Amsalu Neme MSC Thesis... Edited

AMBO UNIVERSITY
COLLEGE OF AGRICULTURE AND VETERINARY SCIENCES
DEPARTMENT OF PLANT SCIENCES
Seed borne Mycoflora of Faba bean (Vicia fabae L.) seeds in Ambo District and
Evaluation of Plant Extract and Trichoderma species for their Managements
By
Amsalu Neme Boru (ID. No: PGR/30162/11)
A Thesis submitted to the Department of Plant Science in Partial Fulfillment of the

Requirements for the Degree of Master of Science in Plant Pathology
MAJOR ADVISOR: Dr. Ararsa Leta
CO ADVISOR: Dr. Amin Mohommed
February, 2020
Ambo, Ethiopia
APPROVAL SHEET
SCHOOL OF GRADUATE STUDIES
AMBO UNIVERSITY
As members of the Examining Board of the Final M.Sc., open defense, we certify that we have
read evaluated the thesis prepared by Amsalu Neme Boru entitled “Seed Borne Mycoflora of
Faba Bean (Vicia fabae L.) seeds in Ambo District and Evaluation of plant Extract and
Trichoderma Species for their Managements’’ and recommended that it is accepted as
fulfilling the thesis full requirements for the award of Degree of Master of Science in Plant
Pathology
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DEDICATION
I dedicated this thesis to my beloved parents and next to my brother Mr. Degebasa Neme for his
desirable love and persistent encouragement for my success, who inspired me to higher ideas of
my life and they will forever live in my dreams and also for their nursing, affection and love,
which is contributed to the success of my life.
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STATEMENT OF THE AUTHOR
First, I declare that this thesis is my work and all sources of materials used for preparation of this
thesis have been duly acknowledged. This thesis has been submitted in partial fulfillment of the
requirements for the M.Sc., degree in Plant Pathology at Ambo University and deposited at the
University library to be made available to borrowers under rules of the library. I solemnly
declare that this thesis is not submitted to any other institution anywhere for the award of any
academic degree, diploma, or certificate.
Brief quotation from this thesis is allowable without special permission provided that accurate
acknowledgement of the source is made. Request for permission for extended quotation from or
duplication of this manuscript in the whole or in part may be granted by the Head of the
Department of Plant Sciences or the Dean of the School of Graduate Studies, when in his or her
judgment the proposed use of material is in the interests of scholarship. In all other instances,
however, permission must be obtained from the author.
Name: AMSALU NEME BORU Signature: _______________________
Place: Ambo University, Ambo, Ethiopia
Date of submission: May, 2021
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BIOGRAPHY
The author, Amsalu Neme Boru was born on January 1978G.C in Bebela, Jima Rare Oromia
Regional State, Ethiopia from his father, Neme Boru and his mother, Degitu Guyasa. He
attended his Elementary School education at Babela Elementary School from 1985 to 1993G.C
and secondary school education at Gedo Senior Secondary School from 1994 to 1997G.C in
Gedo town. After completion of the Ethiopian School Leaving Certificate Examinations, then, he
was joined in Ambo University for his Under Graduate Studies in General Agriculture Dipiloma
from September 1998 to 1999G.C, and he continued in Ambo University for his Under Graduate
Studies in Plant Science from October 2000 to 2002 G.C. After that, he has been appointed as
Junior Expert from September 1999 and then work at different position in Ambo District
Administrative Office until he joined School of Graduate Studies, Ambo University, on October
2019 G.C to pursue his M.Sc. degree studies in Plant Pathology
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ACKNOWLEDGEMENTS
First and foremost I praise the almighty God for I did everything through him who gave me
strength and made me stand firm in my all ups and downs during my research work.
It is my great pleasure to express my heartfelt appreciation and special gratitude to my Major

advisor Dr. Ararsa Leta and Co-advisor Dr. Amin Mohommed for their patience and thoughtful
guidance throughout my study. With the deepest gratitude, I wish to thank them for their support,
professional expertise and advice from the very planning of this work to its execution and critical
review of the manuscript and made it scientifically sound.
I extend my gratitude to the Head, Department of Plant Sciences and School of Graduate
Studiesof Ambo University for the facilities provided during my course work. My special thanks
go to Dr. Gudeta Napir, Plant Science Department Head for their cooperation during my course
work.
I would like to extend my special thanks to all Mycology, Pathology, Veterinary and Soil
Science Laboratory Instructors at Ambo University College of Agriculture and Veterinary
Science for their unreserved material support during Laboratory work. I also express my sincere
gratitude to my lovely wife Alemnesh Tadele for her taking care of my kid Meti, Simbo and
Dibora Amsalu during my absence and for all rounded support throughout my study.
It gives me immerse pleasure to express my deep sense of gratitude to Ambo District

Administrative Office for allowing me to upgrade my career through the provisions of monthly
salary and their financial support.
Finally, the unreserved support of my brother, Degebasa Neme who stands with me in all
rounded financial, spiritual and moral support from the beginning to the end for the success of
my aim is ever unforgettable.
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Acronyms and Abbreviations
ANOVA…………………….....Analysis of Variance
APPRC……………………......Ambo Plant Protection Research Center
BRR..........................................Black Root Rot
CRD.........................................Completely Randomized Design
CSA………………………....Central Statistical Authority
DZARC……………………. Debre Zeit Agricultural Research Center
EEPA………………………..Ethiopian Export Promotion Agency
EIAR………………………..Ethiopian Institute of Agricultural Research
FAO…………………………Food and Agricultural Organization
FBNYV...................................Faba Bean Necrtic Yellow Virus
HARC ....................................Holeta Agricultural Research Center
ICARDA….International Center of Agricultural Research Development on Dry Areas
ICRISAT………….International Crops Research Institute for the Semi-Arid Tropics
IDM.......................................Integrated Disease Management
LSD………………………...Least Significant Difference
ISTA......................................International Seed Testing Assossation
MoARD…………………….Ministry of Agriculture and Rural Development
ORARI……………………. Oromia Regional Agricultural Research Institute
PDA………………………...Potato Dextrose Agar
PPRC……………………......Plant Protection Research Center
SAS………………………....Statistical Analysis System
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Table of Contents
Title Pages
Dedication.......................................................................................................................................iv
Statement of the Author.................................................................................................................v
Biography.........................................................................................................................................v
Acknowledgements.........................................................................................................................vi
Acronyms and Abbreviations......................................................................................................vii
Table of contents....................................................................................................................viii-xi
List of Tables...............................................................................................................................xii
List of Figures.............................................................................................................................xiii
Abstract.........................................................................................................................................xiii
CHAPTER ONE..............................................................................................................................1
1. INTRODUCTION....…………….….……………….....………………………………...…….1
1.1. Back Ground....................……………........…………………….....……………………....…1
1.2. Statement of the Problem…......................................................................................................2
1.3. Research Questions...................................................................................................................3
1.4. Significance of the Seed Testing Study………………...…........………………………….....3
1.5. General Objective.………...…….......………….…………….………........…........................5
1.6. Specific Objectives …..............................................................................................................5
CHAPTER TWO.............................................................................................................................6
2. LITERATURE REVIEW.............................................................................................................6
2.1.Faba bean, production................................................................................................................6
2.2. Production constraints of faba bean.......................................................................................6-7
2.2.1.Seed borne mycoflora of faba borne....................................................................................7-9
2.2.1.1.Black root tot (Fusarium solani)..........................................................................................9
2.2.1.1.1. Economic significance.....................................................................................................9
2.2.1.1.2. Ecology and epidemiology..............................................................................................9
2.2.1.1.3. Managements black root rot of faba bean..................................................................9-10
2.2.1.1.3.1. Cultural practice..........................................................................................................10
2.2.1.1.3.2. Biological control........................................................................................................10
2.2.1.1.3.3. Host resistance............................................................................................................10
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2.2.1.1.3.3. Chemical control....................................................................................................10-11
2.2.1.1.3.4. Integrated disease management..................................................................................11
2.2.2.2. Fusarium wilt (Fusarium oxsporium)...............................................................................12
2.2.2.2.1. Economic significance...................................................................................................12
2.2.2.2.2. Ecology and epidemiology............................................................................................12
2.2.2.2.3. Managements black root rot of faba bean................................................................13-14
2.2.2.2.3.1. Cultural practice..........................................................................................................14
2.2.2.2.3.3. Host resistance............................................................................................................14
2.2.2.2.3.4. Chemical control.........................................................................................................14
2.2.2.3. Damping off (Rihoctonia solani)......................................................................................15
2.2.2.3.1. Economic significance...................................................................................................15
2.2.2.3.2. Ecology and epidemiology.......................................................................................15-16
2.2.2.3.3. Managements black root rot of faba bean.....................................................................16
2.2.2.3.3.1. Cultural practice....................................................................................................16-17
2.2.2.3.3.3. Host resistance.......................................................................................................17-18
2.2.2.3.3.4. Chemical control.........................................................................................................18
2.2.2. Botanicals for disease managements..............................................................................19-20
2.2.2.1. Garlic (Allium sativam).....................................................................................................20
2.2.2.2. Argissa (Aloe vera)......................................................................................................20-21
2.2.2.3. Turmeric (Curcuma longa L.)...........................................................................................21
2.2.2.4. Tobacco (Nicotina tabacum).............................................................................................22
2.2.2.5. Nicandra physalodes.............................................................................................................
2.2.2.6. Maytenus senegalensis.......................................................................................................
2.2.2.7. Phytolacca dodecandra........................................................................................................
2.2.3. Bio-agents for disease managements..............................................................................22-23
CHAPTER THREE.......................................................................................................................24
3. MATERIALS AND METHODS...............................................................................................24
3.1. Description of study area................................................................................................. ......24
3.2. Collection of seed samples......................................................................................................24
3.3. Isolation of fungi from faba bean seeds.............................................................................24-25
3.4. Determination of isolated seed borne fungi............................................................................25
3.5. Identification of fungi........................................................................................................25-26
3.6. Determination of effect of seed borne myco-flora on seed germination................................26
3.7. Determination of effect of seed borne myco-flora on disease transmission......................26-27
3.8. In vitro assay for the management of seed borne pathogens..................................................27
3.8.1. Evaluation of botanical plants..............................................................................................27
3.8.1.1. Collection of botanical plant materials........................................................................27-28
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3.8.1.2. Preparation of the plant aqueous extract...........................................................................28
3.8.1.3. Effect of plant extracts on mycelium growth of test fungi..........................................28-29
3.8.2. Evaluation of bio-agents......................................................................................................29
3.8.2.1. Source of Pure cultures of Trichoderma isolate................................................................29
3.8.2.2. Preparation of Pure culture Trichoderma isolate.........................................................29-30
3.8.2.3. Effects of Trichoderma spp. on mycelium growth of test fungi..................................30-31
3.9. Data Analysis.........................................................................................................................31
CHAPTER FOUR..........................................................................................................................32
4. RESULTS AND DISCUSSION................................................................................................32
4.1. Results.....................................................................................................................................32
4.1.1. Isolated fungi from faba bean seed samples........................................................................32
4.1.2. Determination of seeds infected with fungi....................................................................32-36
4.1.3. Identification of fungi based on morphological and cultural characteristics..................36-38
4.1.4. Determination effects of seed-borne fungi on germination of seeds..............................38-39
4.1.5. Determination effects of seed borne fungi on disease transmission...............................39-42
4.1.6. Antifungal activities of plant aqueous extracts...............................................................42-46
4.1.7. In vitro evaluation of Trichoderma species against test fungi........................................46-47
4.2. Discussion..........................................................................................................................48-49
CHAPTER FIVE...........................................................................................................................50
5. CONCLUSION AND RECOMMENDATION.........................................................................50
5.1. Conclusion..............................................................................................................................50
5.2. Recommendation....................................................................................................................50
Reference..................................................................................................................................51-57
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List of Tables
Table 1: Botanical plants used for antifungal activities..................................................27
Table 2: List of Fungi Isolated from 50 Faba Bean Seed Samples by agar plate method......32
Table 3: Per cent association of seed borne fungi (%) in locations.........................................33
Table 4: Cultural and morphological characteristics of Selected isolate fungi................35
Table 5: Frequency of occurrence on faba bean seed samples................................................37
Table 6: Analysis of Variance for the frequency of occurrence of fungi.................................38
Table 7: Effects of seed-borne fungi on the germination of seeds.........................................39
Table 8: Seed to seedlings transmission efficacy....................................................................41
Table 9: In vitro evaluation of plants extracts with cold and hot water against
F. oxsporium.................................................................................................................43
F. solani........................................................................................................................44
R. solani....................................................................................................................... 45
Table 12: In vitro evaluation of Tricoderma spp. against test fungi.......................................46
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List of Figures
Figure.1 Antagonistic test between Fusarium sp. and Trichoderma spp ...............................29
Figure.2 Antagonistic test between Rhizoctiona solani and Trichoderma spp.......................29
Figure 3: Infected seed (%) of faba bean seeds by major seed borne fungi .........................34
Figure 4: Incubated seeds showing infection of fungal pathogens on agar plate method.......35
Figure 5: F. oxsporium. grown on PDA media culture plates.................................................36
Figure 6: F. solani grown on PDA media culture plates.........................................................36
Figure 7: R. solani grown on PDA media culture plates........................................................37
Figure 8: Frequency of occurrence on faba bean seed samples..............................................38
Figure 9: Effects of seed borne mycoflora on seed germinations of faba bean seeds........... 40
Figure 10: Damping off Root rot symptoms from 7 days plate method test of faba bean...42
Figure 11: Damping off Root rot symptoms from 21 days plate method test of faba bean...42
Figure 12: Evaluation of Trichoderma spp. against mycelial growth of test fungi.................47
13. ABSTRACT
Faba bean is one of the most important food legumes due to its high nutritive value both in
terms of energy and protein. In spite of its wide cultivation and huge importance, the average
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yield of faba bean is quite low and far below the potential because of several limiting biotic and a
biotic constraints. Diseases are the most important biotic factors limiting the production of faba
bean. The aim of this laboratory studies were carried out to isolate and identify seed borne
mycoflora associated with faba bean seed samples and to determine their effects on seed
germination, diseases transmit ion as well as to evaluate the antimicrobial activities of seven
plant extracts and four trichoderma species against the pathogen recovered from the seed. Fifty
seed samples were collected from different farmers’ saved seeds of five major faba bean
producing kebele’s in Ambo district were tested by agar plate methods as recommended by
ISTA. A total of 7 fungal species belonging to 6 genera viz. Fusarium oxysporum
(Schlechlendahl), Fusarium solani (Mart.) Sacc., Aspergillus spp. Penicillium spp. Botrytis spp.
Rhizoctonia solani (Kühn) and Alternaria spp. were observed and identified. Among them
Fusarium spp., Aspergillus spp, and Penicillium spp. were the most predominant fungi to all seed
samples. Seed to seedling transmit ion test result confirmed that, F.oxysporum, F.solani and
R.solani were the common causal pathogens causes the root rot and damping off disease of faba
beans and transmitted from seed to seedlings. The presence of the mycoflora significantly affects
the seed germination. The maximum seed germination rate was observed in Golja-GF002 (97%)
and minimum in Kure Gatira-KF0038 (81%).The seven botanical plants extracts. In vitro
evaluation of aqua’s plant extract and Trichoderma species against F.oxysporum, F.solani and
R.solani results revealed that, all the tested plant extracts at 5%, 10% and 20% concentrations
significantly inhibited the mycelia growth of all test fungi, and the four trichoderma species was
also exhibited antagonistic activity. The best inhibitory effect on the three test fungi were
obtained by T.longibrachiaium (T14) (70.45%, 60.22% and 57.95%), followed by T.atroviride
(T5) (61.36%, 54.54% and 48.86%), T. Virense (T13) (53.97%, 45.45% and 44.31%) and T.
harzianum (T1) (42.04%, 43.75% and 38.63%), respectively. The inhibitory action of the
aqueous plant extracts on mycelia growth increased with increase in concentrations and the hot
water extracts giving high effects/toxicity than cold extract in all test fungi. Results highlight the
highest inhibitory effect of A. sativum extracts with hot water at 20% concentration on mycelia
growth of the three test fungi (79.54%, 62.19% and 60.22%) respectively, but Nicandra
physalodes extracts at the same concentration showed lowest inhibitory effects on three test
fungi (69.31%, 51.22% and 51.20%) respectively.
Keywords: Seed borne mycoflora, faba bean, agar plate method, plant extract, trichoderma spp.
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CHAPTER ONE
1. INTRODUCTION
1.1 1.1. Background

Faba bean (Vicia fabae L.) is an important diploid (2n = 12 chromosomes) Fabaceous pulse crop
with common names including broad bean, horse bean, tic bean and field bean. It is one of the
earliest domesticated food legumes in the world, probably in the late Neolithic period (Metayer,
2004; Dagne et al., 2016). It is the most important pulse crop in terms of both area coverage and
volume of annual production in Ethiopia.
Faba bean is one of the most important food legumes due to its high nutritive value both in terms
of energy and protein contents 24-30% (Crepona et al., 2010) and also is an excellent nitrogen
fixer (Sahile et al., 2008). It is ranking in the world fourth after garden pea, chickpea and lentil.
Faba bean production in the world is concentrated in nine major agro-ecological regions, namely;
northern Europe, Mediterranean, the Nile valley, Ethiopia, Central Asia, East Asia, Oceana,
Latin America, and North America (Bond et al., 1985).
Ethiopia is the first producer of faba bean in Africa and the second in the world next to China
(Mussa and Gemechu, 2006), its share is only 6.96% of world production and 40.5% of Africa.
The crop is considered as the secondary center of diversity (Torres et al., 2006) and mainly
cultivated in mid and high altitude areas, with an elevation ranging from 1800-3000 meters
above sea level (Temesgen and Aemiro, 2012).
The average national yield of faba bean is about 2.1 t ha -1 (CSA, 2018), which is very low
compared to the average yield of 3.7 t ha -1 in major producer countries (FAO, 2017). Faba bean
is an important high land pulse crop of Ethiopia, which covered 520,519 ha of cultivated land
with annual production of 930,633 tons (FAO, 2019). The crop takes the largest share of the area
under pulses production in Ethiopia. The growing importance of faba bean as an export crop in
Ethiopia has led to a renewed interest by farmers to increase the area under production (Samuel
et al., 2008).
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In spite of its wide cultivation and huge importance, the average yield of faba bean is quite low
in Ethiopia and the productivity of the crop is far below the potential because of several limiting
biotic and abiotic constraints (Sahile et al., 2008, Mussa et al., 2008 and EIAR, 2011).
According to Samuel et al. (2008), diseases are the most important biotic factors limiting the
production of faba bean in Ethiopia. Faba bean is attacked by more than 100 pathogens
(Hebblethwaite, 1983). More than 17 diseases causing pathogens are reported in Ethiopia (Dereje
and Tesfay, 1995).
Numerous diseases are affecting faba bean production and productivity, but only a few of them
have economic significance. About 16% annual crop losses due to plant diseases, at least 30%
loss is incurred due to seed-borne diseases. Among these, fungi are the largest and perhaps the
most important groups affecting all parts of the plant at all growth stages (Negussie et al., 2008).
Diseases such as chocolate spot (Botrytis fabae), rust (Uromyces fabae), black root rot
(Fusarium solani), and foot rot (Fusarium avenaceum) are among the fungal groups that
contribute to the low productivity of the crop (Berhanu et al., 2003, Negussie et al., 2008). Other
diseases are less significant, although aschochyta blight (Ascochyta fabae) and faba bean
necrotic yellow virus (Dereje and Tesfaye, 1994).
The most common seed-borne fungal pathogens were isolated from faba bean seeds such as
Alternaria spp., Ascophyta fabae, Aspergillus spp., Botrytis cinerea, B. fabae, Cephalosporium
sp., Cladosporium sp., Epicoccum sp., Fusarium spp., Mucor sp., Penicillium spp., Rhizoctonia
sp., Rhizopus sp., Stemphylium sp., Trichothecium sp., and Verticillium sp. (Neergard, 1979;
Abdel-Hafez, 1988; El-Wakil et al., 2009). These fungi are transmitted by seeds and can be
preserved as conidia in the coat or as mycelia at the seeds surface (Gargouri et al., 2000).
As the report of Neergard (1979), seed abortion, shrunken seeds, reduction of seed size, seed rot,
seed necrosis and seed discoloration, reduction in germination capacity and physiological
alterations in seed are the symptoms caused by the seed borne fungal pathogens. Moreover,
many diseases are affecting faba bean but only a few of them have either major or intermediate
economic significance in Ethiopia.
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Management of plant diseases is important for most crops, and it is particularly critical for the
production of high-quality seeds. Plant pathogens can reduce the quantity and quality of the seed
harvested, and in addition they can be preserved in seed lots in the case of seedborne pathogens.
In this way, seeds can inadvertently provide an efficient means of plant pathogen dissemination
(Mancini and Romanazzi, 2014).
Attempts have been made to reduce seed-borne fungi by chemical treatment of the seeds and
some successes have been reported. Seed dressings are used to eliminate most surface infestation
of seeds but have relatively little effect on internally borne organisms (Jackson, 1963). An urgent
need for alternatives to fungicides for the control of seed-borne fungi is important in recent
years; much attention has been given to non-chemical systems for seed treatment to protect them
against seed-borne pathogens. Plant extracts have played a significant role in the inhibition of
seed-borne pathogens and in the improvement of seed quality and field emergence of plant seeds.
Many authors reported the effective and safe use of plant extracts for controlling seed-borne
fungi (El-Metwally et al., 2010; Yoon et al., 2011; Ammar et al., 2013; Perello et al., 2013; Baka
2014b).
2.1 1.2. Statement of the Problem

In spite of faba bean cultivation is increasing from 520,519.72 ha of land in 2017/18 to
26,573,001 ha of land in 2018/19 with total production from 8.55 million tons to 9.30 million
tons, but productivity remains low 1.99 tone ha–1(CSA, 2018) despite many efforts to increase
productivity. Low productivity is attributed to many biotic and abiotic factors.
Among biotic factors chocolate spot (Botrytis fabae Sard.), rust (Uromyces Vicia fabae), black
root rot (Fusarium solani), and foot rot (Fusarium avenaceum) contributes to the low
productivity of the crop (Berhanu et al; 2003 and Nigussie et al; 2008).
According to Vijendra and James (1987), Ascochyta fabae, Fusarium oxysporum, Botrytis fabae,
B. ricini, Alternaria alternata and Uromyces fabae are considered as seed borne fungi of faba
beans all over the world.
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Hence studies on effects of seed borne fungi on seed germination, seed to seedlings transmission
and their managements is important to guarantee the improvement of the crop.
Therefore, this study is initiated to assess the status of seed borne fung in farmers saved seed in
Ambo district, test the effect of seed borne fungi of faba bean on seed germination, seed to
seedlings transmission and evaluate their management options.
3.1 1.3. Research Questions

The basic question of this research was, therefore, whether that seed borne fungi inoculum
resides in seed passes to the seedlings thereby influences the production and productivity of faba
bean around Ambo districts. The specific questions include; Does seed borne fungi survives in
seed? How much passes from seed to seedling? Is there a relationship between seed infection
level and seedling infection? And does eradicating the seed inoculums with botanical plant
extract and trachoderma possible?
4.1 1.4. Significance of the Seed Testing Study

 To detect the most important seed-borne fungal pathogens.
 Testing seed before sowing identifies potential disease problems and allow steps taken to
reduce the disease risk.
 Uncontrolled movement of infected seed can result in the rapid expansion of the area
affected by these diseases.
 Therefore, laboratory testing is usually required, as infected seed may often have no visible
disease symptoms.
5.1 1.5. General objectives

To determine the important seed borne fungal pathogens associated with Faba bean (Vicia fabae
L.), and their management using in botanicals and Trichoderma spp.
6.1 1.6. Specific objectives

 To assess the fungal pathogens associated with farmers saved seeds of faba bean in the
study areas.
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 To determine the level of seed infection by fungal pathogens associated with farmers
saved seeds of faba bean in the study area.
 To determine the effect of seed infection on seed germination and disease transmissions
and
 To evaluate the efficacy of some plant extracts and Trichoderma spp against fungi
associated with faba bean seeds.
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CHAPTER TWO
2. LITERATURE REVIEW
7.1 2.1. Faba bean production

Faba bean (Vicia fabae L.) is one of the earliest domesticated food legumes in the world,
probably in the late Neolithic period (Metayer, 2004; Dagne et al., 2016). It is the fourth food
legume in production after peas, chickpea and lentil (FAOSTAT 2014, Kaur et al., 2014). Faba
bean production in the world is concentrated in nine major agro-ecological regions, namely;
northern Europe, Mediterranean, the Nile valley, Ethiopia, Central Asia, East Asia, Oceana,
Latin America, and North America (Bond et al., 1985). It is cultivated in temperate and
subtropical regions of the world (Torres et al., 2006). The leading faba bean producing countries
are China, Ethiopia and Egypt (Tekalign, 2014; FAOSTAT, 2017).
Ethiopia is one of the major faba bean producing countries in the world (FAO, 2015). It is the
fourth largest faba bean exporting country next to France, Australia, and the United Kingdom
(FAO, 2016). Faba bean is the most important pulse crop in terms of both area coverage and
volume of annual production in Ethiopia. It takes the largest share of area (443,966 ha) and
production (848655 tones) of the pulses grown in the country (CSA, 2015). Faba bean plays a
key role in improving food and feed security of smallholder farmers and soil fertility. The crop
usually grows in Nitisol and Vertisol dominated areas of Ethiopia mixed with cereals and field
peas. The average national yield of faba bean is about 2.1 t ha -1 (CSA, 2018) which is very low
compared to the average yield of 3.7 t ha-1 in major producer countries (FAOSTAT, 2017).
8.1 2.2. Production constraints of faba bean

The yield of faba bean is rather low in Ethiopia due to number of biotic and abiotic factors
(Agegnehu and Fessehaie, 2006). Diseases are among the most important biotic factors causing
faba bean yield reduction (Yohannes, 2000). Numerous diseases are affecting faba bean but only
few of them have either major or intermediate economic significance. These include chocolate
spot (Botrytis fabae), rust (Uromyes fabae), black rot (Fusarium solani), aschochyta blight
(Ascochyta fabae) and faba bean necrotic yellow virus (FBNYV) (Dereje and Tesfaye, 1994).
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Fungi including Ascochyta fabae, Botrytis cinerea, Botrytis fabae, Alternaria alternata,
Fusarium spp. and Rhizoctina solani have been found associated with faba bean and among
these, Fusarium spp is the most destructive pathogen of faba bean. Faba bean foot rot and wilts
caused by Fusarium spp. is one of the major seed borne fungal disease of faba bean.
1.1.1 2.2.1. Seed-borne fungi of faba beans

Many faba bean pathogens were found to be seed-borne and seed transmitted. Therefore, several
methods have been developed for testing seeds for associated microorganisms, which were
discussed in various reviews (De Tempe, 1961, 1963, 1964; Agarwal, 1976; Neergard, 1977;
Agarwal and Sinclair, 1987; Gaur and Dev, 1988).
According to Vijendra and James (1987), Ascochyta fabae, Fusarium oxysporum, Botrytis fabae,
Botrytis ricini, Alternaria alternata and Uromyces fabae are considered as seed-borne fungi of
faba beans (Vicia faba. L) all over the world. Gärber, et al (1993) suggested the possibility of
systemic infection with Ascochyta fabae in seed of faba beans. Simay (1998) reported that
Alternaria spp, Fusarium spp,Trichothecium roseum Ascochyta fabae, A. pinodes, Botrytis
cinerea and B. fabae were observed on several seed samples of faba beans and some pathogenic
fungi were also observed.
Kaiser, (1998) reported that Ascochyta blights of faba beans is caused by Ascochyta fabae which
is an important seed-borne pathogen. Neergard (1979) reported that the most common seed-
borne fungi listed on faba bean are: Ascochyta fabae, which causes leaf and pod spot; Botrytis
cinerea, the cause of grey mould; Botrytis fabae, the cause of chocolate spot; Fusarium sp., the
cause of foot rot and wilts; and Rhizoctonia solani, the cause of damping-off of seedlings. Seed
abortion, shrunken seeds, reduction in seed size, seed rot, seed necrosis, seed discoloration,
reduction in germination capacity and physiological alterations in seed are the symptoms caused
by these pathogens.
Abdel-Hafez (1988) isolated Aspergillus, Penicillium, Rhizopus, Mucor and Fusarium from faba
bean seeds. Of the 69 sp. and 4 vars. of pathogens isolated, he found that the most frequent
species were A. niger, A. flavus, A. nidulans, A. terreus, A. flavus var. columnaris, M.
7
chrysogenum, P. citrinum, P. funiculosum, R. stolonifer, M. hiemalis and F. moniliforme
(Gibberella fujikuroi).
Dubey and Patel (2000) reported that Alternaria alternata, the cause of blight of faba bean,
remained viable in diseased plant debris or seeds for over one year. They showed that infected
plant debris, infested soil or infected seeds were the primary pathogen sources for new
infections. Recently, Dubey and Patel (2001) have shown the mode of perpetuation and spread of
Alternaria blight of board beans. Simay (1996) found that the germination of faba beans seeds
was affected less by Fusarium pallidoroseum and most Fusarium oxysporum.
Rauf (2000) isolated 24 fungi belonging to different genera from legume seeds in Pakistan using
the blotter paper method. Alternaria alternata, Ascochyta sp., Colletotrichum sp., Fusarium sp.
and Macrophomina phaseolina were the most frequent isolated fungi, all of which are known as
common pathogens in these legumes.
Moreover, Lenti (1993) reported that Cladosporium herbarum was seed-borne of faba beans in
Hungary. Gärber, et al (1993) suggested the possibility of systemic infection with Ascochyta
fabae in seed of faba beans. Simay (1998) reported that Alternaria spp, Fusarium spp,
Trichothecium roseum Ascochyta fabae, Ascochyta pinodes, Botrytis cinerea and Botrytis fabae
were observed on several seed samples of faba beans and some pathogenic fungi were also
observed.
In Egypt, faba bean was susceptible to a number of fungal diseases (Aspergillus flavus,
Aspergillus niger, Aspergillus ochraceus, Penicillium digitatum, Penicillium italicum, Alternaria
alternata, Botrytis faba, Cephalosporium sp., Cladosporium cladosporioides, Epicoccum nigrum,
Fusarium oxysporum, Fusarium semitectum, Fusarium solani, Fusarium verticillioides
(moniliforme), Rhizoctonia solani, Rhizopus stolonifer, Stemphylium globuliferum,
Trichothecium roseum and Verticillium dahlia) which decrease production and lower the quality
of seeds (M.A. Elwakil, et al. 2009).
Awgechew (1999) reported several fungi associated with seed of local and improved faba bean
varieties collected from Arsi, Bale, Gojam, Gonder and Wwestern Shewa. The major fungi
8
isolated were Ascochyta fabae, Botrytis fabae, Fusarium avenaceum, F. oxysporum and F.
culmorum. Most of these fungi cause major diseases of faba bean in Ethiopia.
2.2.1.1. Black root tot (Fusarium solani)
1.1.1.1.1 2.2.1.1.1. Economic significance

The fungus Fusarium solani has been encountered on a large number of hosts in Ethiopia,
including on faba bean causing black root rot (PPRC, 1998). Black root rot (BRR) is the second
most important disease of faba bean. Complete crop losses could occur in severe infection
conditions and when favorable conditions prevail. Faba bean black root rot can cause complete
crop losses (Negussie et al., 2008). In farmers’ fields, under severe conditions a yield loss of
about 45% -70% was estimated due to this disease (Stewart and Dagnachew, 1967; Habtamu and
Dereje, 1985; PPRC, 1996).
2.1.1.1.1 2.2.1.1.2. Ecology and epidemiology

In Ethiopia, Fusarium solani is the most commonly isolated fungal pathogens causing black root
rot faba bean (PPRC, 1998). The disease exclusively occurs in black clay soils (Vertisols) where
water logging is severe. Water logging is a key factor that predisposes faba bean to this disease.
Since the disease develops slowly, infected plants show chlorosis and dark black roots, which
finally disintegrate. Pulling out of plants with symptoms of BRR becomes easy and the black
discoloration of the whole root is easily observed. Death of the plant follows severe rotting
(Dereje and Tesfaye, 1994a; PPRC, 1996). Foot rot disease may also reduce seed size of faba
bean, and if seeds from severely infected plants are used, emergence can be delayed. The
pathogen of Fusarium avenaceum is survives as mycelium on diseased seeds and infected plant
debris. The symptoms of leaves yellowing and characteristic discoloration of xylem vessels of
the root were observed. This is accompanied by a marked reduction in plant vigor and in severe
cases, by death of the growing tip.
3.1.1.1.1 2.2.1.1.3. Managements black root rot of faba bean

Different management options have been developed to reduce the yield losses in faba bean due
to black root rot in the worldwide. These include the use of chemical fungicides, resistant/
tolerant varieties, use of certain cultural practices such as crop residue management and altering
9
planting date (Dereje, 1999; Bretag and Raynes, 2004; Hawthorne, 2004). Integrated pest
management is an important option for the management of faba bean black root rot disease but
there is weak genetic resistance in cultivars of faba beans to black root rot (Lawes et al., 1983).
1.1.1.1.1.1 2.2.1.1.3.1. Cultural practice

Planting crops that are not hosts of Fusarium solani: noug (Guizotian abyssinica), rapeseed
(Brassica napus) and linseed (Linum usitatissimum) (PPRC, 1996) in rotation with faba bean
may reduce inoculum level in soil and seed. However, it is still not known to what extent would
this inoculum reduction suppresses BRR. The time interval occurring between the repeated
cultivation of faba bean (or another susceptible crop such as chickpea) is not determined, as well.
As water logging is a key factor in predisposing plants to this disease, proper drainage of faba
bean fields is essential to minimize the effect of this disease (Dereje and Tesfaye, 1994).
2.1.1.1.1.1 2.2.1.1.3.2. Biological control

The role of Trichoderma viride in protecting plants from BRR infection has been tested on faba
bean under glasshouse conditions (Tesfaye, 1999). The results of this study suggest that the
biological control agent T. viride can play a role in a strategy for the control of BRR in faba
bean.
3.1.1.1.1.1 2.2.1.1.3.3. Host resistance

Planting of either the released faba bean cultivar Wayu (moderately resistant) or local cultivar
(susceptible) with improved drainage system, broad bed and furrow , reduce black root rot
incidence and thereby increase seed yield (ICARDA, 2006). A group action by all farmers of a
region, however, is essential if adoption of specific cultural practices such as BBF is to help in
disease control. The National Faba bean Improvement Program at HARC and APPRC and
RRCS, made efforts to identify sources of resistance to BRR (Tesfaye, 1995; PPRC, 2003) and
thereby develop BRR-resistant varieties possessing high yield.
4.1.1.1.1.1 2.2.1.1.3.4. Chemical control

Chemical control is widely being used in past and present to cope with black root rot disease.
(Pernezny et al., 2008) observed the effects of six fungicides at four different concentrations
through poisoned food technique. There was a significant decrease in mycelial development of
F. solani pathogen with a raise in fungicidal concentration. Obtaining useful levels of black root
10
rot control with seed-applied fungicides can be considered effective. The fungicides like Thiram
and Apron star offers a little protection against root rot (DZARC, 2005). Seed treatment with
broad-spectrum fumigants such as methyl bromide, chloropicrin, or methyl is thiocyanate both
alone or in mixtures controlled root rot of tomato and increased crop yield. However, application
of fungicides does not always prove economic against soil and seed borne pathogens and it has
led to environmental pollution, pathogen resistance and apparition of new pathogen overcoming
resistant genes and increased risk to human and animal health (Landa et al., 2004). In addition,
excessive use of fungicides creates an imbalance in the microbial community in soil (Li et al.,
2012).
5.1.1.1.1.1 2.2.1.1.3.5. Integrated disease management

Integrated Disease Management (IDM) is defined as ‘‘a sustainable approach to managing
diseases by combining biological, cultural, physical, and chemical tools in a way that minimizes
economic, health, and environmental risks’’. The combination of genetic resistance, hygiene and
monitoring of crops for threshold levels of infestation, allows the most economic and effective
use of chemical controls with the result that economic yields can be maximized. Studies on
integrated black root rot management in Ethiopia indicated that the disease epidemics can be
managed by combining early sowing, spray of Mancozeb and Chlorothalonil fungicide, use of
moderately resistant cultivars and cereal intercropping (Sahile et al., 2008).Different
concentrations of the abiotic inducer (0.3 and 0.5 mMbenzothiadiazole) and the biotic inducer (1
× 107 and 2 × 107 spore/ml Trichoderma harzianum) were used alone or in combination to study
their efficiency against faba bean black root rot disease caused by F. solani and their effect on
some chemical analyses (phenylalanine ammonia lyase activity, total flavonoids and peroxidase
isozymes, pectin and lignin content and total chlorophyll content). Application of the tested
inducers as foliar treatment significantly reduced the severity of black root rot disease as
compared with untreated infected plants. The reduction in disease severity was associated with a
gradual increase in phenylalanine ammonia lyase activity.
11
2.2.1.2. Fusarium wilt (Fusarium oxsporium)

The fungus Fusarium oxsporium is a serious seed-borne pathogen of world-wide distribution
encountered in the survey of seed-borne fungi of faba bean. It has been reported in China (Yu
and Fang, 1948), Japan (Ikata, 1951; Yamamoto et al, 1955), Canada (Coulombe, 1957), Russia
(Dunin, 1962). In Egypt, the causal pathogen of broad bean wilt was identified as Fusarium
oxysporum f.sp fabae (Ibrahim and Abdel Rahim 1965; Sahab 1970). In the Sudan, the causal
organism of faba bean wilt was reported as Fusarium oxysporum (Ibrahim, 1978; Ibrahim and
Owen, 1997). Foot rot of faba bean is also caused by Fusarium avenaceum (Corda ex.Fr.)
Sacc.), reduces seed germination by 23% or dropped from 97.2 to 77.4%, germination energy by
35% or decreased from 79.6 to 64.8% and seedling emergence (stand establishment) by 55%
(Dereje, 1996). With regard to seed-borne Fusarium oxysporum, little has been published in the
literature.
However, Elliot and Crowford (1922) reported that the seed transmission of Fusarium
oxysporum frequently occurs when the fungus propagules such as conidia or chlamydosproes,
are carried as surface contaminants on or in seed that remains after harvest. Naturally infected
seed may carry viable pathogen for at least seven months, serving to carry the organism over
from one season to the next. Simay (1996) found that the germination of faba beans seeds was
affected less by Fusarium pallidoroseum and most Fusarium oxysporum.

The pathogen can survive as mycelium and chlamydospores in seed and soil and also on infected
crop residues, roots and stem tissue buried in the soil for more than 6 years, even in the absence
of the host (Sing et al., 2008). Fusarium oxysporum has been reported as causal of cortical rots,
head blights, leaf spots, root rots, fruit rots, cankers, dieback and vascular wilt disease (Mace et
al; 1981). Fusarium wilt is much more common and destructive in warm temperature regions
and in the tropical and sub-tropical zones, becoming less damaging in colder climates (Agrios,
1988). The symptoms produced by Fusarium oxysporum were described by (Hussein, 1982) and
usually appear two to three weeks after sowing. Infection of symptomless dicotyledonous weeds
can enhance survival of the pathogen in fallow soils. Thus, infested soil is a main source of
12
primary inoculum for the development of Fusarium wilt epidemics in faba bean. Temperature
and pH ranges for mycelial growth of the fungus are 7.5 to 35 0C and 4 to 9.4, respectively. The
optimal condition being 25 to 27.50C and 5.1 to 5.90C depending upon the strain (Navas Cortes
et al., 2007). The optimum conidial concentration to cause infection on the host ranges between
10-12 ml (Duro Almazan, 2000). For a given temperature, isolates of the yellowing pathotype
grow at a higher rate compared with that of wilting isolates. The mycelium is interred and
intracellular hyaline, branched geniculate and septate. The reproduction takes place by asexual
methods only. There are three types of spores; microconidia, macrocondidia and
chlamydospores.
6.1.1.1.1 2.2.1.2.3. Management of Fusarium wilt of faba bean

Management of Fusarium wilt of faba bean is difficult to achieve and no single control measure
is fully effective (Chandar et al., 2016 and Landa et al., 2004). But, developing resistant varieties
is the best alternative to manage this disease. Fusarium wilt of faba bean is a monocyclic disease
in which development is driven by the pathogen’s primary inoculum. Therefore, management of
the disease should be targeted to exclusion of the pathogen as well as by reducing the amount
and efficiency of the initial inoculum (Jiménez-Díaz et al., 2015). Management of soil and seed
borne pathogens with fungicides has been attempted for long time. However, it is difficult to
manage these diseases economically with fungicides alone because of its’ soil and seed borne
nature and wide host range. For such goal, measure of control should include:
6.1.1.1.1.1 2.2.1.2.3.1. Host resistance

Currently, the use of resistant varieties appears to be the most practical and economically
efficient control measure for management of faba bean Fusarium wilt. Resistant faba bean
varieties represent a key component in integrated disease management programs that involve the
use of additive or synergistic combinations of biotic, cultural, and chemical control measures
(Jimenz-Diaz et al., 2011). Development of plant lines resistant to Fusarium wilt is the most
effective approach to the management of the disease. Breeding of resistant lines and
identification of DNA markers for resistance to Fusarium wilt has been achieved in chickpea
(Sharma et al., 2005). It is important to note that in some cases, resistant plant varieties are only
suitable for use against certain Fusarium wilt races (Jiménez-Gasco et al., 2004).
13
7.1.1.1.1.1 2.2.1.2.3.2. Cultural practices
Advancing the sowing date of chickpea crops from early spring to early winter can decelerate the
development of Fusarium wilt epidemics and increase faba bean seed yield (Landa et al., 2004).
Environmental factors such as temperature, nutrients and soil pH can significantly influence the
development of Fusarium wilt diseases, and proper choice of cultural practices that take
advantage of such an influence can contribute to good management of Fusarium. Merkuz et al.,
(2010) reported that Fusarium wilt incidence was reduced with different doses of green manure
and dried plant residue and none of the treatments showed complete disease suppression. This
heating releases biocidal products after microbial degradation of the plant material incorporated
into soil, which together with the anaerobic and strongly reducing soil conditions that develop
are effective against fungal propagules (Kirkegaard, 2009).

There is no commercial biological control agents that directly attack Fusarium pathogens are
currently available. However, potential biological agents have been identified for control of these
Fusarium wilt diseases. Plant growth promoting rhizobacteria such as Pseudomonas and Bacillus
strains can be a suitable approach in plant disease control (Schmidt et al., 2004). These bacterial
genera are the major root colonizers (Manikanda et al., 2010) and increase yield crops based on
diverse mechanisms. Similar results have been found for the control of F. oxysporum, with
Trichoderma spp reducing plant mortality when applied to seed and sown in the field. Numerous
other micro-organisms have been reported as potential bio-control agents of F. oxysporum
including Rhizobium. Trichoderma species are more effective when integrated with moderately
susceptible or resistant cultivars controlled Fusarium wilt by 30–46% (Arfaoui et al., 2007).
However, this method has been given no or little attention in managing faba bean wilt in
Ethiopia.

Faba bean seed treatment with thiram + pentachloronitrobenzene or thiram + carboxin reduced
the incidence of the disease (Sultana and Abdul, 2013). For faba bean obtaining useful levels of
fusarium wilt control with seed-applied fungicides can be considered effective. The fungicides
like Thiram and Apron star offers a good protection against wilt (DZARC, 2005).
14
It is an ecology-based approach aiming minimizing damage caused by diseases through the
combined use of all available disease control measures either simultaneously or in a sequence.
Management of Fusarium wilt in faba bean would be best achieved if those disease control
measures are used within an integrated management strategy whereby their use is combined
either simultaneously or in a sequence (Jimenez-Díaz and Jimenez-Gasco, 2011). Promising
results were observed that combination of effective microorganism, Apron Star, stubble free and
resistant variety become suppressive to Fusarium wilt of faba bean and integration of effective
microorganism, neem seed extracts and resistant variety had significant effects on yield and yield
components (DZARC, 2009). Merkuz and Getachew (2012) reported that raised bed preparation,
tolerant variety and optimum time of planting managed the wilt incidence and reduce mortality
of wilt. Integrated management strategies should include solution to maintain plant health. These
strategies should include minimum use of chemicals for checking the pathogen population,
encouragement of beneficial biological agents to reduce pathogen inoculums, modification of
cultural practices and use of resistance varieties (Moradi et al., 2012).
2.2.1.3. Damping off (Rhizoctonia solani)

The fungus Rhizoctonia solani is common fungal pathogen that cause root rot diseases of faba
beans throughout the world. It is one of the most economically important root diseases of beans.
It has a broad host range that includes most annual and many perennial plants. Sikora et al (2004)
reported that R. solani is one of the most economically important root and hypocotyl diseases in
the world. Rhizoctonia solani can cause seedling damping-off and root rot in bean (Hagedorn &
Hanson 2005) and a number of other major crops including sugarbeet, soybean, cotton, potato,
etc. (Sneh et al. 1991).

Rhizoctonia solani survives between crops as sclerotia or as fungal mycelia in seed and soil.
Young plants are more susceptible to infection than older plants. It has showed a varied
morphological variation in colony color, sclerotia color and size, and distribution pattern of
sclerotia. The mycelia growth was rapid in the majority of isolates. The growth was rapid if on
15
the third day of mycelia has grown reach on Petri dish diameter (90 mm), slow if on the third day
less than 90 mm(M. Jayaprakashvel and N. Mathivanan, 2012). The genus Rhizoctonia is
considered as a complex mixture of filamentous fungi, having in common the possession of a
non-spored imperfect state, usually referred to as the Rhizoctonia anamorph. The Rhizoctonia
anamorph is characterized by several common features present among members of the entire
Rhizoctonia species complex. The phytopathological studies in the complex have represented the
major contingent of contributions in the group, especially in the case of R. solani. Further,
molecular characterization is needed for understanding of biology, physiology and systemic
study. Symptoms on diverse hosts include seed rot, root rot, hypocotyl rot, crown rot, stem rot,
limb rot, pod rot, stem canker, black scurf, seedling blight, and pre- and post-emergence
damping off. Seedling disease symptoms on soybean range from seed rot and pre-emergence
damping off, especially under high inoculums density, to root or hypocotyl rot, depending on
which anastomosis group is present at the time of infection.
9.1.1.1.1 2.2.1.3.3. Management of Damping off- of faba bean

Management of Damping off of faba bean is difficult to achieve and no single control measure is
fully effective (Chandar et al., 2016 and Landa et al., 2004). But, developing resistant varieties is
the best alternative to manage this disease. Damping off-of faba bean is a monocyclic disease in
which development is driven by the pathogen’s primary inoculum. Therefore, management of the
disease should be targeted to exclusion of the pathogen as well as by reducing the amount and
efficiency of the initial inoculum (Jiménez-Díaz et al., 2015). Management of soil and seed
borne pathogens with fungicides has been attempted for long time. However, it is difficult to
manage these diseases economically with fungicides alone because of its’ soil and seed borne
nature and wide host range. For such goal, measure of control should include:
11.1.1.1.1.1 2.2.1.3.3.1. Host resistance

Currently, the use of resistant varieties appears to be the most practical and economically
efficient control measure for management of faba bean Damping off. Resistant faba bean
varieties represent a key component in integrated disease management programs that involve the
use of additive or synergistic combinations of biotic, cultural, and chemical control measures
(Jimenz-Diaz et al., 2011). Development of plant lines resistant to Damping off is the most
effective approach to the management of the disease. Breeding of resistant lines and
16
identification of DNA markers for resistance to Damping off has been achieved in chickpea
(Sharma et al., 2005). It is important to note that in some cases, resistant plant varieties are only
suitable for use against certain Damping off races (Jiménez-Gasco et al., 2004).
12.1.1.1.1.1 2.2.1.3.3.2. Cultural practices

Advancing the sowing date of faba bean seeds from early spring to early winter can decelerate
the development of Damping off epidemics and increase faba bean seed yield (Landa et al.,
2004). Environmental factors such as temperature, nutrients and soil pH can significantly
influence the development of Damping off diseases, and proper choice of cultural practices that
take advantage of such an influence can contribute to good management. Merkuz et al., (2010)
reported that Damping off incidence was reduced with different doses of green manure and dried
plant residue and none of the treatments showed complete disease suppression. This heating
releases biocidal products after microbial degradation of the plant material incorporated into soil,
which together with the anaerobic and strongly reducing soil conditions that develop are
effective against fungal propagules (Kirkegaard, 2009).

Application of the fungicides is not economical in the long time because they pollute the
environment, leave harmful residues and can lead to the development of resistant strains of the
pathogen with repeated use (Vinale et al., 2008). Replacement of fungicides with bio-control
agents is an alternative mean to; manage the plant pathogens, produce safety food and reduce the
environment pollution (Barakat and Al-Masri, 2005). One of the most important bio-control
agents is Trichoderma spp. that the most frequently isolated soil fungi and present in plant root
ecosystems (Harman et al., 2004). The antagonistic activity of the genus Trichoderma to R.
solani has been widely demonstrated (Lewis et al., 1998). Trichoderma harzianum protected the
bean seedlings against pre-emergence damping off infection, reduced the disease severity and
increased the plant growth in the presence of R. solani pathogen (Paula et al., 2001). El-Kafrawy
(2002) reported that the Trichoderma spp. inhibited the radial mycelial growth of R. solani in
vitro test from 59.6 to 78.4 %. Seed treatment with Trichoderma spp. gave the maximum
protection against pre- and post-emergence Gonzalez et al. (2005) showed that the field
application with T. viride and/or T. harzianum as soil application gave the same effectiveness
(99%) against R. solani pathogen, comparing with the seed immersion. Seed amendments with
17
T. harzianum significantly increased the heights and weight of plants and significantly reduced
the R. solani infection (Malik et al., 2005). Application of T. harzianum as seed treatment
significantly reduced the incidence of damping-off diseases some leguminous crops, i.e. faba
bean, lentil, and chickpea, when planted in a soil naturally infested with R. solani (Abou-Zeid et
al., 2003). Trichoderma spp., as seed dressing, improved the seedling emergence and health of
runner bean (Pieta et al., 2003). Seeds of common bean were dressed, prior to sowing; with
conidia of T. harzianum protected the germinating seedlings and plants against infection by soil
and seed borne pathogenic fungi, i.e. Fusarium spp. and R. solani (Pieta and Pastucha, 2004).

Chemical control is widely being used in past and present to cope with Damping off disease.
Subhani et al., (2011) observed the effects of six fungicides at four different concentrations
through poisoned food technique. There was a significant decrease in mycelial development of
Rhioctonia solani pathogen with a raise in fungicidal concentration. Obtaining useful levels of
Damping off control with seed-applied fungicides can be considered effective. The fungicides
like Thiram and Apron star offers a little protection against wilt (DZARC, 2005). Seed treatment
with broad-spectrum fumigants such as methyl bromide, chloropicrin, or methyl is thiocyanate
both alone or in mixtures controlled Damping off of tomato and increased crop yield. However,
application of fungicides does not always prove economic against soil and seed borne pathogens
and it has led to environmental pollution, pathogen resistance and apparition of new pathogen
overcoming resistant genes and increased risk to human and animal health (Landa et al., 2004).
In addition, excessive use of fungicides creates an imbalance in the microbial community in soil
(Li et al., 2012).

It is an ecology-based approach aiming minimizing damage caused by diseases through the
combined use of all available disease control measures either simultaneously or in a sequence.
Management of Damping off in faba bean would be best achieved if those disease control
measures are used within an integrated management strategy whereby their use is combined
either simultaneously or in a sequence (Jimenez-Díaz and Jimenez-Gasco, 2011). Promising
results were observed that combination of effective microorganism, Apron Star, stubble free and
resistant variety become suppressive to Damping off in faba bean and integration of effective
18
microorganism, neem seed extracts and resistant variety had significant effects on yield and yield
components (DZARC, 2009). Merkuz and Getachew (2012) reported that raised bed preparation,
tolerant variety and optimum time of planting managed the wilt incidence and reduce mortality
of wilt. Integrated management strategies should include solution to maintain plant health. These
strategies should include minimum use of chemicals for checking the pathogen population,
encouragement of beneficial biological agents to reduce pathogen inoculums, modification of
cultural practices and use of resistance varieties (Moradi et al., 2012).
2.1.1 2.2.2. Botanicals for disease managements

Today, there is a growing movement in many countries to reduce the amount of chemicals being
released into the environment. Among the notable hazards of misuse and overuse of pesticides
are the induction of resistance in plant pathogens, occupational hazards, environmental pollution,
destruction of natural enemies (ecological hazards) and residues in food which are poisonous
and/or carcinogenic, mutagenicity, genetic damage and teratogenicity, acute and chronic residual
toxicities and severe and acute mammalian toxicities.
Keeping in view the draw back of chemical management of plant disease the use of plant extracts
and Trichoderma species in the management of plant disease is gaining importance (Makovitzki
et al., 2007, Okereke et al., 2007, Joseph et al., 2008). Both Plant extracts and Trichoderma
species have played significant role in the inhibition of seed-borne fungal pathogens and in the
improvement of seed quality and field emergence of plant seeds. Contrary to the synthetic drugs,
antimicrobials of plant origin and Trichoderma species are not associated with many side effects
and have an enormous therapeutic potential to heal many infectious diseases (Patel, 2007).
Several plant families like Acanthaceae, Amranthaceae, and Magnoliaceae are known for their
antifungal properties (Ashraf and Javaid, 2007). Plants are rich in a wide variety of secondary
metabolites such as tannins, terpenoides, alkaloids and flavonoid, which have been found in vitro
to have antimicrobial properties. Extracts of many plants are now known to exhibit antimicrobial
activity (Dewanjee et al., 2007, Rani et al., 2008). Although there is a growing interest in the use
of medicinal plants to control plant diseases, only about 2,400 plant species among more than
250,000 higher plants have been screened for phytoactivity (Nduagu et al., 2008).
19
2.2.2.1. Garlic (Allium sativam)
Garlic has anti-feedant (insect stop feeding), anti-bacterial, fungicidal, insecticidal,
nematicidaland repellent properties. Among the natural fungicide substance, garlic (Allium
sativam) extract has been found active in various trials in vitro and to a less degree in vivo.
Allium sativam L. show highest inhibition on germination of Phytophthora infestance. The
inhibition rate on germination of sporangia and zoospores increased with increasing
concentration. Garlic extract from fresh bulbs had stronger inhibiting effect against the
germination of sporangia and zoospores. However, there was no significance difference between
the extract of immediately made and those which was kept for a week under 4 oc (Ke-Qiang,
2001). The turmeric and garlic extracts were most effective in inhibiting the growth of Fusarium
spp. (Patel and Vala, 2004; Assadi and Behroozin, 1987). Garlic clove extract and neem leaf extract
were most effective for the growth inhibition of F. solani (Mallesh et al., (2009).
2.2.2.2. Aloe vera

Aloe vera belongs to the family of Asphodelaceae (Liliaceae) and is a xerophytic, succulent,
shrub plant. It is found generally in Africa, America and Europe. In India it is grown in
Rajasthan, Gujrat, Tamil Nadu, Maharashtra and Andhra Pradesh (Surjushe et al., 2008). Aloe
vera contain over 75 nutrient and 200 active compounds, including vitamins, enzymes, minerals,
sugar, lignin, anthraquinones, saponins, salicylic acid and amino acids ( Park & Jo, 2006,
Shelton, 1991). Aloe vera has been used in herbal medicine all over the world since ancient times
and helps in skin irritation, cuts, skin burning, skin swellings, facial wrinkles, damaged skin cell
repairing and antimicrobial (Rajeswari et al., 2012). Aloe vera inhibits the growth and
development of fungal pathogen such as Candida (Pathak and Sharma, 2017, Christaki and
Florou-Paneri, 2010). Aloe vera was evaluated on Rhizoctonia solani, Colletotrichum coccodes
and Fusarium oxysporum fungi. It was seen to successfully inhibit the growth of these fungal
species (Sahu et al., 2013). An in vitro study of Aloe vera gel performed antifungal activity at
higher concentration also showed that it can be used as an antifungal agent (Jain et al., 2017).
The antifungal activity of crude extract of Aloe vera inhibited Candida albicans and Fusarium
oxysporum (Waithaka et al., 2018).
20
2.2.2.3. Turmeric (Curcuma longa L.)
Curcuma (Curcuma longa L.) belongs to the family Zingiberaceae and is a popular spice with a
strong taste and remarkable yellowish color which has been increasingly interesting considering
its possible to replace synthetic preservatives for containing compounds, such as curcumin, with
high antifungal and antimicrobial activities. It is largely cultivated in Asian countries and not
restricted only to its use as food, but also present in many different areas of industry, medicine,
and agriculture (Vilella & Artur, 2008). The properties of curcuma are related to both the
prevention and treatment of diabetes, cancer, and inflammatory processes, as mentioned by
Tripathi and Misra (2016), in addition to degenerative processes such as osteoarthritis (Chin et
al., 2016), antimicrobial activity against Staphylococcus aureus (Teow et al., 2016), Exserohilum
turicicum, Fusarium oxysporum, Colectrotrichum cassiicola. Pseudomonas aeruginosa, Bacillus
subtilis, Escherichia coli, Candida albicans (Petnual et al.,2010) and Aspergillus flavus (Hu et
al., 2015).
Curcumin content was determined in the rhizome powder through the adapted method described
by Fulekil & Francis (1968). Total curcumin content was expressed in percentage. Research by
Kumar et al. (2016) used curcuma essential oil at 2.45 mg/ml with activity inhibitory on the
growth of Fusarium graminearum.
2.2.2.4. Tobacco (Nicotina tabacum)

Tobacco (Nicotiana tabacum) is an herbaceous perennial plant, native to tropical and subtropical
America and cultivated worldwide (Rukangira E, 2001). Plant extracts have been found useful
and less harmful to man and animals as well as the environments reports (Ma.WW.Anderson
et.al.1989) has shown that compounds from plant sources are moderately toxic and are suitable
as fungicides. The antifungal activity of crude extracts of Tobacco leaf showed complete
inhibition growth of Aspergillus viridae and Pencillium digitatum and progressive retardation on
Rhizopus vegetative growth (M.N. Suleiman, 2011). Historical successes recorded in the use of
nicotine from tobacco plant (Nicotiana tabacum) as biopesticides have spurred scientists to
search for alkaloids, flavonoids, terpenoids and other secondary compounds, as bio-pesticides
which are like to prove effective and environmentally safe. Tobacco Plant (Nicotiana tabacum)
was reported to be used for the control of plant diseases being ecologically friendly (Olufolaji,
21
1999). Study also showed that, tobacco as medicinal plant, posse’s potential antifungal properties
which inhibit fungal mycelila growth of Fusarium oxsporium of sunflower wilt under in vitro
condition (A.Keerio et. 2017)
2.2.2.5. Nicandra physalodes

1 Nicandra physalodes belongs to the family of solanaceae. The literature survey of the plant
possessed various pharmacological activities like anti-diabetic, diuretic, antimicrobial etc. It
also shows in-vitro cyto-toxic activity. Traditionally it is used as Analgesic, anthelmintic,
antibacterial, anti-inflammatory and febrifuge and its regular use also increases bodily
vigour. It is also used in the treatment of contagious disorders, toothache, and intestinal pain
from worms and in impotence. On the basis of their traditional use and scientific
investigation, this plant is considered to be very important according to medicinal point of
view. Methanolic and aqueous extract of leaves and roots was active against fungi, Candida
albicans and Aspergillus flavus. The Nicandra physalodes extract, both methanolic and
aqueous showed growth inhibition for fungi, Candida albicans and Aspergillus flavus
(Mann As, et.al. 2008). It’s responsible for the in- vitro cyto-toxicity of Nicandra
physalodes extracts activity has also been reported (Devi P, et.al. 2010).
2.2.2.6. Maytenus senegalensis

Maytenus senegalensis which belongs to the family of Celastraceae (Bekele Tasemma,2007).
Different plant parts of this species are largely used in traditional medicinal for infectious and
inflammatory diseases treatment. Several studies have been reported for this species. The leaf is
used in East Africa to treat different dieases such as inflammations, respiratory diseasis and
sores, (http;//www.metafro.be/prelude). Tritepenes and phenol compounds were dectected on the
leaf and invitro antiplasmodial, antileishmanial and antibacterial activities were reported for this
part of the plant (El.Tahir A, et.al. 1999 and Hussein, G, et al.1999). Leaves of Maytenus
senegalensis used to treat toothaches, in India (Mueller M. and Mechier E., 2005).
2.2.2.7. Phytolacca dodecandra

Phytolacca dodecandra which belongs to the family of Phytolaccaceae, it is commonly called
African soapberry plant P.dodecandra locally called ‘’Edndod’’ produces a series of triterpenoid
22
saponins that possess very potent and use full biological properties including anti-fungal, anti-
protozoan, spermicidal and insecticidal properties (Essar, Semagn and Wolde-yohannes, 2003).
Previously, Tadag, Mohommed, Asres and Gebre-mariam(2005) used P.dodecandra extracts to
evaluate their efficacy against same human bacterial and fungal strains causing skin infections
and should that this plant hand antimicrobial property.
3.1.1 2.2.3. Bio-agants for disease managements

The use of biological control method using four Trichoderma species: Trichoderma harzianum
(T1), Trichoderma longibrachiaium (T14), Trichoderma atroviride (T5), and Trichoderma
Virence (T13) is generally favoured as a method of plant disease managements. Biological
control method has been preferred in some cases because it is selective with no side effect and is
cheap. Babu et al. (2000) evaluated the efficacy of six Trichoderma species on early blight of
tomato. Among the six species, T. harzianum exerted the highest inhibition of the mycelial
growth (50.22%) of the pathogen over control followed by T. viride.
The antagonistic activity of the genus Trichoderma to F. solani and R. solani has been widely
demonstrated (Lewis et al., 1998). Trichoderma harzianum protected the bean seedlings against
pre-emergence damping off infection, reduced the disease severity and increased the plant
growth in the presence of R. solani pathogen (Paula et al., 2001). El-Kafrawy (2002) reported
that the T. harzianum, Trichoderma hamatum, Trichoderma pseudoknonningii and Trichoderma
polysporum inhibited the radial mycelial growth of R. solani in vitro test from 59.6 to 78.4 %.
Serious soil and seed borne pathogens, i.e. Pythium debaryanum, P. ultimum, P. dissotocum, P.
oligandrum, P. violae, P. aphanedermatum, Macrophomina phaseolina, Rhizoctonia solani,
Fusarium solani, F. semitectum, Aphanomyces euteiches, Sclerotinia sclerotiorum and many
species of Verticillium and Cladosporium., can cause damping-off and root rot diseases,
resulting in great economic losses in crop yield and quality.
The use of fungicides for control of these pathogens has met moderate success and their future
use is in question due to increased regulatory restrictions. So, the modern approach in plant
disease control is directed toward minimizing the fungicidal use to decrease environmental
pollution and finding alternatives to chemical fungicides. Hence, in recent time application of
23
plant extracts as well as plant metabolites for plant disease management has become important
viable component of Integrated Pest Management, as plant metabolites are eco-friendly where
botanicals place an important role (Sahayaraj et al., 2009). Several investigation studies have
been conducted in order to screening different plants for their antifungal properties (Stephan et
al., 2005 and Satish et al., 2010) and biochemical compounds that these plants have. Studies
revealed a highly significant antifungal activity of some water extracts or essential oils of plants.
Since some plants are already known to possess several biological activities (Amin et al., 2009
and Belabid et al., 2010).
24
CHAPTER THREE
3. MATERIALS AND METHODS
9.1 3.1. Description of the study area

The seed samples were collected from major faba bean growing kebeles of of Ambo district and
the laboratory study was conducted in Ambo University College of Agriculture and Veterinary
Science. Ambo is located 120 km west of Addis Ababa at 8 098’ South latitude and 37083’ North
longitude. It has a total geographical area of 83,598.69 sq. km.,with elevation ranging from
1380-3300 m. a. s. l. Annual rainfall ranged from 900-1100 mm and temperature ranged from
10-270C, with an average of 180C. The soil type of the study site is vertisol with a pH value of
6.8 (EARO, 2004).
10.1 3.2. Seed Sample Collection

A total of 50 faba bean seed samples were collected in polythene envelopes from different
farmers’ saved seeds of five major faba bean producing kebeles namely; Golja, Jijigu Weransa,
Ya’i Cabo, Kure Gatira and Dase Akililo in Ambo woreda with consults of the district
agricultural office and brought to the Plant pathology laboratory of the Ambo University Gudar
Campus for isolation identification and further test. Purposive sampling method was used from
selected kebele’s according to (ISTA, 2008) sampling procedure. Each sample was enclosed in
paper bags with proper labeling, taken to the laboratory and then kept in refrigerator at 40C for
further consecutive studies.
11.1 3.3. Isolation of fungi from faba bean seeds

Four hundred faba bean seeds were randomly taken from each seed samples for mycoflora study.
Four replicates of 100 seeds per sample were surface sterilized using 0.5% sodium hypochlorite
(NaOCl) solution for 10 minutes with constant agitation. Seeds were rinsed three times in sterile
distilled water for a minute then placed on sterile what man filter paper to dry and inoculated 10
seeds per Plate aseptically in (90 mm diameter) Petri dish containing PDA using a sterile pair of
forceps.
25
Ten of such Petri dishes were plated with 100 seeds of faba bean to represent the replicates of a
treatment and these were arranged in completely randomized design. Incubate all the petri-dishes
at 25+20C for 7days under alternating cycles of 12 hours near Ultra-Violet (UV) light and 12
hours darkness for sporulation of fungi. After incubation the growth characters as well as
percentage of infection were recorded. The isolated colony culture of fungi was maintained on
Potato Dextrose Agar (PDA) medium. The fungi were identified after reference to Booth (1971),
Barnett and Hunter (1972), and Nelson et al. (1983).
12.1 3.4. Determination of seeds infected with fungi

Precentage of seeds with fungal infection were determined by counting the infected seeds and
dividing by the total number of seeds and expressed as a percentage. Thus;
Percent of seeds infected= No of seed infected with fungi x 100
Total no of seed per plate
A colonies obtained from each infected seed were subjected to cultural and microscopic
morphological characterization for identification. Occurrence of fungi was determined by
counting the number of times each individual fungus occurred divided by the total number of
fungi and expressed as a percentage. Thus;
Percentage occurrence of fungi= No of times each fungi occurred x 100
Total no of fungi per plate
13.1 3.5. Identification of fungi

The identification of the fungi was based on the cultural characteristics, mainly on the growth
patterns and pigmentation. Further microscopic examinations were carried out for mycelial and
conidia structures based on using identification manual of illustrated genera of fungi (Barnett &
Hunter, 1972 and Watanabe, 2002). The morphological characteristics was carried out by taking
small amount of mycelium from ten days old pure culture plates using a sterile needle and
transferred on to a cleaned glass slide. The culture was taken from five different positions of the
26
culture to plate, four from adjacent side and one from middle. The mycelium was stained with
0.1 % lacto phenol cotton blue and observed under compound microscope.
14.1 3.6. Determination of effect of seed borne myco-flora on

seed germination
The effect of seed myco-flora on seed germinations were tested by blotter method as
recommended by (ISTA, 2008). In this method, three pieces of filter paper were soaked in
sterilized distilled water and placed at the bottom of 90 mm diameter Petri dish. One hundred
seeds were taken randomly from each sample and then placed on the moist filter paper at the rate
of 10 seeds per Petri dish. The Petri dishes were then incubated at 25+2 0c for seven days under
12 hour alternating cycle of light and darkness. The seeds were kept moistened by adding (about
3ml) sterilized distilled water every two days throughout the incubation period, examine the seed
germination and seed borne fungal infections grown on seeds were recorded. Germ inability of
the seeds was determined by visual observation. Seed germination was assessed by counting the
seeds with seed leaf and dividing by the total number of seeds in the Petri plates expressed as a
percentage. Thus;
Percentage germination= Number of seed germinated x 100
Total no of seed per plate
15.1 3.7. Determination of effect of seed borne myco-flora on

disease transmission
To examine seed to seedling transmission, seedling bioassay were carried out in the growth
chamber. The sand used in the studies were sterilized in autoclave at 121°C and pressure of 15
lb; for one hour after which it was ready for use and allowed to dry. One hundred seeds were
randomly taken from each sample, were surface sterilized by soaking in 0.5% sodium
hypochlorite (NaOCl) solution for 10 min with constant agitation followed by rinsing in three
changes of sterile distilled water for a minute and then dried between two layers of sterilized
what man filter paper. Hundred treated seeds were placed in a tray containing sterilized sand
aseptically by using a sterilized pair of forceps at equidistance to avoid cross contamination and
27
then placed in growth chamber at 28 + 20C in alternating cycles of 12 hours darkness and 12
hours light. The seeds were kept moistened by adding (about 3ml) sterilized distilled water every
two days throughout the planting period. The progress of the disease and symptoms development
were monitored every day in the plated plants and disease data were recorded. Finally, the
seedling infection percentages per plate were calculated by the formulae described by Lević et
al., (2011).
Seedling infection percentages = No. of seedlings affected by a pathogen x 100
Total number of seed sown
The incidence of the fungi in coleoptiles and root tissues of seedlings were determined on the 7 th,
14th and 21st day after plating in sterilized sand. In addition, progress of the disease and
symptoms developed were monitored in the plated plants. Transmission efficiency (TE) of fungi
from seeds to seedling was estimated from the incidence data with the following formula:
TE = C X 100
Notice: TE is transmission efficiency of fungi from seeds to seedling
C is infection percentage of seedlings by fungi in the transmission study
S is seed infection percentage of the by fungi during laboratory assay on agar.
16.1 3.8. In vitro assay for the management of seed borne

pathogens
In vitro antimicrobial assay of the plant extracts and the bio-agents were done in plant pathology
laboratory of Ambo University College of Agriculture and Veterinary Science.
28
4.1.1 3.8.1. Evaluation of botanical plants
3.8.1.1. Collection of botanical plant materials

The fresh aerial and under graund plant parts of Garlic clove (Allium sativum L.), Kombolcha
leaf (Maytenus senegalensis), Argissa (Aloe vera gel.), Abshoo leaf (Nicandra physalodes),
Tabacoo leaf (Nicotiana tabacum), Turmeric rhzome clove (Curcuma longa L.) and Endod seed
(Phytolacca dodecandra) used in this study were collected during the vegetative stage in
December 2019.
Allium sativum and Maytenus senegalensis were collected from Ambo woreda and Amaro areas.
Aloe vera, Nicandra physalodes and Nicotiana tabacum were collected from Toke Kutaye
woreda and Guder areas. Curcuma longa were collected from Weast Wellaga Zone (Gimbi),
Phytolacca dodecandra were collected from Jibat state forest, Weast Shewa as shown in (Table
1). The samples were separated in to its selected parts (leaf, clove and seed) washed thoroughly
under tap water followed by sterilized distilled water, cut in to smaller size of about 1-3cm long
and air-dried under shade at room temperature for 1 to 2 weeks and then pounded using sterile
mortar and pistil in to fine powder and kept in refrigerator at 40C until use (Selvaraj and
Narayanasamy, 1993, Singh et al., 2007).
Table 1: plants used for antifungal activities assay in the present study
S Plant used for the experiment Pathogen

No inTypes of
test
Local Name Scientific Name Part used Local use
1 Garlic Allium sativum L. Clove Malaria Anti-fungal
treatment
2 Kombolcha Maytenus Leaf Diarrhea, Anti-fungal
senegalensis worms
3 Argissa Aloe vera Gel Antibacterial Anti-fungal
(keesoo)
4 Abshoo Nicandra Leaf Liver problem Anti-fungal
physalodes
5 Tabccoo Nicotiana tabacum Leaf Chewed, Anti-fungal
Smoked
6 Irdii Curcuma longa L. Rhzome Healing wound Anti-fungal
29
(Turmeric)
7 Endod Phytolacca Seed Assoap, Rabies, Anti-fungal
dodecandra Malaria vector
3.8.1.2. Preparation of the plant aqueous extract

First, crude plant leaf extract was obtained by socking/infusing 50 g of each air-dried powdered
plant material were soaked in 500 ml hot and cold distilled water separety to give (1:10 w/v) in a
1000 ml conical flask and kept on rotary shaker for 30 min. Extraction then took place under
cold conditions for 24 hours (Rivillas-Acevedo and Soriano-Garcia 2007). After that, plant
extracts were filtered through double layers of muslin cloth and followed with Whatman No 1
filters paper. Finally, the aqueous extract at a concentration of 10% was used as an original
concentration in the antifungal activity test and stored in air tight bottle in refrigerator for further
usage (Naduagu, et al., 2008 and Rani, et al., 2008).
3.8.1.3. Effect of plant extracts on mycelium growth of test fungi

Seven plant parts (leaf, clove, gel and rhzome seed) were evaluated against mycelial growth of
seed borne test fungi Fusarium solani, F.oxsporum and R. solani by using poisoned food
technique, These plant extracts were used at 5, 10 and 20% concentrations for which 5, 10 and
20 ml of stock solution was mixed with 95, 90 and 80 ml of sterilized molten PDA media. The
medium was thoroughly shaken for uniform mixing of leaf extract. 20 ml of agar media was
poured into sterile petriplates and allowed to solidify. Five mm of agar disk of test fungi were cut
from 7days old culture plate by using sterile cork borer and placed in the centre of petri-plate
containing different concentration of plant extract. The experiment was conducted in complete
randomized design (CRD), with 87 treatments and 3 replications for each test pathogen. The
petriplates containing only PDA medium without plant extracts serve as control. All these
inoculated Petri plates were incubated at 25+20C for seven days and the data of mycelial growth
of the fungus was recorded after 24 hours of inoculation till 8 days of inoculation. The
percentage inhibition of mycelial growth was calculated as per formula given by (Vincent, 1947)
%I = (C-T) X 100
30
Where: - I: Percent inhibition of test fungi
C: Average increase in mycelial growth in control plate and
T: Average increase in mycelial growth in treatment plate.
5.1.1 3.8.2. Evaluation of bio-agents
3.8.2.1. Source of Pure cultures of Trichoderma isolate

Purified culture of antagonistic Trichoderma isolate Trichoderma harzianum (T1), Trichoderma
longibrachiaium (T14), Trichoderma atroviride (T5), and Trichoderma Virence (T13) used in
the this study were obtained from Plant Pathology Department of Ambo University College of
Agriculture and Veterinary Sciences, was cultured by (Amin Mohammad, 2019), Stock cultures
of Trichoderma isolate were contain the pertinent information regarding how they were isolated
from natural environment and maintained on Petri dishes contain PDA medium in a refrigerator
at 40C for subsequent use.
3.8.2.2. Preparation of Pure culture Trichoderma isolate

Stock cultures of Trichoderma isolate were re-cultured in sucrose peptone broth (SPB) for
multiplication, as recommended by Kumar (2004). The propgules (colony forming unit, cfu)
suspension of each Trichoderma harzianum (T1), Trichoderma longibrachiaium (T14),
Trichoderma atroviride (T5), and Trichoderma Virence (T13) was prepared in sterile distilled
water from 7-days-old-culture on PDA (Rojo et al., 2007). The fungal inoculum was harvested
by flooding the culture with SDW and then rubbing the culture surface with a sterile glass rod.
The fungal propgules concentration in each suspension was determined by counting using a
haemocytometer slide and adjusted at 108 cfu / ml and used in dual culture test.
3.8.2.3. Effects of Trichoderma spp. on mycelium growth of test fungi

Antagonistic effect of Trichoderma harzianum (T1), Trichoderma longibrachiaium (T14),
Trichoderma atroviride (T5), and Trichoderma Virence (T13) against isolate test fungi of
Fusarium oxsporum, Fusarium solani and Rhizoctonia solani were evaluated by using dual
culture technique as described by (Rahman et al. 2009, Coskuntuna and Özer, 2008, Dhingra and
Sinclair, 1995). All the isolate test fungi and Trichoderma spp.were grown on sterilized standard
31
PDA (Potato Dextrose Agar) at 25+20C in an incubator for 5 days, separetly, in order to obtain
juvenile colonies for the studies of antagonism. After the incubation period of 5 days, five
millimeter diameter mycelial plugs of each isolated test fungi were cut by using sterile cork borer
and placed at the periphery of culture plates amended with tetracycline (0.1g/L) by leaving 2 cm
away from the edge side of the Petri dish and on the same day Antagonist Trichoderma
harzianum (T1), Trichoderma longibrachiaium (T14), Trichoderma atroviride (T5), and
Trichoderma Virence (T13) were placed with equal distance on the opposite side of the same
previous petri- dishes , separately. For each treatment, three replications were used. Control plate
was maintained by inoculating the medium with the pathogen only. The plates were incubated at
25±2°C in an incubator (Morton & Stroube, 1955). The growth of the pathogen in both the test
and control experiments was recorded. The percent inhibition of the isolated test fungi was
calculated by using the following formula of Vincent (1947).
%I = (R1-R2) x 100
R1
Where, I: Percent inhibition of test fungi
R1: The Radial colony growth of test fungi in control plate (mm)
R2: The Radial colony growth of test fungi in Dual culture plate (mm)
And the width of zone of inhibition (ZI) measured as the smallest distance between the colonies
in the dual culture plate (Royse and Ries, 1977; Whips 1987; Reddy and Hynes 1993).
17.1 3.9. Data Analysis

Experiments were conducted in completely randomized design (CRD). All Data obtained from
this study was subjected to analyzed by using Analysis of Variance (ANOVA). Treatment
means were separated by Least Significant Difference (LSD) at p=0.05 and Fishers tests.
32
33
CHAPTER FOUR
4. RESULTS AND DISCUSSION
18.1 4.1. Results
6.1.1 4.1.1. Isolated fungi from faba bean seed samples

Seed borne mycoflora of faba bean was tested by using agar plate method as recommended by
ISTA. Of the 50 samples of farmer’s saved seed samples were collected from the five major faba
bean producing kebele’s in Ambo District, a total of 7 fungal species Fusarium oxysporum,
Fusarium solani, Aspergillus, Botrytis, Penicillium, Rhizoctonia solani and Alternaria belonging
to 6 fungal genera were Fusarium oxysporum, Fusarium solani, Aspergillus, Botrytis,
Penicillium, Rhizoctonia solani and Alternaria were isolated and identified.
7.1.1 4.1.2. Determination of seeds infected with fungi

Among the isolated fungi F.oxysporum, F.solani and Aspergillus spp. Have the maximum
incidence was recorded from all locations in this experiment (12.92%, 12.33% and 9.77%)
respectively, followed by Penicillium (8.52%) and Botrytis spp. (6.47%) and the minimum
incidence were of Rhizoctonia solani (6.39%) and Alternaria (6.34%). Samples from Kure
Gatira kebele showed maximum infection percentage (88.25%), whereas; samples collected from
Golja kebele showed minimum infection percentage (31.25%) (Table2).
34
Table 2. List of Fungi Isolated from 50 Faba Bean Seed Samples by agar plate method
(Drown 400 seeds from each sample were used in the experiments)
PAS Seed Seed infection Percentage infection Total Infecte Germ
Sampl seed d seed inatio
e infecti (%)ag n
Code on e (%)a
ge
F.oxs F. Aspeg Peneci Botry Rhizo Altern
p solan il llum tis ctonia aria
orum i us spp. spp. spp. spp. spp.
G olja
GF001 62 23 20 0 0 16 38 167 41.75 93

GF002 14 16 29 0 51 15 0 125 31.25 96
GF003 56 50 0 29 15 35 20 205 51.25 100
GF004 68 20 0 46 0 20 58 212 53 96
GF005 43 50 60 0 0 0 0 153 38.25 93
GF006 62 61 32 0 20 0 0 175 43.75 98
GF007 42 52 20 53 58 0 50 275 68.75 94
GF008 43 62 21 0 57 49 27 259 64.75 100
GF009 17 60 27 0 60 23 20 207 51.75 100
GF001 62 53 0 39 43 38 52 287 71.75
0 100
Jijigu Weransa
JF001 48 40 0 30 58 60 48 284 71
1 75
JF001 55 26 0 52 60 0 0 193 48.25
2 100
JF001 39 26 40 0 38 61 54 258 64.5
3 84
JF001 52 18 38 46 51 0 0 205 51.25
4 100
JF001 45 20 30 29 30 0 20 174 43.5
5 84
JF001 62 39 30 45 59 0 67 302 75.5
6 100
JF001 67 30 30 20 0 0 26 173 43.25
7 70
JF001 38 32 60 60 60 0 50 300 75
8 100
JF001 32 44 71 40 51 40 62 340 85
9 100
JF002 54 53 0 55 53 0 67 282 70.5
0 75
Ya’i Cabo
YF002 72 45 39 60 30 0 39 285 71.25

1 78
YF002 55 55 45 30 44 0 0 229 57.25
2 96
YF002 46 57 46 0 0 66 0 215 53.75
3 94
YF002 52 60 30 51 0 0 0 193 48.25
4 100
YF002 24 52 56 30 57 0 58 277 69.25
5 95
YF002 52 45 62 68 10 20 58 315 78.75
6 76
YF002 72 56 0 75 0 63 20 286 71.5
7 78
YF002 42 46 38 0 65 58 54 303 75.75
8 75
YF002 30 52 68 36 0 20 0 206 51.5
9 94
YF003 69 52 74 48 36 68 0 347 86.75
0 64
Kure Gatira
KF003 72 60 74 60 0 0 70 336 84
1 100
KF003 31 51 57 0 0 56 0 195 48.75
2 68
KF003 74 68 0 56 0 0 0 198 49.5
3 75
KF003 73 60 50 63 16 58 0 320 80
4 67
KF003 72 63 74 0 68 0 0 277 69.25
5 83
KF003 55 70 72 53 0 30 0 280 70
6 76
KF003 56 72 55 44 0 50 29 306 76.5
8.1.1
700 F.oxysporum
F.solani
600 Aspegillus spp.

Penecillum spp.
In fectedseeds (% )
Botrytis spp.
500 R. solani
Alternaria spp.
400
300
200
100
0
Golja Jijigu Weransa Ya’i Cabo Kure Gatira Dase Akililo
Five major faba bean producing kebeles
Figure 3 Infected seed (%) of faba bean seeds by major seed borne fungi
9.1.1 4.1.3. Identification of fungi based on morphological and cultural

characteristics
In this study, the morphological and cultural characteristics of fungal pathogens commonly
causes root rot and damping off disease of faba bean were observed on PDA media culture plates
as follows. Vvisual and microscopic observation was used to characterize the selected colony
cultures. Details of the hypha and spore characteristic of fungi are noted (Table 3). The above
results gave an idea of morphology and mycelium characteristics of isolated strain which would
aid in identification of the isolated fungi strains in the future. Based on their colony morphology,
mycelial growth, pigmentation and microscopic observations from the total of 12,545 cultures
were; Fusarium oxysporium (2584), were Fusarium oxysporium, Fusarium solani (2465),
Fusarium solani, Aspegillus spp. (1953), Aspegillus spp., Penecillum spp. (1703), Penecillum
spp., Botrytis spp. (1294), Botrytis spp., Rhizoctonia solani (1278 ) Rhizoctonia solani and
Alternaria spp. (1268 Alternaria spp.).
Table 3: Cultural and morphological characteristics of test fungi
2006 Leslie and anceRefer

ReIsolat Cultural characteristics Morphological characteristics Suggested
es Mycelial Pigmentation Septation Conidia Chlamydo genus
growth spore
Summerell,
GF001 Fluffy White Present Macro conidia Present F.oxysporum
JF0015 Fluffy White Present Macro conidia Both F.oxysporum
YF0023 Fluffy White Present Macro conidia Present F.oxysporum
KF0032 Fluffy White Present Macro conidia Present F.oxysporum
DF0044 Fluffy White Present Macro conidia Present F.oxysporum
Leslie and Summerell, 2006
GF003 Velvet Read Present Macro conidia Absent F.solani

JF0017 Velvet Light pinkish Present Both Absent F.solani
YF0025 Fluffy Read Present Both Present F.solani
KF0038 Velvet Grey purple Present Macro conidia Present F.solani
DF0046 Fluffy Read Present Both Absent F.solani
GF008 Appressed Black Absent - - R. solani

JF0019 Fluffy Dark brown Absent - - R. solani
YF0028 Appressed Black Present - - R. solani
KF0040 Fluffy Dark brown Absent - - R. solani
DF0049 Fluffy Dark brown Absent - - R. solani
Watanabe, 2002; Srinivas, 2016
Apparently healthy seeds
Agar plate method
Figure 4: Incubated seeds showing infection of fungal pathogens

Plate 4. Upper surface of Plate 5. Lower surface of
F. oxsporium grown on PDA F. oxsporium grown on PDA
Plate 6. Microscopic features of F. oxsporium

under compound microscope 400x
Figure 5: Plate 4, 5 and 6 F. oxsporium isolated from faba bean seeds

F. solani grown on PDA F. solani grown on PDA
Plate 3. Microscopic features of F. solani

Figure 6: Plate 1, 2 and 3 F. solani isolated from faba bean seeds
R. solani grown on PDA R. solani grown on PDA
Plate 9. Microscopic features of R. solani

Figure 7: Plate 7,8 and 9 R. solani isolated from faba bean seeds
10.1.1 4.1.4. Frequency of Occurrence of Fungi

Frequency of occurrence of fungi ranged from 6.34-12.94% as shown in table 4. There was no
significant difference in the frequency of occurrence of fungi isolated from the various sampling
locations as shown in table 5 figure 1. Out of 12545 fungal colonies isolated from the faba bean
seed samples in the present study, 6343 (50.56%?) are pathogenic fungi; which belonged to the
genus F.oxysporum, F.solani and Botrytis species (12.92%, 12.33% and 9.77%) respectively.
Saprophytic fungi were 6202(49.44%?) which belonged to the genus Aspergillus, Penicillium,
Alternaria and Rhizoctonia solani (9.77%, 8.52%, 6.39% and 6.34%) respectively (Table 4).
Table 4: Percentage number of occurrence of fungi isolated
Fungi isolated Locations Infection

%
Golja Jijigu Ya’i Kure Dase
Werans Cabo Gatira Akililo
a
F. oxysporum 469 492 514 613 496 12.92
F. solani 447 328 508 658 524 12.33
Aspegillus spp. 210 299 470 502 472 9.77
Penecillum spp. 187 358 398 370 390 8.52
Botrytis spp. 279 480 242 135 158 6.47
R. solani 204 161 295 296 322 6.39
Alternaria spp. 265 394 230 208 171 6.34
Table 5: Analysis of Variance in the frequency of occurrence of fungi
Locations Fungi isolated

F.oxys F.solani Aspegill Penecill Botrytis R. Alterna
porum us spp. um spp. solani ria spp.
spp.
Golja 469 447 210 187 279 204 265
Jijigu Weransa 492 328 299 358 480 161 394
Ya’i Cabo 514 508 470 398 242 295 230
Kure Gatira 658 613 502 370 135 296 208
Dase Akililo 496 524 472 390 158 322 171
LSD (0.05) ** ** ** ** ** ** **
Means followed by same letter(s) are the same at P=0.05
Key: * - significant difference at %5 level

11.1.1
700 Golja Jijigu Weransa Ya'I Cebo Kure Gatira Dase Akililo
600
500
400
300
200
100
0
F.oxsporium F.soani Aspergilus Pencillium Botryties spp. R.solani
spp. spp.
Figure 8. Frequency of occurrence of fungi isolated.
12.1.1 4.1.5. Determination eEffects of seed-borne fungi on germination

of seeds
Effect of the seed borne fungi on germination of faba bean seed samples were carried out as
described above, Aafter 7 days of incubation,, viabilityResults from germination percentage of
seed samples recorded reveals that the germination percentage ranges from 81% to 97%. A
significant difference in germination percentage among the seed samples was observed. The seed
samples obtained from Golja kebele showed highest percentage of germination (97%). A
significant difference in germination percentage among the seed samples was observed. The
germination percentage of faba bean seeds differed significantly from location to location and
also farmer to farmer. Seed germination of a major faba bean producing kebeles, Golja was
significantly higher (97%) followed by Jijigu Weransa (89%) but Kure Gatira has lowest
germination percentage (81%) as shown in (Table 6).
Table.6: Effects of seed-borne fungi on the germination of seeds
Locations Germina Per cent association of seed borne fungi (%) Total
tion (%) F.oxys F. Aspegill Penecillu Botryti R. Alternar
porum solani us spp. m spp. s spp. solani ia spp.
Golja 97 469 447 210 187 279 204 265 2061
Jijigu Weransa 89 492 328 299 358 480 161 394 2512
Ya’i Cabo 85 514 508 470 398 242 295 230 2657
Kure Gatira 81 613 658 502 370 135 296 208 2782
Dase Akililo 86 496 524 472 390 158 322 171 2533
Seed collected from Golja kebele in the farmer of seed sample code GF002, showed the highest
germination (97%), followed by the seed sample code GF005, GF001 and JF0017 with
germination (96%, 94% and 93%), respectively, but the seed collected from Kure Gatira kebele
in the farmer of seed sample code KF0038 showed the lowest germination (81%), followed by
the seed sample code YF0026, DF0048 and KF0037 with germination (82%, 82% and
83%),respectively. The also reveal that sample with the highest fungal effect of the fungal
prevalence reflected theresulted in the lowest germination. as recorded in which significantly
maximum prevalence were recorded the fungi species of Fusarium, Aspergillus, and Penicillium.
The study results, of the seed borne fungal organisms were in agreement with the information of
seed borne nature of the pathogen reported by Marley and Gbenga (2004).
100 Germination (%)
90 Seed infection (%)
80
P e r c e n t(a%g )e
70
60
50
40
30
20
10
0
Golja Jijigu Weransa Ya’i Cabo Kure Gatira Dase Akililo
Five major faba bean producing kebeles
Figure 9: Effects of seed borne mycoflora on seed germinations of faba bean seeds
13.1.1 4.1.6. Determination eEffects of seed borne fungi on disease
transmission
The seed borne pathogen invasion reduced germination, and nutritional and also
responsible in producing mycotoxins and loss quality (Youssef, 2009). Dereje et al., 2012
reported that seed-borne diseases serve as primary inocula for the infection of the next
developing crops there by reducing yields and qualities of the produces and also play a role
in spreading the diseases to new areas. The seed borne pathogens associated with seeds
externally or internally may cause various infections like seed necrosis, reduction or
elimination of germination capacity, as well as seedling damage resulting in development of
disease at later stages of plant growth by systemic infection (Khanzada et al., 2012).
Infected seeds play a key role in the dissemination of plant pathogens and disease
establishment (Raj et al., 2007).
Whereas, the seed borne fungal diseases are transmitted by seeds and the fungi can survive
as conidia or mycelia on the seed coat or surface (Gargouri et al., 2000). As a result, seed
procured from blighted plants results in poor germination and show severe disease
development (Kumar et al., 1983). Sattar, 1933 reported that the surface contamination of
seed with fungus spores and their role in causing infection. He found that 50% of such
spores survived on seed for 5 months at 25-30°C. Transmission of seed-borne pathogens from
seeds to seedlings was also detected by seedling symptom except Phoma, Colletotrichum
graminicola, Aspergillus flavus, Aspergillus niger, Penicilium spp., Rhizopus spp. and
Trichoderma spp. These pathogens can therefore be a concern for post harvest storage of faba
bean seeds. Among the seed borne fungi, transmitted from seed to seedlings, distinct symptoms
of damping off, seed rot and seedling blight were observed for Fusarium spp., Alternaria spp.,
Botrys spp., Rhizoctonia solani and Cladosporium spp. All the transmitted seed-borne fungi may
serve as primary source of infection to the faba bean crop. The seedling growth was most
affected by Exserohilum rostratum, Aspergillus flavus, A.niger and Penicillium spp. These fungi
serve as primary inoculum for spread of diseases and also have epidemiological significance. A
widespread distribution of Fusarium, Aspergillus and Pencillium species may be attributed to the
wide occurrence of these fungi on a wide range of substrates and their efficient spore dispersal
mechanism. In the present study Fusarium oxysporium and Fusarium solani were isolated from
almost all seed samples with high frequency but Rhizoctonia solani is rarely occurred(table???).
As observed from the results, both pathogens are isolated from seed and commonnely causes of
root rot and damping off disease of faba bean that transimitted from seed to seedlings in this
Experiment. The result of this studies are similar to those obtained by earlier workers and
show that Fusarium is the most dominant species isolated from maize (Rasool et. al., 2014;
Tsedaley and Adugna, 2016; Dawood and Elshamry, 2015) and in Egypt, the main
pathogens responsible for damping-off and wilt incidence of bean are R. solani (Kühn) and
F. oxysporum f. sp. phaseoli (El- Mougy et al., 2007).
19.1
Table 7. List of Fungi Isolated from 50 Faba Bean Seed Samples by agar plate method
P Seed Drown 400 seeds per sample and total 20,000 100 seeds per sample and total 5000
A Samp seeds were used as experiments Seedling Infection TES
S le Rate??
Code Seed infection Percentage infection Wilt Ro Damp FO FS RSO
ot ing
rot off
FO FS ASS PS BS RSO ALS
G olja
GF001 62 23 20 0 0 16 38 21 0 11 33.87 0 68.75

GF002 14 16 29 0 51 15 0 0 0 0 0 0 0
GF003 56 50 0 29 15 35 20 16 1 23 28.57 34 65.71
7
GF004 68 20 0 46 0 20 58 0 0 0 0 0 0
GF005 43 50 60 0 0 0 0 0 2 0 0 40 0
0
GF006 62 61 32 0 20 0 0 25 2 0 40.32 34.43 0
1
GF007 42 52 20 53 58 0 50 20 1 0 47.62 23.08 0
2
GF008 43 62 21 0 57 49 27 0 2 24 0 38.71 48.98
4
GF009 17 60 27 0 60 23 20 0 0 0 0 0 0
GF001 62 53 0 39 43 38 52 32 0 21 51.61 0 55.26
0
Jijigu Weransa
JF001 48 40 0 30 58 60 48 18 2 20 37.5 50 33.33

1 0
JF001 55 26 0 52 60 0 0 25 0 0 45.45 0 0
2
JF001 39 26 40 0 38 61 54 0 0 31 0 0 50.82
3
JF001 52 18 38 46 51 0 0 15 0 0 28.85 0 0
4
JF001 45 20 30 29 30 0 20 24 1 0 53.33 50 0
5 0
JF001 62 39 30 45 59 0 67 22 9 0 35.48 23.08 0
6
JF001 67 30 30 20 0 0 26 11 1 0 16.42 43.33 0
7 3
JF001 38 32 60 60 60 0 50 0 1 0 0 40.63 0
8 3
JF001 32 44 71 40 51 40 62 0 2 14 0 54.55 35
9 4
JF002 54 53 0 55 53 0 67 14 1 0 25.93 28.3 0
0 5
Ya’i Cabo
YF002 72 45 39 60 30 0 39 23 1 0 31.94 42.22 0

1 9
YF002 55 55 45 30 44 0 0 0 2 0 0 47.27 0
2 6
YF002 46 57 46 0 0 66 0 20 2 21 43.48 38.6 31.82
3 2
YF002 52 60 30 51 0 0 0 16 1 0 30.77 26.67 0
4 6
YF002 24 52 56 30 57 0 58 0 0 0 0 0 0
5
YF002 52 45 62 68 10 20 58 15 0 0 28.85 0 0
6
YF002 72 56 0 75 0 63 20 0 1 33 0 28.57 52.38
7 6
YF002 42 46 38 0 65 58 54 27 1 18 64.29 30.43 31.03
8 4
YF002 30 52 68 36 0 20 0 20 1 0 66.67 34.62 0
9 8
YF003 69 52 74 48 36 68 0 0 0 20 0 0 29.41
0
Kure Gatira
KF003 72 60 74 60 0 0 70 32 3 0 44.44 53.33 0

1 2
KF003 31 51 57 0 0 56 0 11 2 16 35.48 41.18 28.57
2 1
KF003 74 68 0 56 0 0 0 24 1 0 32.43 26.47 0
3 8
KF003 73 60 50 63 16 58 0 23 1 18 31.51 16.67 31.03
4 0
KF003 72 63 74 0 68 0 0 22 3 0 30.56 52.38 0
5 3
KF003 55 70 72 53 0 30 0 19 1 0 34.55 24.29 0
6 7
KF003 56 72 55 44 0 50 29 27 2 20 48.21 27.78 40
7 0
KF003 76 59 39 48 29 59 43 26 0 21 34.21 0 35.59
8
KF003 52 45 56 20 39 0 72 20 0 0 38.46 0 0
9
KF004 47 56 20 43 0 65 0 0 0 23 0 0 35.38
0
Dase Akililo
DF004 45 71 62 68 19 16 0 0 2 0 0 29.58 0
1 1
DF004 49 61 42 0 0 50 0 19 1 15 38.78 26.23 30
2 6
DF004 40 46 49 57 0 30 49 20 2 0 50 50 0
3 3
DF004 48 31 60 30 60 39 0 18 0 19 37.5 0 48.72
4
DF004 65 48 30 47 0 40 0 26 1 24 40 37.5 60
5 8
DF004 47 65 48 10 62 0 58 0 1 0 0 26.15 0
6 7
DF004 64 65 55 10 0 0 0 16 2 0 25 38.46 0
7 5
DF004 51 70 46 70 0 14 58 21 2 0 41.18 40 0
8 8
DF004 50 59 30 22 0 37 0 10 0 18 20 0 48.65
9
DF005 42 50 67 59 0 74 0 0 0 27 0 0 36.49
0
14.1.1
15.1.1 Where: FO- Fusarium oxsporum, FS-Fusarium solani, ASS-Aspergillus species,

PS-Pencillium species, BS-Botryits species, ROS-Rihzoctonia solani, ALS-Alterneria
species and TES-Transmission efficience.
16.1.1
Figure 10:Damping off and Root rot symptoms from 7 days plate method test of faba bean
Figure 11:Damping off and Root rot symptoms from 21 days plate method test of faba bean
17.1.1 4.1.7. Antifungal activities of plant aqueous extracts

In vitro the antifungal effects of seven plants extracted with hot and cold water against the
mycelia growth of Fusarium oxsporium, Fusarium solani and Rhizoctina solani of test fungi by
poisoned food technique was shown as follows. The results presented in (Table.8 - 10) revealed
that all plant extracts were significantly inhibited the mycelial growth of the three tested fungi at
(P=0.05) in all tested concentrations and both aqueous extract. However, the effects varied with
the concentration of the extracts and the test fungal pathogens varied. The mycelial growth of
test fungi increased as the concentration percentage of plants increased in both hot and cold
water extracts, but the greatest inhibitory effect was shown by hot water extract than cold water.
Based on this, impacts of various treatments on mycelium growth of test fungi in comparison
with control/ untreated plate was assessed at 5%, 10% and 20% concentrations when compared
with the control. The mycelium growth of the tested fungi has been influenced differently by the
plant extracts as shown in (Table 8 - 13).
Table 8: Effects of seven plants extracts with cold water against on Fusarium oxsporium
Treat Concentration %
ments 5% 10% 20%
Mycelia Percent of Mycelia Percent of Mycelia Percent
growth inhibition growth inhibition growth of
(mm)* (%) (mm)* (%) (mm)* inhibition
(%)
Allium sativum 46.50abcd 47.15 34.00abcd 61.36 21.50a 75.56
Curcuma longa 48.00abcd 45.45 35.50abcd 59.65 23.00ab 73.86
Phytolacca 49.50abcd 43.75 37.00abcd 57.95 24.50abcd 72.15
dodecandra
Aloe vera gel 51.00abcd 42.04 38.50abcd 56.25 26.50abcd 69.88
Nicotiana tabacum 52.50bcd 40.34 40.00abcd 54.54 27.50abcd 68.75
Maytenus senegalensi 54.00cd 38.63 41.50abcd 52.84 29.00abcd 67.04
Nicandra physalodes 55.50d 36.93 43.00abcd 51.13 30.50abcd 65.04
Control (Untreated) 88.00e 0.00 88.00e 0.00 88.00e 0.00
*Mean values are mean of three replicates followed by same alphabets are not significantly
different from each other, analyzed by using LSD at 0.05 = 10.13
Results in (Table. 8) showed that, plant extracted with cold water at 20% concentration, clove of
Allium sativum caused highest inhibition mycelial growth of F.oxsporium (75.56%) followed by
clove of Curcuma longa (73.86%), and seed of Phytolacca dodecandra (72.15%), whereas the
lowest inhibition (65.04%) of mycelial growth was recorded at the same concentrations leaf
extract of Nicandra physalodes as compared to control.
80
70
60
50
40
30 20%
10%
20
5%
10
Figure 10:Effects of plant extracts with cold water against mycelial growth of F.oxsporium
Table 9: Effects of seven plants extracts with hot water against on Fusarium oxsporium
ments 5% 10% 20%
Mycelia Percent 0f Mycelia Percent 0f Mycelia Percent 0f
growth inhibition growth inhibition growth inhibition
(mm)* (%) (mm)* (%) (mm)* (%)
Allium sativum 43.00h 51.13 30.50de 65.34 18.00a 79.54
Curcuma longa 44.50hi 49.43 32.00e 63.63 19.50ab 77.84
Phytolacca dodecandra 46.00i 47.72 33.50ef 61.93 21.00b 76.13
Aloe vera gel 47.50ij 46.02 35.00f 60.22 23.00bc 73.86
Nicotiana tabacum 49.00j 44.31 36.50fg 58.52 24.00c 72.72
Maytenus senegalensi 50.50jk 42.61 38.00g 56.81 25.50cd 71.02
Nicandra physalodes 52.00k 40.90 39.50gh 55.11 27.00d 69.31
Control (Untreated) 88.00l 0.00 88.00l 0.00 88.00l 0.00
different from each other, analyzed by using LSD at 0.05= 0.74?
Results in (Table. 9) showed that, plant extracted with hot water at 20% concentration, clove of
Allium sativum caused highest inhibition mycelial growth of F.oxsporium (79.54%) followed by
clove of Curcuma longa (77.84%), and seed of Phytolacca dodecandra (76.13%), whereas the
lowest inhibition (40.90%) of mycelial growth was recorded at 5% leaf extract of Nicandra
physalodes as compared to control.
80
70
60
50
40
30 20%
10%
20
5%
10
Figure 11:Effects of plant extracts with hot water against mycelial growth of F.oxsporium
Table 10: Effects of seven plants extracts with cold water against on Fusarium solani
ments 5% 10% 20%
Mycelia Percent of Mycelia Percent of Mycelia Percent of
(mm)* (%) (mm)* (%) (mm)* (%)
Allium sativum 37.50cd 54.26 35.00abc 57.31 32.50a 60.36
Curcuma longa 39.00cde 52.43 36.50bcd 55.48 34.00ab 58.53
Phytolacca dodecandra 40.50cdefg 50.60 38.00cd 53.65 35.50abcd 56.70
Aloe vera gel 42.00fghi 48.78 39.50cdef 51.82 37.00bcd 54.88
Nicotiana tabacum 43.50hij 46.95 41.00defgh 50.00 38.50cd 53.04
Maytenus senegalensi 45.00jk 45.12 42.50ghij 48.17 40.00cdefg 51.21
Nicandra physalodes 46.50kl 43.29 44.00ijk 46.34 41.50efghi 49.39
Control (Untreated) 82.00l 0.00 82.00l 0.00 82.00l 0.00
different from each other, analyzed by using LSD at 0.05= 0.74
Results in (Table.10) showed that, plant extracted with cold water at 20% concentration, clove of
Allium sativum caused highest inhibition mycelial growth of F.solani (60.36%) followed by
clove rhizome of Curcuma longa (58.53%), and seed of Phytolacca dodecandra (56.70%),
whereas the lowest inhibition (49.39%) of mycelial growth was recorded at 5% leaf extract of
Nicandra physalodes as compared to control.
70
60
50
40
30
20%
20 10%
5%
10
Figure 12:Effects of plant extracts with cold water against mycelial growth of F. solani
Table 11: Effects of seven plants extracts with hot water against on Fusarium solani
ments 5% 10% 20%
(mm)* (%) (mm)* (%) (mm)* (%)
A.sativum 36 abcdef 56.09 33.5ab 59.14 31a 62.19
C. longa 37.5 bcdefgh 54.26 35 abcd 57.31 32.5a 60.36
P.dodecandra 39 defghij 52.43 36.5abcdefg 55.48 34abc 58.53
Aloe vera gel 40.5 ghijk 50.60 38 cdefghi 53.65 35.5 abcde 56.71
N. tabacum 42 ijkl 48.78 39.5 efghijk 51.82 37 abcdefgh 54.87
M.senegalens 43.5 kl 46.95 41 hijkl 50.00 38.5 cdefghij 53.04
i
N. physalodes 45 lm 45.12 42.5 jkl 48.17 40 fghijk 51.22
C(Untreated) 82 m 0.00 82 m 0.00 82 m 0.00
Results in (Table.11) showed that, plant extracted with hot water at 20% concentration, clove of
Allium sativum caused highest inhibition mycelial growth of F.solani (62.19%) followed by
clove of Curcuma longa (60.36%) and seed of Phytolacca dodecandra (58.53%), whereas the
70
60
50
40
30
20%
20 10%
5%
10
Figure 13:Effects of plant extracts with hot water against mycelial growth of F. solani
Table 12: Effects of seven plants extracts with cold water against on Rhizoctonia solani
ments 5% 10% 20%
(mm)* (%) (mm)* (%) (mm)* (%)
A.sativum 41.00 hijk 53.40 38.75 klm 55.96 36.5m 58.52
C. longa 42.25 fghi 51.98 40.00 ijkl 54.54 37.75 lm 57.10
P.dodecandra 43.50 efg 50.56 41.25 ghij 53.31 39.00 jklm 55.68
Aloe vera gel 44.75 de 49.14 42.50 efgh 51.70 40.25 hijkl 54.26
N.tabacum 46.00 cd 47.72 43.75 defg 50.28 41.75 fghij 52.55
M. senegalensi 47.25 bc 46.30 45.00 cde 48.86 43.00 efgh 51.13
N. physalodes 48.50 ab 44.88 46.25 bcd 47.44 44.25 def 49.71
C (Untreated) 88.00a 0.00 88.00a 0.00 88.00a 0.00
different from each other, analyzed by using LSD at 0.05=0.74
Results showed in (Table 12) plant extracted with cold water at 20% concentration, clove of
Allium sativum caused highest inhibition mycelial growth of R.solani (58.52%) followed by
60
50
40
30
20%
20 10%
5%
10
Figure 14: Effects of plant extracts with cold water against mycelial growth of R. solani
Table 13: Effects of seven plants extracts with hot water against on Rhizoctonia solani
ments 5% 10% 20%
(mm)* (%) (mm)* (%) (mm)* (%)
A.sativum 39.50 jklmno 55.11 37.25 nop 57.67 35.00p 60.22
C.longa 40.75 ghijklm 53.69 38.50 lmnop 56.25 36.25 op 58.80
P.dodecandra 42.00 efghij 52.72 39.75 ijklmn 54.82 37.50 mnop 57.38
Aloe vera gel 43.25 cdefg 50.85 41.00 fghijkl 53.40 38.75 klmno 55.96
N. tabacum 44.50 bcde 49.43 42.25 defghi 51.98 40.00 hijklm 54.54
M.senegalens 45.75 bc 48.01 43.50 cdef 50.56 41.25 fghijk 53.12
i
N. physalodes 47.00 ab 47.72 44.75 bcd 49.14 42.50 defgh 51.20
C (Untreated) 88.00a 0.00 88.00a 0.00 88.00a 0.00
Results showed in (Table 13), plant extracted with hot water at 20% concentration, clove of
Allium sativum caused highest inhibition mycelial growth of R.solani (60.22%) followed by
70
60
50
40
30
20%
20 10%
5%
10
Figure 15: Effects of plant extracts with hot water against mycelial growth of R. solani
4.1.7.2. In vitro evaluation of Trichoderma species against three test fungi

The inhibitory effect of T.harzianum (T1), T.longibrachiaium (T14), T.atroviride (T5), and
T.Virense (T13) against the mycelia growth of the three test fungi F.oxysporum, F.solani and R.
solani in dual culture method are shown in (Table 14). The antagonistic effects of Trichoderma
spp. against F.oxsporium were in the range of 43.75– 60.22%. T.longibrachiaium (T14) gave the
highest effect about 60.22%, followed by T.atroviride (T5) (54.54%), T.Virense (T13) (43.75%)
and T.harzianum (T1) (37.50%), respectively. Results showed that the growth inhibition of F.
solani by Trichoderma spp were in the range of 38.63–57.95%. T.longibrachiaium (T14) also
highly inhibited the mycelial growth of F. solani, where the growth inhibition was 57.95%,
followed by T.atroviride (T5) (48.86%), T.Virense (T13) (44.31%) and T.harzianum (T1)
(38.63%), respectively. Results showed that the growth inhibition of R.solani by Trichoderma
spp were in the range of 42.04–70.45%. T.longibrachiaium (T14) inhibited the mycelial growth
of R. solani, by 70.45%, followed by T.atroviride (T5) (61.36%), T.Virense (T13) (53.97%) and
T.harzianum (T1) (42.04%), respectively. Results showed that the best growth inhibition against
the three isolated test fungi was obtained by T.longibrachiaium (T14), while the lowest one was
obtained by T.harzianum (T1) (Table 14).
Table 14: Effects of four Trichoderma spp. against on the three isolated test fungi
Trichoderma Antagonistic effect against

species Fusarium oxsporium Fusarium solani Rhizoctonia. solani
Mycelial Growth Mycelial Growth Mycelial Growth
(mm)* (%) (mm)* (%) (mm)* (%)
T.longibrachiaium 35.00d 60.22 37.00c 57.95 26.00d 70.45
(T14)
T.atroviride (T5) 40.00cd 54.54 45.00bc 48.86 34.00cd 61.36
T.Virense (T13) 48.00bc 45.45 49.00bc 44.31 40.50bc 53.97
T.harzianum(T1) 49.50ab 43.75 54.00ab 38.63 51.00ab 42.04
Tc (Control) 88.00a 0.00 88.00a 0.00 88.00a 0.00
L.S.D at 5% 0.80 0.00 1.63 0.00 1.37 0.00
Where;- T: Trichoderma, Tc: Without Trichoderma, *Mean of three replications. The values are
significantly different from the control. Mycelial growth was measured in (mm). Means in each
column followed by the same letter are not significantly different according to LSD test
(P=0.05).
80
70
60
50
40 T.longibrachiaium (T14)
T.atroviride (T5)
30
T.Virense (T13)
T.harzianum (T1)
20
Tc (Control)
10
0
F.oxysporum F.solani R. solani
Figure12:Evaluation of Trichoderma spp. against mycelial growth of test fungi
20.1 4.2. DISCUSSION

According to the experimental work on the seed borne fungi associated with faba bean seeds,
seven species of fungi were isolated and identified. Faba bean is susceptible to a number of
fungal diseases which decrease production and lower the quality of seeds. Because of this, the
researchers studied the seed-borne fungi of faba bean which affect production and seed quality.
The agar plate methods were used (ISTA, 2008) ith same modification. Use of agar plate method
results in rapid saprophyte growth, which often impairs the detection of parasitic fungi and
inhibit spore germination of some important seedborne fungi (Neergard, 1979). Results of the
study revealed that F.oxysporum, F.solani, Aspergillus spp. Penicillium spp. Botrytis spp.
R.solani and Alternaria spp. were observed and identified. These results are in agreement with
the findings of other researchers (M.A. Elwakil, et al. 2009, Negussie et al., 2008, Berhanu et
al., 2003,; Dubey and Patel, 2000; Rauf, 2000, Awgechew, 1999, Abdel-Hafez, 1988). Among
the present study isolated fungi of Fusarium and Aspergillus species were the most predominant
fungi to all seed samples. In vitro plate method Seed to seedling transmission test indicated that,
F.oxysporum, F.solani and R.solani isolates were the most fungal isolates significantly induced
damping off and root rot disease on faba bean plants. and conifirmed these three isolated test
fungi were the common causal pathogens causes the root rot and damping off disease of faba
beans and transmitted from seed to seedlings. This result is similar to the findings of (Elliot and
Crowford, 1922), who found these fungal pathogenic organisms to be the most devastating rot
causing organisms in bean crops in different locations in the World (Abd-El-Khair, et. al.,2010).
As results showed that, several root rot and wilt pathogens such as F. oxysporum, R.solani and
F.solani are reported to attack faba bean roots and stem base causing serious losses in seed
germination and seedlings. These results agree with those recorded by (Abdel-Kader MM., et.al
2011).
Biological control of the three isolated test fungi by seven plant extract and four Trichoderma
species have been evaluated. in this study, to considerably a more natural and environmentally
acceptable alternative than the existing chemicals. These results agree with those recorded by
(Baker and Paulitz, 1996). The inhibitory action of the aqueous plant extracts on mycelial growth
increased with increase in concentrations and the hot water extracts giving high effects/toxicity
than cold extract in all test fungi. Results highlight the highest inhibitory effect of A. sativum
extracts with hot water at 20% concentration on mycelial growth of the three test fungi (F.
oxysporum, F.solani and R.solani) (79.54%, 62.19% and 60.22%) respectively, but Nicandra
physalodes extract at the same concentration showed lowest inhibitory effects on three test fungi
(69.31%, 51.22% and 51.20%) respectively. The present results showed that the mycelial growth
of the three fungi decreased with increase concentrations. The results revealed that, all the tested
plant extracts at 5%, 10% and 20% concentrations significantly inhibited the mycelial growth of
all test fungi, but the four trichoderma species exhibited the strongest Antagonistic activity.
These results revealed that antifungal activities of the extracts were enhanced by increasing the
concentration from 5 to 20 % (w/v); hence the inhibition activities of the extracts were
concentration dependent. This is in agreement with the report of Ilondu (2012), and Chiejina and
Ukeh (2013), Jasso et al., (2005) who indicated that increase in the antifungal activities had
corresponding increase in concentration of plant extracts.
The antagonistic effects of Trichoderma spp against the test fungi also indicate significant
inhibitory activities. The best inhibitory effect on the three test fungi were obtained by
T.longibrachiaium (T14) (70.45%, 60.22% and 57.95%), followed by T.atroviride (T5) (54.54%,
53.97% and 48.86%), T.Virense (T13) (61.36%, 45.45% and 44.31%) and T.harzianum (T1)
(43.75%, 42.04% and 38.63%), respectively. These results agree with those recorded by El-
Kafrawy (2002) and (Lewis et al., 1998). The antagonistic activity of Trichoderma sp. against
Fusarium spp. also concides with the result reported by Morsy et al., (2009). The inhibitory
action of the aqueous plant extracts on mycelial growth increased with increase in concentrations
and the hot water extracts giving high effects/toxicity than cold extract in all test fungi. Results
highlight the highest inhibitory effect of A. sativum extracts with hot water at 20% concentration
on mycelial growth of the three test fungi (F. oxysporum, F.solani and R.solani) (79.54%,
62.19% and 60.22%) respectively, but Nicandra physalodes extract at the same concentration
showed lowest inhibitory effects on three test fungi (69.31%, 51.22% and 51.20%) respectively.
The present results showed that the mycelial growth of the three fungi decreased with increase
concentrations.
CHAPTER FIVE
5. CONCLUSION AND RECOMMENDATION
21.1 5.1. Conclusions

The results obtained from this study showed that plant extracts of different medicinal plants
screened exhibit antifungal effects against F.oxysporum, F.solani and R.solani. In particular,
hot water extracts of Allium sativum, Maytenus senegalensis, Aloe vera gel, Nicandra
physalodes, Nicotiana tabacum, Curcuma longa and Phytolacca dodecandra offer effective
bioactive compounds for growth inhibition of the test fungi. The tested Trichoderma spp also
show good antagonistic effect against three test fungi. These indicate that plant extract and
Trachoderma spp can be potential alternative management option against plant disease and can
be used as seed treatment or seed couting.
 Hot water extracts of all screened and tested plants revealed relatively more effective against
the pathogen than cold water extracts.
 Active compounds of all test plants are extractable with water and most extracted with hot
water.
 The results of this study revealed that cold and hot aqueous extracts of all the plants possess
diverse antifungal activity against the test fungi.
 The differentiating activities against the selected isolate of these plant extracts encourage
developing broad spectrum antifungal in the future.
22.1 5.2. Recommendation

All the tested plant extract and Trichoderma spp contains antifungal agents and can be
used against seed borne fungi in faba bean and but need further purification for better
efficacy.
The result also confirem that A continuous effort to search good botanical extracts and
antagonistic microorganisms is primarily needed.
The potential benefits of using mixture of fungal isolate and plant extracts to suppress
diseases under green house trial and field experiments are needed for the future.
The result of the present study can be further exploited and tested under green house and
field experiment for formulating integrated disease management schedule of root rot and
damping off disease of Faba bean.
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AMBO UNIVERSITY

COLLEGE OF AGRICULTURE AND VETERINARY SCIENCES

DEPARTMENT OF PLANT SCIENCES

Amsalu Neme Boru (ID. No: PGR/30162/11)

A Thesis submitted to the Department of Plant Science in Partial Fulfillment of the

CO ADVISOR: Dr. Amin Mohommed

__________________________ _________________ _____________

Chairman Signature Date

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Main Advisor Signature Date

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External Examiner Signature Date

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Co-advisor Signature Date

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Dean, School of Graduate Studies Signature Date

Name: AMSALU NEME BORU Signature: _______________________

Place: Ambo University, Ambo, Ethiopia

Date of submission: May, 2021

It is my great pleasure to express my heartfelt appreciation and special gratitude to my Major

It gives me immerse pleasure to express my deep sense of gratitude to Ambo District

1.1. Back Ground....................……………........…………………….....……………………....…1

1.2. Statement of the Problem…......................................................................................................2

1.3. Research Questions...................................................................................................................3

1.4. Significance of the Seed Testing Study………………...…........………………………….....3

1.5. General Objective.………...…….......………….…………….………........…........................5

1.6. Specific Objectives …..............................................................................................................5

Table 3: Per cent association of seed borne fungi (%) in locations.........................................33

Table 4: Cultural and morphological characteristics of Selected isolate fungi................35

Table 5: Frequency of occurrence on faba bean seed samples................................................37

Table 6: Analysis of Variance for the frequency of occurrence of fungi.................................38

Table 7: Effects of seed-borne fungi on the germination of seeds.........................................39

Table 8: Seed to seedlings transmission efficacy....................................................................41

Table 12: In vitro evaluation of Tricoderma spp. against test fungi.......................................46

Figure.2 Antagonistic test between Rhizoctiona solani and Trichoderma spp.......................29

Figure 5: F. oxsporium. grown on PDA media culture plates.................................................36

Figure 6: F. solani grown on PDA media culture plates.........................................................36

Figure 7: R. solani grown on PDA media culture plates........................................................37

Figure 8: Frequency of occurrence on faba bean seed samples..............................................38

1.1 1.1. Background

2.1 1.2. Statement of the Problem

3.1 1.3. Research Questions

4.1 1.4. Significance of the Seed Testing Study

5.1 1.5. General objectives

6.1 1.6. Specific objectives

7.1 2.1. Faba bean production

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