since the sprouting stage at fortnightly intervals in the
eld could signicantly protect Amorphophallus campanulatus against infection by virus. Maximum reduction in disease incidence and symptom severity was exhibited by plants which received six sprays. Maximum plant
growth along with considerable increases in corm weight was also observed
in such plants (Khan and Awasthi, 2006).
It appears that antiviral phytoproteins trigger the host defence
mechanism in a specic manner either by signal transduction, as demonstrated in the case of Phytolacca antiviral protein (PAP), and/or by increased
synthesis of antiviral proteins in host plants treated by systemic resistance
inducers as for C. aculeatum. While success has been achieved in developing
transgenics that have genes for mutant PAP without RIP activity (Smirnov
et al., 1997), it remains to be seen how these transgenics can be utilized under
eld conditions. On the other hand, SRIs such as CA-SRI show a potential for
use under eld conditions. However, a larger quantity of SRIs would be
required for their wider application. Cloning of the genes for CA-SRI and
their expression has been achieved in an Escherichia coli expression vector.
The expressed protein has been shown to inhibit protein synthesis (Kumar
et al., 1997). A detailed study is still required to determine whether or not
a transgenic with native and mutant CA-SRI genes would be able to
show systemic protection against virus infection. Although the C. aculeatum
protein has been demonstrated to have very high antiviral activity and
to be extremely useful as a plant immunizing agent, it has not been
commercialized so far. The proteins from a few other non-host plants have
also been recognized as good defence stimulants, but they have not been
developed into products for disease control, because the industry nds
it easier to patent newly synthesized compounds than natural plant
products.
Singh (2006) reported the anti-proliferative property of phytoproteins
from B. diffusa and C. aculeatum for the retardation of proliferation of human
breast cancer cell lines as well as the inhibition of activity of the Semliki
Forest virus in mice.
167
7.6 Conclusion
With the precipitous withdrawal of some of the toxic protectants, it may be
protable to explore natural plant products as alternatives, particularly
against virus diseases where all other methods fail. The phytoproteins or
their smaller peptides may prove valuable as lead structures for the development of synthetic compounds. It would pay us to explore this rich source
of antivirals more thoroughly. The value of these antiviral proteins is unlimited because they are quite safe, non-toxic even after repeated and prolonged
use, and substantially enhance plant growth and yield. Antiviral phytoproteins may be useful if they are integrated with other strategies of virus disease
management.
Botanical resistance inducers can be classied as ideal virus-suppressing
agents, as they encompass all the characteristics of an ideal antiviral compound. Induced resistance operates through the activation of natural defence
mechanisms of the host plant and induces systemic resistance to viral multiplication in plants. The active products present in these extracts have no
direct effect on viruses; their antiviral activity is mediated by host cells in
which they induce the antiviral state. Systemic resistance inducers obtained
from plants have been shown to be effective against a wide range of viruses.
Plant extracts or the semi-puried proteins from these plants stimulate the
hosts to produce a virus inhibitory agent, which spreads to surrounding
tissues and other plant parts.
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Phytochemicals as Natural
Fumigants and Contact
Insecticides Against
Stored-product Insects
MOSHE KOSTYUKOVSKY AND ELI SHAAYA
Agricultural Research Organization, the Volcani Center, Israel
Abstract
For centuries, traditional agriculture in developing countries has used effective methods of insect pest control using botanicals. In order to make them a cheap and simple
means of insect control for users, their efcacy and optimal use still need to be
assessed. Currently, the measures to control pest infestation in grain, dry stored food
and cut owers rely heavily on toxic fumigants and contact insecticides. In recent
years, the number of pesticides has declined as health, safety and environmental concerns have prompted authorities to consider restricting the use of toxic chemicals in
food. Lately a new eld is developing on the use of phytochemicals in insect pest
management, such as edible and essential oils and their constituents. The aim here
has been to evaluate the potential use of edible oils obtained from oil seeds and
essential oils, and their constituents obtained from aromatic plants, as fumigants and
contact insecticides for the control of the legume pest Callosobruchus maculatus
F. (Coleoptera: Bruchidae). The most active edible oils as contact insecticides were
crude oils from rice, maize, cottonseed and palm, and the fatty acids capric acid and
undecanoic acid. The essentials oils and their constituents were found to have higher
activity as fumigants than contact insecticides. From our studies, to elucidate the
mode of action of essential oils, it was possible to postulate that essential oils may
affect octopaminergic target sites.
8.1 Introduction
Insect damage in stored grains and other durable commodities may amount
to 1040% in developing countries, where modern storage technologies
have not been introduced (Raja et al., 2001). Currently, food industries rely
mainly on fumigation as an effective method for insect pest control in grain
and other dry food commodities. At present only two fumigants are still in
use: methyl bromide and phosphine. The rst one is mostly being phased
out in developed countries due to its ozone depletion effects (WMO, 1995;
(ed. N.K. Dubey)
175
176
M. Kostyukovsky and E. Shaaya
Shaaya and Kostyukovsky, 2006). In addition, there have been repeated

indications that certain insects have developed resistance to phosphine,
which is widely used today (Nakakita and Winks, 1981; Mills, 1983; Tyler
et al., 1983). It should be mentioned that although effective fumigants and
contact synthesized insecticides are available, there is global concern about
their negative effects on non-target organisms, pest resistance and pesticide
residues (Kostyukovsky et al., 2002a; Ogendo et al., 2003). In recent years,
attention has been focused on the use of botanicals as possible alternatives
to toxic insecticides. Lately, there has been a growing interest in the use of
plant oils and their bioactive chemical constituents for the protection of
agriculture products due to their low mammalian toxicity and low persistence in the environment (Raja et al., 2001; Papachristos and Stamopoulos,
2002). Plant oils have repellent and insecticidal (Shaaya et al., 1997;
Kostyukovsky et al., 2002a; Papachristos and Stamopoulos, 2002), nematicidal
(Oka et al., 2000), antifungal (Paster et al., 1995; Srivastava et al., 2009), antibacterial (Matasyoh et al., 2007), virucidal (Schuhmacher et al., 2003), antifeedant and reproduction inhibitory (Raja et al., 2001; Papachristos and
Stamopoulos, 2002) effects.
Numerous plant species have been reported to have insecticidal properties capable of controlling insects (Grainge and Ahmed, 1988; Arnason et al.,
1989). The toxicity of a large number of essential oils and their constituents
has been evaluated against a number of stored-product insects. Essential oils
extracted from Pogostemon heyneanus, Ocimum basilicum and Eucaluptus
showed insecticidal activities against Sitophilus oryzae, Stegobium paniceum,
Tribolium castaneum and Callosobruchus chinensis (Deshpande et al., 1974;
Deshpande and Tipnis, 1977). Toxic effects of the terpnenoids d-limonene,
linalool and terpineol were observed on several coleopterans damaging postharvest products (Karr and Coats, l988; Coats et al., 1991; Weaver et al., 1991).
Fumigant toxic activity and reproductive inhibition induced by a number of
essential oils and their monoterpenenoids were also evaluated against the
bean weevil Acanthoscelides obtectus and the moth Sitotroga cerealella (Klingauf
et al., 1983; Regnault-Roger and Hamraoui, 1995). Our earlier investigations
on the effectiveness of the essentials oils extracted from aromatic plants,
showed great promise for the control of the major stored-product insects.
Several of them were found to be active fumigants at low concentrations
against these insects (Shaaya et al., 1991, 1993, 1994).
The use of edible oils as contact insecticides to protect grains, especially
legumes, against storage insects is traditional practice in many countries in
Asia and Africa. The method is convenient and inexpensive for the protection of stored seeds in households and in small farms. Many different edible
oils have been studied as stored grain protectants against insects (Oca et al.,
1978; Varma and Pandey, 1978; Pandey et al., 1981; Santos et al., 1981; Messina
and Renwick, 1983; Ivbijaro, 1984; Ivbijaro et al., 1984; Pierrard, 1986; Ahmed
et al., 1988; Don Pedro, 1989; Pacheco et al., 1995).
This chapter evaluates the efcacy of essential and edible oils as fumigants and contact insecticides to suppress populations of the stored-product
insects, mainly the legume pest insect Callosobruchus maculatus. The pulse
Phytochemicals as Natural Fumigants
177
beetle, Callosobruchus maculatus F. (Coleoptera: Bruchidae), is one of the major

pests of stored cowpea, lentils and green and black gram in the tropics
(Sharma, 1984; Raja et al., 2007). The infestation often begins in the eld on
the dry ripe seeds before harvest. The damage to the seeds can reach up to
50% after 6 months of storage (Caswell, 1980) and up to 90% annually, according to the International Institute of Tropical Agriculture (IITA, 1989).
8.2 Studies with Edible Oils and Fatty Acids: Biological Activity
and Repellency
The biological activity of a number of crude and distilled edible oils and a
number of straight-chain fatty acids, which contain from C5 to C18, was evaluated in laboratory tests against the common legume pest C. maculatus. All
the edible oils tested were found to have different degrees of activity at a
concentration of 1 g/kg (= 1 kg/ton) chickpea seeds (Table 8.1).
The most active oils were crude oils from rice, maize, cottonseeds and
palm. Of the eggs laid, 9096% did not develop to larvae and only 01% developed to F1 adults in the seeds treated with these oils (Table 8.1). In addition,
some of the oils tested were found to prevent oviposition. The most active oil
in this regard was the rice crude oil: only 50 eggs were laid on seeds treated
with 1 g/kg oil compared to 287 eggs on the control seeds (Table 8.1).
Table 8.1.
Biological activity of various edible oils against Callosobruchus maculatus.
Oil
Crude rice
Crude maize
Refined maize
Crude cotton seed
Refined cotton seed
Crude palm
Refined palm
Crude soya bean
Refined soya bean
Crude coconut
Distilled peanut
Distilled safflower
Crude olive
Refined olive
Refined sunflower
Distilled kapok
Control
Adult emergence
Number of
eggs laid
Egg mortality
(%)
Number
50
137
150
212
275
375
237
212
262
125
150
287
175
300
187
387
287
100
96
97
99
89
99
84
85
96
95
95
97
74
86
82
88
5
0
1
3
2
22
4
38
21
10
6
8
9
46
18
30
39
270
0
1
2
1
8
1
16
10
4
5
5
3
26
6
16
10
94
Each oil was used at a concentration of 1 g/kg chickpea. The required amount of the oil was first mixed
with acetone (50 ml/kg seeds) and the acetone was evaporated under a hood. Five males and five females
were introduced to 5 g seeds. The data are the average of three experiments, each one was in triplicate.
178
To obtain an insight into the nature of the activity of the oils, the activity
of straight-chain fatty acids ranging from C5 to C18 at a concentration of
4 g/kg was studied against C. maculatus (Table 8.2). The results showed that
C9C11 acids were the most active in preventing oviposition at this concentration, with the C11 acid the most active: only 13, 13 and 0 eggs were found
on the treated seeds, respectively (Table 8.2).
C12C16 fatty acids were less effective and activity was remarkably
decreased for the lower C5C7 and higher C17C18 acids. At lower concentrations of 1.6, 0.8 and 0.4 g/kg, the C11 acid was found to be the most active;
fewer eggs were laid than with C9 or C10 and no eggs developed to adults.
The data presented in Table 8.3 show clearly that C9C11 acids are strongly
repellent to C. maculatus, but they have no lethal effect on the adults.
Field tests using crude palm kernel and rice bran oils showed that both
oils were effective in controlling C. maculatus infestation providing full protection for the rst 45 months of storage at a rate of 1.53.0 g/kg seeds. They
persisted in controlling insect infestation for up to 15 months. The numbers of
adult insects found in the treated seeds after 15 months of storage were only
about 10% of that in the control samples (results not shown).
8.3 Studies with Essential Oils as Contact Insecticides

The efcacy of a large number of essential oils has been evaluated for the
control of C. maculatus at a concentration of 400 ppm. The various oils tested
were found to have a toxic effect on the eggs laid and on the development of
the eggs to adults (Table 8.4).
Table 8.2. Biological activity of straight-chain fatty acids C5C18 against
Callosobruchus maculatus.
Adult emergence
Fatty acid
Pentanoic(C5)
Hexanoic (C6)
Heptanoic (C7)
Octanoic (C8)
Nanonoic (C9)
Decanoic (C10)
Undecanoic (C11)
Dodecanoic (C12)
Tridecanoic (C13)
Tetradecanoic (C14)
Pentadecanoic (C15)
Hexadecanoic (C16)
Heptadecanoic (C17)
Octadecanoic (C18)
Control
Number of eggs laid
Number
185
130
112
105
13
13
0
32
32
62
72
65
120
140
287
110
92
66
22
0
0
0
22
20
45
56
37
88
115
272
59
71
59
21
0
0
0
69
62
73
77
57
73
82
95
Each fatty acid was used at a concentration of 4 g/kg chickpea. The data are the average of three
experiments, each of which were in triplicate.
179
Table 8.3. Toxicology and repellency of the fatty acids C9C11 tested against
Callosobruchus maculatus.
Fatty acid
C9
Control
C10
Control
C11
Control
Adult emergence
Concentration
(g/kg)
Number of
eggs laid
Undeveloped
eggs (%)
Number
1.6
0.8
0.4
0
1.6
0.8
0.4
0
1.6
0.8
0.4
0
60
67
100
360
65
27
62
300
20
20
22
300
100
95
72
3
100
92
78
5
100
100
100
3
0
3
18
345
0
1
7
280
0
0
0
287
0
4
18
96
0
4
11
93
0
0
0
96
The data are the average of three experiments, each of which were in triplicate.
Table 8.4. Contact activity of a number of essential oils on egg laying, egg development
and F1 of Callosobruchus maculatus.
Eggs laid
Essential oil
Number
Syrian marjoram
171
Lemon grass
144
Geranium
119
Vistria
157
Basil
51
Clary sage
40
Orange
67
Grapefruit
120
Lemon
172
Caraway
97
Cumin
148
Thyme
120
Celery
239
Thyme leaved savory
98
Ruta
109
Rosemary
123
Peppermint
159
SEM76 oil
67
Control
150350
% of control Eggs developed

to larvae (%)
value
83
70
34
57
78
22
34
64
88
51
59
69
179
68
75
68
92
28
87
88
14
77
93
75
84
77
91
79
74
68
79
89
76
79
83
93
8595
No. of
Eggs developed
adults F1
to adults (%)
0
5
8
15
23
22
45
49
50
62
55
46
169
75
51
64
54
27
120280
0
3.5
6.7
9.6
45
55
67
41
29
64
37
38
71
77
47
32
34
17
8085
Each essential oil was tested at a concentration of 400 ppm and an exposure time of 24 h.
Twenty unsexed adults were introduced to the treated and untreated seeds 24 h after treatment.
180
In the case of seeds treated with Syrian marjoram, the number of eggs
laid on the treated seeds was 83% of the control, but 0% of the eggs developed to adults. Other oils were found to cause a reduction in the number of
eggs laid on the treated seeds but were less effective on egg to adult development. In the case of clary sage oil, only 40 eggs were laid on the treated seeds
(= 22% of control), but 55% of the eggs developed to adults. In contrast, the
essential oil SEM76, obtained from Labiata plant species, was found effective
on both the reduction of number of eggs laid (28% of control) and on the
number of eggs that developed to adults (17% of control) (Table 8.4).
8.4 Studies with Essential Oils as Fumigants Against

Stored-product Insects
In order to isolate active essential oils we rstly screened a large number of
essential oils extracted from aromatic plants and isolated their main constituents, using space fumigation (Shaaya et al., 1991, 1993, 1994). The most active
compounds are summarized in Table 8.5.
Among the tested compounds, the essential oil SEM76 and its main constituent were found to be the most potent fumigants. To obtain the LC90 of all
insect species tested at adult stage, SEM76 oil was required at the concentration
of 0.61.2 l/l air (Table 8.5). The main constituent of the oil, which accounts for
approximately 80% of the oil, was also found to have high activity, a little higher
than the oil, as expected (Table 8.5). Space fumigation studies with SEM76
against various developmental stages of C. maculatus showed that eggs and
young larvae before they penetrated into the seeds were the most susceptible
to the compound. A concentration of 0.5 l/l air was enough to cause 100%
mortality of the eggs and the rst instar larvae. After the larvae penetrated
inside the seeds, they became more tolerant (Table 8.6). Only pupae 12 days
before adult emergence again became sensitive to the compound (Table 8.6).
Table 8.5. Fumigant toxicity of the most active monoterpenes tested on stored-product
insect pests in space fumigation tests.
Oryzaephilus
surinamensis
Rhizopertha
dominica
Sitophilus
oryzae
Tribolium
castaneum
Compound
LC50
LC90
LC50
LC90
LC50
LC90
LC50
LC90
1,8-Cineol
Carvacrol
Limonene
Linalool
Pulegone
SEM-76
SEM-76 oil
Terpinen-4-ol
Terpineol
3.1
>15.0
3.0
1.7
3.9
4.3
7.3
6.0
2.8
<1.0
<1.0
11.4
12.7
2.5
>15.0
6.7
6.0
2.8
0.4
0.5
1.3
>15.0
4.0
10.3
8.5
4.5
0.6
0.8
2.0
7.2
>15
>15
10.1
0.7
1.2
>15
14.2
19.8
1.4
<0.5
<1.0
5.2
7.5
>15.0
7.6
>15
2.5
0.7
0.9
2.5
>15
8.5
8.6
3.2
1.2
1.4
3.3
LC50 and LC90 values are l/l air. Exposure time was 24 h.
181
Table 8.6. Fumigant activity of the essential oil SEM76 extracted from Labiatae species on
various developmental stages of Callosobruchus maculatus.
F1
Stage treated
Age
Concentration
(l/l)
No. of
eggs
No. of
adults
Eggs developed to
adults (%)
Egg
Larvae outside
the seed
Larvae inside
the seed
Larvae
2024 h
01 day
0.5
0.5
20
20
0
0
0
0
2 days
Larvae
7 days
Larvae
11 days
Pupae
45 days
before
emergence
12 days
before
emergence
1.0
1.5
1.5
3.0
1.5
3.0
1.5
3.0
1.5
3.0
20
20
20
20
20
20
20
20
20
20
3.5
4.5
10.5
8
14
13
17.5
16.5
12
11
17
22
53
40
70
63
88
83
60
55
0.5
1.5
3.0
0
20
20
20
20
4
1
0
17
20
5
0
85
Pupae
Control
3 days
Twenty unsexed adults were used for each test. The data are an average of three duplicate
experiments. Exposure time was 24 h.
8.5 Distribution of a-Terpineol in the Fumigation Chamber

Most of the research to study the potential of essential oils and their constituents (terpenes) for the control of insect pests was done using space fumigation, without paying attention to the amount of the fumigant available to the
treated insects. As a model, we studied the distribution of the terpenoid
-terpineol in the fumigation chamber among the air in the chamber space,
lter paper and the ask walls. Using concentrations of 3, 5, 10 and 15 mg/l
air and stirring at 20C, only minor differences in the amount of the terpenoid in the chamber space was measured: 0.710, 0.676, 0.782 and 0.897 mg/l
air, respectively. The rest was found on the lter paper and the ask walls
(Table 8.7). At a higher temperature of 25C, a higher amount of the terpenoid, 5060%, was recovered in the chamber space compared to the amount
recovered at 20C (Table 8.7). No pronounced change of the terpenoid in the
chamber space was measured at a different intensity of stirring or when no
stirring was applied (Table 8.8). It should be mentioned that stirring caused
a higher concentration of the terpenoid on the glass walls compared to the
lter paper, and the opposite if no stirring was applied (Table 8.8). Using
stirring at 20C, we could show that after 2 h of fumigation the air in the
fumigation chamber was already saturated (Table 8.9).
182
Table 8.7. The distribution of -terpineol in the chamber space, filter paper and the
fumigation chamber walls at different temperatures, using stirring.
Amount recovered (%)
Temperature
(C)
Amount of
- terpineol
in the chamber
space
(mg/l air)
20
0.710
85.0
(2.682)
20
0.676
25
1.106
10
20
0.782
10
25
1.165
15
20
0.897
81
(4.265)
84
(4.366)
105
(10.932)
91
(9.53)
112
(17.591)
Concentration
used (mg/l air)
Total of
- terpineol
Chamber
recovered (%)
space
Filter
paper
Flask
walls
27.8
(0.745)
10.7
(0.287)
61.6
(1.65)
16.6
(0.710)
26.4
(1.16)
7.4
(0.820)
12.8
(1.22)
5.2
(0.921)
18.9
(0.805)
6.1
(0.266)
41.1
(4.54)
32.8
(3.13)
62
(10.89)
64.5
(2.75)
67.4
(2.96)
51.5
(5.68)
54.4
(5.18)
33
(5.78)
The numbers in brackets are the value of -terpineol in mg, recovered after 24 h of space fumigation.
The volume of the fumigation chamber is 1050 ml. The sensitivity of the GC measurements was 15%.
Table 8.8. Effect of stirring on the distribution of -terpineol in the chamber space, filter
paper and fumigation chamber walls.
Concentration Stirring time

used (mg/l) air and intensity
5
15
Amount recovered (%)

Amount of
- terpineol in
the chamber
Total of
space
- terpineol Chamber
Filter
Flask
(mg/l air)
recovered (%)
space
paper
walls
24 h moderate
0.965
24 h slow
0.856
15 min slow
0.800
0.858
24 h moderate
0.684
0.639
85.0
(4.549)
93
(4.265)
99
(5.200)
103
(5.320)
107
(16.898)
111
(17.490)
22.7
(1.010)
16.6
(0.710)
16.2
(0.840)
16.9
(0.901)
4.3
(0.718)
3.8
(0.671)
6.7
(0.278)
18.9
(0.805)
63.5
(3.30)
76.7
(4.080)
51.5
(8.70)
90.9
(15.90)
71
(3.160)
64.5
(2.750)
20.4
(1.060)
6.4
(0.339)
44.3
(7.480)
5.3
(0.919)
The numbers in brackets are the value of -terpineol in mg, recovered after 24 h of space fumigation.
The volume of the fumigation chamber was 1050 ml. The fumigation was conducted at a temperature
of 20C. The sensitivity of the GC measurements was 15%.
183
Table 8.9. Amount of -terpineol measured in the fumigation chamber space at various time
intervals of fumigation, using stirring.
Concentration
used (mg/l) air
3
5
10
15
The amount of -terpineol (mg) measured in the fumigation

chamber space, hours following fumigation
2
24
0.685
0.877
0.900
0.838
0.678
0.804
0.922
0.769
0.865
0.767
0.731
0.701
0.821
0.857
The volume of the fumigation chamber was 1050 ml.

The fumigation was conducted at a temperature of 20C.
8.6 Possible Mode of Action of Essential Oils

The essential oil SEM76 and pulegone were found to be highly toxic against
all stored-product insects tested (Shaaya et al., 2001, 2002). The effect of these
two compounds on acetylcholinesterase and the octopamine systems in
insects was studied in order to elucidate their mode of action.
In our studies with acetylcholinesterase (Greenberg-Levy et al., 1993;
Kostyukovsky et al., 2002b), using acetylcholinesterase extracted from Rhizopertha dominica, a stored-product insect, we showed that the inhibitory activity
of these two terpenoids on acetylcholinesterase was only evident when high
concentrations of these compounds (103 M) were applied. These doses were
at such high levels that they cannot account for the toxic effects observed in
vivo by these compounds on the same insect species (Shaaya et al., 2001, 2002),
which were obtained at much lower concentration (106 M = 1.5 l/l of air).
The failure of these biologically active compounds to produce a stronger
enzyme inhibition indicated that acetylcholinesterase was probably not the
main site of action of essential oils.
Another possible target for neurotoxicity of essential oils is the octopaminergic system in insects. Octopamine is a multifunctional, naturally occurring biogenic amine that plays a key role as a neurotransmitter, neurohormone
and neuromodulator in invertebrate systems (Evans, 1981; Fig. 8.1), with a
physiological role analogous to that of norepinephrine in vertebrates.
The octopaminergic system in insects represents a biorational target for
insecticidal action and has been targeted by various insecticides in the past,
e.g., formamidines (Haynes, 1988; Perry et al., 1998). Many of the physiological functions of octopamine appear to be mediated by a class of octopamine
receptors, specically linked to a protein coupled to the enzyme adenylate
cyclase. These physiological actions have been shown to be associated with
elevated levels of cyclic-AMP (Evans, 1984). Using an insect in-vitro cuticular
tissue preparation, octopamine was found to induce a signicant increase in
the intracellular messenger cyclic AMP (Rafaeli and Gileadi, 1995). SEM76
and pulegone directly affect the intracellular response of these tissues by
mimicking the octopamine response, thereby increasing the production of
184

Nerve impulse
HOCHCH2NH2
O
CH3
CH3
N
CH3
OH
CH3
Acetylcholine
Octopamine
Muscular
junction
Post-synaptic neuronal
membrane
Nerve impulse
Hemolymph
Muscular contraction,
other physiological,
modulatory functions
Fig. 8.1. Neurotransmitters in insect systems as possible sites of action of essential

oil toxicity.
Table 8.10. Effect of essential oils in the absence and presence of the octopamine
antagonist, phentolamine (105 M), on intracellular cyclic-AMP levels.
Treatment
Without
phentolamine
With
phentolamine
Concentration
(M)
Intracellular cyclic AMP levels

pmol/abdominal segment
Octopamine
SEM76
0
108
107
0
108
107
106
Pulegone
d-limonene
0.023
0.88
0.02
0.2
0.39
0.58
0.027
0.42
0.18
0.05
0.11
0.17
0.17
0.01
0.14
0.2
cyclic-AMP at low physiological concentrations of 108 M (Table 8.10). Moreover, addition of the octopamine antagonist phentolamine strongly antangonized the response to octopamine, as well as to the essential oil SEM76 and
pulegone, were strongly antagonized and no elevation in cyclic-AMP was
observed (Shaaya, et al., 2001; Kostyukovsky et al., 2002b). These effects were
observed at low dilutions of the essential oils (estimated at 107 and 108 M),
levels that induced the overt behavioural toxicity responses in vitro (Table
8.10; Kostyukovsky et al., 2002b). It should be mentioned that d-limonene,
185
which was found to have very low toxicity to stored-product insects, did not
show any effect on the production of cyclic-AMP (Table 8.10).
Because the essential oil response is strongly insect specic, as is octopamine neurotransmission, the essential oils can mimic the action of octopamine at low doses, and phentolamine, an octopamine inhibitor, has an
inhibitory action on the essential oils, we can postulate that the essential oils
may affect octopamonergic target sites.
8.7 Discussion
Studies with edible oils and fatty acids showed that crude oils of rice, maize,
cottonseed and palm were found the most potent against C. maculatus compared to the other oils tested. The puried forms of these oils were found to
be less effective. In eld studies, rice and palm crude oils at concentrations of
1.5 and 3 g/kg, protected chickpeas completely from insect infestation for a
period of 45 months and partially for up to 15 months. Studies by Khaire
et al. (1992) showed that adult emergence of C. chinensis was completely prevented for up to 100 days in pigeonpea treated with 1% neem oil or karanj oil.
In addition, theses oils were found to have no adverse effect on seed germination. Boeke et al. (2004) showed that a number of botanical products might
provide effective control of C. maculatus in cowpea.
The mode of action of edible oils was rst attributed to interference with
normal respiration, resulting in suffocation of the insects. The action of the
oil, however, is more complex, since insects deprived of oxygen survived
longer than those treated with oils (Gunther and Jeppson, 1960). From this
study and others (Wigglesworth, 1942; Ebeling and Wagner, 1959; Ebeling,
1976; Shaaya and Ikan, 1979), it may be postulated that the biological activity
of the edible oils is attributed to both their physical and chemical properties
such as viscosity, volatility, specic gravity and hydrophobicity.
Among the straight-chain fatty acids ranging from C5 to C18 carbon
atoms, it was found that C9C11 acids were the most effective in preventing
oviposition of C. maculatus on the treated seeds, but have no lethal effect on
the adults. In earlier studies (Shaaya et al., 1976), we showed that wheat seeds
treated with C10 fatty acid, at a concentration of 4 g/kg, repelled Sitophilus
oryzae L, but that forced contact of the beetles with the treated seeds was
found to have no effect on mortality rates of the beetles (Shaaya et al., 1976).
It should be mentioned that C. maculatus adults do not consume food and
live approximately 1 week only, whereas S. oryzae adults feed as long as they
live several months. We postulate, therefore, that fatty acids act as repellents and the beetles die because of starvation.
Essential oils as contact insecticide were found to have low toxicity. A
high concentration of 400 ppm of the most active essential oil from Syrian
marjoram was needed to prevent the development from egg to adult. On
the contrary, essential oils and their constituents as fumigants showed
high activity in controlling young larvae and pupae prior to emergence
of C. maculatus. The contact and fumigant toxicity of ve essential oils,
186
cardamom, cinnamon, clove, eucalyptus and neem were also investigated

against the cowpea weevil (Mahfuz and Khalequzzaman, 2007). In space
fumigation, at a concentration of 1 l/l air, Ocimum gratissimum oil and its
constituent eugenol caused 100% mortality of C. chinensis 24 h after treatment (Ogendo et al., 2008). The essential oils of citrodora and lemongrass
were found to have insecticidal and ovicidal activities against adults and
eggs of C. maculatus (Raja and William, 2008). Van Huis (1991), AdabieGomez et al. (2006), Henning (2007), Boateng and Kusi (2008) have reported
the efcacy of Jatropha seed oil as contact insecticide against C. maculatus.
Similarly, Kita et al. (2001) reported that kaolin powder aromatized with
pure oil of Ocimum spp. at 6.7 l/g of pea seeds provided complete protection
over 3 months of storage.
In space fumigation studies to evaluate the potency of the essential oils
and their constituents against insect pests, special attention has to be given
to not using a higher amount of the fumigant than needed to saturate the
air in the fumigation chamber at constant temperature. It should be mentioned that the amount of the various test materials needed to saturate the
air in the fumigation chamber might be different, depending on the physical and chemical properties of the fumigant. Our ndings, as well as results
of other researchers, suggest that certain essential oils, and particularly
their terpenoids, are highly selective to insects, since they are probably
targeted to an insect-selective octopaminergic receptor, which is a nonmammalian target. The worldwide availability of plant essential oils and
their terpenoids, and their use as avouring agents in food and beverages
is a good indication of their relative safety to humans. In our earlier studies,
we showed that essential oils have a high potential as alternative fumigants
to methyl bromide for the control of stored-product insects and cut owers
(Shaaya, 1998; Wilson and Shaaya, 1999; Kostyukovsky and Shaaya, 2001;
Kostyukovsky et al., 2002). The reduction in the use of conventional synthetic toxic pesticides gives to these bioactive chemicals the potential to be
used as a complementary or alternative method in crop production and
integrated pest management.
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Prospects of Large-scale
Use of Natural Products
as Alternatives to Synthetic
Pesticides in Developing
Countries
D.B. OLUFOLAJI
Department of Crop, Soil and Pest Management, The Federal University
of Technology, Akure, Nigeria
Abstract
The incidence of pests and diseases on crops in developing countries has caused a lot
of food insecurity and negatively affected the agro-allied industries. The use of synthetic pesticides has been the only means of combating the pests and diseases to
achieve large-scale food production in developing countries due to lack of appropriate technology to formulate their own pest control measures. The use of these chemicals is associated with a lot of problems. The problems of mammalian toxicity,
pollution of the environment and high cost of the few available chemical pesticides
are enormous. The Environmental Protection Agency (EPA) worldwide reacts against
these problems and encourages research into a better alternative to these synthetic
pesticides. The natural products as alternatives are available at all times of the year
due to their location in the tropical regions, they are easy to formulate, are not toxic to
mammals, are less costly and are environmentally friendly. These attributes assist in
the production of cheap and safe food, so the botanicals are assets to developing
countries and encourage their large-scale usage. Extracts from plants such as Azadirachta indica, and Ocimum spp. are used for the control of various crop pests such as
cowpea beetles (Callosobruchus maculatus), and diseases such as wet rot of Amaranthus
spp. (Choanephora curcubitarum) and anthracnose of soybean (Colletotrichum
truncatum).
9.1 Introduction
About one third of the worlds agricultural produce in terms of plants and animals are destroyed by a combination of both pests and diseases. In Africa, where
most of the developing countries are located, most of the food crops are attacked
by one pest or another, thereby causing high crop loss (Table 9.1; Ogbalu, 2009).
(ed. N.K. Dubey)
191
192
D.B. Olufolaji
Table 9.1.
Estimate of crop losses due to insect attacks in some African countries.
Crop
Cereals
Sorghum
Rice
Maize
Pests
Chad
Nigeria
Ethiopia
Nigeria
Ghana
Chad
Ethiopia
Busseola fusca
Busseola fusca
Stemborers
Stemborers / weevils
Stemborers
Stemborers
Stemborers
Weevils
Weevils
Maruca testulalis
Busseola fusca
Thrips
Stemborers
Stemborers
54
47
25
3560
2530
3555
3255
4855
68
4368
2455
55
37
24
Swaziland
Ethiopia
Kenya
Chad
Weevils
Pod borers
Pod borers
Hemipterans
Ophiomyia phaseoli
Pod borers
3058
6072
2048
3857
2843
4854
Nigeria
Nigeria
Ghana
Chad
Nigeria
Nigeria
Scale insects
Zonocerus variegatus
Zonocerus variegatus
Yam beetles
Yam beetles
Cylas puncticollis
34
2831
1527
2448
54
6776
Nigeria
Nigeria
Nigeria
Nigeria
Dipterous pests
Dipterous / bugs
Flea beetles
Lixus cameranus
Diacrisia maculosa
Z. variegatus
D. maculosa
4590
3553
4560
5468
5587
5582
Nigeria
Maize tassels
Legumes
Cowpea
Roots and tubers

Cocoyam
Cassava
Yam
Sweet potatoes
Vegetables
Tomatoes
Peppers
Okra
Bitterleaf
Fluted pumpkin
Estimated crop
loss (%)
Country
Nigeria
Ethiopia
Cameroun
Nigeria
Nigeria
Source: Ogbalu, 2009.
Knowledge of natural products or botanicals as pesticides is as old as

agriculture and local usage of the plants for both medicinal and pesticidal
purposes also predates history. However, the advent of chemical pesticides
in the mid-20th century has relegated the use of botanicals to the background, the striking negative side effect of these synthetic pesticides not
withstanding (Lale, 2007). Until now the synthetic pesticides still record
Prospects of Large-scale Use of Natural Products
193
very formidable success in their control attributes on both pests and diseases
in plants. The discovery of residues of synthetic pesticides in the food chain
and their effect on the environment have led to agitation by environmentalists, ecologists and the Environmental Protection Agencies (EPA) worldwide
to look inward for a better alternative in order to produce safe food for
everybody, and maintain a friendly environment. The natural products
which can also be referred to as botanicals and their products have and will
continue to play important roles in alleviating human suffering and also
control the losses of farm products experienced in most developing countries (Edeoga and Eriata, 2001; Aworinde et al., 2008). Crop production and
protection are being supported by the use of botanicals in developing countries in Africa and some third-world countries (Lale, 2007; Isman 2008).
Researchers in Nigeria and some other African countries have investigated
several plant species for their efcacy as pesticides for protecting their crops
against pest degradation especially with the storage pests and diseases
(Boeke et al., 2001; Ofuya et al., 2007; Olufolaji, 2008).
Until recently, conventional pesticides had been used to curb these pests
and, in most cases, quite efciently. However, the technicalities involved in
the formulation, standardization and distribution of the botanicals as pesticides to reach the teeming population in the developing countries still remain
problems for their usage (Lale, 2007). There are the attendant problems of
resistance, environmental pollution, and adverse effect on health and climate
change traceable in part to the high levels of chloro-uoro-carbon being used
as carriers in aerosol pesticides, which is affecting the ozone layer. This led to
caution in the continued use of persistent synthetic pesticides, especially
those in the class of chlorinated hydrocarbons and the highly toxic organophosphorus pesticides (Olaifa, 2009).
9.2 History of Natural Products in the Developing Countries

and their Use by Local Farming Communities
Developing countries have been endowed by nature with large quantities
and diverse collections of many such natural products, which are more of
plant origin (botanicals). The population of inhabitants in developing
countries is fed by farm produce from the local peasant farmers. Thus the
major users of botanical pesticides are the local farmers who are less educated, very poor and most of them cannot afford the expensive synthetic
pesticides.
The tropical forests, characteristic of most developing countries, are the
abode of naturally occurring genera and species of most of these botanicals.
Over centuries, evolution of these botanicals has occurred to produce various biotypes that are in good use today as medicines and pesticides (Table
9.2). A survey of the use of the botanicals in local farms revealed that around
50% of about 500 peasant farmers in India and Pakistan who stored food
grains for more than 6 months used neem (Azadirachta indica) leaves to get rid
of storage pests, whereas the relatively afuent and better educated farmers
194
D.B. Olufolaji
used neem cake for nematode control (Ahmed and Stoll, 1996). These farmers buy neem cake in the market because they nd no synthetic pesticides to
be as equally potent, cheap and readily available. One of the few reported
cases of the use of the botanical pesticidal materials for pest management in
Nigeria is the application of chilli pepper (Capsicum spp.) and tobacco (Nicotiana tabacum) leaf extracts by some farmers in the south western states of
Nigeria (Nworgu, 2006), and the use of Chromolaena odorata to solve some
pest and disease problems e.g. anthracnose of soybean caused by Colletotrichum truncatum has also been reported (Ajayi and Olufolaji, 2007). There are
many other plants (Eichhornia crassipes, Vernonia amygdalina, Piper guineense,
etc.) used by farmers as botanical pesticides for various pests and diseases,
even in their crude form, and they have been found to be very effective (Olufolaji, 1999a, 2006, 2008; Ajayi and Olufolaji, 2007). Those farmers have
adopted the use of botanicals in the control of pests and diseases on their
crops due to desperation for survival, and since they are dependent on an
agrarian economy, they evolved various method of formulating and using
the botanicals without sourcing the orthodox expertise and technicalities that
are either not within their reach or expensive to adopt.
Table 9.2.
Some bioactive plants reported for the control of agricultural pests.
Pests
Bioactive plants
References
Maize weevil
Sitophilus orazae (L.)
Neem seed
Eugenia aromatica
Callosobruchus maculatus
Piper guineense
Acrae epomia
Several plants
Monodora tenuifolia
Zanthoxylum zanthoxyloide
Azadirachta indica
Lippia adoensis
Ivbijaro, 1983
Lale, 1992, 1994;
Aranilewa et al., 2002
Olaifa and Erhum,1988
Ofuya and Dawodu, 2002
Adedire and Lajide, 1999
Adedire et al., 2003
Ogunwolu and Idowu, 1994
Lale and Abdulrahman, 1999
Olaifa et al., 1987
Dysdercus superstittiosus
Ootheca mutabilis
Riptortus dentipes
Acrae epomia
Dysdercus superstittiosus
Ootheca mutabilis
Riptortus dentipe
Grasshopper
(Zonocerus variegates)
Tetrapteura tetraptera
Field pests of cowpea, maize,
sorghum, cassava, banana
Source: Olaifa, 2009.
Clausena anisata
Azadirachta indica
Piper guineense
Azadirachta indica
Azadirachta indica
Azadirachta indica
Tephrosia vogelli
Okunade and Olaifa, 1987

Olaifa and Akingbohungbe,
1987a
Olaifa and Adenuga,
1988a,b
Olaifa et al., 1991a
Olaifa et al., 1991b
Adebayo, et al., 2007
195
9.3 Availability of Natural Products in Large and Required

Quantities
Most countries in the developing world are favoured by very good climatic
conditions and agro-ecosystems that support the growth and development
of most of the botanicals used for the production of the botanical pesticides.
The farmers have a locational advantage in that the botanicals are available
in abundance in their ecosystem and exist throughout the year in both rainy
and dry seasons. For example, most of these popular plants grow in the wild
and are regarded as weeds, and some of them are not useful for food or raw
materials for the industries (Table 9.2).
However, some are now being cultivated to prevent the extinction of
useful species. In recent times, most countries in the developing world nd
it easy to incorporate the botanicals into their agro-forestry programmes
and this has increased and maintained the existence of the botanicals in
large quantities (Ogunnika, 2007). This has further strengthened the largescale use of the botanicals as a means of pest and pathogen control in the
sub-region. Furthermore, germination of the seeds of most of these botanicals is not tied down by an unnecessarily long dormant period, which is
characteristic of some botanicals in developed world (Ogunnika, 2007). The
activities of the farming population in the tropics have included intercropping and mix-cropping of main crops with the botanicals. These involve
planting Manihot esculenta (cassava) with Ocimum gratissimum (camphor
basil), Theobromae cacao (cocoa) with Carica papaya (pawpaw) and so on
(Ogunnika, 2007). It is worth noting here that some of these botanicals when
mixed or intercropped with the main crop could serve as a control for pests
and diseases in the form of allelopathy. N. tabaccum and some other plant
species, especially grasses, have been found to possess this attribute
(Florentine et al., 2003, Ogunnika, 2007). Eucalyptus camaldulensis and some
other botanicals are also used as weed control in farmlands to maximize
yields (Oyun and Agele, 2009). Many farm crops are protected by this means
without the need to carry out formulation and application processes for pest
and disease control. There is the availability of rain and moisture throughout the year and no occurrence of snow or completely dry period in most
developing countries close to the tropics. Thus, plants can grow throughout
the year and there is no threat of extinction of most of these botanicals, both
wild and cultivated species.
The fact that almost half of the worlds forest that houses these botanicals
is in Africa is an indication of the availability of the much needed plants for
botanical pesticides (Owolabi and Olanrewaju, 2007). Farnsworth and
Soejarto (1991) estimated that 28% of the total inventories of the worlds plant
species are used in ethnomedicines. Groombridge (1992), and Lange and
Schippmann (1997) reported that over 21,000 plant taxa (including synonyms) are being used worldwide for pesticidal purposes, which amounted
to about US$800 million in 19951996. If these plants had mainly resided in
the developed world, then developing countries would not benet immensely
196
D.B. Olufolaji
from this since they are mostly poor. Despite the indiscriminate deforestation
in developing countries in which these botanicals exist, rapid and large
recovery of most of these botanicals still occurs. It has been observed that
most of the botanicals obtained in the tropical forests of developing countries
sprout easily and survive through the little water stress experienced during
the dry season. These attributes support the possibility of large-scale use of
these botanicals as pesticides.
9.4 Formulations of Botanicals in Developing Countries

Local formulation by peasant farmers
Methods adopted by farmers in the developing countries for the formulation
and delivery of botanical pesticides vary depending on the parts of plants
used and the target pests. The plant parts could be either pulverized fresh
with a pestle and mortar or grinding stones before soaking in water (hot or
cold) or sun-dried before pulverizing into powdered form. They are then
soaked and extracted before use. The powdered form could also be used as
dust to prevent incursion of pests and diseases on the stored farm produce or
for on-farm dusting for pest and disease control. The formulated botanicals
can be buried near the crop plants such that they can diffuse into the soil and
protect the root or move in a systemic way to control pests and diseases on
the farm. Termites, soil insects and some other soil pathogens such as Pythium
spp. could be controlled through this method.
Some botanicals are effective when they are burnt and the fumes are
directed into storage silos or into the soil as in the orthodox fumigation
practice.
Scientific formulations
Researchers in most of the developing countries have investigated more
than 100 different species of botanicals for their efcacy in crop protection
against pest degradation, especially the storage pests and diseases (Boeke
et al., 2001; Ofuya et al., 2007; Olufolaji, 2008). Some researchers have suggested that a few botanicals used as pesticides in Nigerian ora with
conrmed pesticidal attributes against stored-product insects merit scientic formulations (Lale, 2001; Ofuya, 2003). However, pesticidal formulations of most botanicals which have been found effective and may be
recommended for use in crop protection in Nigeria are rather simple, and
easy to make in solid and liquid formulations (Ofuya, 2009). Solid formulations are mainly powders, whereas liquid formulations include oils and the
crude extracts prepared in water and organic solvents. These methods of
formulation of the botanicals as pesticides also make their large-scale use
easy and expansive in the developing countries because not much
technicality is involved.
197
Methods of formulation
Formulations of the botanicals in developing countries are also of immense
importance in their large-scale use. Due to low-level scientic technicality
and non-availability of sophisticated equipment associated with the production of synthetic pesticides, the type of formulation associated with the production of the botanical pesticides in developing countries is the type that
the majority of the agrarian community and scientists will be able to handle
and utilize for effective botanical pesticide formulation.
Powders
These are prepared by harvesting the plant materials, which are then sundried and pulverized into ne powder. The powders have been investigated
undiluted for stored-product protection against insects and fungi. The
required quantity of powder is admixed with an appropriate quantity of
commodity prior to storage. Powders have also been extracted with water
(water-extractable powders), ltered and applied as aqueous solutions for
protecting eld crops and grains (Table 9.3; Ofuya, 2009).
Crude water extracts
These are crude extracts obtained by using water as a solvent, and may be
obtained simply by pressing out juices and then diluting in water or through
maceration (steeping in water for prolong periods). They may also be
obtained by infusion (the immersion of plant parts in boiling water for prolonged periods). Such aqueous extracts or solutions have mostly been investigated against eld-crop insect pests and diseases (Table 9.3) (Olufolaji, 2006;
Ofuya, 2009).
Oils
Oils are usually extracted from pulverized medicinal plant parts by means of
organic solvents and are used very much like concentrate liquids. Organic
Table 9.3.
Some medicinal plants in Nigeria and potential formulations in crop protection.
Botanical
Potential formulation
Azadirachta indica
Piper guineense
Eugenia aromatica
Dementia tripetalia
Tephrosia vogelli
Nicotaina tabacum
Allium sativum
Zingiber officinale
Dust, water-extractable powder, emulsifiable concentrate, liquids

Water-extractable powder, emulsifiable concentrate, liquids
Dust, water-extractable powder
Source: Ofuya, 2009.
198
D.B. Olufolaji
solvents, particularly methanol, ethanol, acetone, hexane, petroleum ether,

diethyl ether, chloroform or methyl chloride, have been used (Table 9.3)
(Olufolaji, 2006; Ofuya, 2009).
Mixed formulations
Using herbal mixtures for crop protection is traditional with some farmers
(Kitch et al., 1997). Efcacy of mixed formulations of medicinal plant powders for stored-grain protection has been subjected to empirical verication
(Ogunwolu and Idowu, 1994; Dawodu and Ofuya, 2000; Emeasor et al., 2007).
Overall pesticidal activity of each material was not mitigated by mixing the
two against the test insects. However, synergistic or additive effects would
be desirable to enhance efcacy. Lale (2002) reported that mixing different
essential oils from plants, in some cases, provided much better control than
single use (Table 9.3) (Ofuya, 2009; Olufolaji, 2006, 2008).
Use of adjuvants
Ofuya et al. (2007) have demonstrated the possibility of using organic
ours from yam, cassava and plantain as diluents in the formulation of
insecticidal dusts from buds of Eugenia aromatica and dry fruits of Piper
guineense. Filtrates that are obtained in the preparation of aqueous neem
extracts can be improved after dilution by adding brown sugar, which
improves the adherence of the ltrate on leaves and other plant parts
(Jackai, 1993).
9.5 Attributes of the Botanicals Versus Conventional

Synthetic Pesticides
The botanicals have an array of good attributes over the synthetic pesticides,
which is encouraging because the low technicality required for use has
enabled the developing world to achieve desirable advantages from their
use. Ahmed and Stoll (1996) highlighted the various problems caused by
synthetic pesticides including toxicity to non-target organisms such as man
and benecial macro/microbes, pollution in the agro-ecosystem and development of resistance by the target organisms. However, the botanical pesticides possess some special characteristics and may be used as better
alternatives in managing different agricultural pests:
The materials used for the production of the botanical pesticides are
easily available since they are usually weeds in the agro-ecosystem.
Botanical pesticides are usually inherently less harmful than conventional pesticides.
Botanical pesticides generally affect only the target pest and closely
related organisms, in contrast to broad-spectrum conventional
pesticides that may affect organisms as different as birds, insects and
mammals.
199
Botanical pesticides are often effective in very small quantities and

often decompose quickly, thereby resulting in lower exposure and
largely avoiding the pollution problems caused by conventional
pesticides.
When used as a component of integrated pest management (IPM)
programmes, botanical pesticides can greatly decrease the use of
conventional pesticides, while crop yields remain high.
9.6 The Views of Environmental Protection Agency and the Policy

of Most Developing Countries
The view of the EPA at national and international levels is not to support
the use of synthetic pesticides. These views have made some advanced
and developed countries to promulgate laws against the usage of some of
these synthetic pesticides. In Nigeria, the body vested with the authority
of pesticides registration and control is the National Agency for Food and
Drug Adminstration and Control (NAFDAC) (Olaifa, 2009). Despite this,
some pesticides such as DDT and some mercuric-based pesticides, which
had been banned from use due to the residue left on crops for consumption
and because they constitute a health hazard, are still found in pesticides
markets. The presence of heavy metals in most pesticides have caused
untold hardship and death to the consumer of the crops on which it has been
used.
Most of the developing countries have formulated various policies
on the usage of the synthetic pesticides. The policy emanated from the
effect which the pesticides have unleashed on the crops due to their
phytotoxicity, mammalian toxicity and carcinogenic effects on both the
crops and man.
In 1994, the Botanical Pesticides and Pollution Prevention Division was
established in the Ofce of Pesticide Programmes to facilitate the registration
of botanical pesticides. This division promotes the use of safer pesticides,
including botanical pesticides, as components of IPM programmes. The division also coordinates the Pesticide Environmental Stewardship Programme
(PESP).
Since botanical pesticides tend to pose fewer risks than conventional
pesticides, the EPA generally requires much less data to register a botanical
pesticide than to register a conventional pesticide. In fact, new botanical pesticides are often registered in less than a year, compared with an average of
more than 3 years for conventional pesticides.
While botanical pesticides require less data and are registered in less
time than conventional pesticides, the EPA always conducts rigorous reviews
to ensure that pesticides will not have adverse effects on human health or the
environment. For the EPA to be sure that a pesticide is safe, the agency
requires that registrants submit a variety of data about the composition,
toxicity, degradation and other characteristics of the pesticide.
200
D.B. Olufolaji
9.7 The Attitudes of the Poor Resource Farmers in the

Developing Countries to the Use of Botanical Pesticides
The poor resource farmers in the developing countries have been relieved by
the use of botanicals as it affords them the advantages of low pest control
cost, easy pest control strategies, minimum pollution in their agro-ecosystem
and, ultimately, higher yield and prot. They are now in search of any available information regarding new innovations on botanicals to control the
pests on their farm lands. They are also ready to pay for the services because
botanicals cost less than the conventional synthetic pesticides with all the
safe attributes to food production in the developing world. The farmers are
already trying to combine the research ndings of scientists with their agelong practice to arrive at formidable control measures on their farm lands.
Interestingly, the farmers are now adopting the principle of IPM in order to
maximize yields on their farms. Moreover, the botanists who engage in oriculture and horticulture are included in the use of botanicals to preserve
most of their important species of plants from the scourge of pests and diseases. The farmers have already formed cooperatives and through them
approach government extension agencies for more enlightenment on the use
of botanicals for their benet.
9.8 The Future of the Use of Botanicals

The future of botanicals as alternatives to synthetic pesticides is bright, in that
more people are using them due to the availability of the raw materials, accessibility of the raw materials and easy use of the botanicals, with minimum
demand for specialized application and equipment that is characteristic of synthetic pesticides. Most of the research on pest and disease control in both developed and developing countries are now centred on the use of botanicals as
alternatives to synthetic pesticides. The World Health Organization has also
encouraged the use of botanicals because there is a prevalence of diseases traceable to chemicals used for the control of plant diseases because the consumed
plant materials possess high degree of residues that cause carcinogenic diseases
and other health issues. It is also an issue with environmentalists concerned
about the threat of global warming because most of the pollution accredited to
cause global warming is traced to the pollutants produced and discharged in
the developing countries (Olufolaji, 2006). The present economic recession has
even made the purchase of synthetic pesticides more expensive and thus difcult for the poor and even the enlightened farmers in the developing countries,
hence the adoption of an alternative, which is the use of botanical pesticides.
9.9 Conclusion
The botanicals have solved enormous problems of farmers in the developing
world who are faced with an array of pests and diseases that reduce their
yields and make farming a harder process.
201
Acknowledgement
The author is grateful for the opportunity given by the coordinator of this
book collection, Prof. N.K. Dubey, to be a contributor to this book. I also
appreciate the management of The Federal University of Technology, Akure,
Nigeria, with whom I am residing to carry out this research work.
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10
Current Status of Natural

Products in Pest Management
with Special Reference to
Brassica carinata as a
Biofumigant
MARA PORRAS
Department of Crop Protection, IFAPA Centro Las Torres Tomejil,
Sevilla, Spain
Abstract
This chapter covers the use of natural products in plant disease management in the
form of fungicides and bactericides, and personal experience of the development and
optimization of biofumigation with Brassica carinata and soil solarization as alternatives
to the traditional use of chemicals in strawberry production. The potential of biofumigation as a component of the integrated management of soil pathogens has been demonstrated in various agricultural systems. The presence of high amounts of glucosinolates,
and of the enzyme myrosinase that catalyses their hydrolysis, linked to the high biocidal activity of some glucosinolate enzymatic hydrolysis derivative products have been
suggested for a practical use of amending soil with these natural biocidal compounds
through cultivation and green manure of selected species of the family Brassicaceae.
10.1 Introduction
Chemical pesticides such as methyl bromide are being phased out globally
because of their impact on the ozone layer (European Parliament, 2000).
Since 2005, the use of methyl bromide has been banned in European Union
countries. Chemical, physical and biological alternative methods for pathogen control have been evaluated in crop production to replace the compounds lost due to the new registration requirements (Duniway, 2002; Martin
and Bull, 2002; Porras et al., 2007b). Natural-product-based pesticides can
sometimes be specic to the target species and have unique modes of action
with little mammalian toxicity (Duke et al., 2003). Modern instrumentation
and improved methods should increase interest in natural-product-based
pesticide discovery research (Duke et al., 2002).
(ed. N.K. Dubey)
205
206
M. Porras
Naturally occurring substances found in fungi, bacteria and plants are

important sources of molecules with different biological properties. They
may be developed either as products per se or used as starting points for
synthesis to optimize specic properties. Low mammalian toxicity, low environmental impact, low levels of residues in food and compatibility with integrated pest management (IPM) programmes are important considerations in
the election of fungicides for development (Knight et al., 1997).
This chapter covers the use of natural products in plant disease management (fungicides and bactericides), and personal experience of Brassica
carinata as a biofumigant in strawberry elds.
10.2 Natural Products Used as Fungicides and Bactericides in

Agriculture
The natural products used as agrochemicals have been well documented
(Knight et al., 1997; Warrior, 2000; Copping and Duke, 2007; Kim and Hwang,
2007; Dayan et al., 2009). Microorganisms can synthesize secondary metabolites of versatile chemical structures with diverse biological activities that
exceed the scope of synthetic organic chemistry (Porter, 1985). Pesticidal
potency is not the only factor for formulating a plant-based product as a
commercial pesticide. In-eld chemical stability adequate to reduce the
application times to an economical level, and lowered volatility for sufcient
retention on the surface of host plants are also important factors for microbial
fungicides to be efciently applied in agriculture (Kim and Hwang, 2007).
Therefore, only a few microbial metabolites have been successfully developed into commercial fungicides. The excellent fungicidal activity of these
microbial metabolites and their potential as lead candidates for further fungicide development continue to stimulate research and screening for antifungal
microbial metabolites (Kim and Hwang, 2007; Dubey et al., 2009). Table 10.1
summarizes the microbial metabolites, and plant- or animal-derived products
used as fungicides or bactericides in agriculture.
Biological control agents (including both bacteriological or fungus origin), plants, natural compounds and preparations have been described with
activity against bacterial or fungal plant pathogens. Furthermore, plants protect themselves from microbial attacks with both constitutive antimicrobials
and compounds induced by the attacking pathogens (phytoalexins). Natural
products have been used to protect plants indirectly from pathogens by
inducing systemic acquired resistance (SAR). The SAR-inducing compounds
are termed elicitors. Pathogens cannot evolve resistance directly to the elicitor because the activity is indirect, making such products excellent candidates for integrated disease management. Nevertheless, elicitors are generally
not as effective as chemical fungicides, partly because the timing of elicitor
application and threat to the crop by a pathogen is crucial, but difcult to
maximize (Dayan et al., 2009).
Disease suppression by biocontrol agents is the sustained manifestation
of interactions between the plant, the pathogen, the biocontrol agent, and the
Natural Products as Alternatives to Synthetic Pesticides

Table 10.1.
207
A summary of natural products used as agrochemicals.
Species
Products
Bacteria
Bacteria-derived
products
Blasticidin-S
Streptomyces
griseochromogenes
Erwinia amylovora
(Burrill) Winslow
Harpin protein
Streptomyces
kasugaensis
Hamad et al.
Kasugamycin
Streptoverticillium
rimofaciens
Mildiomycin
Streptomyces
natalensis
Struyk, Hoette, Drost,
Waisvisz, Van Eek
& Hoogerheide
S. chattanoogensis
Burns & Holtman
Natamycin
Target pathogens
References
Kim and
Pyricularia oryzae
Hwang,
Cavara; perfect stage
2007
Magnaporthe grisea
(Hebert) Barr
Copping and
Xanthomonas campestris
Duke, 2007
(Pammell) Dowson
Pseudomonas syringae
Van Hall
Pseudomonas solanacearum
(Smith) Smith
Fusarium sp.
Phytophthora sp.
Magnaporthe
salvinii (Cattaneo)
Krause & Webster
Rhizoctonia solani
Khn (Pellicularia
sasakii Ito)
Venturia inaequalis
(Cooke) Winter
Erwinia amylovora
Botrytis sp.
Guignardia bidwellii (Ellis)
Viala & Rivas
Diplocarpon rosae Wolf
Anon, 2005;
P. oryzae
Copping and
Cercospora spp.
Duke, 2007
Venturia spp.
Cladosporium fulvum
Erwinia atroseptica
Xanthomonas campestris
Copping and
Erysiphe spp.
Duke, 2007
Uncinula necator
(Schwein) Burrill
Podosphaera spp.
Sphaerotheca spp.
Fusarium oxysporum
Copping and
Schlecht
Duke, 2007
Continued
208
Table 10.1.
M. Porras
Continued.
Species
Products
Target pathogens
References
Streptomyces rimosus
Sobin et al.
Oxytetracycline
Erwinia amylovora
Pseudomonas spp.
Xanthomonas spp.
Copping and
Duke, 2007
Streptomyces
cacoai var.
asoensis
Isono et al.
Polyoxins:
polyoxin B
polyoxorim
(polyoxin D)
Streptomyces
griseus
(Krainsky)
Waksman & Henrici
Streptomycin
Streptomyces
hygroscopicus
(Jensen) Waksman &
Henrici var. limoneus
Pseudomonas
fluorescens
Validamycin
Pyrrolinitrin
Copping and
Duke, 2007
Sphaerotheca spp. and
other powdery mildews
Botrytis cinerea Pers.
Sclerotinia sclerotiorum
De Bary
Corynespora melonis Lindau
Cochliobolus miyabeanus
(Ito & Kuribay) Drechsler
ex Dastur
Alternaria alternata (Fr.)
Keissler and other
Alternaria spp.
R. solani
Nectria galligena
Bresadola (Diplodia
pseudodiplodia Fuckel)
Drechslera spp.
Bipolaris spp.
Curvularia spp.
Helminthosporium spp.
Bacteria, particularly
effective against:
Xanthomonas oryzae
Dowson
X. citri Dowson
Pseudomonas
tabaci Stevens
P. lachrymans Carsner
Rhizoctonia spp.
B. cinerea
M. grisea
(In vitro and in the
greenhouse, not in
field because of
photosensitivity)
Copping and
Duke, 2007
Copping and
Duke, 2007
Haas and
Keel, 2003;
Kim and
Hwang,
2007
Continued

Table 10.1.
209
Continued.
Species
Fungi
Saccharomyces
cerevisiae
Meyer ex Hansen
Strobilurus
tenacellus
Oudemansiella
mucida
Plants
Cassia tora L.
(also known as
Cassia obtusifolia L.)
The two active

ingredients are
found in virtually all
living organisms and
in their pure form
Simmondsia
californica Nutt. and
S. chinensis Link.
Laminaria
digitata (Hudson)
Products
Target pathogens
Fenpiclonil and
Fludioxonil
(Phenylpyrroles
derivates to
enhanced
photostability)
Fusarium graminearum
Schwabe
Gerlachia nivalis
Gams & Mll
Botrytis spp.
Monilinia spp.
Sclerotinia spp.
Fungus-derived
products
Yeast extract
hydrolysate
ni
Copping and
Duke, 2007
Ishii et al.,
2001;
Balba, 2007
Strobilurin A
Oudemansin A
From which synthetic Resistance to
strobilurins such as strobilurins has
already evolved
azoxystrobin and
kresoxim-methyl
have been
developed
Plant-derived
products
Cinnamaldehyde
Verticillium spp.
Rhizoctonia spp.
Pythium spp.
Sclerotinia homeocarpa
Bennett
Fusarium moniliforme
var. subglutinans
Wollenw. & Reinking
Powdery mildew
L-glutamic acid and
-aminobutyric
acid (Auxien has
registered the
combination product
containing both
active indredients)
Jojoba oil
Powdery mildew
Laminarine
References
Fungal pathogens of
cereals, particularly
septoria and powdery
mildews
Copping and
Duke, 2007
Copping and
Duke, 2007
Copping and
Duke, 2007
Copping and
Duke, 2007
Continued
210
M. Porras
Table 10.1.
Continued.
Species
Products
Target pathogens
References
Reynoutria
sachalinensis
(Fr. Schm.) Nakai
Macleaya
cordata
R. Br.
Extract of giant
knotweed
Copping and
Duke, 2007
Rosmarinus
officinalis
Thymus
vulgaris
Azadirachta
indica
Rosemary oil
Botrytis spp.
Powdery mildews
Xanthomonas spp.
The target pathogens
are those causing
foliar fungal diseases,
such as powdery
mildew, alternaria
leaf spot
and septoria leaf spot
ni
Thyme oil
ni
Clarified
hydrophobic
extract of
neem oil
Cottonseed oil with
garlic extract
ni
Gossypium
hirsutum and
Allium sativum
Animals
Cow
Dried, crushed
crustacean
exoskeletons
Pink plume poppy

extract
Copping and
Duke, 2007
Dayan et al.,
2009
Dayan et al.,
2009
Dayan et al.,
2009
ni
Dayan et al.,
2009
Animal-derived
products
Milk
Powdery mildews
Poly-D-glucosamine
or chitosan
Powdery mildews
Botrytis spp.
Copping and
Duke, 2007
Copping and
Duke, 2007
ni, information not provided in reference.
microbial community on and around the plant and the physical environment
(Handelsman and Stabb, 1996). Relevant biocontrol microorganisms of the
rhizosphere include Pseudomonas spp., Bacillus spp., Streptomyces spp., Trichoderma spp., and non-pathogenic Fusarium spp. (Cook, 1993; Paulitz and
Belanger, 2001; Whipps, 2001). Different species of Trichoderma are studied
primarily for their ability to control plant disease through antagonism, rhizosphere competence, enzyme production, induction of defence responses in
plants, metabolism of germination stimulants, and benecial growth of the
host following root colonization (Weindling, 1934; Zimand et al., 1996; Bailey
and Lumsden, 1998; Howell, 2003; Porras et al., 2007a,b). Determination of
these effects depends on many interactions that take place in the soil among
Trichoderma spp., other microorganisms, the plant root, and the soil environment (Bailey and Lumsden, 1998).
211
10.3 Biofumigation with Brassica

The term biofumigation has been defined as the suppression of soil-borne
pathogens and pests by Brassica rotation or green manure crops, through
liberation of isothiocyanates from hydrolysis of the glucosinolates that
characterize the Brassicaceae (Kirkegaard and Matthiessen, 2004).
Biofumigation is based on the action of volatile compounds, especially
isothiocyanates, produced by the hydrolysis of glucosinolates. Glucosinolates are secondary metabolites produced by plants belonging to the Capparales
order (Gimsing and Kirkegaard, 2009). Of the many hundreds of cruciferous
species investigated, all are able to synthesize glucosinolates, and at least
500 species of non-cruciferous dicotyledonous angiosperms have been
reported to contain them. Most glucosinolate-containing genera are clustered
within the Brassicaceae, Capparaceae and Caricaceae. Among the Brassicaceae,
the genus Brassica contains a large number of the commonly consumable
species (Fahey et al., 2001). The enzyme myrosinase, produced by all plants
that produce glucosinolates, is physically separated from the glucosinolates
in the intact plant tissue. Glucosinolates and myrosinase are reported to be
located in vacuoles, and are not colocalized in the same cells (Andrasson et
al., 2001) (Fig. 10.1). Upon tissue disruption the glucosinolates and the myrosinase come into contact (Fig. 10.2) and the myrosinase hydrolyses the glucosinolates to form hydrolysis products such as isothiocyanate, nitrile and
thiocyanate (Gimsing and Kirkegaard, 2009) (Fig. 10.3).
The different chemical side-chain structures of the isothiocyanates
determine important chemical and physical properties such as hydrophobicity and volatility, and their toxicity (Brown and Morra, 1997). Biofumigation can be achieved by incorporating fresh plant material (green manure),
seed meals (a by-product of seed crushing for oil), or dried plant material
treated to preserve isothiocyanate activity (Kirkegaard and Matthiessen,
2004; Lazzeri et al. 2004; Matthiessen and Kirkegaard, 2006).
A field experiment conducted in Huelva (South-west Spain; the most
important area of strawberry production in Europe) contributes to the development and optimization of biofumigation with Brassica and soil solarization
as alternatives to the traditional use of chemicals in strawberry production
(Porras et al., 2008).
Solarization is a process that employs solar radiation to heat soil under a
transparent plastic film to temperatures that are detrimental to soilborne
pathogens. Increased soil temperatures can decrease populations of plant
pathogens (Katan, 1981). Solarization can enhance the effectiveness of other
pest management approaches (Katan, 1981; Ben-Yephet et al., 1987; RamirezVillapudua and Munnecke, 1988; Porras et al., 2007a,b) and has the additional
advantages of being a non-chemical alternative method for pathogen control
(Batchelor, 2002).
The soil in the field experiment had never been previously treated with
methyl bromide, and was naturally infested by Phytophthora spp. Treatments
were soil solarization (S), biofumigation + solarization (B + S), and the
untreated control (C). Treatments were applied in the same plots each year
212
M. Porras
Myrosinase
Glucosinolate
Fig. 10.1. Intact vegetal tissue with the myrosinase enzymes and the glucosinolates
physically separated (within vacuoles).
(20052006 and 20062007). Biofumigation was done with Brassica carinata in

the siliqua stage that showed the major suppressive effect in previous in vitro
experiments (Romero et al., 2008). B. carinata was cut, because upon tissue
disruption the substrate and the enzyme come into contact and the myrosinase hydrolyses the glucosinolates to form hydrolysis products. The
chopped material was incorporated into the soil to a depth of 10 cm using a
rotary tiller. The soil was covered using clear 50-m low-density polyethylene
mulch, and drip-irrigated from July to September (Fig. 10.4).
Plots were covered to avoid the escape of isothiocyanates from the soil by
volatilization and to increase soil temperatures. Drip irrigation was applied to
enhance the formation of isothiocyanates in soil (Matthiessen et al. 2004).
213
Myrosinase
Glucosinolate
Fig. 10.2. Upon tissue disruption the glucosinolates and the myrosinases come into contact
and the myrosinase hydrolyses the glucosinolates to form hydrolysis products.
Glucose
Isothiocyanate
Thiocyanate
S
C
R
NOSO3
Nitrile
Glucosinolates
Fig. 10.3. Glucosinolates and hydrolysis products originated by the activity of the enzyme
myrosinase.
214
M. Porras
(a)
(b)
(c)
(d)
Fig. 10.4. Brassica carinata (a) used as biofumigant was chopped (b), buried (c),
and covered and drip-irrigated (d).
Biofumigation and solarization increased foliar surface and strawberry yield the most each year, and differences were observed relative to
solarization alone and the untreated control (Tables 10.2 and 10.3).
Biofumigation and solarization reduced Phytophthora spp. soil population relative to the untreated control, but it did not totally eliminate the
pathogen. According to previous experiments (Porras, 2005; Porras et al.,
2007a), the results indicate that solarization may serve as a component in
an integrated, sustainable approach to the management of Phytophthora
spp. in strawberry.
The presence of high amounts of glucosinolates, and of the enzyme
myrosinase that catalyses their hydrolysis, linked to the high biocidal
activity of some glucosinolate enzymatic hydrolysis derivative products
(mainly isothiocyanates and nitriles) have suggested the practical
possibility of amending soil with these natural biocidal compounds
by the cultivation and green manure of selected species of the family
Brassicaceae (Lazzeri et al., 2004). The potential of biofumigation as a

Table 10.2.
215
Foliar surface (cm2) in December and January 20052006 and 20062007.

20052006
16th December
20062007
13th January
19th December
9th January
B+S
S
C
295.90 a
229.91 b
204.74 b
502.49 a
413.58 b
350.57 c
367.04 a
301.07 b
208.32 c
435.14 a
346.31 b
228.03 c
F2,374
29.00*
20.83*
36.10*
53.48*
Letters indicate a significant difference, Tukey HSD test; *, P<0.001.
Table 10.3. Total accumulated strawberry yield (g/plant) until 31st March 2006 and 2007.
20052006
20062007
B+S
S
C
349.31 a
300.17 b
252.84 c
318.66 a
261.45 b
148.13 c
F2,38
11.12*
63.70*
Letters indicate a significant difference, Tukeys multiple range test; ***, P<0.001.
component of the integrated management of soil pathogens has

been demonstrated in various agricultural systems (Gimsing and
Kirkegaard, 2009).
10.4 Conclusion
Naturally occurring substances found in fungi, bacteria and plants are
important sources of molecules with pesticidal properties. They may be
developed either as products per se or used as starting points for synthesis to
optimize specic properties. Low mammalian toxicity, low environmental
impact, low levels of residues in food, and compatibility with IPM programmes are important considerations in formulation of plant-based products as pesticides. In addition, chemical pesticides such as methyl bromide
are being phased out globally because of the impact on the ozone layer.
Chemical, physical and biological alternative methods for pathogen control
have been evaluated in crop production to replace the compounds lost due
to the new registration requirements. Natural-product-based pesticides can
sometimes be specic to the target species and have unique modes of action
with little mammalian toxicity. Modern instrumentation and improved
methods would increase interest in natural-product-based pesticide
discovery research.
216
M. Porras
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A.S., Rask, L. and Meijer, J. (2001) Different myrosinase and idioblast distribution
in Arabidopsis and Brassica napus. Plant
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epa.gov/opprd001/factsheets/kasugamycin.pdf
Bailey, B.A. and Lumsden, R.D. (1998) Direct
effects of Trichoderma and Gliocladium on
plant growth and resistance to pathogens.
In: Harman, G.E. and Kubicek, C.P. (eds)
Trichoderma and Gliocladium: Enzymes, biological control and commercial applications.
Taylor and Francis, London, United
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Balba, H. (2007) Review of strobilurin fungicide
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and Health part B - Pesticides Food Contaminants and Agricultural Wastes 42, 441451.
Batchelor, T.A. (2002) International and
European Community controls on methyl bromide and the status of methyl
bromide use and alternatives in the European Community. In: Batchelor, T.A. and
Bolivar, J.M. (eds) Proceedings of International Conference on Alternatives to Methyl
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Ben-Yephet, Y., Stapleton, J.J., Wakeman, R.J.
and DeVay, J.E. (1987) Comparative effects of soil solarization with single and
double layers of polyethylene lm on
survival of Fusarium oxysporum f. sp.
vasinfectum. Phytoparasitica 15, 181185.
Brown, P.D. and Morra, M.J. (1997) Control
of soil-borne plant pests using
glucosinolate-containing plants. Advanaces in Agronomy 61, 167231.
Cook, R.J. (1993) Making greater use of introduced microorganisms for biological control of plant pathogens. Annual Review of
Copping, L.G. and Duke S.O. (2007) Natural
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Dayan, F.E., Cantrell, C.L. and Duke, S.O.
(2009) Natural products in crop protec-
tion. Bioorganic and Medicinal Chemistry

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Dubey, N.K., Kumar, A., Singh, P., and
Shukla, R. (2009) Exploitation of natural
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Gullino, M.L. (eds) Recent Developments
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A.M., Schefer, B.E., Tellez, M.R., Wedge,
D.E., Schrader, K.K., Akey, D.H., Arthur,
F.H., De Lucca, A.J., Gibson, D.M., Harrison,
H.F., Peterson, J.K., Gealy, D.R.,
Tworkoski, T., Wilson, C.L. and Morris,
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Duke, S.O., Dayan, F.E., Rimando, A.M.,
Schrader, K.K., Aliotta, G., Oliva, A. and
Romagni J.G. (2002) Chemicals from
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Duniway, J.M. (2002) Status of chemical alternatives to methyl bromide for pre-plant
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(2001) The chemical diversity and distribution of glucosinolates and isothiocyanates
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Gimsing, A.L. and Kirkegaard, J.A. (2009)
Glucosinolates and biofumigation: fate
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11
Fungal Endophytes:
an Alternative Source
of Bioactive Compounds
for Plant Protection
R.N. KHARWAR1 AND GARY STROBEL2

1Department
of Botany, Banaras Hindu University, India; 2Department of

Plant Sciences, Montana State University, Bozeman, USA
Abstract
Endophytes are a group of microorganisms that represent an abundant and
dependable source of bioactive and chemically novel compounds with potential
for exploitation in a wide variety of applications. The mechanisms through which
endophytes exist and respond to their surroundings must be better understood in
order to be more predictive about which higher plants to seek, study and employ
in isolating their microoral components. This may facilitate the natural product
discovery process. Endophytic fungi are now attracting great interest from
researchers for an alternative way of controlling plant pathogens.
11.1 Introduction
The bioactive compounds of natural origin have been under ever-increasing
demand to solve various problems related to human health and agriculture.
Plants have provided humans with resources for healing purposes for millennia. Some representative and well known medicines derived from plants
are quinine, digitalin, taxol, aspirin, reserpine and many more (Wani et al.,
1971; Hoffman et al., 1998). Some plants have been under great threat as a
result of the enormous pressure brought upon them by virtue of their healing
and biocontrol properties.
In some cases, plant-associated fungi are able to make the same bioactive compounds as the host plant itself and one of the best examples of this
is the discovery of the gibberellins in Fusarium fujikuroi in the early 1930s
(Borrow et al., 1955). Eventually it was learnt that the gibberellins are one of
only ve classes of phytohormones that are to be found in virtually all
plants. Endophytic (endon = inner; phyton = plants) fungi are a group
of microorganisms that reside in healthy and functional inner tissues of
the plants without causing any detectable symptoms (Petrini, 1991).
218

(ed. N.K. Dubey)
Fungal Endophytes
219
The observation that fungi make the same compounds as their host led
Stierle et al. (1993) to examine the prospect that endophytic fungi associated
with Taxus brevifolia may also produce taxol, the most promising natural
bioactive molecule discovered against cancer. This signicant discovery
established that the endophytic fungi which reside in the living healthy tissues of the plant, may produce one or more bioactive principles or natural
products with either medicinal/therapeutic or biocontrol potential which
were previously known to be produced by the host plant (Wani et al., 1971;
Stierle et al., 1993; Dimetry et al., 1995; Hoffman et al., 1998; Gahukar, 2005).
Taxol itself is the worlds rst billion dollar anticancer drug and its main
source is Taxus spp. Potentially, a fungal source of taxol would reduce its
price and save the plant, in some areas, from extinction. The success of nding fungal taxol has produced a paradigm for other bioactive compounds
to be found in endophytic fungi. This might have happened during the
course of evolution of symbiosis between fungus and host plant, where
reciprocal gene transfer has occurred. As observed in the case of Agrobacterium tumefaciens, the t-DNA of the bacterial plasmid was transferred and
incorporated into the genome of the host plant. This is a ne example of the
transaction of genetic materials between prokaryote and eukaryote. However, in case of the fungal endophytehost relationship, it is a eukaryote to
eukaryote transaction.
As the fungi are a less studied group of microbes, only a few studies
can be exemplied. Alkaloids synthesized by Neotyphodium sp. in its grass
hosts have been implicated in fescue toxicosis in rangeland animals. The
chemistry and biology of this and other grass endophytes are reviewed
elsewhere. Unfortunately, because this work was so comprehensive, one
may be led to the conclusion that endophytes only produce toxic compounds in their respective hosts and that they hold no promise for any
medicinal applications. It turns out that this is simply not the case. Endophytes examined from a plethora of sources show that an overwhelming
number of them produce natural products with promising potential for
many applications.
Bacon and White (2000) gave an inclusive and widely accepted denition of endophytes: Microbes that colonize living, internal tissues of plants
without causing any immediate, overt negative effects (Fig. 11.1). While the
usual lack of adverse effect nature of endophyte occupation in plant tissue
has prompted a focus on symbiotic or mutualistic relationships between
endophytes and their hosts, the observed biodiversity of endophytes suggests they can also be aggressive saprophytes or opportunistic pathogens
(Promputtha et al., 2007).
Both fungi and bacteria are the most common microbes existing as endophytes. It would seem that other microbial forms most certainly exist in
plants as endophytes such as mycoplasmas, rickettsia, streptomycetes and
archebacteria. In fact, it may be the case that the majority of microbes existing
in plants are not culturable with common laboratory techniques, making
their presence and role in plants even more intriguing. The most frequently
isolated endophytes are the fungi (Redlin and Carris, 1996). However, at the
220
R.N. Kharwar and G. Strobel
Fig. 11.1.
A prominent endophytic fungal mycelium lying between the cellular spaces.
outset, it is important to note that the vast majority of plants have not been
studied for any endophytic association. Thus, enormous opportunities exist
for the recovery of novel fungal forms, including genera, biotypes, as well as
species in the myriad of plants yet to be studied. Hawksworth and Rossman
(1987) estimated there may be as many as 1.5 million different fungal species,
yet only about 100,000 have been described. As more evidence accumulates,
estimates keep rising as to the actual number of fungal species. For instance,
Dreyfuss and Chapela (1994) have estimated at least 1 million species of
endophytic fungi alone. It seems obvious that endophytes are a rich and
reliable source of genetic diversity and may represent many previously
undescribed species.
Out of 300,000 higher plants that exist on the earth, only a handful,
primarily grass species, have been studied relative to their endophytic
biology. Consequently, there is enormous opportunity to nd new and
interesting endophytic fungi amongst the myriad of plants in different settings and ecosystems. The intent of this chapter is to provide insights into
the occurrence of endophytes in nature, the products that they make, and
how some of these organisms show their potential for human and plant
use. The chapter discusses the rationale for study, methods used, and
examples of some of the endophytes isolated and studied over the course
of time. This chapter, however, also includes some specic examples that
illustrate the work done in this emerging eld of bioprospecting and
sourcing the endophytic fungi.
Fungal Endophytes
221
11.2 Diversity in Endophytes

Of the myriad of ecosystems on Planet Earth, those having the greatest general biodiversity of life seem to be the ones that also have the greatest number
and most diverse endophytes. Tropical and temperate rainforests are the
most biologically diverse terrestrial ecosystems. The most threatened of these
spots cover only 1.44% of the lands surface, yet they harbour over 60% of the
worlds terrestrial biodiversity. In addition, each of the 2025 areas identified
as supporting the worlds greatest biodiversity also support unusually high
levels of plant endemism (Mittermeier, 1999). As such, one would expect,
that with high plant endemism, there should also exist specific endophytes
that may have evolved with the endemic plant species. Biological diversity
implies chemical diversity because of the constant chemical innovation that
is required to survive in ecosystems where the evolutionary race to survive
is most active. Tropical rainforests are a remarkable example of this type of
environment. Competition is great, resources are limited and selection pressure is at its peak. This gives rise to a high probability that rainforests are a
source of novel molecular structures and biologically active compounds
(Redell and Gordon, 2000).
Bills et al. (2002) describe a metabolic distinction between tropical and
temperate endophytes through statistical data which compares the number
of bioactive natural products isolated from endophytes of tropical regions to
the number of those isolated from endophytes of temperate origin. Not only
did they find that tropical endophytes provide more active natural products
than temperate endophytes, but they also noted that a significantly higher
number of tropical endophytes produced a larger number of active secondary metabolites than did fungi from other substrata. This observation suggests the importance of the host plant as well as the ecosystem in influencing
the general metabolism of endophytic microbes.
11.3 Techniques for Isolation, Preservation and Storage of

Endophytes
Cultures for product isolation
Detailed techniques for the isolation of microbial endophytes are outlined in
a number of reviews and technical articles (Strobel et al., 1993; Strobel, 2002;
Strobel and Daisy, 2003; Strobel et al., 2004; Castillo et al., 2005). If endophytes
are being obtained from plants growing in polar regions, the dry tropics, or
some temperate areas of the world, one can expect to acquire from none to
only one or two endophytic cultures per plant sample (0.510.0 cm limb
piece). However, from the wet tropics this number may rise to 2030 or even
more microbes per plant piece. Given limited fermentation capabilities, it is
critical to label and store cultures of freshly isolated microbes for work in the
future and for patent and publication purposes.
222
Generally, preservation in an aqueous 15% glycerol solution at 70C is

an exceedingly good procedure for saving cultures until work on them can
proceed at a later date (Strobel et al., 1993; Strobel, 2002; Strobel and Daisy,
2003; Strobel et al., 2004).
Growing fungal cultures on sterilized barley or other grains with
subsequent storage at 70C is an effective alternative to the glycerol storage
solution.
Plant selection norms and ethics

It is important to understand that the methods and rationale used to guide
plant selection seem to provide the best opportunities to isolate novel endophytic microorganisms at the genus, species or biotype level. Since the number of plant species in the world is so great, creative and imaginative strategies
must be used to quickly narrow the search for endophytes displaying
bioactivity (Mittermeier et al., 1999).
A specic rationale for the collection of each plant for endophyte isolation and natural product discovery is used. Several hypotheses govern this
plant selection strategy:
1. Plants from unique environmental settings, especially those with an
unusual biology, and possessing novel strategies for survival are seriously
considered for study.
2. Plants that have an ethnobotanical history (use by indigenous peoples)
that are related to the specic uses or applications of interest are selected for
study. These plants are chosen either by direct contact with local people or
via local literature. This approach may also provide benets to the local/
tribal people by documenting their help under a participatory research programme. The ethics of science restrict the researcher drawing the monetary
and intellectual benets alone, especially when knowledge of local people is
used. However, it often happens that at the time of documentation, scientists
dont give proper importance to the knowledge source or the local people,
and this must be checked to maintain the sanctity of the research community.
Ultimately, it may be learned that the healing powers of the botanical source,
in fact, may have nothing to do with the natural products of the plant, but of
an endophyte inhabiting the plant.
3. Plants that are endemic, having an unusual longevity, or that have
occupied a certain ancient land mass, such as Gondwanaland, are also more
likely to lodge endophytes with active natural products than other plants.
4. Plants growing in areas of great biodiversity, it follows, also have the
prospect of housing endophytes with great biodiversity.
Distinct environmental settings are therefore considered to be a promising source of novel endophytes and their compounds, and so too are plants
with an unconventional biology. For example, an aquatic plant, Rhyncholacis
penicillata, was collected from a river system in south-west Venezuela where
that plant faced constant beating by virtue of rushing waters, debris, and
Fungal Endophytes
223
tumbling rocks and pebbles. These environmental insults created many

portals through which common phytopathogenic oomycetes could enter the
plant. Still, the plant population appeared to be healthy, possibly due to the
protection provided by an endophytic product. This was the environmental
biological clue used to choose this plant for a comprehensive study of its
endophytes.
An example of plants with an ethnobotanical history is the snakevine
Kennedia nigriscans, from the Northern Territory of Australia, which was
selected for study since its sap has traditionally been used as a bush medicine. In fact, this area was selected for plant sampling as it has been home
to the worlds longest standing human civilization, the Australian Aborigines. The snakevine is harvested, crushed and heated in an aqueous brew
by local Aborigines in South-west Arnhemland to treat cuts, wounds and
infections. As it turned out, the plant contains an entire suite of streptomycetes (Castillo et al., 2005). One in particular, designated Streptomyces NRRL
30562, has unique partial 16S rDNA sequences when compared to those in
GenBank (Castillo et al., 2002). It produces a series of actinomycins including
actinomycins D, Xo, and X2. It produces novel, broad-spectrum peptide
antibiotics called munumbicins. This seems to be an excellent example illustrating the potential benets of knowledge of indigenous peoples and
researchers.
Furthermore, some plants generate bioactive natural products and have
associated endophytes that produce the same natural products. Such is the
case with taxol, a highly functionalized diterpenoid and famed anticancer
agent that is found in Taxus brevifolia and other yew species (Taxus spp.) (Wani
et al., 1971). In 1993, a novel taxol-producing fungus Taxomyces andreanae was
isolated and characterized from Taxus brevifolia (Strobel et al., 1993).
While combinatorial synthesis produces compounds at random, secondary metabolites, dened as low molecular weight compounds not
required for growth in pure culture, are produced as an adaptation for
specic functions in nature (Demain, 1981). Schulz (2001) points out that
certain microbial metabolites seem to be characteristic of certain biotopes,
both at an environmental as well as an organism level. Accordingly, it
appears that the search for novel secondary metabolites should centre on
organisms that inhabit unique biotopes. Thus, it behoves the investigator to
carefully study and select the biological source before proceeding, rather
than to take a totally random approach in selecting the source material.
Careful study also indicates that organisms and their biotopes that are subjected to constant metabolic and environmental interactions should produce even more secondary metabolites (Schulz at al., 2002). Endophytes are
microbes that inhabit such biotopes, namely higher plants, which is why
they are currently considered as a wellspring of novel secondary metabolites offering the potential for exploitation of their medical benets (Tan
and Zou, 2001). Moreover, novel compound-screening experiments have
already proved that endophytes are considerably ahead of soil isolates,
with 51% new structure compounds compared with 38%, respectively
(Schulz et al., 2002).
224
Molecular techniques provided unexpected and thrilling results with

Pestalotiopsis microspora
It is of some compelling interest how the genes for taxol production may have
been acquired by endophytes such as Pestalotiopsis microspora (Long et al.,
1998). Although the complete answer to this question is not at hand, some
other relevant genetic studies have been performed on this organism.
P. microspora Ne 32 is one of the most easily genetically transformable fungi
that has been studied to date. In vivo addition of telomeric repeats to foreign
DNA generates chromosomal DNAs in this fungus (Long et al., 1998). Repeats
of the telomeric sequence 5-TTAGGG-3 were appended to non-telomerictransforming DNA termini. The new DNAs, carrying foreign genes and the
telomeric repeats, replicated independently of the chromosome and expressed
the information carried by the foreign genes. The addition of telomeric repeats
to foreign DNA is unusual among fungi. This nding may have important
implications in the biology of P. microspora Ne 32 since it explains at least one
mechanism as to how new DNA can be captured by this organism and eventually expressed and replicated. Such a mechanism may begin to explain how
the enormous biochemical variation may have arisen in this fungus. Also, this
initial work represents a framework to aid in the understanding of how this
fungus may adapt itself to the environment of its plant hosts and suggests
that the uptake of plant DNA into its own genome may occur. In addition, the
telomeric repeats have the same sequence as human telomeres which points
to the possibility that P. microspora may serve as a means to make articial
human chromosomes, a totally unexpected result.
11.4 Natural Products and Phytochemicals from Endophytes

It has been suggested that the reason some endophytes produce certain phytochemicals, originally characteristic of the host, might be related to a genetic
combination of the endophyte with the host that occurred in evolutionary
time (Tan and Zou, 2001). This is a concept that was originally proposed as a
mechanism to explain why Taxomyces andreanae may be producing taxol (Stierle et al., 1993). All aspects of the biology and interrelationship of endophytes
with their respective hosts is a vastly under-investigated and exciting eld
(Strobel, 2002; Strobel and Daisy, 2003; Strobel et al., 2004). Thus, more background information on a given plant species and its microorganismal biology would be exceedingly helpful in directing the search for bioactive
products (Lane et al., 2000). Presently, no one is quite certain of the role of
endophytes in nature and what appears to be their relationship to various
host plant species. While some endophytic fungi appear to be ubiquitous
(e.g. Fusarium spp., Pestalotiopsis spp., Aspergillus spp. and Xylaria spp.), one
cannot denitively state that endophytes are truly host specic or even systemic within plants any more than assume that their associations are chance
encounters (Promputtha et al., 2007). Frequently, many endophytes of the
same species are isolated from the same plant, and only one or a few biotypes
Fungal Endophytes
225
of a given fungus will produce a highly biologically active compound in

culture. A great deal of uncertainty also exists about what an endophyte produces in culture and what it may produce in nature. It does seem possible
that the production of certain bioactive compounds by the endophyte in situ
may facilitate the domination of its biological niche within the plant or even
provide protection to the plant from harmful invading pathogens, insects
and environmental stresses (Omacini, et al., 2001; Rodriguez, et al., 2008). Furthermore, little information exists on the biochemistry and physiology of the
interactions of the endophyte with its host plant. It would seem that many
factors changing in the host depending on the season, and other factors
including age, environment, and location, may inuence the biology of the
endophyte. Indeed, further research at the molecular level must be conducted
in the eld to study endophyte interactions and ecology. All of these interactions are probably chemically mediated for some purpose in nature. An ecological awareness of the role these organisms play in nature will provide the
best clues for targeting particular types of endophytic bioactivity with the
greatest potential for bioprospecting.
There is a general call for new antibacterial, antifungal, antiviral and chemotherapeutic agents that are highly effective and possess low toxicity. This
new approach of search is driven by the development of resistance in infectious microorganisms (e.g. Staphylococcus, Mycobacterium, Streptococcus, Bacillus, Aspergillus spp.) to existing drugs and by the menacing increase of
naturally resistant organisms. The ingress to the human population of disease-causing agents such as AIDS, Ebola and SARS requires the discovery
and development of new drugs to combat them. Not only diseases such as
AIDS require drugs that target them specically, but new therapies are
needed for treating ancillary infections that are a consequence of a weakened
immune system. Furthermore, others who are immunocompromised (e.g.
cancer and organ-transplant patients) are at high risk of infection by opportunistic pathogens, such as Aspergillus, Cryptococcus and Candida sp., that are
not normally major problems in the human population. In addition, more
drugs are needed to treat efciently parasitic protozoan and nematodal infections such as malaria, leishmaniasis, trypanomiasis, incephalitis and lariasis. Malaria alone is more dangerous in causing human death each year than
any other single infectious agent with the exception of AIDS and tuberculosis
(NIAID, 2001). However, the enteric diseases as a group claim the most lives
each year of any other disease complex and, unfortunately, the victims are
mostly children (NIAID, 2001).
Endophytic fungi are now attracting great interest from researchers as an
alternative source in controlling plant pathogens. The control of late blight,
caused by Phytophthora infestans, is very difcult by chemical means, because
of the high virulence of the pathogen and increasing resistance to available
fungicides (Grifth et al., 1992). The fermentation broths of 52.3% of endophytic fungi displayed growth inhibition of at least one pathogenic fungus,
such as Neurospora sp., Trichoderma sp. and Fusarium sp. (Huang et al., 2001).
In a similar study, fermentation broths of nine (4.8%) out of the 187 endophytic fungi isolated from mainly woody plants were highly active against
226
P. infestans in tomato plants (Park et al., 2005). Induced resistance was

generated in tomato plant against fusarium wilt by endophytic Fusarium
oxysporum (Duijff et al., 1998). Sclerotinia sclerotiorum is a common root-,
crown- and stem-rot-causing pathogen in cabbage, common bean, citrus, celery, coriander, melon, squash, soybean, tomato, lettuce, cucumber and so on.
Cyclosporine is characterized as a major antifungal substance against S. sclerotiorum from the fermentation broth of F. oxysporum S6 (Rodriguez et al.,
2006). F. oxysporum strain EF119, isolated from roots of red pepper, showed
the most potent in vivo anti-oomycete activity against tomato late blight and
in vitro anti-oomycete activity against several oomycete pathogens (Kim
et al., 2007). Out of 510 endophytic fungi screened, 64 strains exhibited antifungal activities against Candida albicans, Candida glabrata, Candida krusei,
Cryptococcus neoformans, Aspergillus fumigatus, Aspergillus flavus, Rhizopus
oryzae, Trichophyton rubrum and Microsporum canis (Anke et al., 2003).
Narisawa et al. (2000) found that the root endophytic hyphomycete Heteroconium chaetospira suppressed Verticillium yellows in Chinese cabbage in
the eld. Verticillium wilt is one of the most destructive diseases of aubergine. Eleven out of 123 isolates of endophytic fungi, especially Heteroconium
chaetospira, Phialocephala fortinii, Fusarium, Penicillium, Trichoderma and mycelium radicis atrovirens (MRA), after being inoculated onto axenically reared
aubergine seedlings, almost completely suppressed the pathogenic effects of
a post-inoculated, virulent strain of Verticillium dahliae (Narisawa et al., 2000).
Out of 39 endophytes of Artemisia annua investigated, 21 showed in vitro antifungal activity against crop-threatening fungi Gaeumannomyces graminis var.
tritici, Rhizoctonia cerealis, Helminthosporium sativum, Fusarium graminearum,
Gerlachia nivalis and Phytophthora capsici (Liu et al., 2001). The extracts of
endophytic Alternaria sp., isolated from medicinal plants of Western Ghats of
India, inhibited the growth of C. albicans (Raviraja et al., 2006). About 41.2%
of all the isolated endophytic fungi from rice plants showed antagonism
against Magnaporthe grisea, Rhizoctonia solani and Fusarium moniliforme. Colletotrichum gloeosporioides (Penz) Penz & Sacc. was isolated as an endophyte
from healthy leaves of Cryptocarya mandioccana and had antifungal activity
against phytopathogenic fungi Cladosporium cladosporioides and Cladosporium
sphaerospermum (Inacio et al., 2006). Fungal endophytes Chaetomium spp.,
Phoma sp., isolated from asymptomatic leaves of wheat, reduced the number
of pustules and the area of pustules of Puccinia recondita f. sp. Tritici (Dingle
and Mcgee, 2003). An in vitro study showed 40%, 65% and 27% antagonistic
interaction by endophytic morphospecies against cacao pathogens Moniliophthora roreri, Phytophthora palmivora and Crinipellis perniciosa, respectively,
while in-eld endophytic C. gloeosporioides produced a signicant decrease in
pod loss (Mejia et al., 2008).
For novel natural products, exciting possibilities exist for those who are
willing to venture into the wild and unexplored territories of the world to
experience the excitement and thrill of engaging in the discovery of endophytes, their biology, and potential usefulness. It is evident that naturalproduct-based compounds have an immense impact on modern medicine.
For instance, about 40% of prescription drugs are based on them, and well
Fungal Endophytes
227
over 50% of the new chemical products registered by the US Food and Drug
Administration (FDA) as anticancer agents, antimigraine agents and antihypertensive agents from 1981 to 2002 are natural products or their derivatives
(Newman et al., 2003). Excluding biologics, between 1989 and 1995, 60% of
approved drugs and pre-new-drug-application (NDA) candidates were of
natural origin. From 1983 to 1994, over 60% of all approved and pre-NDA
stage cancer drugs were of natural origin, as were 78% of all newly approved
antibacterial agents (Concepcion et al., 2001). The discovery and development of taxol is a modern example of a natural product that has made an
enormous impact on medicine (Wani et al., 1971; Suffness, 1995; Schulz and
Christine, 2005).
Antibiotics from endophytic fungi

Generally, the most commonly isolated endophytic fungi are in the group of
Fungi Imperfecti or Deuteromycotina. Also, it is quite common to isolate
endophytes that produce no fruiting structures whatsoever such as MyceliaSterilia.
Cryptosporiopsis cf. quercina is the imperfect stage of Pezicula cinnamomea,
a fungus commonly associated with hardwood species in Europe. It was isolated as an endophyte from Tripterigeum wilfordii, a medicinal plant native to
Eurasia (Strobel et al., 1999). On Petri plates, C. quercina demonstrates excellent antifungal activity against some important human fungal pathogens
including C. albicans and Trichophyton spp. Cryptocandin, a unique peptide
antimycotic related to the echinocandins and the pneumocandins (Walsh,
1992), was isolated and characterized from C. quercina (Strobel et al., 1999).
This compound contains a number of peculiar hydroxylated amino acids
and a novel amino acid, 3-hydroxy-4-hydroxymethyl proline (Fig. 11.2). It is
generally true that not one but several bioactive and related compounds are
produced by an endophytic microbe. So it is that other antifungal agents
related to cryptocandin are also produced by C. quercina. Cryptocandin is
also active against a number of plant pathogenic fungi including S. sclerotiorum and Botrytis cinerea. Cryptocandin and its related compounds are currently being considered for use against a number of fungi causing diseases of
the skin and nails.
Cryptocin, a unique tetramic acid, is also produced by C. quercina
(Fig. 11.3) (Li et al., 2000). This unusual compound possesses potent activity
against Pyricularia oryzae, the causal organism of rice blast, one of the worst
plant diseases in the world, as well as a number of other plant pathogenic
fungi (Li et al., 2000). The compound was generally ineffective against an
array of human and plant pathogenic fungi. Nevertheless, with minimum
inhibitory concentrations against P. oryzae at 0.39 g/ml, this compound is
being examined as a natural chemical control agent for rice blast and is being
used as a model to synthesize other antifungal compounds.
Pestelotiopsis microspora is a common rainforest endophyte (Strobel, 2002;
Strobel and Daisy, 2003). It turns out that enormous biochemical diversity
228

OH
3,4-Dihydroxy-homotryosine
HO
Glutamine
O
H2N
OH
N
H
O
OH
4-Hydroxyproline
H
N
N
O
O
O
OH
HO
3-Hydroxy-4-hydroxy
methyl-proline
NH
HN
OH
4,5-Dihydroxy
ornithine
OH
Threonine
NH
O
(CH2)14
Palmitic acid
Fig. 11.2 Cryptocandin A, an antifungal lipopeptide obtained from the endophytic fungus
Cryptosporiopsis cf. quercina.
CH3
O
N
OH
O
CH3
H
CH3
O
CH3
H3C
H
Fig. 11.3 Cryptocin, a tetramic acid antifungal compound found in Cryptosporiopsis

cf. quercina.
does exist in this endophytic fungus and many secondary metabolites are
produced by various strains of this widely dispersed organism. One such
secondary metabolite is ambuic acid, an antifungal agent, which has been
recently described from several isolates of P. microspora found as representative
Fungal Endophytes
229
isolates in many of the worlds rainforests (Fig. 11.4; Li et al., 2001). As an

interesting spin-off to endophyte studies, ambuic acid and another endophyte product, terrein, have been used as models to develop new solid-state
NMR tensor methods to assist in the characterization of molecular
stereochemistry of organic molecules (Harper et al., 2001).
A strain of P. microspora was isolated from the endangered tree Torreya
taxifolia and produces several compounds that have antifungal activity,
including pestaloside, an aromatic -glucoside, and two pyrones, pestalopyrone and hydroxypestalopyrone (Lee et al., 1995a,b). These compounds also
possess phytotoxic properties. Other newly isolated secondary products
obtained from P. microspora (endophytic on Taxus brevifolia) include two new
caryophyllene sesquiterpenes, pestalotiopsins A and B (Pulici et al., 1996a,b),
2-hydroxydrimeninol and a highly functionalized humulane. Variation in
the amount and types of products found in this fungus depends on both the
culture conditions and the original plant source from which it was isolated.
Pestalotiopsis jesteri is a newly described endophytic fungal species from
the Sepik river area of Papua New Guinea, and it produces the highly functionalized cyclohexenone epoxides, jesterone and hydroxy-jesterone, which
exhibit antifungal activity against a variety of plant pathogenic fungi (Li and
Strobel, 2001). Jesterone has subsequently been synthesized with complete
retention of biological activity (Fig. 11.5; Hu et al., 2001). Jesterone is one of
H
17
H 3C
13
15
12
16
11
10
14
4
5
O
6
19
COOH
1
18
CH3
H
H
OH OH
Fig. 11.4. Ambuic acid, a highly functionalized cyclohexenone epoxide produced by a

number of isolates of Pestalotiopsis microspora found in rainforests around the world.
O
O
H
H
OH
Fig. 11.5. Jesterone, a cyclohexenone epoxide from Pestaliotiopsis jesteri, has

antioomycete activity.
230
only a few products from endophytic microbes for which total synthesis has
been successfully accomplished.
Phomopsichalasin, a metabolite from an endophytic Phomopsis sp., represents the rst cytochalasin-type compound with a three-ring system replacing the cytochalasin macrolide ring. This metabolite exhibits antibacterial
activity in disc diffusion assays (at a concentration of 4 g/disc) against Bacillus subtilis, Salmonella gallinarum and Staphylococcus aureus. It also displays a
moderate activity against the yeast Candida tropicalis (Horn et al., 1995).
An endophytic Fusarium sp. isolated from Selaginella pallescens collected
in the Guanacaste Conservation Area of Costa Rica was screened for antifungal activity. A new pentaketide antifungal agent, CR377, was isolated
from the culture broth of the fungus and showed potent activity against
C. albicans in agar diffusion assays (Brady and Clardy, 2000). Colletotric
acid, a metabolite of Colletotrichum gloeosporioides, an endophytic fungus isolated from Artemisia mongolica, displays antibacterial activity against bacteria as well as against the fungus Helminthsporium sativum. Antimicrobial
products have been identied from another Colletotrichum sp. isolated from
Artemisia annua, a traditional Chinese herb that is well recognized for its
synthesis of artemisinin (an antimalarial drug) and its ability to inhabit
many geographically different areas. Not only did the Colletotrichum sp.
found in A. annua produce metabolites with activity against human pathogenic fungi and bacteria, but also metabolites that were fungistatic to plant
pathogenic fungi (Lu et al., 2000).
A novel antibacterial agent, guignardic acid, was isolated from the endophytic fungus Guignardia sp.; the organism was obtained from the medicinal
plant Spondias mombin of the tropical plant family Anacardiaceae found in
Brazil. The compound was isolated by UV-guided fractionation of the fermentation products of this fungus and is the rst member of a novel class of
natural compounds containing a dioxolanone moiety formed by the fusion
of 2-oxo-3-phenylpropanoic acid and 3-methyl-2-oxobutanoic acid, which
are products of the oxidative deamination of phenylalanine and valine,
respectively (Fig. 11.6; Rodriguez-Heerklotz et al., 2001).
Another antibacterial compound javanicin is produced by endophytic
fungus Chloridium sp., under liquid- and solid-media culture conditions
O
O
H3C
CH3
Fig. 11.6. Guignardic acid from Guignardia sp. obtained from Spondias mombin, an
Anacardiaceaeous plant in Brazil.
Fungal Endophytes
231
(Kharwar et al., 2009). This highly functionalized naphthaquinone exhibits

strong antibacterial activity against Pseudomonas spp., representing pathogens to both humans and plants. The compound was crystallized and the
structure was elucidated using X-ray crystallography. The X-ray structure
conrms the previously elucidated structure of the compound that was done
through standard spectroscopic methods (Fig. 11.7).
Volatile antibiotics from fungal endophytes

Muscodor albus is a newly described endophytic fungus obtained from small
limbs of Cinnamomum zeylanicum (Worapong et al., 2001). This xylariaceaous
(non-spore-producing) fungus effectively inhibits and kills certain other fungi
and bacteria by producing a mixture of volatile compounds (Strobel et al.,
2001). The majority of these compounds have been identied by gas chromatography / mass spectrometry (GC/MS), synthesized or acquired, and then
ultimately formulated into an articial mixture that mimicked the antibiotic
effects of the volatile compounds produced by the fungus (Strobel et al., 2001).
Individually, each of the ve classes of volatile compounds produced by the
fungus had some antimicrobial effects against the test fungi and bacteria but
none was lethal. However, collectively they acted synergistically to cause
death in a broad range of plant and human pathogenic fungi and bacteria.
The most effective class of inhibitory compounds was the esters, of which
isoamyl acetate was the most biologically active; however, in order to be lethal
it needs to be combined with other volatiles (Strobel et al., 2001). The composition of the medium on which M. albus is grown dramatically inuences the
kind of volatile compounds that are produced (Ezra and Strobel, 2003).
The ecological implications and potential practical benets of the mycofumigation effects of M. albus are very promising given the fact that soil
fumigation utilizing methyl bromide will soon be illegal in the USA. Methyl
bromide is not only a hazard to human health but it has been implicated in
causing destruction of the worlds ozone layer. The potential use of mycofumigation to treat soil, seeds, and plants will soon be a reality as AgraQuest of
Davis, California, has EPA approval to release this organism for agricultural
uses. The articial mixture of volatile compounds may also have usefulness
OH
CH3
H 3C
O
O
CH3
OH
Fig. 11.7. Javanicin, an antibacterial compound isolated from endophytic fungus

Chloridium sp.
232
in treating seeds, fruits and plant parts in storage and while being transported. In addition, M. albus is already in a limited market for the treatment
of human wastes. Its gases have both inhibitory and lethal effects on such
faecal-inhabiting organisms as Escherichia coli and Vibrio cholera. It will be
used for this purpose in coming years. Studies are underway that show its
promise to fumigate buildings, thus removing the potential for fungi to
contaminate building surfaces and cause health risks.
Using M. albus as a screening tool, it has now been possible to isolate other
endophytic fungi producing volatile antibiotics. The newly described M. roseus
was twice obtained from tree species found in the Northern Territory of
Australia. This fungus is just as effective in causing inhibition and death of
test microbes in the laboratory as M. albus (Worapong et al., 2002). Other interesting M. albus isolates have been obtained from several plant species growing
in the Northern Territory of Australia and the jungles of the Tesso Nilo area of
Sumatra, Indonesia (Ezra et al., 2004; Atmosukarto et al., 2005).
A non-muscodor species (Gliocladium sp.) has also been discovered to
produce volatile antibiotics. The volatile components of this organism are
totally different from those of either M. albus or M. roseus. In fact, the most
abundant volatile inhibitor is [8]-annulene, formerly used as a rocket fuel
and discovered here for the first time as a natural product. However, the
bioactivity of the volatiles of this Gliocladium sp. is not as good or as comprehensive as that of the Muscodor spp. (Stinson et al., 2003). Due to the volatile
antibiotic producing properties of these fungi, they could be used against
several soil plant pathogens to reduce their inoculum. The latter, G. roseum,
has already shown its potential through the production of a series of hydrocarbons and hydrocarbon derivatives as mycodiesel (Strobel et al., 2008).
Interestingly, another novel species of Muscodor sp., M. crispans, has been
reported to produce a mixture of strong volatile compounds (VOCs) that
were effective against a wide range of plant pathogens, including the fungi
Pythium ultimum, Phytophthora cinnamomi, S. sclerotiorum and Mycosphaerella
fijiensis (the black sigatoka pathogen of bananas), and the serious bacterial
pathogen of citrus, Xanthomonas axonopodis pv. citri (Mitchell et al., 2010).
Antiviral compounds from endophytic fungi

Another fascinating use of products from endophytic fungi is the inhibition
of viruses. Two novel human cytomegalovirus (hCMV) protease inhibitors,
cytonic acids A and B, have been isolated from solid-state fermentation of the
endophytic fungus Cytonaema sp. Their structures were elucidated as
p-tridepside isomers using MS and NMR methods (Guo et al., 2000). It is
apparent that the potential for the discovery of compounds having antiviral
activity from endophytes is in its infancy. The fact, however, that some compounds have been found already is promising. The main limitation to compound discovery to date is probably related to the absence of common
antiviral screening systems in most compound discovery programs. Since no
antiviral compound isolated from endophytes has been tested against
Fungal Endophytes
233
virulent plant pathogens, an intensive exploration is therefore needed to

identify some potential antiviral compounds which could be very effective
against variety of pathogenic plant viruses.
Antioxidants from fungal endophytes

Two compounds, pestacin and isopestacin, have been obtained from culture
uids of Pestalotiopsis microspora, an endophyte isolated from a combretaceaous plant, Terminalia morobensis, growing in the Sepik River drainage system of Papua New Guinea (Strobel et al., 2002; Harper et al., 2003). Both
pestacin and isopestacin display antimicrobial as well as antioxidant activity.
Isopestacin was attributed with antioxidant activity based on its structural
similarity to the avonoids (Fig. 11.8). Electron spin resonance spectroscopy
measurements conrmed this antioxidant activity showing that the compound is able to scavenge superoxide and hydroxyl free radicals in solution
(Strobel et al., 2002). Pestacin occurs naturally as a racemic mixture and also
possesses potent antioxidant activity, at least one order of magnitude more
potent than that of Trolox, a vitamin E derivative (Harper et al., 2003). The
antioxidant activity of pestacin arises primarily via cleavage of an unusually
reactive CH bond and, to a lesser extent, through OH abstraction (Harper
et al., 2003). Endophytes also help plants to keep t through balancing the
generation and elimination of reactive oxygen species (Rodriguez and
Redman, 2005). The use of chemical antioxidants is being slowly prohibited
due to their small carcinogenic effect, and thus the demand for natural
OH
NH
H
OH O
OH
Fig. 11.8. Isopestacin, an antioxidant produced by an endophytic Pestalotiopsis microspora

strain, isolated from Terminalia morobensis growing on the north coast of Papua New Guinea.
234
antioxidant compounds from natural resources has increased, so that the

shelf life of stored crop produce and plant health could be enhanced to cater
for a hungry population, and endophytes may be one of the novel natural
resources to achieve this goal (Tejesvi et al., 2008).
Immunosuppressive compounds from fungal endophytes

Immunosuppressive drugs are used today to prevent allograft rejection in
transplant patients and in the future they could be used to treat autoimmune
diseases such as rheumatoid arthritis and insulin-dependent diabetes. The
endophytic fungus, Fusarium subglutinans, isolated from Tripterygium wilfordii, produces the immunosuppressive, but non-cytotoxic diterpene pyrones,
subglutinols A and B (Lee et al., 1995). Subglutinols A and B are equipotent in
the mixed lymphocyte reaction (MLR) assay and thymocyte proliferation
(TP) assay with an IC50 of 0.1 M. The famed immunosuppressant drug
cyclosporin A, also a fungal metabolite, was roughly as potent in the MLR
assay and 104 more potent in the TP assay. However, the lack of toxicity associated with subglutinols A and B suggests that they should be explored in
greater detail as potential immunosuppressants (Lee et al., 1995).
11.5 Conclusion
Geographical, oristic and signicant seasonal variations that exist in different parts of globe provide conducive/or adverse conditions for the luxuriant
growth of microbes in a wide range of different habitats, including living
tissues of higher plants where they grow as endophytes. Due to great variation in plant biodiversity and seasonal changes in tropical and subtropical
regions, there is a need to collect/isolate various types of promising endophytic fungi, especially from rainforests and mangrove swamps, which may
be able to produce an enormous variety of potential bioactive natural compounds. The fungi, as a group, hold enormous potential as sources of antimicrobials. The observations prove that this group of organisms resides inside
the healthy plants tissue, as endophytes, without causing any detectable
symptoms. Therefore, we strongly feel that there is a need to accelerate and
focus the research to exploit the maximum potential of the promising endophytes for natural-product discovery, which could at least facilitate some
existing problems of the huge population.
The past history of endophytic research in India especially with fungi is
not so encouraging. It seems that workers who have started this research in
India are still actively involved in advancing their research manifesto with
this under-studied group of microbes, and have not advanced to the elds
and forests of the countryside looking for novel microbeplant associations.
Prof. T.S. Suryanarayanan and his group (Chennai) have initiated biodiversity
and distribution patterns of fungal endophytes with some medicinal plants
in India and have published several papers along this line. They have also
Fungal Endophytes
235
isolated some bioactive compounds and melanin from endophytic fungi

(Suryanarayanan et al., 2004). To date, the overall situation in India has not
drastically improved, as is the case in other countries such as China, Japan
and Brazil (Verma et al., 2009). However, several research groups have started
paying more attention to various aspects of endophytic fungi. No more than
a dozen research groups at different places in India are vigorously involved
either with biodiversity or in natural-product discovery from this untapped
and alternative resource. It has become obvious to many workers throughout
the world that endophytic microbes have enormous potential for solving
many of humankinds problems. Thus, with the discovery of new compounds, we can protect our interests in agricultural and medical industries
as well as plant health (Omacini et al., 2001; Gunatilaka, 2006; Gimenez et al.,
2007). Currently, some focused research on the different aspects of endophytes
is being carried out and valuable results have been published (Shankar et al.,
2003; Seena and Sridhar, 2004; Amna et al., 2006; Verma et al., 2006, 2007, 2008,
2009; Gond et al., 2007; Tejesvi et al., 2007; Gangadevi and Muthumarry, 2008;
Kharwar et al., 2008, 2009).
It is important for all involved in this work to realize the importance of
acquiring the necessary permits from governmental, local, and other sources
to collect and transport plant materials (especially from abroad) from which
endophytes are to be eventually isolated. In addition to this aspect of the
work is the added activity of producing the necessary agreements and
nancial-sharing arrangements with indigenous people or governments in
case a product does develop an income stream.
Another concern is that if endophytic fungi may produce a natural product of host origin, a host plant may also acquire the ability to produce some
mycotoxins or other secondary metabolites of fungal origin. The need for
new and useful compounds to provide assistance and relief in all aspects of
the human condition is ever growing. Drug resistance in bacteria, the appearance of new life-threatening viruses, the recurrent problems of diseases in
people with organ transplants, a variety of severe diseases affecting plants
and the tremendous increase in the incidence of fungal infections in the
worlds population all underscore our inadequacy to cope with these problems. Environmental degradation, loss of biodiversity, and spoilage of land
and water also add to problems facing us, and each of these in turn can have
health- and hunger-related consequences.
The researchers who are interested in exploiting endophytes should have
access to, or have some expertise in, microbial taxonomy and this includes
modern molecular techniques involving sequence analyses of 16S and 18S
rDNA. Currently, endophytes are viewed as an outstanding source of bioactive natural products because there are so many of them occupying literally
millions of unique biological niches (higher plants) growing in so many
unusual environments. Thus, it would appear that a myriad of biotopical
factors associated with plants can be important in the selection of a plant for
study. It may be the case that these factors govern which microbes are present
in the plant as well as the biological activity of the products associated with
these organisms.
236
Certainly, one of the major problems facing the future of endophyte biology and natural-product discovery is the rapidly diminishing rainforests,
which hold the greatest possible resource for acquiring novel microorganisms and their products. The total land mass of the world that currently supports rainforests is about equal to the area of the USA (Mittermeier et al.,
1999). Each year, an area the size of Vermont or greater is lost to clearing, harvesting, re, agricultural development, mining or other human-oriented
activities (Mittermeier et al., 1999). Presently, it is estimated that only a small
fraction (1020%) of what were the original rainforests existing 10002000
years ago are currently present on the earth (Mittermeier et al., 1999). The
advent of major negative pressures on them from these human-related activities appears to be eliminating entire mega-life forms at an alarming rate. Few
have ever expressed information or opinions about what is happening to the
potential loss of microbial diversity as entire plant species disappear. It can
only be guessed that this microbial diversity loss is also happening, perhaps
with the same frequency as the loss of mega-life forms, especially since certain microorganisms may have developed unique symbiotic relationships
with their plant hosts. Thus, when a plant species disappears, so too does its
entire suite of associated endophytes. Consequently all of the capabilities that
the endophytes might possess to provide natural products with medicinal
potential are also lost. Multi-step processes are needed now to secure information and life forms before they continue to be lost. Areas of the planet that
represent unique places housing biodiversity need immediate preservation.
Countries need to establish information bases of their biodiversity and at the
same time begin to make national collections of microorganisms that live in
these areas. Endophytes are only one example of a life form source that holds
enormous promise to impact many aspects of human existence. The problem
of the loss of biodiversity should be one of concern to the entire world.
Acknowledgements
The authors express appreciation to the NSF, USDA, NIH, the R&C Board of
the State of Montana and the Montana Agricultural Experiment Station for
providing nancial support for some of the work reviewed in this report.
RNK expresses his sincere thanks to DST, New Delhi, for nancial
assistance.
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12
Suppressive Effects
of Compost Tea
on Phytopathogens
MILA SANTOS, FERNANDO DINEZ

AND FRANCISCO CARRETERO
Plant Production Department, University of Almera, Almera, Spain
Abstract
Suppression of soilborne and airborne diseases of horticultural crops by compost has
been attributed to the activities of antagonistic microorganisms. A great diversity of
biological control agents naturally colonize compost. The knowledge of mechanisms
for biological control through the action of compost or its water extracts (compost tea)
is necessary in order to increase the efciency of the suppressing power. Modes of
action of biocontrol agents include: inhibition of the pathogen by antimicrobial compounds (antibiosis), competition for iron through production of siderophores, competition for colonization sites and nutrients supplied by seeds and roots, induction of
plant resistance mechanisms, inactivation of pathogen germination factors present in
seed or root exudates, degradation of pathogenicity factors of the pathogen such as
toxins, parasitism that may involve production of extracellular cell wall-degrading
enzymes, for example, chitinase and -1,3 glucanase that can cause lysis of pathogen
cell walls. None of the mechanisms are necessarily mutually exclusive and frequently
several modes of action are exhibited by a single biocontrol agent. Indeed, for some
biocontrol agents, different mechanisms or combinations of mechanisms may be
involved in the suppression of different plant diseases. The healthy development of
plants, as well as the biological control of soilborne fungi, originates from many
microorganisms, partly due to the production of siderophores under iron-restricting
conditions. The siderophores are natural chelators that keep iron available for plants
in soil. Iron also plays a major role in nutrient competition among pathogens and
benecial microorganisms in infection sites.
12.1 Introduction
Suppressive soils or suppressive substrates are characterized by a very low
level of disease development even though a virulent pathogen and susceptible
host are present. Biotic and abiotic elements of the soil environment contribute
to suppressiveness; however, most dened systems have identied biological elements as primary factors in disease suppression. Many soils possess
242

(ed. N.K. Dubey)
Suppressive Effects of Compost Tea on Phytopathogens
243
similarities with regard to microorganisms involved in disease suppression,

while other attributes are unique to specic pathogen-suppressive soil systems. Modes of action of biocontrol agents include: inhibition of the pathogen by antimicrobial compounds (antibiosis); competition for iron through
the production of siderophores; competition for colonization sites and nutrients supplied by seeds and roots; induction of plant-resistance mechanisms;
inactivation of pathogen germination factors present in seed or root exudates;
degradation of pathogenicity factors of the pathogen such as toxins; parasitism
that may involve production of extracellular cell-wall-degrading enzymes,
for example, chitinase and -1,3 glucanase that can cause lysis to pathogen
cell walls (Keel and Dfago, 1997; Whipps, 1997). None of the mechanisms
are necessarily mutually exclusive and frequently several mode of action are
exhibited by a single biocontrol agent. Indeed, for some biocontrol agents,
different mechanisms or combinations of mechanisms may be involved in
the suppression of different plant diseases (Whipps, 2001). So the organism
operatives in pathogen suppression do so via diverse mechanisms including
competition for nutrients, antibiosis and induction of host resistance. Nonpathogenic Fusarium spp. and uorescent Pseudomonas spp. play a crucial
role in naturally occurring soils that are suppressive to Fusarium wilt. Suppression of take-all of wheat (Triticum aestivum), caused by Gaeumannomyces
graminis var. tritici, is induced in soil after continuous wheat monoculture
and is attributed, in part, to selection of uorescent Pseudomonads with the
capacity to produce the antibiotic 2,4-diacetylphloroglucinol. Cultivation of
orchard soils with specic wheat varieties induces suppressiveness to Rhizoctonia root rot of apple (Malus domestica) caused by Rhizoctonia solani AG 5.
Wheat cultivars that stimulate disease suppression enhance populations of
specic uorescent pseudomonad genotypes with antagonistic activity toward
this pathogen. Methods that transform resident microbial communities in a
manner that induces natural soil suppressiveness have the potential as
components of environmentally sustainable systems for the management of
soilborne plant pathogens (Mazzola, 2002).
During the past decades, compost prepared from a variety of organic
waste has been utilized successfully in container media for suppressing soilborne diseases (Table 12.1). Examples are composts prepared from tree barks,
liquorice (Glycyrrhina glabra) root waste, grape pomace, separated cattle
manure, and municipal sewage sludge (Kuter et al., 1998). Suppressive soils
or substrates are characterized by a very low level of disease development
even though a virulent pathogen and susceptible host are present. Diseases
that have been shown to be effectively suppressed by compost use include
those caused by Fusarium, Phytophthora, Pythium and R. solani. Disease
suppression in compost has been attributed mainly to elevated levels of
microbial activity (Chen et al., 1988b, Santos et al., 2008). There are several
mechanisms involved in the suppression: the competition among microbial
populations for available carbon or nitrogen, iron competition, mycoparasitism, production of inhibitors or hydrolytic enzymes by microorganisms and
interactions with some saprophytes. Since 1991, several reports have shown
that some rhizosphere microorganisms can induce systemic resistance in
244
M. Santos et al.
Table 12.1.
Different composted material suppressive to soilborne disease.
Composted
material
Pathogens
References
Tree bark
Phytophthora cinnamomi
Hoitink et al., 1977; Sivasithamparam, 1981;

Spencer and Benson, 1981, 1982;
Blaker and MacDonald, 1983;
Hardy and Sivasithamparam, 1991
Spencer and Benson, 1981;
Daft et al., 1979; Chen et al., 1987;
Chen et al., 1988 a,b
Daft et al., 1979; Stephens et al., 1981;
Nelson and Hoitink,1983
Chef et al., 1983; Cebolla and Pera, 1983;
Trillas-Gay et al., 1986
Malek and Gartner, 1975;
Mc Sorley and Gallaher, 1995
Sivasithamparam, 1981;
Spencer and Benson, 1981, 1982
Gugino et al., 1973; Zhang et al., 1996
Chef et al., 1983; Pera and Calvet, 1989
Mandelbaum et al., 1988
Gorodecki and Hadar, 1990
Gorodecki and Hadar, 1990;
Hadar and Gorodecki, 1991
Pera and Calvet, 1989
Phytophthora citricola
Phytophthora drechleri
Phytophthora nicotianae
Pythium ultimum
Rhizoctonia solani
Fusarium oxysporum
Nematodes
Pine bark
Phytophthora spp.
Grape marc
Pythium spp.
Fusarium oxysporum
Pythium aphanidermatum
Rhizoctonia solani
Sclerotium rolfsii
Olive marc
Fusarium oxysporum f. sp.

dianthi
Pythium spp.
Municipal
sewage sludge
Municipal solid
Rhizoctonia spp.
residues
Cattle manure
Phytophthora nicotianae
Rhizoctonia solani
Sclerotium rolfsii
Fusarium oxysporum
Beer industry
sludge
Liquorice roots
Lumsden et al., 1983

Mathot, 1987
Pythium graminicola
Mandelbaun et al., 1988

Szczech et al., 1993
Garibaldi, 1988;
Szczech et al., 1993
Craft and Nelson, 1996
Hadar and Mandelbaun, 1986
plants to root and foliar disease (Han et al., 2000). This systemic resistance
was found in compost by Zhang et al. (1996).
Most of the composts are naturally suppressive against root rot disease
caused by Phytophthora and Pythium (Santos et al., 2008), nearly 20% are naturally suppressive against damping off caused by Rhizoctonia (Hoitink and
245
Boehm, 1999), less than 10% of the compost induced systemic resistance in
plants (Zhang et al., 1996, 1998; Hoitink and Boehm, 1999).
There are two primary mechanisms by which the colonies of biocontrol
organisms in compost combat disease, general suppression and specic
suppression.
General suppression occurs when a high-microbial activity environment
is created in which the germination of pathogen propagules is inhibited.
General suppression occurs when many different organisms compete with
pathogens for nutrients (as root or seed exudates) and/or produce general
antibiotics that reduce pathogen survival and growth. In compost there is a
slow release of nutrients which supports benecial activity of this microbiotic. Biocontrol agents that colonize composts include bacteria such as
Bacillus, Enterobacter, Flavobacterium balusstinum, and Pseudomonas; actinomycetes such as Streptomyces; and fungi such as Trichoderma and Gliocladium
(Hoitink et al., 1991b).
General suppression owes its activity to the total microbial biomass in
the soil or substrate and is not transferable between them. Whether soil
organic matter can support biological control depends on its decomposition
level and the types of biocontrol agents present on the substrate (Hoitink and
Boehm, 1999). The carrying capacity of organic matter in the substrate limits
suppressiveness to pathogens that depend on exogenous sources of nutrients
(root exudates) for germination and infection. Excessively stabilized organic
matter such as dark peat has a limited ability to sustain activity of the general
microbial biomass in soil (Hoitink et al., 1993). Dark decomposed sphagnum
peat is consistently conductive to Pythium root rot, whereas light sphagnum
peat harvested from the surface of peat bogs is less decomposed and has a
higher microbial activity (Hoitink et al., 1991a). Light peat moss is suppressive against Pythium for a short time (67 weeks).
Specic suppression involves the action of one or a few specic microbial
agents in suppressing a specic pathogen (Hoitink, 1993). They exert hyperparasitism on the pathogen or induce systemic resistance in the plant to specic pathogens. Specic suppression owes its activity to the effects of
individual or select groups of microorganisms and is transferable. This can
be achieved by inoculating the compost with the desired microbial agent
(Hoitink, 1993).
The suppression effect on pathogens is linked to the type of pathogens.
Those pathogens that have a small propagule size, such as Pythium and Phytophthora species, are susceptible to general suppression (nutrient-dependent
pathogens). They have small nutrient reserves and need to rely on an external carbon source and other nutrients. Pathogens with a large propagule size,
such as Sclerotium rolfsii and R. solani, are susceptible to specic suppression.
Structures like sclerotia are less susceptible to microbial competition but specic hyperparasites such as Trichoderma species will colonize the sclerotia.
Composting conditions as well as the materials that are composted are
critical, with the type of the material impacting on the sort of active microora. Thus the composting of lignocellulosic wastes will induce a specic
suppression of Rhizoctonia by Trichoderma species, while Penicillium fungi
246
M. Santos et al.
predominate in grape pomace (high sugar, low cellulose) in suppressing

S. rolfsii. In this medium, Trichoderma spp. were absent and would have been
ineffective even if applied. Rhizoctonia (a common nursery pathogen) is also
a very active saprophyte in fresh wastes but not in low-cellulose mature composts. Trichoderma, by contrast, colonizes both. However, the hyperparasitic
activity and antibiotic production by Trichoderma is repressed in fresh compost because of the high glucose level. Saline composts (for instance overfertilized media) enhance Pythium and Phytophthora, whereas composts with
low C/N ratios that release nitrogen, especially as ammonia, promotes the
growth of Fusarium on host species. The appropriate control of the potting
medium with a pH greater than 5.0 and moisture content (oven dried)
maintained between 40 and 50% is also important (Hoitink et al., 1997).
12.2 Competition Among Microbial Populations

Microbiostasis
There is a competition for nutrients in exudates such as sugars and amino
acids which leak out of seeds during germination or out of root tips as plants
grow through the soil. Pathogens that grow or swim to these sources of nutrients must compete with the benecial microora at the infection site on the
surface of the seed or root (Hoitink and Changa, 2004). This type of competition plays a major role in general suppression and with nutrient-dependent
pathogens such as Pythium and Phytophthora species. The competition includes
microbial competition for nutrients and competition for infection sites and
root colonization. Limited nutrient availability to the germinating spores or
invading hyphae is the most common explanation given for the microbial
cause of fungistasis. On the other hand, fungistasis has been attributed to the
presence of antifungal compounds of microbiological origin. The production
of antifungal compounds is correlated with available carbon, which is not
surprising, as microbial production of antibiotics can be induced by interspecic competition for substrates (Ellis et al., 2000; Slattery et al., 2001). The sensitivity of plant pathogenic fungi to fungistasis is thought to protect them
from germinating and initiating growth under unfavourable conditions (e.g.,
the absence of a host plant) (Garrett, 1970; Lockwood, 1977). However, this
protection comes at a cost, since the viability of resting structures decreases
during prolonged incubation in soils (Lockwood, 1986). The negative effects
of fungistasis on the inoculum density of plant pathogenic fungi has been
suggested as a mechanism to explain the commonly found correlation
between fungistasis and disease suppressiveness (Lockwood, 1977; Hornby,
1983; Larkin et al., 1996; Knudsen et al., 1999).
Iron competition also plays a major role in nutrient competition among
pathogens and benecial microorganisms in infection sites (Hoitink and
Changa, 2004). Ionic forms of iron, especially iron (III), its most common state,
are very insoluble, especially when plants are grown on calcareous soils or
soils with high pH. Iron is only available to organisms at concentrations at or
247
below 1018 M in soil solutions at neutral pH (Handelsman and Stabb, 1996).

Iron salts that have low solubility such as iron oxides, carbonates, phosphates,
hydroxides and some forms of insoluble chelates are created in certain soil
types that make iron not readily available to plants. To circumvent the solubility problem, many microorganisms synthesize and utilize very specic
low molecular weight (5001000 Da) iron chelators called siderophores (that
can bind iron and are sent out by the cells to absorb iron, as well as being used
inside the cell to store it). When grown under iron-decient conditions, many
microbes will synthesize and excrete siderophores in excess of their own dry
cell weight to kidnap and solubilize iron and made it unavailable to the pathogens. The typical system involves a siderophore, which is an iron-binding
ligand, and an uptake protein, which transports the siderophore into the cell.
The uorescent pseudomonads produce a class of siderophore known as the
pseudobactins, which are structurally complex iron-binding molecules. Analyses of mutants lacking the ability to produce siderophores suggest that they
contribute to the suppression of certain fungal and oomycete diseases (Duijff
et al., 1994; Buysens et al., 1996). An interesting aspect of siderophore biology
is that diverse organisms can use the same type of siderophore. Microorganisms may use each others siderophores if they contain the appropriate uptake
protein (Koster et al., 1993; Raaijmakers et al., 1995), and plants can even
acquire iron from certain pseudobactins (Duijff et al., 1994).
Microorganisms in compost produce siderophores that keep iron in an
available form for plants in the soil, even at high pH. Compost also produces
water-soluble humic substances, including fulvic acids, which keep iron, zinc,
manganese and other trace elements in solution (Chen and Inbar, 1993).
Antibiosis
Antibiosis is dened as antagonism mediated by specic or non-specic
metabolites of microbial origin, by lytic agents, enzymes, volatile compounds
or other toxic substances (Jackson, 1965; Fravel, 1988). Antibiotic production
appears to be important to the survival of microorganisms through the elimination of microbial competition for food sources, which are usually very
limited in soil (Ellis et al., 2000; Slattery et al., 2001). Antibiotic production is
very common among soil-dwelling bacteria and fungi. Inhibition in the Petri
dish may be the result of antibiosis, but it is not easy to show that this antibiosis is actually responsible for disease suppression. First, the antibiotic
must be extracted, puried and identied chemically. Then it is necessary to
show that the microorganism grows in the microhabitat of the pathogen, and
that the antibiotic is produced in the right place, at the right time, and in sufcient amounts to control disease. It is also necessary to demonstrate that the
pathogen is sensitive to the antibiotic. Genetic analyses have been particularly informative in determining the role of antibiotics in biocontrol, in part
because mutants can be screened easily in vitro for changes in antibiotic
accumulation, providing the means to conduct thorough genetic analyses
(Handelsman and Stabb, 1996).
248
M. Santos et al.
Antibiotic production by strains of the bacteria Pseudomonas and Bacillus

has been shown to be important to the successful biocontrol of several crop
diseases. The antibiotics zwittermicin A and kanosamine, produced by the
biocontrol agent Bacillus cereus UW85, appear to be important in the biocontrol of oomycetes such as Phytophthora (Silo-Suh et al., 1994; Milner et al.,
1996). The antibiotic phenazine derivatives, produced by Pseudomonas fluorescens strain 2-79 and P. aureofaciens strain 30-48, control take-all of wheat
(Weller and Cook, 1983; Brisbane and Rovira, 1988).
Trichoderma and Gliocladium are closely related fungal biocontrol agents.
Each one produces antimicrobial compounds and suppresses disease by
diverse mechanisms, including the production of the structurally complex
antibiotics gliovirin and gliotoxin (Howell et al., 1993). Mutants with
increased or decreased antibiotic production show a corresponding effect on
biocontrol (Howell and Stipanovic, 1983; Handelsman and Stabb, 1996).
Hyperparasitism
Hyperparasitism is parasitism on another parasite. The mycelium and
resting spores (oospores), hyphae or sclerotia of several pathogenic soil
fungi such as Pythium, Phytophthora, Verticillium, Rhizoctonia, Sclerotinia and
Botrytis (Fig. 12.1) are invaded and parasitized (mycoparasitism) or are lysed
(mycolysis) by several non-pathogenic microbes.
Fig. 12.1.
cinerea.
Microscopic photography ( 400) of Trichoderma saturnisporum catching Botrytis
249
Mycoparasitism is parasitism of a pathogenic fungus by another fungus.

These events require specic interactions between the parasite and fungal
host. The process involves direct contact between the fungi, resulting in death
of the plant pathogen and nutrient absorption by the parasite. Trichoderma
spp. parasitize fungal plant pathogens. The parasite extends hyphal branches
toward the target host, coils around and attaches to it with appressorium-like
bodies, and punctures its mycelium (Chet et al., 1981; Goldman et al., 1994).
Mycoparasites produce cell-wall-degrading enzymes, which allow them to
bore holes into other fungi and extract nutrients for their own growth. But
many so-called mycoparasites also produce antibiotics, which may rst
weaken the fungi they parasitize. So the digestion of host cell walls is accomplished by a battery of excreted enzymes, including proteases, chitinases and
glucanases. These enzymes often have antifungal activity individually and
are synergistic in mixtures or with antibiotics (Di Pietro et al., 1993; Lorito
et al., 1993, 1994; Handelsman and Stabb, 1996). By manipulating their activity through the construction of overproducing mutants, enzyme-negative
mutants or even transgenic plants expressing the enzyme, a role for their
production in biocontrol has been implied (Whipps, 2001).
The best documented examples of hyperparasitism involve the mycoparasitic Trichoderma spp. on R. solani. Highly competitive as a saprophyte,
R. solani, can utilize cellulose and colonize fresh bark but cannot colonize the
low-in-cellulose mature bark compost. However, isolates of Trichoderma that
function as biological agents for R. solani are capable of colonizing mature
compost. Biological control does not occur in fresh, undecomposted organic
matter because both fungi grow as saprophytes and R. solani remains capable
of causing disease. In mature compost, on the other hand, sclerotia of R. solani
are killed by the hyperparasites and biological control prevails (Hoitink and
Fahy, 1986; Chung and Hoitink, 1990). One way to ensure specic suppression is to inoculate the compost with the appropriate benecial microorganisms, fungus like Trichoderma sp., Talaromyces flavus, non-pathogenic Fusarium
oxysporum, or bacterial like Bacillus subtilis and Streptomyces griseoviride,
which leads to increased levels of disease suppressiveness.
Induced resistance
The natural resistance of plants to pathogens is based on the combined effects
of preformed barriers and induced mechanisms. In both cases, plants use
physical and antimicrobial defences against the invaders. In contrast to constitutive resistance, induced resistance relies on recognition of an invader
and subsequent signal transduction events leading to the activation of
defences (Mauch-Mani and Mtraux, 1998). Plants possess active defence
mechanisms against pathogen attack; some biotic and abiotic stimuli increase
their tolerance to infection by a pathogen, by activation of these active defence
mechanisms. This phenomenon is known as induced resistance. Induced
resistance was dened by Kloepper et al. (1992) as the process of active resistance dependent on the host plants physical or chemical barriers, activated
250
M. Santos et al.
by biotic or abiotic agents. Induced defence responses are regulated by a

network of interconnecting signal transduction pathways in which the
hormonal signals salicylic acid (SA), jasmonic acid (JA) and ethylene (ET)
play a major role (Pieterse and Van Loon, 1999; Glazebrook, 2001), and other
hormones such as brassinosteroids and abscisic acid can also be involved
(Nakashita et al., 2003; Ton and Mauch-Mani, 2004). The phenotypic effects of
activated defences are quite similar when the stimuli are biotic or abiotic
agents, but the biochemical and mechanistic changes appear to be subtly
different (Van Loon et al., 1998; Whipps, 2001). This has resulted in the term
induced systemic resistance (ISR) for bacterially induced resistance and
systemic acquired resistance (SAR) for that induced by abiotic agents or
microorganisms that cause localized damage (Pieterse et al., 1996).
SAR confers long-lasting protection against a broad spectrum of microorganisms. SAR requires the signal molecule SA and is associated with the
accumulation of pathogenesis-related (PR) proteins such as chitinase, -1-3glucanases or proteinase inhibitor, which are thought to contribute to resistance (Durrant and Dong, 2004). One of the major differences between SAR
and ISR is that PR proteins are not universally associated with bacterially
induced resistance (ISR) (Hofand et al., 1995) and SA is not always involved
in the expression of ISR.
Several reports suggest that compost and compost-amended soil may
alter the resistance of a plant to disease. This was observed in airborne diseases such as powdery mildew of wheat and barley (Trnkner, 1992), early
blight and bacterial spot of tomato (Lycopersicum esculentum) (Roe et al., 1993)
or Anthracnose and Pythium root rot in cucumber (Cucumis sativus) (Zhang
et al., 1996). A select few benecial microorganisms of compost can induce
mechanisms of ISR. These microorganisms activate biochemical pathways in
plants leading to ISR to root as well as some foliar diseases. This mechanism
helps explain the often heard statement that plants raised on healthy organic
soils are more able to resist diseases (Zhang et al., 1998). Plants produced in compost-amended mixes harbouring biocontrol agents that induce systemic resistance have higher concentrations of proteins related to host defence mechanisms
(Zhang et al., 1998). The activation of plant defence produces changes in cellwall composition, de novo production of PR proteins such as chitinases and
glucanases, and synthesis of phytoalexins, although further defensive compounds are likely to exist but remain to be identied (Heil and Bostock, 2002).
Zhang et al. (1996, 1998) showed that composted pine-bark-amended
potting mix provide Pythium root rot and Anthracnose control in cucumber by
inducing systemic resistance (reported as SAR) utilizing split-root techniques. Experiments showed that when only some of the roots of a plant are
in compost-amended soil, while the other roots are in diseased soil, the entire
plant can still acquire resistance to the disease. On those plants, the systemic
protection induced by compost was accompanied by increased peroxidase
activity in leaf tissue. Acid peroxidase was previous reported as a putative
molecular marker of SAR in cucumber.
ISR plays a role in the suppression of plant pathogens that colonize aerial
and soil parts (Pharand et al., 2002). Krause et al. (2003) demonstrated that
251
less than 2% of 80 different batches of compost tested induced systemic resistance in radish (Raphanus sativus) against bacterial leaf spot. The effect was
due to the activity of specic biocontrol agents in the batches of compost that
suppressed bacterial leaf spot. They identied Trichoderma hamatum 382
(Bonord.) Bainer (T382) as the most active inducer of ISR in radish (Khan et al.,
2003). Another species of the genera Trichoderma (Trichoderma harzianum Rifai
T-203) was reported as an inducer of ISR in pepper (Capsicum annum) seedlings
against Phytophthora capsici when the seeds were previously treated with this
biological agent (Ahmed et al., 2000). Khan et al. (2003) report how Phytophthora
root and crown rot of cucumber caused by P. capsici was suppressed signicantly in cucumber transplants produced in a composted cow-manureamended mix compared with those in a dark-sphagnum-peat mix. In split-root
bioassays, Trichoderma hamatum 382 (T382) inoculated into the compostamended potting mix signicantly reduced the severity of Phytophthora root
and crown rot of cucumber caused by P. capsici on paired roots in the peat
mix. This effect did not differ signicantly from that provided by a drench
with benzothiadiazole (BTH) or mefenoxam (Subdue MAXX).
12.3 Suppression of Plant Pathogens by Compost Tea

The disease-suppressive characteristic of organic tea was reported as early as
1973 by Hunt et al. immobilizing the sting nematode (Belonolaimus longicaudatus). Compost tea or compost water extract can be dened as a watery
extract of compost produced through a deliberate process. The goal is to
enhance populations of benecial microbes that can then exert a biological
control over pathogens. The terminology used is not clear because there are
several different methods used for making compost teas, those where tea is
aerated during the production of the tea, and where no aeration is used and
compost is just passively steeped with little agitation. The review of Scheuerell
and Mahaffee (2002a) claried the numerous terms that have been used to
describe composts fermentation; many are synonymous or easy confused
with other concepts. Terms used include compost tea, aerated compost tea,
organic tea, compost extracts, watery fermented compost extract, amended
extracts, steepages and slurries. Compost tea is produced by mixing compost
with water and culturing for a dened period, either actively aerating (aerated compost tea; ACT) or not (non-aerated compost tea; NCT) and with or
without additives that are intended to increase microbial population densities during production (Scheuerell and Mahaffee, 2002a). These teas have
been shown to act as a natural fungicide; they contain populations of various
biofungicidal microbes and organic chelators Teas can be used as a foliar
spray to inhibit late blight caused by Phytophthora infestans on tomatoes
and potatoes, the suppressive effect of organic teas are of a living microbial
nature and the sterilized or micron-ltered tea had little ability to impact on
pathogens (Weltzien and Ketterer, 1986; Weltzien, 1989).
There are several reports on the control of plant pathogens or plant diseases with organic teas including airborne and soilborne diseases; compost
252
M. Santos et al.
teas coat plant surfaces (foliar application) or roots (liquid drench application) with living microorganisms and provide food for benecial microbes.
Plasmopara viticola or downy mildew of grape leaves (Weltzien and Ketterer,
1986), Botrytis cinerea or grey mould (Elad and Shtienberg, 1994; Dinez, 2005;
Kon et al., 2010), Phytophthora cinnamomi (Hoitink et al., 1977), Fusarium
oxysporum f.sp pisi or Fusarium wilt of peas (Khalifa, 1965), Fusarium oxysporum f.sp cucumerinum or Fusarium wilt of cucumber (Ma et al., 1999) Pythium
ultimum or damping-off in pea (Pisum sativum) (Trnkner, 1992).
Multiple modes of activity are involved in suppressing plant disease
with NCT, whereas no studies have determined the mechanism involved
with ACT (Scheuerell and Mahaffee, 2002a). The microbes in compost teas
can suppress diseases in several ways: induced resistance, antibiosis and
competition (Brinton, 1995; Scheuerell and Mahaffee, 2002a), and direct
destruction of pathogens structures (Ma et al., 2001).
The microbiotic of NCT (Weltzien, 1991) and ACT (Ingham, 2003) had
been described as being dominated by bacteria. It is important to know how
the manipulation of the compost tea production process enriches and/or
selects for individual microbe populations. Scheuerell and Mahaffee (2002b)
studied the use of ACT and NCT produced with and without nutrient additives, to drench peat-perlite growing media that was inoculated with
Pythium ultimum and planted with cucumber seeds. They used different
nutrient additives, fungal nutrients (soluble kelp, humic acids and rock
dust) and bacterial nutrients (molasses-based nutrients solution). The most
consistent compost tea formula for suppression damping-off in cucumber
was ACT produced with the fungal nutrients, whereas the disease was not
suppressed with ACT produced with the bacterial nutrients and without
nutrients.
12.4 Grape Marc Compost

Grape marc is the waste from wine production; once the juice has been
extracted, the skin 7% (w/w), stalks 5% (w/w) and seeds 4% (w/w) are all
redundant. In total, 1520% of wine production is waste, comprising thousands of tonnes. The amount of marc that is generated from fruit is known to
vary for a number of reasons, including whether the grapes have been irrigated and the type of equipment used to press the grapes (Jordan, 2002).
Spanish production of grapes for wine production is near 5 million tonnes;
up to 700,000 tonnes of marc is estimated to have been generated throughout
Spain. The marc, if not treated effectively, can cause a number of environmental hazards ranging from surface and groundwater pollution to foul
smells. In the European Union all by-products of wine production (grape
marc and wine lees) are obliged to be distilled; the legal basis for distillation
measures in the EU is given in chapter II of title III, Art.27 of regulation (EC)
N1493/1999. In European countries grape marc is rst distilled to recover
alcohol, followed by washing for tartrate recovery, then seed separation and
nally the remainder is either burnt for energy recovery or composted along
253
with sludge from distillation and tartrate recovery to produce fertilizers for
agricultural use (Johnston, 2001).
In general, grape-marc compost (GMC) has low nutrient status and conductivity; this compost has low water-holding capacities. It has a high content
of lignin and cellulose and a low content of water-soluble carbohydrates.
There are a few reports about the disease-suppressive effects of GMC.
Oka and Yermiyahu (2002) tested in pot and in vitro experiments the suppressive effects of GMC, on the root-knot nematode Meloidogyne javanica. Very
few root-galls were found on tomato roots grown in soil containing 50%
grape marc compost. Significant reductions in galling index were also found
in tomato plants grown in soils containing smaller concentrations of this
compost. The water extract of GMC showed weak nematicidal activity to the
juveniles and eggs.
GMC and its water extracts have been reported to suppress fungal diseases such as B. cinerea on tomato and pepper (Elad and Shtienberg, 1994).
GMC suppressed the soilborne disease caused by R. solani and S. rolfsii
(Gorodecki and Hadar, 1990; Hadar and Gorodecki, 1991). Hadar and
Gorodecki (1991) found an inhibitory effect of GMC on sclerotial germination and viability, and associated this effect with high numbers of Penicillium
isolated from sclerotia. Penicillium and Aspergillus spp. have been reported to
colonize GMC; Trichoderma spp. hyperparasites of R. solani were not recovered from this compost (Gorodecki and Hadar, 1990; Hadar and Gorodecki,
1991). The age of the composted grape marc had a major effect on suppression; immature GMC (3 months of composting) failed to inhibit sclerotial
germination (Hadar et al., 1992). Suppression of Pythium aphanidermatum was
reported on grape marc compost, the beneficial effect of the compost was
negated when the medium was autoclaved, and restored when compost that
had not been autoclaved was mixed with the sterile one (Hadar et al., 1992).
Compost can provide natural biological control of diseases of roots as
well as the foliage of plants. Its water extracts (compost tea) has been proposed as a substitute for synthetic fungicides (Zhang et al., 1998). Most of the
papers published on the control of pathogens by means of compost tea have
studied pathogens from the aerial part of the plants, the number of trials that
use NTC being higher. Research into the control of soilborne pathogens by
means of compost tea has been lower, although this practice is common in
ecological agriculture (Scheuerell and Mahaffee, 2002a).
Our research focuses on soilborne disease suppression by GMC. The
grape marc compost was produced in the University of Sevilla. It was composted in 40 m3 windrows and turned each week. Composting took 5 months.
During composting, the grape marc pile was fertilized with ammonium
nitrate, superphosphate, iron sulfate and magnesium sulfate. Microbiological analysis of our grape marc was performed using the dilution plate technique (Wakelin et al., 1998) on different agar growth media: water agar (WA)
pH11 (actynomicetes), tryptose soy agar (TSA) 1/10 (bacteria), glucose peptone medium (GP) (yeast) and malt extract agar (MEA) (fungi). All the plates
were incubated at 25C, and WA and TSA plates were also incubated at 40C,
for detection of thermophylic bacteria and actynomicetes. Two analyses of
254
M. Santos et al.
Thermophilic
actinomycete
13 %
Fungi 5%
Yeast 2%
Thermophilic
actinomycete
25%
Fungi 3%
Yeast 1%
Mesophilic
bacteria 30%
Mesophilic
bacteria 31%
Mesophilic
actinomycete
24%
Mesophilic
actinomycete
9%
Thermophilic
bacteria 25%
Thermophilic
bacteria 32%
Fig. 12.2. Distribution of the different morphologies found in the first (left) and second (right)
analysis of grape marc compost microflora.
the same compost were done, 6 months apart. Microbiological analysis of

GMC showed a high number of microbial morphologies in the compost. In
the rst analysis, 192 different morphologies were found, and 240 in the second, 6 months later. Most of the morphologies present in the rst and second
analyses were bacteria, with the average percentages being 31% mesophylic
bacteria, 28% thermophylic, 16% mesophylic actinomycetes and 20% thermophylic actinomycetes, and only a few moulds 4% and yeasts 1%; the
distribution of morphologies found in both assays is shown in Fig. 12.2.
Most microorganisms, both aerobic and anaerobic, respond to conditions
that restrict iron ions due to the production of siderophores, whereby ions
are kidnapped or assayed thus preventing their availability. Siderophores
can act as growth factors and some as powerful antibiotics (Neilands, 1981).
Many studies have described suppressiveness in agricultural soil due to siderophores. Thus, the addition of different species of Pseudomonas to a conductive soil infected with Fusarium oxysporum f. sp. lini makes this soil
suppressive, preventing the development of the disease. The addition of
Fe-EDTA reverses this situation (Kloepper et al., 1980). This principle also
applies to Gaeumannomyces graminis var. tritici, and several species of Pythium
(Becker and Cook, 1984; Weller et al., 1986).
The presence of siderophores produced by the microorganisms present
in the grape marc aerated compost tea and their involvement in the development of eight phytopathogenic fungi in the soil and one mycopathogenic
fungus was studied.
The results obtained from the in vitro analysis of the inhibiting effect of
ACT on fungal development are shown in Fig. 12.3. The effect of the incubation time of ACT on the efciency of the in vitro fungal suppression of the
nine fungi tested highlighted an increase in inhibition percentages as the
time of incubation of the compost increased (1, 7 or 14 days) being 80100%
for extract F (Filtered), in extractions after 1 and 7 days, and 100% after
255
14 days, for all the cases examined. In the case of extract C (microltered), the
results vary depending on the fungus, with the same inhibition tendency
percentage increasing as the incubation time and the extract concentration
added to the medium increase, reaching 100% inhibition after 14 days of trial
for most of the fungi, except for the two races of Fusarium oxysporum f. sp.
lycopersici (Fig. 12.3). From the results obtained, we need to highlight the
abrupt change in inhibition percentages for the fungi R. solani and Pythium
aphanidermatum, using the microltered tea after 7 and 14 days incubation.
This effect is the result of measuring the radial growth of the fungus, but it
1 day ACT
1 week ACT
2 weeks ACT
120
Phytophthora parasitica (8)
% Inhibition
100
80
60
40
20
0
Fa8 Fb8 Fc8 Ca8 Cb8 Cc8 Ea8 Eb8 Ec8 Fa8 Fb8 Fc8 Ca8 Cb8 Cc8 Ea8 Eb8 Ec8 Fa8 Fb8 Fc8 Ca8 Cb8 Cc8 Ea8 Eb8 Ec8
120
+ FeCl3
100
% Inhibition
80
60
40
20
0
Fa8 Fb8 Fc8 Ca8 Cb8 Cc8 Ea8 Eb8 Ec8 Fa8 Fb8 Fc8 Ca8 Cb8 Cc8 Ea8 Eb8 Ec8 Fa8 Fb8 Fc8 Ca8 Cb8 Cc8 Ea8 Eb8 Ec8
*Vertical T bars represent standard errors
Fig. 12.3.
Treatment
In vitro analysis of the inhibiting effect of ACT on fungal development.
256
M. Santos et al.
does not reect the effect on the density of the mycelium, which can only be
observed under a microscope with a clear inhibition by the compost extracts,
although this cannot be expressed in the graph.
The sterilization of compost water extracts annuls the suppressing effect
on fungal growth shown by non-thermally treated extracts, except for
Verticillium dahliae and V. fungicola, for which inhibition values reach 60%,
in compost tea incubated for 1 day at concentration levels of 10 and 15%.
The detection of siderophores on GMC tea, which can affect the development of the fungal mycelium, occurred equally in the extract obtained simply by ltering (F), in those that were microltered (C ) and in those that were
sterilized (E), with the addition of FeCl3 to the medium. In these, the biological component has not been eliminated, so besides the potential inhibition
that siderophores can cause, we must bear in mind other antagonistic effects
such as competition for nutrients, space, and so on.
These results suggest that the microorganisms present in GMC produce
siderophores, which grow outside the cells and kidnap iron, stopping its
availability, thus preventing the in vitro development of the phytopathogens
studied.
This study conrms the in vitro inhibition of the growth of eight pathogens and one mycopathogen, as well as the important role of siderophores in
this suppression. In our previous studies, the inhibiting power of GMC had
been veried against the nine fungi tested. As can be expected, the suppressiveness shown by GMC extracts is a combination of various factors, such as
competition for nutrients, antibiosis, and production of lytic enzymes outside the cells and of low molecular weight molecules that are capable of
degrading the fungus wall.
The difference between the use of soil-applied composts and watery
compost extracts perhaps is best summarized as that the teas give immediate
but very short-term control of surface-spreading pathogens, while soil compost acts more slowly over a longer period of time and requires much larger
amounts (York and Brinton, 1996).
12.5 Conclusion
Diseases that have been shown to be effectively suppressed by compost use
include those caused by Fusarium, Phytophthora, Pythium and R. solani. Several reports suggest that compost and compost-amended soil may alter the
resistance of plants to disease. The microbes in compost teas can suppress
diseases in several ways: induced resistance, antibiosis and competition, and
direct destruction of pathogens structures. The effect is due to the activity of
specic biocontrol agents in the batches of compost that suppress the disease.
There are several reports on the control of plant pathogens or plant diseases
with organic teas including airborne and soilborne diseases. GMC and its
water extracts have been reported to suppress fungal diseases. Compost can
provide natural biological control of diseases of roots as well as the foliage of
plants. Its water extracts (compost tea) have been proposed as substitutes for
257
synthetic fungicides. There are several mechanisms involved in the suppression, including competition among the microbial population for available
carbon or nitrogen, iron competition, mycoparasitism, production of inhibitors
or hydrolytic enzymes by microorganisms and interactions with some saprophytes. The microorganisms present in GMC produce siderophores, which grow
outside the cells and kidnap iron, stopping its availability, thus preventing the
development of the phytopathogens. The knowledge of mechanisms for biological control through the action of compost or its water extracts (compost tea)
is necessary in order to increase the efciency of the suppressing power.
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13
Biotechnology: a Tool for

Natural Product Synthesis
SANATH HETTIARACHI
Department of Botany, University of Ruhuna, Matara, Sri Lanka
Abstract
Molecular biology is arguably the fastest growing eld in all biological sciences. New
techniques are discovered and they soon nd applications. The search for natural
products has a long history, as illustrated by the dependence of traditional medicine
on botanicals. With the nding that microorganisms can produce useful products
such as penicillin, scientists took the challenge to explore the microbial world for new
natural products. Although the search continues with increasing intensity, nding
new and more useful products would not have matched with the effort without the
support of biotechnology. Although the most powerful approach is genetic manipulation, other techniques such as mutagenesis, breeding and protoplast fusion and the
relatively old biotechnology of plant tissue culture are very useful. These also include
even more simple approaches such as optimizing culture conditions and design of
fermenters. The combination of technologies together with innovative ideas has
already increased the production level of already existing natural products and
expanded the diversity of products obtainable from biological sources. In addition to
harvesting products from living organisms either in the wild or in cultivation, the
developments in metagenomics have also paved the way to harness the bioproductforming ability of unculturable microorganisms.
13.1 Introduction
By perusing the various denitions given by different authorities and in
dictionaries, it becomes apparent that natural products are predominantly
chemical compounds of biological origin and are extracted from plants and
animals that produce them during secondary metabolism. None the less,
naturally occurring mineral compounds may also be categorized as natural
products. Among the myriad of natural compounds, many have pharmaceutical and other similar applications such as in pest and disease control in
agriculture. Furthermore, natural antioxidants, bioavours, biopreservatives,
(ed. N.K. Dubey)
263
264
S. Hettiarachi
natural colourings, fragrances and microbial polysaccharides are becoming

the choice to replace artificial chemicals in the food and cosmetic industries.
Several contemporary approaches have widened the usefulness of natural
compounds. For example, a new move employing rhizosphere metabolamics
for bioremediation of polychlorinated biphenyls is suggested (Narasimhan
et al., 2003), where plants exuding high levels of phenylpropanoids have
enhanced rhizosphere populations capable of degrading pollutants.
The study of the chemistry and extraction of natural products is so
important that an entire discipline of chemistry is termed natural product
chemistry. Many natural products have quite complex chemical structures,
including stereochemistry, and hence their chemical synthesis is difficult. Yet
some simpler compounds of natural origin are made by total synthesis and
these are also treated as natural compounds. Scientists are also keen to elucidate the biochemical pathways of natural product formation. This information will lead to the development of technologies for the overproduction of
already existing natural products as well as producing entirely new products
through biological systems.
Plants and animals have coexisted with their pests, pathogens,
grazers/predators and other competitors throughout evolution. In order for
them to succeed in such a hostile atmosphere, coevolution has gifted the
organisms with defence mechanisms of different natures, chemical defence
being one of them. Therefore living beings are natural factories of chemical
warfare. Scientific investigations guided by traditional knowledge have led to
the identification of useful natural compounds for the benefit of humankind
in medicine, veterinary medicine and in agriculture. Members of all classes of
living beings from bacteria to higher plants and animals have been identified
as sources of natural products. According to the accepted definitions of biotechnology, exploiting these organisms either from the wild or by cultivation
for the production and extraction of products comes under biotechnology.
This exploitation has already threatened some valuable plants and animals
with local or even global extinction due to over-exploitation, for example for
medicinal uses (Kala, 2005; Maundu et al., 2006). The use of new biotechnological tools would certainly ease the pressure on natural populations.
Although biotechnology plays an important role in non-biological
natural products, these are not included in this discussion.
13.2 Biotechnology in Genetic Diversity Revelation and

Conservation
It is well known that different varieties / races of the same species have different chemical properties and hence their level of importance as sources of
natural products may be different. Until recent times, the taxonomy and
identification of plants, animals and microorganisms were based on morphological (vegetative and reproductive) and anatomical characters. However,
these features usually cannot differentiate between two closely related
individuals and hence cannot identify those individuals with the same
Biotechnology and Natural Product Synthesis
265
morphology, but differences in their genetic makeup. Molecular biological

tools, based on DNA, protein or secondary metabolites, provide additional
taxonomic markers to supplement information gathered through classical
taxonomical methods for further characterization. This enables the identication of variants not only at species or subspecies levels, but tracing down
to variations between two individuals. Such biotechnological approaches
thus enable the identication of more useful genotypes for exploitation in
natural product synthesis. Identication of more useful genotypes is obviously very useful in the conservation of genetic diversity. Systematic studies
can indicate which genomes to search, sample and study for useful products.
Systematics also provides answers to questions relating to the evolution of
chemical and physical structures and their synthesis or ontogeny.
The biotechnological tools available for systematics are protein proles,
polysaccharides, plasmids, DNADNA hybridization, various PCR-based
techniques and DNA sequencing and alignment. With the assistance of bioinformatics, the molecular data are processed and phylogenetic relationships are generated. This facilitates the search for a better performer within
producers of a known product or the search for new products, by narrowing
down the scope of the search. Certain traits have evolved only once and
therefore will only be present within a single clade or very closely related
clades, rather than being present randomly across diverse organisms. For an
example in bioproducts, taxol is known to be present in the yew family,
Taxaceae. Therefore if one is interested in discovering other organisms
producing taxol, the most logical approach would be to start the quest for
taxol and other taxanes in plants closely related to Taxaceae family. The
other sister family most closely related is Podocarpaceae and taxane has
been found in Podocarpus gracilior for the rst time outside Taxaceae (Stahlhut
et al., 1999). The story of mustard oil or mustard glucosinolates is a
different one. With the exception of the genus Drypetes (a member of
Malpighiales), all the other mustard oil producers are in families belonging
to one ordinal clade, Brasicales. As order Brasicales and genus Drypetes are
phylogentically distant, one can assume that mustard oil biosynthesis
evolved twice (Rodman et al., 1993). This is conrmed by the presence of
two different biosynthetic pathways. Therefore, although the nal product
is the same, the evolution of the biosynthetic pathway is different. This
information is extremely important in the search for alternative pathways
for the synthesis of natural products (i.e. one has to search in distantly
related clades).
Molecular tools are extremely sensitive in revealing minor differences in
closely related genotypes within a species. As discussed elsewhere in this
chapter, identifying genotypes of interest in bioproduct formation is particularly important for exploitation, but not restricted to that. This is equally or
more important in conservation strategies. A genotype once lost shall not be
able to be reconstituted. Exploring possibilities of making use of other biotechnological tools such as plant-cell and organ culture also help preserve
the plants in the eld. In addition, micropropagation using tissue culture
techniques is useful in this context.
266
S. Hettiarachi
Recombinant gene technology in natural products

Recombinant gene technology is perhaps the most wonderful tool a biotechnologist ever had. Conventional breeding of plants and animals can
bring the genes of two different but closely related individuals together
through the fusion of two gametes in sexual reproduction. The nuclear
DNA of eukaryotic organisms is organized into chromosomes and the
incompatibility in chromosome numbers and chromosome morphology is
the main barrier for bringing genes together from different parents to produce a hybrid. However, todays technology is such that the identication
of DNA controlling a particular trait, isolation of that particular DNA, in
vitro modication of that DNA, inserting the modied DNA into a desired
organism to express the trait in the new organism, have become possible
in an appropriately equipped laboratory. This is a straightforward
approach when the nal product is a peptide because engineering the base
sequence in an open reading frame under proper control units is sufcient.
It is a different matter when it comes to more complex molecules, such as
secondary metabolites, which are products of a series of biochemical reactions each mediated by a specic enzyme. The maturation of the molecule
may also involve chemical modications within the environment of the
producer cell. In order to make breakthroughs in the production of natural
products, particularly those that are not peptides, using biotechnology, an
understanding of the regulation of the secondary metabolite pathways
involved at the levels of products, enzymes and genes, including aspects
of transport and compartmentation in eukaryotic cells, are of paramount
importance.
Antibiotics are natural products of microbial origin. However, these are
naturally produced only in minute quantities that are not even detectable in
nature. Overproducing individuals are present in nature and screening large
populations can always yield better producers. Appropriate modications to
culture medium and culture conditions also result in higher production.
Generating mutants articially and screening increase the chances of nding
such overproducers. A big leap in this direction was possible due to the new
biotechnological tools such as genetic engineering. This possibility was
already under consideration for more than two decades (Chater, 1990). There
had been a slow progress as antibiotic synthesis occurs as a combination of
the action of several genes.
While antibiotics are extremely useful in medicine and veterinary medicine, the development of resistance by the target pathogens is an increasing
problem. Therefore heavy investments are made into research leading to the
discovery of new antibiotics. However, the natural antibiotics belong to a few
families and it may not be possible to isolate new and therapeutically
important antibiotics belonging to new families. Once resistance is developed it is possible this resistance extends easily for that particular family of
antibiotics. Therefore the modication of already known antibiotics to generate semisynthetic antibiotics, either chemically or enzymatically, may be
more appealing.
267
As microorganisms can be cultivated in fermenters much more easily than

plant cells and they are more amenable to genetic alterations via mutagenesis
and genetic engineering, they have become factories of products originally
produced by other organisms. This is a case of producing natural products in
an unnatural producer. The development of biotechnological approaches for
enhanced production of such natural products is hindered by the complexity
involving unknown regulatory genes and enzymes (Oksman-Caldentey and
Inz, 2004). In this context gene technology can help in overexpression, constitutive expression, breaking tissue specicity and so on, rather than expression
in a different organism. This is possible when control mechanisms of expression are identied. It may be traced down to a particular molecule which
either keeps the entire process switched off (suppressor) or required for switching on the system (inducer). Once the suppressor is knocked off or the inducer
is constitutively expressed without tissue specicity, complex metabolites
may be produced with a higher yield in the producer itself. One major limitation in plant cell suspension cultures is the tissue specicity of the metabolites.
Therefore breaking tissue specicity should be given more attention in order
to exploit the full potential of this technology.
Although animal cells can also be genetically manipulated, other problems associated with animal-cell and tissue culture are still limiting the formation of new products in animals. Nevertheless pharming, a technology
still in its infancy, to produce pharmaceuticals in farm animals is a focal point
in natural product formation with animals. What is basically done in pharming is to transform animals with genes coding for useful bioproducts, and
allow expression and harvesting, for example by milking. Pharming has
yielded drugs such as growth hormone, blood components such as haemoglobin, and large quantities of certain proteins needed for research. Although
pharming is a novel production platform, it is yet to break through many
hurdles such as regulatory barriers, consumer acceptance, environmental
issues and ethical concerns. The term pharming is not restricted to production through animals, but also includes pharmaceutical production in genetically modied plants. Ramessar et al. (2008) reviewed the progress made in
maize plants, the rst plants used in pharming, as a platform for effective
and safe molecular pharming.
Human insulin is the rst mammalian protein expressed in bacteria and
later became a huge contributor to the pharmaceutical industry. All human
insulin preparations available in pharmacies are of microbial origin, based
on two technologies using Escherichia coli as the expression system and different technology using yeasts to secrete the precursor of insulin (Petrides et al.,
1995). Another industrial success is the production of rennet or chymosin
enzyme, which is important in making cheese. The original source was the
stomach of calves. As the amount extracted from a single stomach is very
low, production could not meet demand and hence the cost was very high.
This was rst cloned and expressed in E. coli (Nishimori et al., 1982). Recombinant chymosin is now produced by two fungi, namely Kluyveromyces lactis
and Aspergillus niger, in addition to E. coli, by different manufacturers
(Neelakanthan et al., 1999).
268
S. Hettiarachi
A comprehensive list of plant proteins that have been expressed in E. coli

and in two yeasts, namely Saccharomyces cerevisiae and Pichia pastoris, is provided by Yesilirmak and Sayers (2009).
Whole plants can serve as factories of desired products not only those
that are present naturally, but also products that are engineered into them.
Edible vaccine production is one such example. Antigenic components of
pathogens are expressed in plants, and when eaten they can act as antigens
to trigger antibody production, thereby developing immunity against the
pathogen in question. Neither purication of the antigen nor formulation
into vaccine is necessary. In engineering Hepatitis B edible vaccine into lupin
(Lupinus luteus) and lettuce (Lactuca sativa), the gene coding for a viral envelope surface protein has been transferred from the viral genome to the plant
genome with necessary modications for expression in plants. When tissues
of transgenic plants were fed to mice and human, they developed Hepatitis
B virus specic antibodies to the plant-derived protein (Kapusta et al., 1999).
A fusion protein of components from two naturally occurring proteins,
cholera toxin B subunit and insulin, has been expressed in potato tubers. This
protein, when ingested with the potato tuber, can enter into the gut-associated
lymphoid tissues, as it is linked to the carboxy-terminal sequence of the
cholera toxin B subunit. When mice, having a diabetic mellitus condition due
to insulin autoimmune disease, were fed with transgenic potato, a substantial reduction in pancreatic islet inammation and a delay in the progression
of clinical diabetes were observed (Arakawa et al., 1998).
Nevertheless, the production of secondary metabolites depends mainly
on pathway engineering / metabolic engineering, rather than engineering
the gene responsible for the nal product. Due to the complex nature and
specicity of reactions at strain level rather than at species or broader taxonomic level, the progress has been slow. Transfer of the putrescine:SAM
N-methyltransferase (PMT) gene from Nicotiana tabacum under the control
of the CaM V 35S promoter into a Duboisia hybrid has increased the
N-methylputrescine concentration in hairy root cultures. The increase has
been reported as 24-fold in transgenic versus wild type. The enzyme catalyses the N-methylation of the diamine putrescine to N-methylputrescine,
which is the rst specic precursor of both tropane and pyridine-type alkaloids. Both of these types are present in Duboisia roots, however the concentrations of these alkaloids did not increase in the transgenic hairy root culture,
despite higher levels of the precursor (Moyano et al., 2002). Although the
nal product formation was not enhanced, this is a step forward. Working
along similar lines it was also possible to engineer the enzyme hyoscyamine
6-hydroxylase from Hyoscyamus niger, under the regulation of the CaMV 35S
promoter into a Duboisia hybrid and found one of their transgenic lines produced 74 mg/l scopolamine (Palazna et al., 2003). The enzyme was known
to catalyse two steps in scopolamine biosynthesis.
In the above section the importance of modications, either enzymatic
or chemical, to natural products was mentioned. These unnatural modications to natural products can be done in a more natural manner by
combinatorial alterations by engineering genes relevant for the enzymes. An
269
example of this approach is reported by McDaniel et al. (1999) who engineered erythromycin polyketide synthase to produce a library of over 50
macrolid antibiotics. Kantola et al. (2003) provided an analysis of available
literature on this subject giving details of the gene clusters involved. Hopwood et al. (1985) was the rst group to produce hybrid antibiotics by transferring genes encoding enzymes making a set of antibiotics in one strain to
another. This results in new antibiotics depending on the substrate specicity
of the enzymes. Thus the possibility of nding more efcient antibiotics is
made possible.
Metabolic engineering or combinatorial synthesis of traits from
bacteria to plants has also been successful. For example, a bacterial gene
encoding p-hydroxycinnamoyl-CoA hydratase/lyase (HCHL) has been
expressed in Beta vulgaris. One line was able to accumulate the glucose
ester of p-hydroxybenzoic acid (pHBA) at a rate of 14% of dry weight
(Rahman et al., 2009).
Combinations of different approaches are of course possible and some
trials have already given positive results. The effect of combining metabolic
engineering with providing elicitors in the culture medium has been tested
by Zhang et al. (2009) in avonoid production by hairy root cultures of Glycyrrhiza uralensis. In metabolic engineering, overexpression of chalcone isomerase was achieved by Agrobacterium-mediated transformation with a 150%
increase of total avonoid over the wild type. When PEG8000 and yeast
extract was added to the medium, the engineered line produced over 300%
total avonoids.
Plant tissue and cell culture in natural products

Many higher plants produce economically important organic compounds
such as oils, resins, tannins, natural rubber, gums, waxes, dyes, avours and
fragrances, pharmaceuticals, and pesticides. Plants have been identied since
ancient times as a source of remedies for ailments. Medicinal plants, even
today, are given the highest importance in the pharmaceutical industry,
despite the fact that some of these chemicals, which were rst identied as
botanicals, are synthesized chemically. It is a general rule in nature that if one
has continued enthusiasm, patience and the right approach, one shall encounter a better performer of the function of interest. Natural product formation
is no exception. Screening of natural plant populations has taken place
throughout civilization and will continue. This is now happening in a more
rigorous manner at the level of genetic diversity and identity with the aid of
molecular biology and biotechnology tools. On the other hand, it is also possible to use the biotechnological tools for the generation of new varieties with
more desirable traits, as discussed in the above section with regard to microorganisms. Once a better performer is identied, the next concern would be
how to maintain the genetic constitution so that it would be used for a long
period without genetic variation. Tissue culture is the rst option available
for the clonal propagation of plants within a short period of time in a small
270
S. Hettiarachi
area. Similarly regenerated plants through micropropagation are extremely

important to relieve the pressure on the wild plants exerted by overexploitation. Depending on the tissue specicity of the product, the harvesting may
be destructive as vital parts, such as roots or bark, of the plant may have to
be removed in harvesting, imposing additional heavy pressure on wild
populations.
As mentioned in the review by Chaturavedi et al. (2007), the micropropagation and eld establishment of Dioscorea floribandu in India has been a success
story. They calculate the number of plantlets that could be obtained within a
year starting from a single nodal cutting as 2,560,000 in comparison to a maximum of 10 plants from a single tuber which can be obtained after 3 years of
growth in the eld. Thus there cannot be any doubt about the power of micropropagation, especially for those plants that cannot be rapidly propagated
otherwise. Even with those plants, the advantage of having propagation of
true-to-type plants cannot be underestimated. The report criticizes the progress made in India in the micropropagation of medicinal plants, despite the
fact that experiments in various institutes have come up with protocols ending
up in the eld of a long list of medicinal plants. This fact is mentioned here in
order to highlight the inefciency of technology transfer.
The extraction of natural products from wild or eld-grown plants, however, suffers from many impediments such as low production, and no uniformity of production from place to place, from time to time and from one plant
variety to another. Riker and Hildebrandt (1958) were probably the rst scientists who had vision of the power of plant tissue culture in natural product
synthesis. As tissue and cell cultures can be maintained under desirable culture conditions, once culture media and culture conditions for optimum
growth and optimum production have been worked out, the above problems
can be mitigated. When tissue is collected from cultivated or wild plants,
there is a long waiting time from plantlet formation to product formation
as extraction may be possible only after reaching maturity or some other
status.
Among several other scientists working in the late 1970s and early 1980s,
Berlin (1984) visualized plant tissue culture as a future potential process for
natural product synthesis and stressed that more research was needed for the
elucidation of regulatory controls in biosynthetic pathways. Alfermann and
Petersen (1995) demonstrated the potential of plant tissue and cell cultures
for natural product synthesis by referring to research by many scientists who
were able to produce various metabolites at the laboratory scale.
It has been possible to manipulate biosynthesis through genetic manipulation, and overexpression and expression of new products has been possible
in cultured cells and organs. Organogenesis may be required for generating
certain tissue-specic substances (Rout et al., 2000). The primary purpose of
plant tissue culture was the exploitation of the totipotency of the plant cell
for mass-scale propagation of useful varieties of plants. The plants obtained
are expected to be genetically identical and hence form a clone. However,
variations occur within the clone due to somaclonal variations as a result of
replication errors in mitotic cell divisions. While it seems undesirable, the
271
generation of somaclonal variations is intentionally done for the further

improvement of the selected plant variety. The screening can be done in
much the same way as for microorganisms in Petri dishes rather than in a
greenhouse or a eld. Therefore overproducers and producers of new metabolites can be easily and rapidly selected. The generation of variants can also
be achieved by somatic fusion of two protoplasts of cells from two different
parents. The parents may be closely or distantly related. This can bring
genetic information required, for example, for synthesis of one metabolite
and an enzyme that can further transform it into a more useful product or
products. This section of science is called combinatorial biosynthesis. The
variants made by either method can usually be regenerated into whole plants
or, if desired, can be maintained in cell or organ cultures.
Certain metabolites are tissue specic. Even for metabolites that are not
tissue specic, the higher yield is given in compacted tissues rather than in
loose-cell suspensions. For this, organogenesis is necessary. A very good
solution is available as plant tissues transformed by Agrobacterium rhizogenes
can grow indenitely as roots in appropriate culture media. Once transformed, the cells can grow as hairy roots without the bacterium. When products are formed and secreted or made to secrete into the medium, product
recovery is easy as it is not contaminated by cells. Further, continuous
removal is also possible. While all these facts are fascinating and encouraging, the published literature shows only two plant metabolites that are
produced on a commercial scale. Shikonin is the rst botanical produced on
a commercial scale using plant cell cultures of Lithospermum erythrorhizon.
The specic productivity of shikonin has been increased 25-fold by simultaneous in situ extraction using n-hexadecane added before 15 days and immobilization of cells in algenate beads, in comparison with production in cell
suspension culture (Kim and Chang, 2004).
Immobilized cells have shown to be better producers in general. When
comparing yields of scopadulcic acid B, a diterpene that has antiviral and
anti-tumour activity, production by Scoparia dulcis in suspension culture and
cells immobilized in Luffa sponge, Mathew and Jayachandran (2009) noted
350.57 mg/g of cells by the 19th day by immobilized cells in contrast to 50.85
mg/g of cells after 30 days of incubation in suspension culture. This is a
sevenfold increase with a shorter time of incubation. Continuous removal
of products and inhibitory substances is an added advantage of using
immobilized cells.
The only other compound produced using plant cell culture that has
successfully entered the market is ginseng saponin originating from Panax
ginseng. Further improvements to the yield have been shown in hairy root
cultures fed with specic nitrogen and phosphorous sources (Jeong and
Park, 2006).
Catharanthus roseus is one of the plants that showed success in the production of alkaloids in cell suspension culture. These are indole alkaloids, of
which the bisindole alkaloids vinblastine and vincristine are antineoplastic
medicines and the monoindole alkaloids (ajmalicine and serpentine) are antihypertension drugs. Since the rst results published 30 years ago (Kurz et al.,
272
S. Hettiarachi
1980), continuous efforts have been made experimenting with various cell
suspension culture techniques with modications of culture conditions in
order to maximize the production of desired compounds. The research focusing on improvements on both cell culture and bioreactor aspects is the subject
of the review by Zhao and Verpoorte (2007). Nevertheless, further research is
necessary to bring products of C. roseus to the commercial scale. Berberine, an
isoquinolene alkaloid, has been produced in cell suspension culture of Tinospora cordifolia at a concentration of 5.5 mg g1 dry wt in 24 days (Rao et al.,
2008). By screening eight cell lines, the authors were able to nd one line that
accumulated 13.9 mg g1 dry weight of berberine. This is a 514-fold increase
in product formation compared to that of the intact plant. This is an example
demonstrating the potential of plant cell cultures and somaclonal variation
for the natural product industry. A tenfold increase brings down the cost of
the product in the market by at least the same magnitude.
Cyclotides are small cyclic peptides stabilized by disulde bonds between
six conserved cystine residues. Their biological activities include anti-HIV,
antimicrobial and insecticidal actions, and hence they are important botanicals in biocontrol. In addition, due to their high stability, cyclotides are very
good candidates for the development of drug delivery systems. Drnenburg
et al. (2008) developed techniques to produce cyclotides using callus, suspension culture and hydroponic cultures of Oldenlandia affinis and evaluated them
for Kalata B1 accumulation. In vitro culture produced only up to 15% of Kalata
B1 in comparison with plants grown in hydroponics. Further improvement is
probable by manipulating culture conditions and the same group reported a
higher rate of cell multiplication in a 25-l photobioreactor (Seydel et al., 2009).
They claim that this approach for harvesting Kalata B1 is more protable than
other methods, such as eld cultivation and chemical synthesis.
Cell suspension culture is also useful in the synthesis of volatile oils. By
manipulating culture conditions, Ishikura et al. (1984) obtained a yield of
0.005% to 0.01% of volatile oils of the fresh weight of the cells of Cryptomeria
japonica cell suspension.
The progress of research in the development of hairy root technology for
metabolite production has been reviewed by Guillon et al. in 2006. The delicate nature of the hairy roots is one of the major problems in maintaining
hairy root cultures. Srivastava and Srivastava (2007) reviewed the problems
and different technologies available to overcome them. The basic types of
reactors are either liquid phase or gas phase or even a combination of the two.
The nature, applications, perspectives and scale up are discussed therein.
The hairy root growth, production of the desired substance(s) and secretion may require different culture conditions. The production can be enhanced
by providing the precursors at the right time and the right concentration. The
addition of cadaverine to hairy root cultures of Nicotiana rustica shifted the
alkaloid production in considerable favour of anabasine with a concomitant
diminution of nicotine (Walton et al., 1988), whereas the addition of menthol
or geraniol at 25 mg l1 to hairy root cultures of Anethum graveolens did not
have an impact on the growth and resulted in the transformation of the
compounds to volatile products (Faria et al., 2009). In a recent experiment,
273
Hernandez-Vazquez et al. (2010) were able to increase the production of

centellosides in cell cultures of Centella asiatica by feeding with -amyrin
together with DMSO to assist the penetration of -amyrin in to the cells.
The secretion can be enhanced by manipulating the culture medium
either by increasing the leakiness of tissues or by introducing a trapping
system to trap the secreted metabolite. For example, Rudrappa et al. (2004)
reported an increased recovery of betalaines (red natural pigment) production by hairy roots of B. vulgaris up to 97.2% by using alumina:silica (1:1) in
the culture medium. The use of elicitors is a useful approach to enhance the
productivity as some secondary metabolites are naturally produced in
response to a signal. This has been shown practically by incorporating both
live and autoclaved bacteria in the conversion of hyoscyamine to scopolamine
by hairy root cultures of Scopolia parviflora (Jung et al., 2003).
Podophyllotoxin is a useful botanical extracted from Podophyllum hexandrum for treating general warts. Chattopadhyay et al. (2002) used a 3-l bioreactor for the cell suspension culture of P. hexandrum and successfully
recovered podophyllotoxin. It has been noted that providing coniferin as a
precursor in the culture medium increases the productivity, but this is economically prohibitive. An innovative method to circumvent this problem is
to co-cultivate P. hexandrum cell suspension with hairy roots of Linum flavum,
which produces coniferin. This approach has increased the nal product
formation by 240% (Lin et al., 2003).
A further step in bringing the substrate and enzymes from different
sources has been made possible through combinatorial biosynthesis. Here,
rather than cocultivating the two individual plants in vitro, the genetic traits
controlling the production of one compound, which will be the precursor of
the nal product, and the enzyme required for the transformation are brought
together into one cell. A somatic hybrid of two Solanum species producing
solanidine and solanthrene (S. tuberosum) and tomatidine (S. brevidens) produced all three steroidal glycoalkaloid aglycones. In addition all hybrids also
produced a totally new compound, demissidine (Laurila et al., 1996). These
authors propose a hypothesis to describe this which explains the production
of demissidine by hydrogenating the double bond at position 5 of solanidine
of S. tuberosum by a hydrogenase enzyme of S. brevidens.
13.3 Expanding Natural Product Diversity

In the quest for natural products for the benet of humankind, it can often be
thought that the existing natural product diversity is not sufcient. The generation of mutants is a tool available for increasing natural product diversity.
Random mutagenesis is stimulated by treating with chemical mutagens or
radiation. Although these are useful and have been employed for making
variants and subsequent screening, with the better understanding of DNA
and development of new biotechnological tools, specic, site-directed
mutagenesis is possible. This has reduced the effort in screening a large
number of mutants for the expected outcome.
274
S. Hettiarachi
In an attempt to restore the ability to produce taxol, an anticancer drug,

to an endophytic fungus, Tubercularia sp., mutagenesis and genome shufing
in protoplasts resulted in no restoration, but the production of new metabolites (Wang et al., 2010). One mutant produced three new and one already
known sequiterpenoids, whereas another one made 18 novel compounds
belonging to different classes and 10 already known compounds which were
not present in the wild-type strain.
Altering the culture condition alone can induce the production of different natural products by an individual organism. If the mechanism of control
of a metabolic pathway is understood, then breaking silence in expression
should be simple. Sometimes the presence of another organism in the
neighbourhood may inuence the induction of a pathway leading to a previously unseen product. Therefore mixed fermentation can result in an
increased concentration of already expressed or undetected or unexpressed
metabolites in crude extract, and the production of new analogues of known
metabolites due to combined or extended pathways (Pettit, 2009).
Each and every step of biochemical pathways are supposed be catalysed
by a specic enzyme. Nevertheless, the size of the total genome, as unravelled by genome-sequencing projects, cannot account for the large number of
metabolites present in organisms. Post-transcriptional modications leading
to the formation of isozymes can be partly responsible for the high diversity
of metabolites, but this has now been mainly attributed to the low specicity
of some enzymes, which means that one enzyme can convert several similar
precursors or intermediates into several products causing ramications in
the next step, and nally ending up with several end products. The number
of end products shall be much higher if there was no compartmentalization
of substrates and enzymes within a cell and in different cells. Bringing a natural substrate in one cell in contact with an enzyme from another cell may
result in entirely new natural products. This is possible through breeding of
sexually compatible individuals, or through protoplast fusion, which is possible between rather distant relatives. This technology is known as combinatorial biosynthesis or metabolic engineering or heterologous expression, and
has been applied to microorganisms as well as to plants.
Polyketides are highly diverse natural products, including those important as antibiotics and other pharmaceuticals such as anticancer agents and
immunosuppressors. The most important producers are actinomycetes, but
other bacteria, fungi, plants and marine animals also contain polyketides.
They are formed by the sequential polymerization of carboxylic acids, and as
such carboxylic acid monomers become a source of variation in polyketides.
The diversity has been possible due the modular nature of the polyketide
synthases (PKSs). Once transcribed, the peptides assemble to make the functional enzyme consisting of different number of polypetides, each having
two modules. The large protein adds one monomer at each module to the
growing chain. Mutagenesis in each module is possible and the outcome of
each mutation and combinations of those can cause a great variety of
polyketides (Kosla, 1997). The rst polypeptide in the assembly of the
megasynthase initiates polymerization and is the minimal PKS containing
275
two enzymatic domains (ketosynthase (KS)_malonyl-CoA:ACP transferase

(MAT) didomain) and an acyl-carrier protein (ACP) domain. Zhang et al.
(2008) removed the minimal PKS from Gibberella fujikuroi in two formats and
engineered it in E. coli cyclized polyketide production was observed. In the
fungus cyclization is C2C7, whereas in E. coli cyclization of C9C14 and
C7C12 were predominant. By expressing one of the two minimal PKSs
(PKS_WJ) and downstream processing enzymes in E. coli they were able to
synthesis nonaketide anthraquinone SEK26 in good amounts. This work also
encourages scientists to investigate further the generation of variety through
heterologous expression.
A very good example is the engineering of a new metabolic pathway in
E. coli by a combination of mixing genes and modifying catalytic functions by
in vitro evolution. In this experiment, phytoene desaturases were assembled
by shufing modules from different bacteria and expressing them in E. coli.
The different strains generated were screened for new carotenoid pigments.
One such chimeric enzyme had introduced six, in place of four, double bonds
into phytoene. Another set of chimeric lycopene cyclases had also been made
and engineered into the cells expressing the above novel phytoene, thus
extending that pathway to produce a variety of coloured compounds. One
such combination produced cyclic carotenoid torulene in E. coli. This
experiment reported by Schmidt-Dannert (2000) suggests the unlimited
possibilities of obtaining novel natural products by rational design.
Apart from the usual ribosomal polymerization of amino acids into
peptides, a mechanism of non-ribosomal enzymatic polymerization of amino
acids similar to that of polyketide synthesis is also present in living systems.
For example, the well known hepatotoxin, microsystine produced by Microsystis spp., is formed by a microcystin synthetase complex consisting of
peptide synthetases, polyketide synthases and hybrid enzymes (Dittmann
et al., 2001). Accordingly, heterologous expression in a similar way to PKS is
possible in view of introducing new variants of microsystins in the quest for
novel natural products.
A great proportion of microorganisms cannot be cultivated and hence no
information on their potential for natural product formation is available.
This portion reaches an astounding 90% or 99% of the total populations in
soil. They belong to bacteria and archaea and also viruses. This also indicates
the presence of an extremely large, yet untouched resource. Although the
organisms are unculturable, their DNA can be isolated from environmental
samples and amplied using PCR techniques. The isolation, amplication
and study of DNA from a total community is referred to as metagenomics, or
environmental or community genomics. Once DNA is amplied, purication and sequencing is straightforward. Sequencing helps in seeing what is
present, and at least how close they are to culturable, known members of the
community. On the other hand, a functional approach, in which the DNA is
expressed in surrogate hosts, is also very useful in nding out what they can
do. When analysing clones of recombinant surrogate hosts for new products,
there is a big chance of nding new bioproducts, perhaps some of which may
be in entirely new families of compounds.
276
S. Hettiarachi
Fragments of DNA can be cloned using articial bacterial chromosome

(BAC) vectors. These are designed to carry larger fragments than those produced by PCR techniques. Rondon (2000) used this technique and revealed a
great deal of information on phylogenetic, physical and functional properties
of the metagenome. Some of the clones showed antibacterial and nuclease
activity and the DNA sequences governing these characteristics were found
to be different from those already known. This directly supports the idea that
unculturable organisms provide a source of novel genes. Other functions
observed were the production of amylase and lipase and hemolysis.
13.4 Conclusion
The search for natural products has been rekindled with realization of the
vast biological diversity and genetic diversity that make biological systems
an unlimited resource. Furthermore, the global society is demanding natural
products and rejecting synthetic chemicals for all possible uses, including
food and additives, drugs, cosmetics and so on. Although plants and animals
had been a source of such products from ancient times, the horizons now
have widened and continue to expand, particularly with the development of
modern biotechnological tools. Man was unaware that he was exploiting
microorganisms as they are invisible to the naked eye. Some products become
redundant over time, as is the case for antibiotics for which resistance has
developed in target (and non-target) organisms. Further new diseases
emerge, new lifestyles demand new products, and biotechnology is needed
to satisfy those demands. As new tools in biotechnology have been found,
one can expect that the trend will continue. Exploring and exploiting new
resources will be feasible in the future, as evidenced by metagenomics in
new product formation which harness the biosynthetic capability of
unculturable microorganisms. However, with all of these developments, the
number of bioproducts that have already conquered the market is not encouraging. Therefore, there is a need for more intensive research on optimizing
production of already identied bioproducts, with simultaneous research
efforts on new product formation. It can also be noted that technology transfer is not occurring effectively, because the already available technologies
remain in the laboratories where they were generated without reaching the
industrial fermenters or industrial-scale farms.
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Index
Page numbers in italic indicate gures, those in bold indicate tables.

active components for antifungal
activity 9596
antifungal active methanol
investigations 96
antimicrobial compounds 95
secondary metabolites 95
useful antimicrobial phytochemicals
95
active packaging 3537
advantages of preservative foodpackaging lms 3536
alternative to modied atmosphere
packaging (MAP) 36
innovative concept 35
effect of MAP packaging tested
3637
mustard essential oil 36
oil in liquid phase 37
use of wax-coated paper and board
36
aerated compost tea (ACT) 251, 252
inhibiting effect on fungal
development 255
aatoxin secretion inhibitors 15
aatoxins 2
produced by species in Aspergillus 24
Africa
crop losses 191, 192
forest 195
agriculture, in developing countries 12
agrochemical industry 81
aliphatics 60
allelochemicals 135136
allomones 135
apneumones 135
kairomones 135
synomones 135
allomones 136137
plant metabolites with negative
effect on insect behaviour
136
antifeedants 136137
anti-ovipositants 136
repellents 136
substances meeting requirements
for new botanical
insecticides 137
allyl isothiocyanate (AITC) 10, 10, 3637
stored in plants as a glucosinolate
10
-terpineol 181, 182, 183
-terthienyl 11, 11
ambuic acid 228229, 229
antibiosis 247248
antibiotics
development of resistance to 266
281
282
Index
antibiotics continued
modication 266
natural products of microbial origin
266
antifeedant action mechanism 138141
biological assays 139140, 140
general procedure adopted
140141
feeding deterrents 138
food choice 138
formula to calculate feeding
deterrence 141
leaf discs 139
products can lose primary efciency
141
use of bioassay techniques 139
antifeedant research 137138
antifeedant efciency 138
history of research 137
recent emphasis 137138
studies showing efcacy 137
antifeedant substances 141143
aromatic hydrocarbons 143
aromatic plants 143
Colorado beetle 143, 144
monoterpenes as feeding deterrents
143
no antifeedant efciency in
commercial products 141
Pongamia genus 142
antifeeding activities 142
repellent activities 142143
present study of poyphenolic
substances 143
prospects for products based on
143145
multi-component tactics 145
non-azadirachtin liminoids 145
use in pest management
programmes 144145
antifeedants 12, 136137
deter phytophagous insects 136
prospects for practical use 136137
antifungal active component (plants)
96106
antifungal agents 22
antimicrobial peptides
biopesticidal peptides and
inhibition 112
generation of antimicrobial
peptides 112
antimicrobial peptides from biocontrol

microorganisms 112113,
113114
classied into groups of linear and
cyclic peptides 112113
efcacy 113, 113114
peptide synthesis 112113
antimicrobial peptides from plants
114115
plant defensins
inhibitory activity 114115
range of biological functions 115
antimicrobial products
applied to foods, research in 2734
from plant extracts, current status
7172
Carvone 71
Milsana 71
new products 71
plant essential oils as organic
fungicides 71
new approach to microbial disease
control via SAR 72
antimicrobials of plant origin 92
aromatic compounds 5359, 6061
alkaloids, examples 59, 59
non-phenolic aromatic compounds
53, 54
OH groups absent 53
tetrapyrrole goup 53, 54
phenolic compounds 5559
avonoids 5759, 57, 61
simple phenols all monomeric
5557, 55
aromatic plant compounds 12
Azadirachta indica see neem (Azadirachta
indica)
azadirachtin 78
commercial products 142
considered non-toxic to mammals
78
highly oxidised triterpenoid 7, 7
primary and secondary antifeedant
activities 142
behavioural insect control 13

benzoquinine 70
-Asarone 13, 14
bioactive compounds 218
bioactive substances 44
Index
283
biocontrol agents 206, 210
biodeterioration 9192
biofumigation with Brassica 211215
biological activity and repellency
177178
activity of fatty acids 178, 178
C9C11 acids 178, 179, 185
activity of some edible oils 177, 177
biological diversity 221
biotechnology in genetic diversity
264273
molecular biological tools and
identication of variants
265
minor differences in closely
related genotypes 265
molecular tools and conservation
strategies 265
plant tissue and cell culture 269273
recombinant gene technology
266269
antibiotics 266
edible vaccine production 268
genetic alteration 267
hepatitis B 268
human insulin preparations
267
importance of modications
268269
metabolic engineering 269
more complex molecules 266
peptide as nal product 266
pharming 267
production of rennet
(chymosin) 267
systematics, uses of 265
biotechnological tools available
265
story of mustard oil
biosynthesis 265
botanical pesticides 4
attitudes of resource-poor farmers
in developing countries to
200
now adopting IPM principles
200
willing to pay for the services
200
environmentally safe 510, 1112
replacement by synthetic pesticides
5
botanical products, historical use 45

plant diseases and disease control
methods 45
botanical resistance inducers 153
botanicals
in developing countries 196198
local formulation by peasant
farmers 196
burnt and fumes used 196
methods of formulation
197198
crude water extracts 197,
197
mixed formulations 197,
198
oils 197198, 197
powders 197, 197
use of adjuvants 198
scientic formulations 196
simple to make, easy to use
196
future use 200
alternative to synthetic
pesticides 200
WHO 200
mechanisms of systemic 152154
use in third world countries
193194
versus conventional pesticides
198199
alternative management
198199
brassinosteroids 51, 51, 80, 81
carvone 10, 10
non-toxic botanical insecticide 10
cereals 2733
mycotoxins 27
chemicals, synthetic 3
coevolution and coexistence 264
combinatorial biosynthesis 269, 271, 273
compost tea 242257
aerated compost tea (ACT) 254256
competition among microbial
populations 246251
antibiosis 247248
hyperparasitism 248249
induced resistance 249251
microbiostasis 246247
grape marc compost 252256
284
Index
compost tea continued
microbes in compost teas 256257
suppression of plant pathogens
251252
aerated compost tea (ACT) 251,
252
clarication of terms 251
compost tea described 251
macrobiotic of NCT and ACT
252
non-aerated compost tea 251,
252
reports on control of plant
pathogens/diseases
251252
cotton leaf curl virus disease 149
crop protectants, non-synthetic 47
cyclosporin 226
defensive plant mechanisms 135

deforestation, and botanicals 195196
developing countries
crop protection a necessity 2
destruction of food in storage 2
farmers/researchers seeking
alternatives to synthetic
pesticides 43
food industries rely mainly on
fumigants 175176
formulations of botanicals in 196198
history of natural products and use
by local farming
communities 193194
Nigeria 194
tropical forest botanicals 193,
194
importance 12
insect damage in stored grains 175
use of natural products 48
eco-friendly control measures 24

edible oils 176
edible oils and fatty acids 177,
1778, 178, 179
crude oils against C. maculata
185
no adverse effect on seed
germination 185
mode of action complex 185
elicitors 119, 120122, 206

SAR-inducing compounds 206
endophyte biology
diminishing rainforests a major
problem 236
microbial diversity loss 236
endophytes 220
denition of 219
diversity in 221
fungi and bacteria 219220
metabolic distinction 221
molecular techniques 224
addition of telomeric repeats
224
genes for taxol production 224
plant selection norms and ethics
222223
collection of Rhyncholacis
penicillata 222223
distinct environmental settings
222223
hypotheses governing plant
selection strategy 222
snakevine Kennedia nigriscans
223
taxol and Taxus brevifolia 223
researchers 235
techniques for isolation,
preservation and storage
221224
cultures for product isolation
2212
storage solutions 222
endophytic fungi
as alternative sources 225226
examples 226
Taxus brevifolia and taxol 219
antibiotics from endophytic fungi
227231
artemisinin 230
colletotric acid 230
Cryptocandin 227, 228
Cryptocin 227, 228
guignardic acid 230, 230
javanicin 230231
Pestalotiopsis jesteri, jesterone
and hydroxy-jesterone
229230, 229
Pestelotiopsis microspora
227229, 229
Index
285
phomopsichalasin 230
antioxidants from endophytic fungi
233234
isopestacin 233, 233
compounds from natural
resources 233234
P. microspora, compounds,
pestacin and isopestacin
from 233
pestacin 233
antiviral from endophytic fungi
232233
call for new effective agents
with low toxicity 225
discovery in its infancy 232233
fungi 234
immunosuppressive
compounds 234
F. subglutinans and subglutinols
A and B 234
natural-based compounds
226227
problem of resistant organisms
225
production of certain bioactive
compounds 225
volatile antibiotics from fungal
endophytes 231232
Gliocladium sp. 232
M. albus 232
M. albus 231
M. crispans 232
M. albus 231232
Environmental Protection Agency (EPA)
and developing countries 199
DDT 199
establishment of Botanical
Pesticides and Pollution
Prevention Division 199
register botanical pesticides 199
use of synthetic pesticides 199
views of 199
enzymes, proteinase inhibitors, lectins
and PR proteins 115119
lectins 117
naturally occurring insecticides
117
lytic enzymes 115117
lysozymes 116117
other applications for chinolytic
enzymes 116
potential for plant-disease

management 116
use on plant pathogens by
biocontrol agents 115
PR proteins 118119
families 118119
inducible defence-related
proteins 118
proteinase (protease) 117118
active against plant pathogenic
nematodes 117
effective against phyto
pathogens in vitro and
in vivo 117
in the green consumerization
context 118
used to engineer virus
resistance in transgenic
plants 118
essential oils 1214, 28, 176
advantage of bioactivity in the
vapour phase 34
antifungal effects and antiaatoxigenic afciency
25
antimicrobial additives 23
chemosterilant activity 1314, 14
cinnamon and clove 24
as contact insecticides and
fumigants 185186
difference in anti-fungal and
aatoxin efcacy 2526
and edible oils 176177
efcacy in inhibiting aatoxin
production 2426
clove, mountain thyme and
poleo antifungal effect
25
C. camphora and A. galanga oils
25
P. graveolens and A. parasiticus
25
zimmu extract and inhibition of
AFB1 26
encapsulation 14
as fumigants against stored-product
insects 180, 180, 181
space fumigation with SEM76
essential oil 180, 181
space fumigation studies/tests
180, 180, 186
286
Index
essential oils continued
important role in plant protection
12
insect repellants 13
in vitro assays against
mycotoxigenic fungi 24
in vitro screening of 37 oils 24
as inhibitors of fungal growth and
aatoxin production 15
lemongrass oil and mycotoxigenic
fungi on maize 2729
assayed on irradiated maize 29
mustard essential oil 36
AITC, fungistatic or fungicidal
3637
octopamine 14
plant essential oils 71, 186
possible mode of action 183185
d-limonene 184
Push-Pull (stimulo-deterrent
diversionary) strategy 13
semio-chemicals 13
and synthetic pesticides 14
therapeutic use of 26
Ethiopian subsistence farming
systems 48
fescue toxicosis 219

avonoids 578, 57, 61
glycosides 61, 61
kinones 589, 58
tannin, hydrolysable and
condensed 58, 58
food, loss during storage 92
fruits 3334
blue mould in apples and pears 33
AITC treatments in pears 33
disease incidence 33
pepperfruit 34
cinnamon essential oil 34
volatile eugenol 3334
fumigants and contact synthesized
insecticides
botanicals as possible alternatives
to 176
global concern over negative effects
176
pest resistance and pesticide
residues 176
fungal endophytes 218236
fungal toxins, controlled by natural

plant products 15
fungi
group of microbes 219
plant associated 218
fungicides, chemical 42
pressure to reduce in foods 34
resistance to accelerating 3
fungicides, natural 47
fungistasis 246
genotypes 265
gibberellins 218
glucosinolates 10, 211
and myrosinase
co-located in same cells 211, 212
formation of hydrolysis
products 211, 213
tissue disruption 211, 213
grape marc compost 252256
age and suppression 253
disease-suppressive effects 253
environmental hazards 252
inhibition of sclerotial germination
253
soilborne suppression and diseases
253256
treatment in Europe 2523
waste from wine production 252
hairy root technology 272273

India 234235
induced antiviral proteins 160166
C. aculeatum and B. diffusa 162166
antiviral proteins 165, 166
efcacy of foliar leaf sprays 166
medicinal properties 162
plant viruses 162163
pre-inoculation spray of SRIs
165
protein occurring in B. diffusa
163
proteinaceous modiers
165166
Index
287
systemic resistance inducer
164165
treatment with SRI from C.
aculeatum leaves 163
virus inhibitory activity
163164
inhibitor of virus replication 161
Samsun plants 161
plant-extract-induced virus
inhibitory agent (VIA)
161162
antiviral agents 161
hypersensitivity to virus
infections 160
puried AVF 160
two categories 161
VIAs 162
virus-induced new antiviral protein
components 160161
induced systemic resistance (ISR)
250251
insect growth reguatory chemicals
(IGRS) 1112
insect pests 2
insect repellents
from rainforest plants 11
volatile terpenoids 12
insecticide effects 135
insecticides, toxicity of 23
integrated pest management (IPM) 43,
68, 200
development 4
in organic agriculture 45
pest control in conventional
cropping systems 44
juvabione 1112, 12
lemongrass essential oil 2729, 32

limonene 51, 51
limonoids 142
non-azadirachtin 145
maize 2731
aatoxin accumulation 27
essential oils tested for fungal
control/mycotoxin
prevention 2931
A. asperrima and A.striate 30

dried basil leaves 30
anise, boldus, mountain thyme,
clove and poleo 2930
effects of cinnamon, clove,
oregano and palma rose
oils studied 30
herb oils 29
onion and garlic 29
oregano oil 30
lemongrass 2729, 28
potential prevention of
biodeterioration 96101
active compound 98, 98
in vitro evaluation antifungal
activity 99100
test fungi for antifungal
activity assay 99
use of poisoned food
technique 99100
in vivo evaluation to prevent
biodeterioration
100101
results 100101
isolation and characterization
of antifungal active
component 989
molecular structure of
bioactive compound
99, 99
P. corylifolia 9698, 97
melanin
antimelanogenic compounds 127
infectivity of plant pathogenic fungi
127
metagenomics 275, 276
methyl bromide (fumigant) 6566, 205
microbial population, competition
among 24651
antibiosis 2478
antibiotic production 248
dened 247
and inhibition in the Petri dish
247
Trichoderma and Gliocladium
248
Trichoderma spp. 249
mycoparasites 249
parasitism on another parasite
248, 248
288
Index
microbial population, competition
among continued
compost/compost-amended
soils 250
dened 24950
ISR role in suppression of plant
pathogens 250251
ISR and SAR 250
microbiostasis 246247
competition for nutrients in
exudates 246
fungistasis 246
iron competition 246247
iron salts, synthesis and use of
siderophores 247
and pseudobactins 247
mungbean yellow mosaic virus (MYMV)
149
mycotoxicosis 22
mycotoxigenic fungi, in vitro assays
against 2324
aqueous extract of Adenocalymma
sativum 2324
extracts of Cynara cardunculus 23
zimmu 23
mycotoxigenic fungi and mycotoxins in
foods
natural preservatives 22
plant products 2227
defence mechanisms against
pathogens evolved 33
mycotoxins 2
in foods 2122
naturally occurring secondary
metabolites 21
signicant impact on
economics 22
production 2427
aatoxins 24
myrosinase 211, 212, 213
natural bio-herbicides 7277

allelopathy
dened 73, 195
release of protective chemicals
73
weed management programmes
73
augmentation biocontrol 73
biological approach to weed control

72
classical biocontrol (inoculative
biocontrol) 73
current status of natural herbicides
7677
commercialized for weed
control in organic
agriculture 77
ecologically more favoured 76
inundative biocontrol 7273
need to control weeds 72
plants with herbicidal properties
7476
allelochemicals inhibiting seed
germination and
growth 7576
bioactivities of plant extracts
75
buckwheat 75
essential oils used for weed
control 76
extracts of lucerne cultivars 74
potential of W. shinensis 74
research into allelopathic
activity of plant extracts
75
natural compounds
from plants with bio-stimulatory
potential 7881
brassino steroids 80, 81, 81
ComCat 8081, 81
effect of liquid seaweed extract
on improving yield of
canola 79
products developed 78
properties in plant extracts 79
prototype product 80
from wild plants 4648
plant allelochemicals 78
replacing articial chemicals in
food 263264
usefulness 264
natural pest controls using botanicals 16
natural plant compounds
biocidal effect 111
for controlling plant pathogens and
pests 110111
with microbial activity 47
non-biocidal suppressors 111
Index
289
natural plant extracts
potential to use as plant growth
regulators/natural
herbicides 4445
use to control pathogens 44
natural plant products
aims of research 47
biodegradable and eco-friendly 82
contribution of biochemicals 44
nal application to food products
37
inhibitory action on fungal cells 26
use in agriculture 4647
natural product chemistry 264
natural product diversity, expansion
of 273276
attempts to restore taxol-producing
ability to an endophytic
fungus 274
combinatorial biosynthesis 274
E. coli, engineering new metabolic
pathway in 275
fragments of DNA can be clones
276
metagenomics 275
nonribosomal enzymatic
polymerization of amino
acids 275
polyketides, formation of 274275
mutagenesis 274
site-directed mutagenesis 273
natural product research 60
natural product synthesis 263276
natural products
availability 195196
cultivation within agro-forestry
programmes 195
growth throughout the year
195
inter-cropping and mixcropping 195
use as weed control in
farmlands 195
biofumigation with Brassica
211215
importance in third-world countries
193
and phytochemicals from
endophytes 224234
plant tissue and cell culture in
269273
and ailments 269

biosynthesis manipulation 270
combinatorial biosynthesis 271,
273
cyclotides (small cyclic
peptides) 272
development of hairy root
technology 272273
immobilized cells 271
indole alkaloids 271272
organogensis 270, 271
plant tissues culture and
comaclonal variations
270271
podophyllotoxin 273
regeneration through
micropropagation 270
shikonin 271
tissue culture 269
use as fungicides and bactericides
in agriculture 206210
factors in formulating plantbased products as
pesticides 106
plant protection from microbial
attack 206
summary of natural products
used as agrochemicals
206, 207210
use of 4748
see also botanicals; natural plant
products
natural substances in fungi, bacteria and
plants, pesticidal properties 215
neem (Azadirachta indica) 68, 15, 25,
47
and integrated pest management
78
biopesticides for pest control 6
insect growth regulators (IGRs) 7
leaf extracts 26
limonoids 142
organic farming 78
potential alternative to chemicalbased pesticides 6
seed extracts 141142
use of leaves 193
use of neem cake 194
nematodes 11
New Zealand 74
nicotine 4, 5
290
Index
nuts 35
controlling aatoxin infection 35
effect of fumingation with essential
oil of mustard 35
inhibition of germination of fungi 35
use of protectants 35
octopamine
biological role in insects 14
as neurotransmitter 183, 184
paddy 101106
active principle 102, 102
antifungal assay 101
comparative efciency studies 102,
104
complete inhibition of test fungi
observed 102, 103
D. hamiltonii 101, 101, 105106
evaluation of the bioactive
compounds 105, 105
in vitro antifungal activity assay 102
pathogen control 215
pathogen suppression 243
pesticides 2
aerosol 193
eco-friendly and natural 106
essential-oil-based 13
natural-product-based 205
persistence in biological systems 3
pesticides, chemical
being phased out globally 205
benets to crop producers 43
and creation of green
consumerization 110
downside of 192193
hazards to environment 43
long-term effect of environmental
contamination 46
novel compounds in place of 11011
results of persistent and
indiscriminate use 92
undesirable impacts and
environmental risks 110
phenolic acids 75
phenols, simple 5557
all monomeric 55, 55
formation of coumarin glycosides
56, 56
development as natural
herbicide 5657
as natural herbicides 55
hydrokinone 55
salicylic acid 55
phenyl propanoids
coumaric acid and caffeic acid
5556, 56
potential for development 56
synthesized from p-coumaric
acid 56
synthesized from phenyl
alanine 5556
phytopathogens 110
plant diseases, problems and
consequences of 4243
plant endemism 221
plant extracts
antimicrobial compounds 5962
chemical defences 59
constitutive and induced
compounds 5960
avonoids and saponins
6162
intensive plant screening
programmes for
bioactivity 60
antimicrobial properties of 5972
aqueous extracts 9394
current status 7172
in vitro evaluation for antifungal
activity 9395
plants with antibacterial properties
6971
plants with antifungal properties
6269
secondary metabolites with
antimicrobial properties
6061
solvent extracts 9495
methanolic and ethanolic bark
extracts 94
petroleum ether and methanolic
extract of E. ayapana
tested 94
testing for antifungal activity
9495
plant oils, effects of 176
plant pathogens, biocontrol of 111125
antimicrobial peptides produced
112115
Index
291
enzymes, proteinase inhibitors,
lectins and PR proteins
115119
green consumerization 119125
proteinaeous compounds 111112
proteinic inducers of plant
resistance 119125
plant products
antimicrobial activity studies and
demonstrated 23
disc diffusion test 23
as pesticides, current worldwide
use 510
use in active packaging 3537
plant species and insecticidal
properties 176
plantinsect relationships 134135
a foodconsumer relationship
135
plants
isolation and identification 96106
problems in managing protection
109110
serious efforts to screen for
biological activities 92
under exploited for plant health
needs 106
use of bioactivity-directed
fractionation and isolation
(BDFI) 96
with antibacterial properties 6971
Hedera helis extract and fire
blight on pome fruits
7071
large-scale screening
programmes 69
testing of in vitro growth
inhibition of E.
amylovora 6970
with antifungal properties 6269
active secondary leaf extract
metabolites 62
antifungal potential 64
antimicrobial activity 65
antimicrobial efficacy of plant
extracts 6869
effective control of grape
downy mildew 6364
significant inhibition of
spore germination
64
effects of some essential oils

against R. solani 67
evaluation of potential of plant
extracts to be applied in
IPM programmes 68
exploiting indigenous
knowledge of medicinal
plants 64
grapefruit extract extract used
to control Phytophthora
spp. 645
inhibition
of mycelial growth of
C. cladosporioides 62
of spore germination of
some fungi by
Ajoene 62
investigation of phenol
composition of two
carnation cultivars 65
methanol extract of C. emoryi
62
natural plant extracts and
soil-borne pathogens
656
neem tree extracts 66
plant seeds 6768
saponins 63
South African plants with
fungitoxic properties 63
PR proteins 118119, 159160
precocenes 12, 12
proteinic inducers of plant resistance
119125
Chitozars 120
cold shock protein (CspD) 120122
harpins 120
induction of plant resistance to
pathogens 119
active defence mechanisms
initiated 119
general and proteinaceous
elicitors 119
MF3 protein 122125
MF3 integration with chitosan
124
MF3 polypeptide chain 123
peptidyl-prolyl cis/trans
isomerases (PPIases)
122
plant FKBPs 122
292
Index
proteinic inducers of plant resistance
continued
MF3 protein continued
PPIase interaction with pathogen
proteins 122123
PPIases and plantpathogen
relations 122123
rape lines with mf3 insertion
124
systemic character of resistance
124
Psoralea corylifolia 96101, 97
Push-Pull (stimulo-deterrent
diversionary) strategy 13
pyrethrum 8, 9, 4647
as insecticide 8
low doses 8
pyrethroids versus pyrethrin 8
quassin and neo-quassin 11
rainforests 221
repellents/repellency 136
ribosome inactivating proteins (RIPs)
150, 154155
rice 3233
plant extracts 3233
rotenone 4, 5
used in commercial insecticides 4
ryanodine 4, 5
sabadilla (cevadilla) 810

saponins 52, 52, 6162, 63
biological activity 61
secondary metabolites 21, 4959, 78, 95,
223
alkaloids 59
with antimicrobial properties 6061
aromatic components 539
non-phenolic aromatic
compounds 53, 54
phenolic aromatic components
5559
isoprenoid components 4953
brassinosteroids 51, 51, 52, 80,
81, 81
diterpenes 50, 51
hemiterpenes 49, 50
isoprene units and repeating

units 49, 49
monoterpentes 50, 50
saponins 52, 52
sesquiterpenes 50, 50
sterols 5152, 51, 52
tetraterpenes 53, 53
triterpenes 51
semio-chemicals 13
siderophores 247, 254, 256, 257
soilborne suppression 253256
detection of siderophobes 256
microbiological analysis 254, 254
production of compost 253
production of siderophores 254
results from in vitro analysis 2546,
255
soil-applied composts and watery
extracts 256
solarization 211
sorghum 32
South Africa 13
Blyde River Canyon 63
determination of antifungal
potential of plants 63
search for plant species with
fungitoxic properties 63
statins 125128
ergosterol 125
and fungal melanin 127
lovastatin and compactin in plant
protection 126
natural inhibitors of sterol
biosynthesis 125
sterols 125
stored food commodities 15
strychnine 11, 11
subsistence farmers 46
suppressive soils/suppressive
substrates
biological elements 242243
composting conditions and
materials 245246
general suppression 245
modes of action of biocontrol agents
243
soilborne disease 243245, 244
specic suppression 245
sustainable agriculture 4
synthetic chemicals
consumer resistance to 46
Index
293
increased production 81
systemic acquired resistance (SAR) 206
endogenously occurring virus
inhibitors 150
induced by infection of plants 150
pathogen-induced 158159
dicotyledonous and
monocotyledonous
plants 159
endogenous SA 159
rst report of virus-induced
SAR 158
pre-inocculation with biotic
inducers 158
and role of PR proteins 159160
systemic induced resistance (SIR) 150,
163
systemic resistance inducers (SRIs)
151158, 163, 167
carnation antiviral proteins
(dianthins) 156
characteristics 151
mechanisms of systemic induced
resistance by botanicals
152154
absence of PR proteins 153
production of VIA sensitive to
actinomycin D 153
suppression of disease
symptoms 154
systemic synthesis of some PR
proteins 153
VIA production in hosts 153
virus inhibitory agents (VIAs)
152
mirabilis antiviral protein (MAP) 1
56157
leaf extract 156157
puried protein 157
obtained from plants 152
pokeweed antiviral proteins (PAPs)
155156
pre-inoculation spray 165
resistance development inhibited
by actinomycin D and
cyclohexamide 152
ribosome inactivating proteins

(RIPs) 154155
PAPs, dianthins and MAPs
belong to type-I RIPs
155
show antivral activity against
both animal and plant
viruses 154155
shown to be N-glucosidases
154
Type-I RIPs 155
Type-II RIPs 155
ricinus (RICIN A) antiviral protein
157
A and B chains 157
type II RIP 157
role of RIPs 157158
virus resistance 157158
biological signicance of RIPs
158
problem using type-I RIPs 158
taxol 265
anticancer drug 219, 227
fungal source 219
terpenoids 7, 7, 12, 60, 186
Trichoderma 210
virus disease resistance

constitutive resistance 150
virus infection 150
virus inhibitors from plants 150151
plant products using local or
systemic resistance 150151
ribosome inactivating proteins
(RIPs) 151
viruses 149
wheat 3132
A. speciosa root isolates 3132
resveratrol 31
wild plants 44, 46
world agriculture 46

Natural Products in Plant Pest Management

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NATURAL PRODUCTS IN PLANT PEST

CABI is a trading name of CAB International

CABI North American Ofce

Tel: +44 (0)1491 832111

Tel: +1 617 395 4056

7. Potency of Plant Products in Control of Virus Diseases of Plants

8. Phytochemicals as Natural Fumigants and Contact Insecticides

9. Prospects of Large-scale Use of Natural Products as Alternatives

10. Current Status of Natural Products in Pest Management

11. Fungal Endophytes: an Alternative Source of Bioactive

12. Suppressive Effects of Compost Tea on Phytopathogens

13. Biotechnology: a Tool for Natural Product Synthesis

J.C. Pretorius, Department of Soil, Crop and Climate Sciences, University

antimicrobials owing to various distinct advantages. Botanicals, being the

I wish to extend my appreciation to all the contributors for their cooperation

Global Scenario on the

N.K. Dubey et al.

1.2 The Need to Search for Eco-friendly Control Measures

Global Scenario and Natural Products

N.K. Dubey et al.

to look for alternative solutions to synthetic pesticides (Sharma and

1.3 Historical Use of Botanical Products in Pest Management

Global Scenario and Natural Products

(a) Rotenone (b) Nicotine (c) Ryanodine.

1.4 Plant Products in Current Worldwide Use as Pesticides

N.K. Dubey et al.

Global Scenario and Natural Products

Neem products also function as insect growth regulators (IGRs). The

N.K. Dubey et al.

Global Scenario and Natural Products

Fig. 1.4. Veratrine.

N.K. Dubey et al.

Global Scenario and Natural Products

1.5 Other Plant Products

(a) -terthienyl (b) Strychnine.

N.K. Dubey et al.

(a) Juvabione (b) Precocenes.

(Varma and Dubey, 1999). Analogues of insect juvenile hormones such as

1.6 Essential Oils

Global Scenario and Natural Products

Repellents and attractants modify the behavioural response of insects.

N.K. Dubey et al.

Global Scenario and Natural Products

1.7 Higher Plant Products as Inhibitors of Aflatoxin Secretions

N.K. Dubey et al.

Global Crop Protection Federation,

Global Scenario and Natural Products

N.K. Dubey et al.

Global Scenario and Natural Products

N.K. Dubey et al.

Plant Products in the Control of

2.1 Mycotoxins in Foods

2.2 Natural Preservatives for Control of Mycotoxigenic Fungi

2.3 Plant Products as Alternatives for Control of Mycotoxigenic

Control of Mycotoxins and Mycotoxigenic Fungi

polygalacturonase-inhibiting proteins and -amylases have been shown to

In vitro assays against mycotoxigenic fungi

In vitro assays for the inhibition of mycotoxin production

Control of Mycotoxins and Mycotoxigenic Fungi