CHOCOLATE SPOT EPIDEMICS ON DIFFERENT FABA BEAN

VARIETIES AND CHARACTERIZATION OF SOME Botrytis fabae

ISOLATES COLLECTED IN DAWURO ZONE, SOUTHWEST
ETHIOPIA

[Link] Thesis

MESELE HAILE ONU

December, 2012
Jimma University
 CHOCOLATE SPOT EPIDEMICS ON DIFFERENT FABA BEAN
VARIETIES AND CHARACTERIZATION OF SOME Botrytis fabae
ISOLATES COLLECTED IN DAWURO ZONE, SOUTHWEST
ETHIOPIA

[Link] Thesis

Submitted to the School of Graduate Studies,

Jimma University College of Agriculture and Veterinary Medicine

In Partial Fulfillment of the Requirements for the Degree of MASTER OF

SCIENCE IN PLANT PATHOLOGY

By
Mesele Haile

December, 2012
Jimma University
 APPROVAL SHEET

School of graduate studies

Jimma University

As thesis research advisor, I hereby certify that I have read and evaluated this thesis prepared,
under my guidance, by Mesele Haile Onu Entitled: Chocolate Spot Epidemics on Different
Faba Bean Varieties and Characterization of Some Botrytis fabae Isolates Collected in
Dawuro Zone, Southwest Ethiopia, I recommend that it be submitted as fulfilling the Thesis
requirement.
Girma Adugna (PhD) _________________ _______________

Major Advisor Signature Date

Fikre Lemessa (PhD) _________________ _______________

Co-advisor Signature Date

As member of the Board of Examiners of the [Link] Thesis Open Defense Examination, We
certify that we have read, evaluated the thesis prepared by Mesele Haile, and examined the
candidate. We recommended that the thesis could be accepted as fulfilling the thesis
requirement for the Degree of Master of Science in Plant Pathology.

______________________ _________________ _______________

Chairperson Signature Date

______________________ _________________ _______________

Internal Examiner Signature Date

______________________ _________________ _______________

External Examiner Signature Date

 DEDICATION

This thesis manuscript is especially dedicated to my son Yididiya Mesele and my wife
Tigist Bezuneh for their love and dedicated partnership in the success of my life.

i
 STATEMENT OF AUTHOR

First, I declare that this thesis is my bonafide work and that all sources of materials used for
this thesis have been duly acknowledged. This thesis has been submitted in partial fulfillment
of the requirements for [Link] Degree at the Jimma University and is 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 quotations from this thesis are allowable without special permission provided that
accurate acknowledgment of source is made. Requests for permission for extended quotation
from or reproduction of this manuscript in whole or in part may be granted by the head of the
Department or the dean of the School of Graduate Studies when in his or her judgment the
proposed use of the material is in the interest of scholarship. In all other instances, however,
permission must be obtained from the author.

Name: Mesele Haile

Signature: ____________

Place: Jimma University, Jimma

Date of Submission: December, 2012

ii
 BIOGRAPHICAL SKETCH

The author, Mesele Haile, was born on 3rd of July 1981 in Mareka District of Dawuro Zone,
Southwest Ethiopia. He attained his junior elementary School at Kawuka Elementary School,
junior secondary school at Tocha and his secondary school at Waka Comprehensive
Secondary School from 1988 to 2000. He joined then Ambo College of Agriculture in 2001
and graduated with Diploma in General Agriculture in July, 2002 and he also awarded [Link] in
Horticulture from Jimma University in November, 2008 in advance standing program. After
graduation he was employed by the Ministry of Agriculture, Ethiopia where he served as
agronomist and team leader within different districts of Dawuro Zone until he joined the
School of Graduate Studies at Jimma University, College of Agriculture and Veterinary
Medicine in April, 2010 to pursue his [Link] study in Plant Pathology.

iii
 ACKNOWLEDGMENTS

First of all, I would like to thank my savoir Lord, the Almighty who gave me everything,
without the help of whom my plans would not have been achieved.

It is my pleasure to express my heartfelt appreciation and special gratitude to my major

adviser Dr. Girma Adugna for his unreserved genuine guidance and constructive comments,
starting from proposal writing to end of the work. The visit made by him to my field
experiment at Dawuro zone was highly appreciated and never to be forgotten. I am grateful to
my co-adviser Dr. Fikre Lemessa for his positive comments and suggestions to undertake this
research. Without their support, patience, and guidance this study would never have matured.

Additionally, appreciation is expressed to members of the JUCAVM, Dawuro Zone

Agricultural department and Mareka ‘woreda’ administration for the material and financial
support during my study. I would like to extend my thanks to staff of Holleta Agricultural
Research Center, Dr. Dereje Gorfu for providing Botrytis fabae isolate for compromising and
Mr. Wendafrash Mulugeta for providing faba bean seeds, to undertake this research. I also
express my appreciation to Dr. [Link] Rao, Mr. Goytom Berihun and W/ro Mulu
Didu for helping me in laboratory experiment, and Dr. Waktole Sori, Mr. Gezahgne Berecha
and Mr. Amsalu Nebyu for commenting the manuscript.

I express my heartfelt appreciation and gratitude to my colleagues, Mr. Shimelis Tamirat, Mr

Lema Leykun, Mr. Solomon Ayele, Mr. Tariku Mesele, Mr. Zeleke Ashango, W/ro Genet
Semela and [Link] Ataro, which have been providing me their genuine and unreserved
support throughout the study period. Thanks goes to Mr. Batisa Wagesho and [Link]
Shenu, without whom I would never make it through graduate school.

Last but by no means least, the moral support and encouragement from my mother Gojam
Kercho, my wife Tigist Bezuneh and [Link] Mamo deserve a special place in my
memories.

iv
 LISTOF ACRONYMS AND ABBREVIATIONS

0
C Degree Celsius
AARC Adet Agricultural Research Center
ANOVA Analysis of Variance
AUDPC Area under the disease progress curve
CSA Central Statistical Authority
CV Coefficient of variation
DAP Days after planting
DSI Disease Severity Index
DZAD Dawuro Zone Agricultural Department
EARO Ethiopian Agricultural Research Organization
FAO Food and Agriculture Organization of the United Nations
FBDA Faba Bean Dextrose Agar
GLM Generalized Linear Model
GPS Global Positioning System
ha Hectare
HSW hundred-seed weight
ICARDA International Center for Agricultural Research in the dry Areas
IR Infection rate
M.a.s.l Meters Above Sea Level
mm Millimeter
MoARD Ministry of Agriculture and Rural Development
NS Non significant
PA Peasant association
RCBD Randomized Complete Block Design
RH Relative humidity
SAS Statistical Analysis System
SNNPRS Southern Nations, Nationalities and Peoples' Regional State

v
 TABLE OF CONTENTS

DEDICATION.............................................................................................................................i

STATEMENT OF AUTHOR.....................................................................................................ii

BIOGRAPHICAL SKETCH.....................................................................................................iii

ACKNOWLEDGMENTS.........................................................................................................iv

LISTOF ACRONYMS AND ABBREVIATIONS.....................................................................v

TABLE OF CONTENTS...........................................................................................................vi

LIST OF TABLES...................................................................................................................viii

LIST OF FIGURES...................................................................................................................ix

LIST OF TABLES IN THE APPENDICES...............................................................................x

LIST OF APENDEX PLATES..................................................................................................xi

ABSTRACT..............................................................................................................................xii

1. INTRODUCTION..................................................................................................................1

1.1. Background of the Study.................................................................................................1

1.2. Statement of the Problem................................................................................................3

1.3. Objectives of the Study...................................................................................................4

1.3.1. General Objective......................................................................................................4

1.3.2. Specific objectives.....................................................................................................4

2. LITERATURE REVIEW.......................................................................................................5

2.1. Faba Bean........................................................................................................................5

2.1.1. Taxonomy and ecology of faba bean.........................................................................5

vi
 TABLE OF CONTENTS (continued)

2.1.2. Economic importance................................................................................................6

2.1.3. Production constraints................................................................................................6

2.2. The Genus Botrytis..........................................................................................................6

2.2.1. The pathogen Botrytis fabae......................................................................................7

2.2.2. Host range..................................................................................................................8

2.2.3. Symptoms and aggressiveness...................................................................................8

2.2.4. Pathogenic variability................................................................................................9

2.2.5. Disease epidemiology................................................................................................9

2.2.6. Economic significance of chocolate spot.................................................................11

2.2.7. Disease management................................................................................................11

[Link]. Cultural practices................................................................................................11

[Link]. Chemical control................................................................................................12

[Link]. Biological control...............................................................................................13

[Link]. Host resistance....................................................................................................14

[Link]. Integrated disease management..........................................................................16

3. MATERIALS AND METHODS..........................................................................................18

3.1. Geographic Location of Dawuro Zone..........................................................................18

3.2. Description of the Study Areas.....................................................................................19

3.3. Disease Survey and Assessment Methodology.............................................................20

vii
 TABLE OF CONTENTS (continued)

3.4. Disease Sample Collections..........................................................................................21

3.5. Field Experiment...........................................................................................................21

3.5.1. Plant material...........................................................................................................21

3.5.2. Experimental Design and Management...................................................................22

3.5.3. Diseaseassessment...................................................................................................23

[Link]. Disease progression and modeling.....................................................................24

3.5.4. Yield and yield components.....................................................................................25

3.5.5. Weather parameters.................................................................................................25

3.6. Studies in the Laboratory..............................................................................................26

3.6.1. Media Preparation....................................................................................................26

3.6.2. Isolation of the pathogen..........................................................................................26

3.6.3. Hyphal tip isolation and preservation......................................................................26

3.6.4. Cultural and morphological characterization of the B. fabae Isolates.....................27

3.6.5. Propagation of the Inoculum...................................................................................28

3.7. Aggressiveness of B. fabae isolates..............................................................................28

3.7.1. Disease assessment..................................................................................................28

3.7.2. Reisolation of the pathogen.....................................................................................29

3.8. Data Analysis..............................................................................................................29

viii
 TABLE OF CONTENTS (continued)

4. RESULTS.............................................................................................................................31

4.1. Prevalence and Severity of Chocolate Spot in Dawuro Zone.......................................31

4.2. Association of Faba Bean Chocolate Spot with Independent Variables.......................33

4.3. Chocolate Spot Epidemics and Progression..................................................................36

4.3.1. Disease onset and symptoms observation................................................................36

4.3.2. Analysis of variance for location.............................................................................38

4.3.3. Chocolate spot progress at test locations.................................................................38

4.3.4. Area under the disease progress curve (AUDPC) ...................................................43

4.3.5. Chocolate spot infection rate...................................................................................45

4.4. Yield and Yield Components........................................................................................47

4.4.1. Number of pods per plant (NPP) and number of seeds per pod (NSP)...................47

4.4.2. Performance of tested faba bean varieties across location.......................................51

4.5. Relation of Chocolate Spot to Faba Bean Yield Components......................................53

4.6. Morphological and Cultural Characterization...............................................................57

4.7. Aggressiveness of the Botrytis fabae isolates...............................................................59

5. DISCUSSION.......................................................................................................................60

6. SUMMARY AND CONCLUSIONS...................................................................................69

7. REFERENCES......................................................................................................................73

8. APPENDIXES......................................................................................................................85

ix
 LIST OF TABLES
Table Page
1. Faba bean germplasm resistant to chocolate spot (Botrytis fabae).......................................16
2. List of faba bean varieties used for the study and their agronomic traits.............................19
3. Six resistance levels used for classification of faba bean varieties.......................................23
4. Mean severity index of faba bean chocolate spot for different independent variables in 2011
cropping season for surveyed fields.....................................................................................23
5. Independent variables used in logistic regression analysis and likelihood ratio statistics for
eleven variables entered first and last into a model.............................................................34
6. Analysis of deviance, natural logarithms of odds ratio and standard error of the selected
independent variables in a reduced model analyzing chocolate spot severity.....................35
7. Grouping of 14 faba bean varieties into reaction groups at three test locations, 2011growing
season...................................................................................................................................42
8. Infection rates of chocolate spot on 14 faba bean varieties at three locations in Dawuro
zone, 2011 growing season..................................................................................................46
9. Mean values of number of pods per plant and number of seeds per pod of faba bean
varieties tested at three locations in Dawuro zone, 2011 cropping season......................... 48
10. Mean values of seed yield (kg/ha) and hundred seed weight (g) of faba bean varieties,
2011cropping season............................................................................................................50
11. Combined mean values for chocolate spot severity and yield related traits of tested faba
bean varieties.......................................................................................................................52
12. Combined mean values for yield and yield components of the tested faba bean varieties
across locations....................................................................................................................53
13. Coefficient of correlation between seed yield and chocolate spot severity for assessment
dates, 2011 growing season.................................................................................................54
14. Coefficient of correlation between 100 seed weight and severity of chocolate spot for
assessment dates, 2011 growing season...............................................................................55
15. Morphological characteristics B. fabae isolates from faba bean fields in Dawuro zone,
southwest Ethiopia (n=44)...................................................................................................58
16. Aggressiveness of B. fabae isolates against faba bean under greenhouse conditions........59

viii
 LIST OF FIGURES

Figure Page

1. Geographic location of Dawuro Zone in Ethiopia................................................................19

2. Mean severity index of chocolate spot in farmers’ field, 2011 cropping season..................31

3. Early infection of chocolate spot on faba bean varieties, 30 Days after planting.................36

4. Aggressive stages of chocolate spot symptoms or signs on local cultivar............................37

5. Comparison of disease progress curves for epidemics of chocolate spot on faba bean
varieties at Tocha, 2011 growing season.............................................................................39

6. Comparison of disease progress curves for epidemics of chocolate spot faba bean varieties
at Mari, 2011 growing season..............................................................................................40

7. Comparison of disease progress curves for epidemics of chocolate spot on faba bean
varieties at Turi, 2011 growing season.................................................................................41

8. Classification of 14 varieties of faba bean according to AUDPC values determined at

Tocha, 2011 growing season................................................................................................43

9. Classification of 14 varieties of faba bean according to AUDPC values determined at Mari,

2011 growing season............................................................................................................44

10. Classification of 14 varieties of faba bean according to AUDPC values determined at Turi,
2011 growing season............................................................................................................45

11A. Linear regression relating AUDPC with pod numbers per plant of faba bean under three
locations, 2011cropping seasons..........................................................................................56

11B. Linear regression relating chocolate spot AUDPC with seed yield of faba bean under
Tocha, Mari and Turi experimental sites, 2011cropping seasons. .......................................57

ix
 LIST OF TABLES IN THE APPENDICES

Appendix Table Page

[Link] features of study areas in four districts of Dawuro zone..............................86

1B. The results of soil analysis at the experimental sites..........................................................86
2. Monthly average minimum and maximum temperature ( 0C) and total rainfall (mm) of the
experimental period, 2011 growing season..........................................................................87
3. ANOVA table for the disease severity index (DSI) of each assessment dates under field
conditions.............................................................................................................................87
4. ANOVA table for AUDPC of fourteen faba bean varieties, 2011 cropping season...........88
5. ANOVA table for infection rate of fourteen faba bean varieties, 2011 cropping season...88
6. ANOVA table for pod number per plant of fourteen faba bean varieties, 2011 cropping
season.................................................................................................................................89
7. ANOVA table for seed number per pod of fourteen faba bean varieties, 2011 cropping
season.................................................................................................................................89
8. ANOVA table for seed yield (kg/ha) of fourteen faba bean varieties, 2011 cropping
season.................................................................................................................................90
9. ANOVA table for hundred seed weight (g) of fourteen faba bean varieties, 2011 cropping
season.................................................................................................................................90
10. ANOVA table for combined yield and related parameters, 2011 cropping season..........91
11. ANOVA table for Mean severity index of chocolate spot at eleven peasant asociations
(PA) of farmers’ field, 2011 cropping season....................................................................91
12. Pearson correlation coefficients between epidemiological measurements and yield and
yield components at three locations, 2011 growing season...............................................92

x
 LIST OF APPENDEX PLATES
Appendix Plate Page

1. Isolation and identification of Botrytis fabae isolates.........................................................94

2. Pathogenicity of Botrytis fabae isolates on var. CS20DK under green house conditions...94

xi
 ABSTRACT

Chocolate spot caused by the fungus Botrytis fabae is the major disease threatening faba
bean (Vicia faba L.) production in Ethiopia. However, the intensity and importance of this
disease are not well studied in the faba bean production areas of southwest Ethiopia. The
present study was conducted to determine the distribution, epidemics of chocolate spot on
different faba bean varieties and to characterize some B. fabae isolates in major agro-
ecologies of Dawuro Zone. A total of 44 faba bean fields were surveyed in 11peasant
associations (PAs) of the zone, and all of the fields were infested with chocolate spot. The
disease severity indices (DSI) varied among PAs, altitude range and crop management
practices. For surveyed fields, the mean DSI ranged from 33.4% in Dali to 69.4 % in Waka
PA. Logistic regression analysis showed that PA, crop variety, crop history and altitude were
significantly associated with DSI in a multiple variable model. Higher DSI was significantly
associated with high altitude (>2200). The field experiments carried at three locations and
experimental plots arranged in RCBD with three replications. The progress of the symptoms
evolved slowly on certain varieties but at much greater rates on other varieties depending on
location. DSI scores almost for all assessment dates and the AUDPC values varied
considerably among the varieties and significant genotypic differences were observed at all
locations. The varieties Hachalu, Kuse, Tumsa, Mesay, Moti, Gebelcho, Walki, Nc-58 and
local cultivar were suffered from the disease (highest AUDPC). Conversely, CS20DK,
Degaga, Bulga-70, Tesfa and Kasa were rated moderately resistant and developed the least
symptoms at all locations. Highly significant differences were observed for number of pods
per plant, seed yield and hundred seed weight among varieties. CS20DK (2100kg/ha), NC-58
(2027kg/ha), Moti (1973kg/ha) and Degaga (1910kg/ha) under Tocha conditions where as
Degaga (1327kg/ha), CS20DK (880kg/ha) and NC-58 (870kg/ha) under Mari conditions were
the top yielding varieties. The correlation between seed yield and DSI for all assessment dates
were negatively and highly significant (p <0.01) having correlation values ranging from -
0.592 to -0.361 at Tocha and Mari. Significant differences were observed in the frequency of
isolates among PA with colony color (X2=35.94, df = 2, p<0.05) and colony growth rate
(X2=38.7, df = 2, p<0.01). According to morphological characteristics all isolates were
identified as B. fabae species (11-14 × 7-10 µm, mean 12.5×7.8 µm). In greenhouse, all
isolates showed typical chocolate spot lesions and differed in their aggressiveness (27%
more, 64% medium and 9% less aggressive). The study revealed high occurrence and
importance of chocolate spot (B. fabae) in the major faba bean growing areas located at high
altitudes and integrated disease management options like use of tolerant and high yielding
varieties with appropriate cultural practices like timely weeding, optimum seeding rate,
repeated ploughing, fallow cropping or crop rotation with cereals are recommended.

Key words: Faba bean, Varieties, chocolate spot, epidemics, Botrytis fabae, Aggressiveness

xii
 1. INTRODUCTION

1.1. Background of the Study

In Ethiopia, faba bean (Vicia faba L.) is grown in the highland area with an altitude ranging
from 1800 to 3000 m.a.s.l. It is believed that the crop was introduced to Ethiopia from the
Middle East via Egypt around 5000 B.C., immediately after domestication (Yohannes, 2000).
Ethiopia is now considered as one of the centers of secondary diversity for faba bean. The
crop occupies the largest area in Ethiopia among other pulses (CSA, 2009). Currently, the
total area under cultivation is estimated to be about 512,067 ha from which 200,000 metric
tonnes are produced (FAO, 2010).

Faba bean is a multi-purpose crop that plays an important role in the socioeconomic life of
farming communities mainly grown as a valuable source of protein (24 - 30 %) and energy for
both human food and animal feed (Sahile et al., 2011). In addition, it is an excellent candidate
crop to provide nitrogen input into agricultural systems; and it makes a significant
contribution to soil fertility restoration as a suitable rotation crop that fixes atmospheric
nitrogen (Noorka et al., 2009; Rubiales, 2010). Despite its wide importance, the average yield
of faba bean is still far below the crop’s potential (>3 tonnes per hectare) (EARO, 2004),
because of many biotic and abiotic constraints (Agegnehu et al., 2006; Sahile et al., 2008b).

Diseases are among the most important biotic factors causing faba bean yield reduction. Such
diseases are chocolate spot (Botrytis fabae Sardina), bean rust (Uromyces viciae-fabae), black
rot (Fusarium solani), aschochyta blight (Ascochyta fabae) and faba ban necrotic yellow virus
(Abraham et al., 2000; Yohannes, 2000; Sahile et al., 2010).

Chocolate spot is one of the important biotic factors threatening the production of faba bean
(Stoddard et al., 2010). It is caused by three species of Botrytis, is incited by the fungus
Botrytis fabae Sard. (Sardina, 1929), B. cinerea (Harrison, 1988) and B. fabiopsis (Zhang et
al., 2010). B. fabae is the only causal agent of chocolate spot in Ethiopia (Dereje, 1999;
Sahile, 2008;Sahile et al., 2012) and the most widespread and destructive, causing yield loss
up to 61% on a susceptible genotype (Dereje and Yaynu, 2001). However, complete crop loss
(100%) may occur under prolonged conducive environment for its development (Torres et al.,

1
2006; Ahmed et al., 2010). It mainly infects the leaf tissue but in severe cases, petioles, stems
flowers and seeds are also infected and its main survival structures are sclerotia (Harrison,
1988; Ayman et al., 2009).

Several mechanisms could facilitate gene flow between populations of B. fabae, apart from
the air dispersal of presumed spores that are dispersible by windblown rain and strong air
currents (Harrison, 1988). Infected faba bean seed (Hawthorne, 2004) and infected stubble or
debris (Dereje, 1999) have long been recognized as important sources of chocolate spot
inoculum and effective means of spreading the disease.

Botrytis classification is largely based on morphological and cultural characteristics. But

many species are morphologically similar and growing conditions significantly influence
variation (Beever and Weeds, 2004). Most their species of the genus have a more restricted
host range (Domsch et al., 1993). Most restricted host specificity occurs on members of
eudicot families like Fabaceae (Jarvis, 1977). Despite the importance of this pathogen, there
have been any studies in southwest Ethiopia, especially regarding its taxonomy.

Since awareness of the existing species is essential for effective disease [Link]
authors have reported differences in virulence among isolates (Hutson and Mansfield, 1980;
Hanounik and Maliha, 1986). Hanounik and Maliha (1986) reported the first evidence of races
in B. fabae populations. Besides, the confirmation of race existence is needed from tests of all
sources of inoculum on the supposed host in the same environment (Bond et al., 1994). Also
wide variation in pathogenicity, cultural characteristics, sclerotial production and infection of
different faba bean genotypes exists among isolates of B. fabae from other regions of Ethiopia
(Dereje, 1996, Sahile et al., 2012).

Farmers in developing countries, particularly resource-poor farmers, are the most affected by
this disease. For instance, chocolate spot may cause 100 % yield loss under favorable
conditions and many farmers have dumped faba bean production due to this disease
(Hanounik and Hawtin, 1982). Aggressive stage of this disease can reduce yield by 50 to 100
% and destroy a crop within hours (Torres et al., 2006). In Ethiopia and other African
countries, losses in yield due to chocolate spot disease can reach 60 to 80 % among

2
susceptible cultivars and up to 34 % among tolerant cultivars (Bouhassan et al., 2004;
ICARDA, 2006; Sahile et al., 2008b).

Many methods of control are practiced for chocolate spot such as the integration of genetic
resistance (Stoddard et al., 2010), adopting various cultural control strategies such as early
sowing (Dereje, 1993), deep ploughing (Dereje, 1999), crop rotation, burying of crop residues
and timely application of appropriate fungicides (El-Sayed et al., 2011).

1.2. Statement of the Problem

The extent of the damage and genetic diversity of the causal agents of chocolate spot in
southwestern parts of Ethiopia is not described and this created gaps to direct integrated
management of the disease for faba bean producers. Until now the productivity of faba bean
under traditional farming system is found to be around 0.9 tonne per hectare (CSA, 2009) in
Dawuro Zone, which is very low. However there is a possibility to improve the situation using
improved varieties (1.8 - 4 tonnes per hectare, which can give better yield than locally grown
land races (EARO, 2010).

Many reasons have been given for the decline in the production as susceptibility to biotic
(Sillero et al., 2009) and abiotic stresses (Link et al., 2010). As preliminary survey carried out
by Dawuro Zone Agriculture Department for three successive years (2008 to 2010), chocolate
spot was found to be the primary bottle-neck problems constraining the production and
adoption of improved faba bean varieties that has been challenging to increase per capita of
individual farmers and food self-sufficiency (DZAD, 2010).

Also crop production under varied agro-ecological conditions of the country would require
modern varieties that fit to diverse ecologies. The existing breeding and seed multiplication
capacity does not fully meet the critical constraints of varieties and seeds leading to stagnated
production and decline in per capita food availability (Mulualem et al.,2012). Farmers as well
as seed producer cooperatives are highly demanding on resistant (tolerant) and better yielding
varieties to maximize their product, and improve the livelihood of their families. However,
unaccessibility of approved varieties for specific production areas was also attributed to low
crop production.

3
Therefore, the use of resistant / tolerant and high yielding varieties for subsistence farming
systems considered as the only solution to maximize their likelihood. As it provides practical,
long-term and environmentally friendly being means of limiting the damage from the diseases
(Wang et al., 2001). Most studies carried out on the resistance to foliar diseases like chocolate
spot in faba bean crop have concentrated on just evaluation or assessment of the crop
genotypes resistance to diseases under diseased conditions (Daniel et al, 2008).
Little is known about the disease distribution, intensity and epidemics, and the fungus
population in the study area. Therefore, this study helps to insight the intensity and
importance of the pathogen, and resistance levels of selected varieties under varying agro-
ecologicay of the southwest Ethiopia.

1.3. Objectives of the Study

1.3.1. General Objective

To study and determine the distribution and epidemics of chocolate spot on different faba
bean varieties in major agro-ecologies of Dawuro Zone, southwest Ethiopia.

1.3.2. Specific objectives

The specific objectives of the study are:

1. to determine severity and prevalence of chocolate spot (Botrytis fabae) in major agro-
ecologies of faba bean at Dawuro Zone.
2. to study the epidemics of chocolate spot on different faba bean varieties under field
conditions, and
3. to characterize some isolates of Botrytis fabae collected from different agro-ecologies
of Dawuro Zone, southwest Ethiopia.

4
 2. LITERATURE REVIEW

2.1. Faba Bean

2.1.1. Taxonomy and ecology of faba bean

Taxonomically, faba bean belongs to Kingdom: Plantae; Division: Magnoliophyta; Class:

Magnoliopsida; Order: Fabales; Family: Fabaceae; Subfamily: Faboideae; Tribe: Vicieae
Genus: Vicia; Species: V. faba; Binomial name: Vicia faba L., Synonyms Faba sativa Moench
(Anonymous, 2012). Vicia faba has a diploid (2n) chromosome number of 12 (Terekhina,
2009).

Vicia faba is an annual legume with one or more rigid, hollow and erect stems (McVicar et
al., 2009). Faba bean is 0.5-1.7 m tall with a square cross-section stem, and has a strong tap
root. The leaves are 10-25 cm long, pinnate with 2-7 leaflets, and do not have tendrils for
climbing over other vegetation. The flowers are 1-2.5 cm long with five petals such as one
standard petal white, two wing petals white with a black spot (true black, not deep purple or
blue), and two keel petals white (Mussa et al., 2008). A flower cluster may produce one to
four pods. The pods are large and green, turning dark at maturity (McVicar et al., 2009).
Three to four oblong/oval seeds are contained within each pod.

Faba bean is grown in temperate regions, subtropics, and tropics. It is not suited to the
lowland tropics, where it may flower well but usually does not produce pods. The optimal
temperature for plant growth is 15 - 20 0C and flowers will abort if temperatures exceed 27 0C
(Anonymous, 2012). The crop grows with rainfall of 700 - 1000 mm per annum (Mussa et al.,
2008). Faba bean is best suited to well-structured soils. It prefers clay and loamy fertile soils
with neutral or sub-acidic pH levels and high water-retention ability (Terekhina, 2009). Faba
beans are vulnerable to soil compaction and hard pans. The common sowing dates are mid-
June in mid-altitude areas and late June to early July in high-altitude areas.

5
 2.1.2. Economic importance

Faba bean is a major grain legume used as alternative crops in the changing environments
(Ahmed et al., 2010). It is a "break" crop, which enhances cereal yield because it decreases
the occurrence of take-all and cereal cyst nematode, which affect cereals (Anonymous, 2012).
Bean is a good honey plant (Terekhina, 2009). Due to its nitrogen fixing capacity, faba bean is
used in crop rotation with the nationally important cereal crops like wheat, teff, and barley
(Jarso and Keneni, 2006; Mussa et al., 2008). This unique ability reduces the dependence of
farmers on extensive use of chemical fertilizers protecting soil and water quality (Sillero et
al., 2009).

2.1.3. Production constraints

The total area in the world dedicated to faba bean cultivation is declining (Jensen et al., 2009).
One of the main reasons is the unreliable yields, mainly due to susceptibility of the crop to
Chocolate spot (Botrytis fabae and B. cinerea ), Ascochyta fabae (Ascochyta fabae ), rust
(Uromyces viciae-fabae), Downy mildew (Peronospora viciae), Cercospora leaf spot
(Cercospora zonata), Alternaria leaf spot (A. alternate and A. tenuissima), wilt (Fusarium
oxysporum), root rot (Rhizocotonia solani), stem nematode (Ditylenchus dipsaci), root-knot
nematode (Meloidogyne incognita and M. javanica), weeds, viruses, water logging, cold and
poor soil fertility (Mussa et al., 2008; Sahile et al., 2010; Stoddard et al., 2010). Chocolate
spot (Botrytis fabae) is one of the economically important diseases that damages the leaves
and reduces faba bean production globally including Ethiopia (Torres et al., 2006; Samuel et
al., 2008a).

2.2. The Genus Botrytis

Taxonomically, the genus Botrytis belongs to division: Deutromycotina (imperfect fungi),

class: Hyphomycetes, order: Hyphales (Moniliales), family: Moniliaceae. The fungus is
usually referred to by its anamorph (asexual) form because the sexual phase is rarely observed
or unknown (Pradier et al., 2007).

6
Botrytis and its teleomorph Botryotinia Whetzel comprise 22 species and 1 hybrid species
(Yohalem et al., 2003). Botrytis classification is largely based on morphological and cultural
characteristics. Species of Botrytis have been named based on host association (Jarvis, 1977).
Features such as sclerotial size and form and conidium size are useful in delimiting some
species, but many species are morphologically similar and growing conditions significantly
influence these characters (Beever and Weeds, 2004). Most species have a worldwide
distribution or occur wherever their host crops are grown (Staats, 2005). The genus Botrytis
comprises one generalist, B. cinerea, infecting over 200 eudicot hosts, especially senescing or
otherwise weakened or wounded plants. All other species are considered specialists with a
narrow host range.

Sardina (1929) in Spain was the first to associate Botrytis with the chocolate spot disease. He
considered a new species, Botrytis fabae Sard., to be a cause of chocolate spot but that
infection by B. cinerea Pers. could be similar although the lesions are not identical. Zhang et
al. (2010) found that inoculation of broad bean leaves with conidia of B. fabiopsis caused
typical chocolate spot symptoms with a similar disease severity to that caused by B. fabae but
significantly greater than that caused by B. cinerea in China. He was also able to distinguish
or identify three species of Botrytis based on morphology of colonies, sclerotia and conidia.
At 20 0C B. fabiopsis grew on potato dextrose agar (PDA) at 12–13 mm d–1, similar to B.
fabae (13 mm d–1), but slower than B. cinerea (17–19 mm d–1). It forms pale gray colonies
with short aerial mycelia and gray to black sclerotia in concentric rings on PDA. B. fabiopsis
producs greater numbers of sclerotia than B. cinerea but fewer than B. fabae. Conidia
produces by B. fabiopsis on broad bean leaves are hyaline to pale brown, elliptical to ovoid,
wrinkled on the surface and are larger than conidia of B. fabae and B. cinerea. In
pathogenicity tests, representative isolates caused typical chocolate spot symptoms and were
reisolated from infected leaves, indicating that B. fabae is the causal agent of chocolate spot in
Ethiopia (Samuel et al., 2012).

2.2.1. The pathogen Botrytis fabae

This species closely resembles B. cinerea but is a specialized pathogen of Vicia faba,
distinguished by higher pathogenicity, somewhat larger spore size, tendency to produce small

7
sclerotia in culture and protein electrophoresis patterns (Harrison, 1988; Sahile et al., 2012).
Hutson and Mansfield (1980) explored the pathogenicity of 15 different macrocondial
lineages from one parent, and found a two-fold difference in lesion diameter hinting at the
possibility the original strain was a heterokaryon or heteroplasmon.

2.2.2. Host range

Botrytis fabae was thought to be a host specialized pathogen but has now been recorded as a
cause of disease on Vicia spp., Pisum spp., Lens spp., and Phaseolus spp., all belonging to
Fabaceae (Staats et al., 2005; Tivoli et al. 2006), and Trifolium dasyurum (You et al., 2008).

2.2.3. Symptoms and aggressiveness

Leaves, stems, flowers and pods can all be infected (Gaunt, 1983), with flowers and pods
being the most susceptible parts (Griffiths and Amin, 1977). On flowers tiny red brown spots
appear (MacLeod and Sweetingham, 2006) while in severe infections the pod might split and
small red brown spots can be seen on the outer surface of the seed. If the disease becomes
severe at an early stage of growth before the fruit matures, the entire crop can be lost
(Matthews, 2003).

Aggressiveness is a property of the pathogen reflecting the relative amount of damage caused
to the host without regard to resistance genes and it is description of the rate at which a level
of disease is reached, with more aggressive pathogens reaching this level faster where as
virulence is the genetic ability of a pathogen to overcome genetically determined host
resistance, which is effective against other races of that pathogen, and cause a compatible
disease (Shaner et al., 1992). According to Richardson and Horsham (2008), two stages of the
chocolate spot disease are usually recognized i.e a non-aggressive phase followed by an
aggressive phase. The terms "aggressive" and "nonaggressive” describe rapidly expanding
and limited lesions, respectively. The non-aggressive lesions, which either do not expand at
all or do so only slowly, are discrete reddish-brown spots over the leaves and stems, and
limited. Under conditions of continuous high humidity, limited lesions become aggressive,
dark in color and rapidly increasing in size and coalesce to form larger grey-brown target
spots that eventually cover the entire plant and lead to loss.

8
 2.2.4. Pathogenic variability

A wide variation in pathogenicity exists among isolates of Botrytis fabae. According to

Dereje (1996) nine isolates of B. fabae from Ethiopia, with a similar conidial size, differed in
their cultural characteristics, sclerotial production and infection of different genotypes. These
variations resembled differences in cultural characteristics and virulence patterns in B. fabae
collected from other locations (Mohammed et al., 1981). Hanounik et al. (1984) observed that
in a collection of different isolates, the more virulent isolates produced fewer conidia and
formed larger sclerotia than the less virulent isolates. Variation in morphology and
aggressiveness between isolates has also been reported by Hutson and Mansfield (1980).
According to Sahile et al. (2012) a preliminary severity of disease test results with 76 isolates
revealed that differences in virulence of the B. fabae isolates from faba bean fields in different
agro-ecological zones of Northern Ethiopia. Gubalafto–Woldia district had the highest
percentage of avirulent isolates, while Yilman– Densa, Gondar–Zuria, Wegera, Ambasel–
Tehulederae and Kutaber districts had no avirulent isolates. Overall, the sub-mid north-east
districts Ethiopia (Dessie Zuria, Kutaber and Ambasel–Tehulederae) had a higher frequency
of virulent isolates than the moist, humid north-western districts of the country.

2.2.5. Disease epidemiology

Several mechanisms could facilitate gene flow between populations of Botrytis fabae, apart
from the air dispersal of putative spores that are dispersible by windblown rain and strong air
currents (Harrison, 1988). Infected faba bean seed (Bretag and Raynes, 2004; Hawthorne,
2004) and infected stubble or debris (Dereje, 1999) have long been recognized as important
sources of chocolate spot inoculum and effective means of spreading the disease in Ethiopia.

Carry-over of B. fabae between crops occurs as mycelium in crop debris (Gaunt, 1983) or as
sclerotia in the soil and in crop debris. Mycelium can survive for over a year in crop debris on
the surface of the soil, but only four months when buried 20 cm (Dereje, 1999), while
sclerotia can survive for up to one year without loss of viability (Harrison, 1978). Sclerotia
buried in soil are less likely to survive (Harrison, 1978). This form of survival is particularly
important when there is a long summer gap between host crops as in Mediterranean climates

9
(Jellis et al., 1998). Alternative hosts and volunteer faba bean plants may also be important
for survival of the fungus between cropping seasons. Seed infection may have a role in
introducing the disease into new areas (Harrison, 1978).

The conidia are released as clumps or singly, and are generally wind-borne (Harrison and
Lowe, 1987), but may be dispersed by rain or insect vectors, particularly thrips (Harrison,
1988). Also it spread by splash droplets (Jarvis, 1962). However, the numbers of conidia
dispersed in splash droplets or released by the puff or tap mechanisms at the onset of rainfall
were small by comparison with the diurnal maxima observed in infected crops in the
experiments of Fitt, Creighton and Bainbridge (Fitt, et al., 1985). They concluded that wind
was the main agent in spore dispersal, even during periods of rainfall, and that the
development of chocolate spot epidemics was associated with wet weather because the high
relative humidities favored sporulation, infection, and lesion development. These highlight the
need to use appropriate sampling methods for splash-dispersed or dry dispersed inoculum and
show how conclusions about methods of spore dispersal may greatly affect our understanding
of the epidemiology of a this disease.

Chocolate spot development is highly dependent upon environmental factors. Frequently the
disease is first noticed on isolated plants that act as foci from which the disease develops
rapidly in humid weather. It spreads via airborne conidia within crops where relative humidity
is above 70 % and at temperatures of 15 to 23 0C (Harrison, 1980; Dereje, 1993), especially in
mornings, warm and rainy (frequent rain) weather conditions are favorable for the growth of
chocolate spot epidemic. With this environment, the epidemic grows with apparent infection
rates ranging from 0.142 to 0.164 disease units per day, which means several chocolate spot
infections within a single growing season (Dereje, 1993). This is an indication for rapid and
dangerous spread of the disease. Obviously, if the pathogen falls short of the above mentioned
weather variables, it will be forced to have short infection period, and this is significant in
spread of an epidemic.

Lesions expand slowly when relative humidity is below 66 % (Harrison, 1980). The optimum
temperature for lesion growth is between 15 to 20 0C, with minimum of 4 0C and maximum of
30 0C. Neither light intensity nor water films on the leaves have an impact on lesion growth

10
(Harrison, 1980) though near ultra-violet light induces sporulation (Harrison and Heilbronn,
1988) and it is common practice to use alternating cycles of 12 h darkness and 12 h of near
ultra-violet light to stimulate sporulation of B. fabae (Dhingra and Sinclair, 1995). Plants
become more susceptible to chocolate spot as they age or suffer from freezing (Creighton et
al., 1985; Heilbronn and Harrison, 1989).

2.2.6. Economic significance of chocolate spot

Chocolate spot, caused by B. fabae, is one of the major diseases of faba bean nationwide
(Dereje and Tesfaye, 1994; Sahile et al., 2012). According to Sahile et al. (2010) chocolate
spot disease has reduced the grain yield and quality by reducing 100 - seed weight. Dereje and
Beniwal (1988) recorded yield losses of up to 61 % for susceptible varieties and 34 % for
tolerant varieties in Ethiopia. Yet, complete crop failure due to the disease is commonly
encountered when a long lasting favorable environment for disease development prevails in
an area (Dereje et al., 1994). In Tigray (Negash areas), seed yield loss of 62 % was estimated
due to chocolate spot in 2000 (MARC, 2002).

2.2.7. Disease management

[Link]. Cultural practices

Avoidance of factors that predispose the crop to disease epidemics can also operate chocolate
spot epidemics. The use of crop rotation to avoid infected debris and volunteer plants, burning
of infected plant debris, burying or grazing stubble and removal of volunteer plants will
minimize the risk of chocolate spot in subsequent faba bean crops (Liang, 1989). Deep
plowing of faba bean fields with high chocolate spot infection immediately after harvest
reduces the risk of disease development (Dereje, 1993). High plant densities that provide a
high humidity environment should be avoided and in Europe it is recommended that early
sowing be avoided to prevent spring frost damage and hence predispose the crop to the
disease (Jellis et al., 1998). The association between early sowing and chocolate spot was also
noted in Syria (Hanounik and Hawtin, 1982). In Ethiopia considerable hold-up and shortening
of chocolate spot epidemics and there by reduction of attack can be achieved by late sowing
of faba bean as the conditions suitable for development of the disease do not exist for a

11
sufficiently long period of time (Dereje, 1993). Planting faba bean in mixture with field pea in
a ratio of 1:2 drastically reduces epidemic development of chocolate spot in faba bean
(Dereje, 1999; Amare, 2009).

Adequate fertilizer, in particular potassium and nitrogen, and good soil drainage is necessary
to prevent early senescence, which would provide dead leaves upon which the fungus
sporulates (Liang, 1989; Jellis et al., 1998). According to El-Bramawy et al. (2010) potassium
is an important element for agronomic characters improvement and for enhancing the
resistance to rust and to chocolate spot diseases. The results showed that the potassium soil +
foliar application (PSFA) at level 3 (171.36 + 3.4 Kg K2O/ha) followed by potassium as foliar
application (PFA) at level 3 (3.40 Kg K2O/ha), increased significantly the majority of studied
characters including the agronomic characters and/or increase resistance to foliar diseases. A
higher incidence of chocolate spot occurred in plants grown on acidic soils (Elliott and
Whittington, 1978) and with increased nitrogen fertilizer (Hegab and Beshir, 1994).

[Link]. Chemical control

There are a range of fungicides available to control Botrytis, and selection of the most
appropriate fungicide could depend on the level of disease pressure present. However, several
applications of fungicide are generally required in situations where the environment remains
favorable for disease development. In Ethiopia many fungicides, both systemic and protectant
were tested for control of chocolate spot in the past (Habtamu and Dereje, 1986; Sahile et al.,
2010), among these chlorothalonil and benomyl with menacozeb were effective against
chocolate spot. Chlorothalonil completely protected faba bean plants from infection when
applied at weekly intervals.

In Australia it is recommended that fungicide be applied from flowering onwards, depending

on conditions. Carbendazim or procymidone are recommended in high disease situations,
while benomyl, chlorothalonil, mancozeb, tebuconazole or copper products are also available
(Panagiotopoulos et al., 2002). Carbendazim was better than mancozeb or chlorothalonil for
control of chocolate spot (Bretag et al., 2002). In Europe benomyl, or tank mixes of
carbendazim plus chlorothalonil or iprodione or vinclozolin are used from early flowering and

12
3 - 4 weeks later (Jellis et al., 1998). Bainbridge et al. (1985) were able to reduce an early
chocolate spot epidemic by using seed treated with benomyl plus thiram. In China, sprays of
carbendazim or thiophanate-methyl at podding or flowering were found to be effective
(Liang, 1989).

Late epidemics appear not to affect yield and fungicides are not warranted in these conditions.
The best indication to farmers of a potentially damaging epidemic appears to be the build-up
of chocolate spot on lower leaves before or during flowering (Creighton et al., 1985).
Resistance to benzimidazole fungicides, such as carbendazim, is a major problem when they
are used frequently (Jellis et al., 1998). According the concept of integrated disease
management, chemical treatment should only be applied as supplement to combat chocolate
spot risk. It is worth remembering that these products are protectants and are most effective if
applied before disease development.

[Link]. Biological control

There is promise in using biological control agents to control chocolate spot diseases;
nevertheless, this strategy has not been fully exploited. Isolate of Trichoderma ovalisporum
and Trichoderma longibrachiatum as effective antagonists of B. fabae for the first time
(Sahile et al., 2011). Trichoderma viride was found effective against chocolate spot in 1990
(Jellis et al., 1998). Jackson et al. (1997) reported that some isolates of Penicillium
chrysogenum inhibited growth and development of B. fabae in vitro and showed the
development of chocolate spot under glasshouse conditions was substantially reduced by this
agent. Similar results were obtained with Penicillium brevicompactum and Cladosporium
cladosporioides (Jackson et al., 1997).

Fourteen Bacillus isolates were found to be antagonistic to B. cinerea and B. fabae and
reduced development of chocolate spot symptoms. However, they were not effective in the
field (Sharga, 1997). In Ethiopia, study has revealed that the biological control agents for
chocolate spot of faba bean and Bacilli are natural residents of faba bean leaves. Thirty
isolates of Bacillus spp. were tested for their effects on B. fabae by dual culture technique on
potato dextrose agar. Sixteen isolates produced 5 mm or higher inhibition zone and out of

13
these, two isolates were the most effective having inhibition zone of 8 and 7 mm. Isolates
reduced the growth of the pathogen colony in dual culture by 23 - 64%. Four Isolates proved
most effective in retarding disease development on two susceptible and one tolerant cultivar
and can be further explored for commercial use for management of chocolate spot disease of
faba bean (Sahile et al., 2009). Another antagonistic bacterium, Erwinia herbicola, which was
isolated from the phylloplane of a chocolate spot-resistant genotype, was able to significantly
reduce disease severity when used as a foliar spray (Abd-El-Moity et al., 1990).

[Link]. Host resistance

The major constraint for faba bean breeding for chocolate spot resistance is the lack of good
sources of resistance. Some resistant germplasm has been reported but most of it has yet to be
introduced into the agricultural market. So far, total resistance has not been reported and only
incomplete resistance is being used (Jensen et al., 2010). The level of resistance to chocolate
spot in most faba bean cultivars is low although varying degrees of resistance have been
reported (Bouhassan et al., 2004; Tivoli et al., 2006; Villegas-Fernandez et al., 2009).

Based on an analysis of variance of host and pathogen effects, Van der Plank (1984) also
determined that resistance in a host could be classified as either horizontal (race nonspecific)
or vertical (race specific) and that corresponding pathogenicity in the pathogen was classified
as aggressiveness and virulence, respectively. Horizontal resistance and aggressiveness were
defined as the main effects of the host and pathogen, respectively. Vertical resistance and
virulence were defined as the interaction effect among host genotypes and pathogen races.

Vertical resistance is also known in plant breeding as “specific”, and is determined by one or
a few genes that impart complete resistance to a certain pathogen. This type of resistance is
also known as “short-term” resistance, and also known as qualitative, monogenic, or strain
specific resistance (Gair et al., 1987). Horizontal resistance, in turn, is usually associated with
several genes and imparts an incomplete type of resistance; the host is attacked by the
pathogen in a higher or lower degree, depending on the number and type of genes involved in
the resistance. This type of resistance is also known as general resistance, field resistance,
non-specific, quantitative, or polygenic resistance (Robert et al., 2000).

14
Resistance is thought to be based on the ability of faba bean to produce, as a post-infection
defence response against B. fabae, low-molecular-weight secondary metabolites, such as
furanoacetylenic phytoalexins (Ingham, 1982). In contrast, the greater ability of B. fabae to
colonize broad bean tissues seems to be related to its capacity to detoxify broad bean
phytoalexins and to reduce their toxic effects (Madeira et al., 1993). B. fabae is able to
produce pectin degrading enzymes, such as polygalacturonase, during development of
chocolate spot (Harrison, 1988). The possible dual role of polygalacturonase isoenzymes from
B. fabae as defense responses elicitors during broad bean colonization (Buzi et al., 2003). It is
very clear that Botrytis is well equipped for the necrotrophic lifestyle and may use different
approaches to disease development depending on the strain or strain–host combination
(Hammerschmidt, 2010).

Different approaches may be taken in order to control chocolate spot (Stoddard et al., 2010).
The use of resistant varieties to chocolate spot is one of the management options. In order to
identify the resistant varieties, the test genotype has to be subjected to a uniform and heavy
disease pressure obtained through natural happening (hot spot), and artificial inoculation
(Mussa et al., 2008).

Faba bean collections have been screened for response to chocolate spot and evaluation
methods improved (Bouhassan et al., 2004). The International Centre for Agriculture
Research in Dry Areas (ICARDA) has incorporated resistance into local germplasm, so new
genotypes have been introduced in Australia, Egypt and Ethiopia, among other countries
(Stoddard et al., 2010). So far several chocolate spot resistant genotypes have been reported
(Table 1). Source of resistance for chocolate spot (BPL-1179-A-1, BPL-1802-1 and BPL-
1179-2) have been identified from introductions of ICARDA to Ethiopia (Mussa et al., 2008).
From the landrace collections, a number of varieties, namely CS20DK, NC-58, Bulga 70 (coll
111/77), Wayu (Wayu 89-5), Selale, Lalo, Dagm and Adet Hana have been nationally or
regionally released for different recommendation domains (Mussa et al., 2008).

15
Table 1. Faba bean germplasm resistant to chocolate spot (Botrytis fabae).

Accession name Material References

LPF 44, LPF 237, LPF 05, LPF 113 Breeding lines Rhaiem et al. (2002)
ILB 1414, ILB 6561 Breeding lines Bayaa et al. (2004)
FRYM167, FRYA58 Breeding lines Bouhassan et al. (2004)
BPL 260, BPL 2282, L82010, L83106, L83108,
L83109, L83117, L83129, L83149, B8838, B8839 Breeding lines Khalil et al. (2004)
Reina Blanca Cultivar Khalil et al. (2004)
Icarus, Nura Cultivars Raynes (2008)
Gladice Cultivar INRA/DPE (2008)
BPL710, BPL 1763, ILB 4726, LPF 120, ILB 4709, Breeding lines Villegas-Fernandez et
ILB 5284, Sel.97 Lat.97 132-1, Sel.97 Lat.97 132-3 al. (2009)

[Link]. Integrated disease management

These are controlled through the use of Integrated Disease Management (IDM), which can be
defined as the use of a range of disease management practices to reduce the impact of plant
diseases. Used alone, an individual management practice may not reduce the level of disease
to an acceptable level, whereas the additive effect of several practices will be done. An
integrated approach is the key to successful management of chocolate spot in faba bean.
Mixed cropping of faba bean with cereal crops and mancozeb spray reduced the disease and
increased the grain yield, as well as the seed weight over sole and mixed cropping with field
pea (Sahile et al., 2008b; Sahile et al., 2010). Similarly Agegnehu et al. (2006) and Gemechu
et al. (2006) also found that cereal mixing with faba bean has advantages over faba bean
mixing with field pea. According to Fernandez-Aparicio et al. (2011) chocolate spot was
significantly reduced when faba bean was intercropped with cereals, but not when
intercropped with legumes. Suppressive effects can be ascribed to a combination of host
biomass reduction, altered microclimate and physical barriers to spore dispersal. Also creation
of a physical barrier in the form of non-host plants prevents some of the dispersed spores from
being deposited on the host tissue by interception (Khalil et al., 2011).

Integrated disease control for faba bean studies conducted on farmers’ fields under different
environmental conditions showed that newly released varieties with resistance to chocolate

16
spot responded less to fungicidal applications. These findings led to the development of
improved disease control packages. The use of new, resistant varieties has reduced the use of
chemicals drastically (Khalil and Erskine, 2001). In addition, early sowing, use of improved
varieties and fungicide protection avoid the occurrence of chocolate spot disease at epidemic
proportions (Dereje, 1993).

17
 3. MATERIALS AND METHODS

3.1. Geographic Location Location of Dawuro Zone

Survey and field experiment were carried out in the 2011 crop growing season in Dawuro
zone, Southern Nations, Nationalities and Peoples’ Regional State (SNNPRS). Dawuro zone
lies in between 60 36’ to 7021’ north latitudes and 36068’ to 370 52’ east longitudes. It is one
of the 13 zones of the region situated between Omo river in the north to south and Gojeb river
in the north to northwest. Tercha is the zone capital, 486 km from Addis Ababa through
Jimma road, and 282 km from Hawassa. The zone is subdivided into five woredas (local
districts) with varying altitudes ranges from 730 to 2850 m.a.s.l. The area has mountainous
topography. The zone receives a total annual rainfall of 1200 to 1800 mm with mean monthly
temperature varying from 15 to 27.5 0C.

The area under cultivation is estimated to be 100,395 ha of the total 446, 082 ha of the zone
and out of which 38 % is lowland (500 – 1500 m), 41 % mid altitude (1501 - 2200 m) and
21% (> 2200m) highland. The dominant crops grown in the study areas include Enset,
Legume crops (faba beans, chickpeas, haricot beans, lentils and field peas), Cereal crops
(wheat, maize, teff, sorghum and barley), and Root crops (taro, sweet potatoes and cassava).

18
Note: 1=Bale, 2=Dali, 3=Sengetsi, 4 = Gozoshasho, 5=Waka, 6=Mari, 7=Kechi,
8= Medanalem, 9=Botori, 10=Gessachare and 11= Tulema indicates surveyed peasant
associations; where as A=Tocha, B=Mari and C=Turi indicates field experimental sites.

Figure 1. Geographic location of Dawuro Zone in Ethiopia.

3.2. Description of the Study Areas

Field surveys were conducted in major areas of faba bean production areas of eleven peasant
associations (Bale, Dali, Sengetsi, Waka, Mari, Gozoshasho, Medhanalem, Botori, Kechi,
Gessachare and Tulema) in four districts (Essera, Mareka, Tocha and Loma) of Dawuro zone
during 2011 growing season (Figure 1). These focused administrative units were selected
based on secondary data obtained from Dawuro Zone Agricultural department, conveniently
with the assistance of development agents and local administrators, and measuring the
location of fields using GPS. The selected study sites were stratified to include two agro-

19
ecological zones, “Wayina Dega” Zone (mid-altitude 1800 to 2200 m.a.s.l, with sub-humid
and warm climate) and “Dega” Zone (high land > 2200 m.a.s.l, with humid and cool climate)
(Appendix Table 1A). From each PAs four faba bean fields, a total of 44 fields were surveyed
from September 15 to October 5, 2011. Depending on their relative faba bean production, 36
% were in mid-altitude and 64 % in highland altitude. During surveyed period, the faba bean
fields were at pre-podding and podding stages (at about 81 to 100 days after planting) at this
growth stage chocolate spot severity expected maximum (Sahile et al.,2008a).

The field experiments were carried at farmers training centers (FTC), farm of Dawuro Zone
Agricultural Department at Tocha (2658 m.a.s.l), Mari (2444 m.a.s.l) and Turi (1805 m.a.s.l)
under rain fed conditions. These experimental sites had been planted with faba bean in the
past years and indigenous chocolate spot populations were considered adequate for disease
development. Soil physico-chemical prosperities for field experimental sites were peresented
in Appendix Table 1B.

3.3. Disease Survey and Assessment Methodology

Selection of surveyed faba bean fields within each agro-ecological zone was varying at least 5
km intervals along main and feeder roads depending on the availability of the farm. In each
sample field, three quadrants (1 m x 1 m) of at least 10 m apart were sampled at diagonal
transects. Eight faba bean plants were assessed for disease severity along an X-shaped
transect were taken as the sample unit. In each quadrate of assessed plants for chocolate spot
severity, averages were taken for each field. Disease symptoms were scored on the basis of a
1 – 9 class visual scale where, 1: no lesions or covering up to 1 % of leaf surface; 3: lesions
covering 1 – 2 % of leaf surface; 5: lesions common (3 – 5 mm in diameter), covering 2 – 5 %
of leaf surface; 7: lesions that cover 5 – 10 % of leaf surface; 9: extensive lesions, covering
more than 10 % of the leaf surface. Disease severity scores were converted into disease
severity index (DSI) for the analysis (Hanounik, 1986; Ayman et al., 2009; Abo-Hegazy et
al., 2012).

20
 ∑(NxV)
Dissease severity index (DSI)% = x100
9N
Where: N = number of infected leaves, V= numerical grade, and 9 = Higher degree in the
category.

During the surveyed period, the plant population (m 2) in each quadrant was counted and the
mean crop population was obtained by averaging the crop population in the three quadrants,
altitude (m) from GPS readings, slope (%) of the farm using Clinometer’s reading, and weed
condition of the farm were recorded for each sampled field. Growers were asked information
on their attitude towards faba bean production, type of varieties they planted, and cultural
practices (time of planting, frequency of ploughing their farm, previous crop history, and
disease control practices) employed.

3.4. Disease Sample Collections

Representative samples of leaves, stems, pods and seeds showing symptoms of chocolate spot
were collected from all the surveyed areas. For each sample reference number, sampling date,
locality and altitude were recorded. Then disease specimens were carefully wrapped
individually in newspaper and taken to Plant Pathology Laboratory of Jimma University, kept
at 4 0C in a refrigerator.

3.5. Field Experiment

3.5.1. Plant material

Thirteen improved varieties released in Ethiopia, up to 1.5 kg of each were obtained from
Holeta Agricultural Research Center (HARC) and a local cultivar being used by farmers in
the study area were assessed for their chocolate spot disease reaction and yield performance.
These varieties incorporated source of resistance for chocolate spot and released for different
recommendation domains. About the information obtained from MoARD (2009) and EARO
(2010), varieties are given in Table 2.

21
Table 2. List of faba bean varieties used for the study and their agronomic traits

Variety Pedigree Year of Altitude Maturity

Yield (qt/ha)
name Release (m) days
Station On-farm TSW
Tumsa EH9965-3 2010 2050-2800 121-176 25-69 20-38 737
Hachalu EH960091-1 2010 1900-2800 122-156 32-45 24-35 890
Walki Bulga-70 x ILB4615 2007 1800-2800 133-146 24-52 20-42 676
Gebelcho Tesfa x ILB4726 2006 1800-3000 103-167 25-44 20-30 797
Moti ILB4432 x Kuse 2-27-33 2006 1800-3000 108-165 28-51 23-35 781
Degaga R-878-3 2002 1800-3000 116-135 25-50 20-45 517
Tesfa 75TA26026-1-2 1995 1800-2400 125-135 20-40 20-45 441
Mesay 74TA12050 x 74TA236 1995 1800-2400 120-130 25-50 20-45 428
Bulga-70 Coll 111/77 1994 2300-3000 143-150 20-40 15-35 440
Kasa NA 1980 1900-2300 120-135 45-55 25-40 428
Kuse Kuse 2-27-33 1979 2300-3000 135-150 20-35 15-25 393
NC-58 NC-58 1978 1900-2300 118-132 20-40 15-35 449
CS20DK CS20DK 1977 2300-3000 145-160 20-40 20-40 476
a
Local NA NA - - - - -
NA = information not available; Local cultivar= obtained from farmers in the study area;
TSW=1000 seed weight

3.5.2. Experimental Design and Management

The experimental fields were prepared by using local oxen drown plough (maresha)
according to farmers conventional farming practices. The fields were ploughed three times,
the first at the beginning of June 2011, the second after three weeks and the third during the
first week of July 2011 before planting. In accordance with the specifications of the design, a
field layout was prepared and each treatment was assigned randomly to experimental units
within a block. Sowing was done on July 16, July 18 and July 20, 2011 at Tocha, Mari and
Turi experimental sites, respectively. Two seeds per hill were planted manually to ensure
germination and good stands of the varieties and then thinned to one plant 15 days after
emergence to achieve 40 plants per plot.

The experimental plots were arranged in randomized complete block design with three
replications. Each experimental plot had a total area of 1.6 m2 (1 m x 1.6 m) with spacing of
40 cm between rows and 10 cm between plants to give a final population density of 250,000

22
plants per hectare (EARO, 2010). Between each block (0.5 m), there was a disease spreader
row of local cultivars. This practice induces early disease development on local checks which
can later serve to spread the inoculums to adjacent test entries (ICARDA, 1986). Local
cultivar was also serving as a standard against which disease rating is done in the nursery for
disease resistance. Each plot had 4 rows from which the central two rows of middle four
plants from each were used for disease assessment and yield components determination where
the other two outermost rows were kept as boarder plants.

3.5.3. Disease assessment

Chocolate severity expressed as the proportion of diseased tissue exhibiting chocolate spot
symptoms, was recorded weekly for each plot using a 1 – 9 scale according to Hanounik
(1986) as described in 3.2, starting from 7 days when first symptoms of chocolate spot were
visible in the experimental plots and were repeated ten times every 7–10 days until the
podding stage where the amount of chocolate spot disease was expected maximum (Sahile et
al., 2008b;Villegas–Fernandez et al., 2012) from eight selected and tagged plants from
middle two rows in each plot (excluding the two outer rows). Disease severity scores were
converted into disease severity index (DSI) for the analysis as described in 3.2.

The response of varieties was expressed as the disease severity index (DSI) values grouped in
six resistance levels described by Abo-Hegazy et al. (2012) as indicated in the Table 3.

Table 3. Six resistance levels used for classification of faba bean varieties.

Reaction group DSI range

Highly resistant (HR) 0 and 2.0
Resistant (R) >2.0-15.0
Moderately resistant (MR) >15.0-40.0
Moderately susceptible (MS) >40.0-60.0
Susceptible (S) >60.0-80.0
Highly susceptible (HS) >80.0-100.0

23
 [Link]. Disease progression and modeling

Disease progress curves (chocolate spot severity indices over time) were plotted for each
variety for each location. These curves provide the basis for the three other epidemiology
measurements: final DSI, area under the disease progress curve (AUDPC) and apparent
infection rate (r).

AUDPC values were calculated for each plot according to the mid-point rule using the
following formula:

𝒏 𝟏
𝒀𝒊 + (𝒀𝒊 + 𝟏)
𝐀𝐔𝐃𝐏𝐂 = 𝑻(𝒊 + 𝟏)_𝑻𝒊)
𝟐
𝒊 𝟏

Where Yi is the Disease severity index (DSI) of chocolate spot at ith assessment, Ti is the time
of the ith assessment in days from the first assessment date and n is the total number of disease
assessments. Because severity was expressed in percentage and time in days, AUDPC was
expressed in %-days.

The apparent infection rate, expressed in disease units per day, was calculated by first
converting DSI values to a proportion on a scale of 1 to 9 and then transforming the data
using Logistic, ln [(Y/1-Y)], (Van der Plank, 1963) and Gompertz, -ln [-ln(Y)], (Berger,
1981) models were used to compare estimation of disease progression parameters, where Y
represents the proportion of infected plants and 1-Y represents the proportion of healthy plants
remaining in the plot. The transformed values (y) were regressed against time (x) to determine
the model (Campbell and Madden, 1990). The goodness of fit of the models was tested based
on the magnitude of the coefficient of determination (R2). The appropriate model was then
used to determine the apparent rate of disease increase (r) and the intercept of the curve.

24
 3.5.4. Yield and yield components

Separating harvesting (boarder and central rows) was made from the middle of November to
first week of January, 2011 depending on the maturity period of each variety. After exposed
to sun drying, the harvested products were threshed and yield and hundred seed weight were
measured from central rows of each plot (Khalil et al., 2011).

At harvest, number of pods/plant, number of seeds/pod, seed yield per hectare and weight of
100 seeds per plot were recorded in the following manner.

1. Number of pods per plant (NPPL): Eight plants were tagged for chocolate spot
severity from each plot was used and their pods were counted and averaged.
2. Number of seeds per pod (NSPL): The seeds of five randomly taken pods from each of
eight randomly pre-tagged plants was counted and averaged.
3. Seed yield (SYD): For seed yield two central rows of each plot were harvested, sun-
dried, threshed, cleaned and weighed with an electronic balance in gram then
converted to kilogram per hectare according to the following equation (Khalil et al.,
2011).
Grain weight (g)
Yield (kg per ha) =
No. of rows (2) X R − R Distance (0.4m) X Row length (0.5 m)
4. Hundred seed weight (HSW): The 100 seed-weight of randomly taken seeds from
each variety at 10% moisture content weighed with electronic balance.

3.5.5. Weather parameters

Monthly total rainfall (mm), relative humidity (%) and the daily minimum and maximum
temperature (0C) for the experimental periods were obtained from the nearby weather stations
in Dawuro zone.

25
 3.6. Studies in the Laboratory

3.6.1. Media Preparation

Botrytis fabae was isolated on faba bean dextrose agar (FBDA). Two hundred gram of faba
bean seeds was weighed out in a 1.5 liter flask to which 1liter of distilled water was added and
boiled for 30 minutes, and also sterilized at 121 0C for 30 minutes in a pressure cooker. Then
after, the extract was separated. Eighteen gram of agar and 5 g of dextrose were added to the
extract, stirred till dissolved and made up the volume to 1 liter with additional water. Then,
this preparation was sterilized at 121 0C for 30 minutes in a pressure cooker, cooled and
poured into Petri dishes (Dereje, 1996).

3.6.2. Isolation of the pathogen

Samples of naturally infected faba bean tissues with symptoms of chocolate spot disease were
cut into small pieces, each with a single lesion. Plant parts, which were not used for the
isolation of the fungus were cut off and discarded (Amare, 2009). Each piece of specimen
were sterilized by soaking in 5 % sodium hypochlorite for 2 to 3 min, rinsed with distilled
water, dried on sterilized filter paper. Five pieces were placed onto each culture placed on
FBDA plate, and the plates were incubated at room temperature five to seven days for
emerging fungal colonies.

3.6.3. Hyphal tip isolation and preservation

Pure isolates were obtained using hyphal-tip techniques (Hanounik and Robertson, 1988).
When the fungal colonies developed from plant pieces, each colony was transferred to another
FBDA plate, by stabbing a flamed loop into the medium, touching the mycelium on growing
edge of a colony and then streaking onto the fresh plate. It was repeated until pure colonies
were obtained. Isolates of each location were maintained on FBDA in sterile screw-capped
test tubes at 4 0C until used for further study. Identification was done using microscopic
examination, comparisons with reference slides with B. fabae obtained from HARC and
identification keys (Marthin and Panela, 1985).

26
 3.6.4. Cultural and morphological characterization of the B. fabae Isolates

The colony appearance (texture, form and pigmentation), growth rate, and conidial
morphology were studied to determine variations among the field collected isolates of B.
fabae from different agro-ecologies. Single colonies of each isolate were initiated by
displeasing a 5-millimeter mycelial plug from the advancing edge of the fungal culture onto
the centre of each Petri dishes containing FBDA amended with 0.01% streptomycin (Sahile et
al., 2012). The plates were incubated at room temperatures under alternative 12 hr white
fluorescent light/12 hr dark cycle. The plates arranged in completely randomized design and
for each isolate three petri-dishes were used as replicate, the colony diameter in two
perpendicular directions were recorded starting from second days on wards, daily until the
colony fully covered the petri dish and used to compute the radial growth rates of the isolates.
On the day that the fungal colony fully covered plate, the color of each isolate colony was
described using Rayner's (1970) mycological color chart. Texture of aerial mycelium, the
nature of colony edges and zonation were also described (Mirzaei et al., 2007).

Colony texture was recorded as either appressed with sparse aerial mycelium, flocculose with
raised and slightly dense aerial mycelium, or floccose with raised and dense aerial mycelium.
Colony color was described as white, white and tending towards gray (grayish white), or gray.
Colony shape was either uniform with smooth edges, irregular with rough edges or banded
with sectors consisting of thin expansive mycelium. Isolates that grew at > 9 millimeter /day
were considered fast growing; those that grew at between 8 and 9 mm /day were considered
medium-growing, while those that grew at < 8 millimeter /day were slowing-growing.

Conidial morphology was studied from ten-days-old cultures of all isolates. Slide preparations
of the conidial suspensions were subsequently made using cotton–blue lactophenol. Conidial
size for each isolate was determined by measuring the length and width of 20 randomly
chosen conidia using an eye-piece micrometer (Sahile et al., 2012).

27
 3.6.5. Propagation of the Inoculum

Mass spore production of B. fabae was carried out on a natural medium of chrysanthemum
(Chrysanthemum sinense Sabine) flower petals (Beniwal and Gorfu, 1989). Twenty five gram
of chrysanthemum flower petals were weighed out and enriched with 5g of dextrose in a flask
and sterilized at 121 0C for 30 minutes in a pressure cooker. A pure culture of B. fabae grown
on FBDA was inoculated into the cool medium and kept under room temperature for 15 days
and then spore concentration (5 × l05 spores /ml) was determined using haeymacytometer
from sporulating media. After the spore concentration was standardized, inoculation test were
carried out.

3.7. Aggressiveness of B. fabae isolates

Seeds from tolerant variety CS20DK (moderately resistant in field evaluation) was sown in
30-cm-diameter pots filled with arable soil, peat and sand ([Link]; v: v: v) (Bouhassan et al.,
2004), and germinated seedlings were thinned to four plants per pot in the greenhouse. Forty-
five days after sowing, the upper side of the leaves was inoculated with 1.5 ml of a spore
suspension containing about 5×105 per ml spores of B. fabae, one droplet on each half of
leaflet by using micropipette and amended with Tween 20 ® (1.2 % v/v) and covered with
polyethylene bags for 24 h to maintain a high relative humidity (Tivoli et al., 2006). The pots
were arranged in completely randomized design and replicated three times (three plants or
leafletes per isolate). Plants sprayed with distilled water were used as a control. The
inoculated and uninoculated plants were maintained under greenhouse conditions.

3.7.1. Disease assessment

Incubation period was determined as the time (in days) between inoculation and the
appearance of first disease symptoms. Disease severity was recorded by calculating the
proportion of leaf surface lesions rated using a 1– 4 scale, where 1: no infection or very small
flecks (1–25% necrosis); 2: necrotic flecks with few small lesions (26–50% necrosis), and
very poor sporulation; 3: medium coalesced lesions (51–75% necrosis) with intermediate
sporulation; 4: large coalesced lesions (76–100% necrosis) with abundant sporulation. Isolates

28
were classified in to 1-3 aggressiveness groups: less = Disease severity ≤ 50% necrosis;
medium = 50 < Disease severity < 75; more = Disease severity ≥ 75% (Tivoli et al., 2006).

3.7.2. Reisolation of the pathogen

The pathogen was reisolated from the leaf lesions. Leaf lesions were cut in to pieces and
surface sterilized with sodium hypochlorite for 2 minute and rinsed thrice with sterile water in
Petri plates. Pieces dried with sterile filter paper and plated on FBDA medium and incubated
at room temperature for 7 days. The fungus was sub cultured to purify, and identification was
by comparison with the respective parent stock culture.

3.8. Data Analysis

For disease survey, primarily descriptive statistics was performed on data collected from each
field. In addition, analysis was conducted to describe the prevalence and association of
chocolate spot severity in relation to independent variables. Contingency X2 tests were used to
compare the frequencies of diseased fields between PAs based on the proportion of fields that
were assigned to a given disease severity class. Where significant difference for disease
severity existed, the mean disease differences were separated using the T-test at P < 0.05
(Zewde et al., 2007).

In the second analysis, the variables classes were categorized based on the frequency of fields.
Disease severity (the response variable) was classified into a distinct class of bi-variate
qualitative data using the SAS software of the univariate procedure of disease severity as
variable. Selected class boundaries were p < 45% and > 45%. Tables for variable classes were
constructed to represent the bivariate distribution of the fields (Fininsa, 2003; Zewde et al.,
2007). The value corresponding to each independent variable represents the frequency of
fields falling in the disease range. The associations of chocolate spot severity with the
independent variables and variables classess were analyzed using a logistic regression model
with the SAS procedure of the GENMOD. The GENMOD procedure estimates the parameters
of the model numerically through an iteractive fitting process and it fits a generalized linear
model to the data by maximum likelihood estimation of the parameter.

29
For field experiment, AVOVA model for individual location articulated the observed
response (𝑦𝑖𝑗) of the variety in the replication and was expressed as:

𝑦𝑖𝑗 = 𝜇 + 𝑉𝑖 + 𝐵𝑗 + ê𝑖𝑗

Where; μ was the mean across replicates, 𝑉𝑖 was effect of variety , 𝐵𝑗 was the effect of
replicate, and êij was the random error assumed to be normally and independently distributed
as mean zero and variance equal to δ2.

The ANOVA model of the combined data expressed the observed mean response over
replicates (Yij) of the ith variety in the jth location with r replicates in each of the (i×j)th cells
was expressed as:

yij = 𝜇 + Vi + Lj + (𝑉𝐿)𝑖𝑗 + 𝐸𝑖𝑗

where µ was the grand mean across all varieties and locations, Vi was the additive effect of
th
the variety, Lj was the additive effect of the j location, VLij was the VLI component for the
ith variety in the jth location, and Eij was the random error assumed to be normally and
independently distributed as (0, σ2/r) (where σ2 was the within locations error variance,
assumed to be constant.

Pearson’s correlation coefficients were calculated to test the strength of relationships among
the parameters. Disease parameters showing significant negative correlation with the yield
and yield components were used in regression analysis.

Chi Square for independence of frequencies in a variable was used to analyze the collected
data for laboratory and greenhouse experiments.

Computer based SAS statistical software version 9.2 (SAS Institute, 2008) was used for
analysis and a significant level of α < 0.05 was used.

30
 4. RESULTS

4.1. Prevalence and Severity of Chocolate Spot in Dawuro Zone

All surveyed faba bean fields were infected with chocolate spot. The results indicated that, the
total fields (44) surveyed were infected with chocolate spot with different levels of severity.
The highest mean chocolate spot severity ranged from 45.8 to 69.4 % at Waka, Bale, Tulema,
Mari, Medhanalem, Botori, Gessachere and Kechi peasant associations (PAs) and lowest
ranged from 33.4 to 44.2 % in Dali, Gozoshasho and Sengetsi PAs (Figure 2).

Bars followed by similar letters are not significantly different (p < 0.05)

Figure 2. Mean severity index of chocolate spot in farmers’ field, 2011 cropping season.

There was significant difference (p < 0.05) in the mean disease severity among the peasant
associations (Appendix Table 11). Among all PAs, the highest disease severity was observed
at Waka and Bale with 64.4 and 63.3 %, respectively. In most cases disease severity varied

31
between altitude ranges within district and it was higher in fields more than 2200 m.a.s.l
(Table 4).

Table 4. Mean severity index of faba bean chocolate spot for different independent variables
in 2011 cropping season for surveyed fields.
Variable Variable class Mean DSI (%) SD (+)
PA Bale 63.3 8.1
Dali 44.2 2.6
Sengetsi 44 4.4
Waka 64.2 6.9
Mari 56.8 6.0
Gozoshasho 42 2.6
Medhanalem 56.2 3.5
Botori 53.2 6.8
Kechi 45.8 2.6
Gessachere 46.7 6.0
Tulema 60.1 2.5
Crop variety Improved 47 9.8
Local 53.2 9.1
2
Crop density <55plants /m 49.8 9.4
2
>55 plants /m 53.3 9.0
Planting date After July,15 50.7 7.2
Before July,15 55.8 12.4
Weed mg’t Weeded 43.9 6.4
Intermediate 50.3 8.7
Not weeded 54.9 8.7
Altitude > 2200 m 57.5 7.4
<2200 m 43.5 4.5
Previous crop Cereals 53.7 9.6
Legumes 50.8 7.5
None (fallow) 40.4 4.0
Land preparation 2xploughing 53.8 9.3
3xploughing 47.6 8.1
Field size < 0.5 ha 49.7 6.8
>0.5 ha 56.7 11.4
Growth stage Pre-podding 49.9 7.9
Podding 55.1 10.2
Slope >8 % 52.3 7.4
<8% 52.4 10.5
DSI (disease severity index), SD (standard deviation)

32
The severity of chocolate spot was higher in local cultivar planted fields and with two times
ploughing than in improved planted and three times ploughed fields. From the cropping
history, it was observed that when bean was planted in rotation with other crops (non
leguminous species) the chocolate spot was less compared with faba bean continuously grown
on the same field of the total fields surveyed; seven fields were continuously planted with
faba bean. Fields with good weed management practices had lower chocolate spot severity
(Table 4). It appeared that the status of weed infestation had an influence on chocolate spot
severity (54.9 %) was recorded in not weeded fields.

4.2. Association of Faba Bean Chocolate Spot with Independent Variables

The association of variables is presented in Table 5. The independent variables such as

peasant association, crop variety, planting time, altitude and crop history were significantly
associated with chocolate spot severity when entered into the logistic regression model as a
single variable. However, when all variables entered last into the regression model, only
peasant association, crop variety, altitude and crop history remained significant in their
association with chocolate spot severity. Among the independent variables, peasant
association (X2 = 1819.0 and 413.37, 10 df), altitude (X2 = 6.75 and 10.91, 1 df) and crop
history (X2=6.81and 8.29, 2df) were the most important variable in its association with
severity when entered first and last in to the model. Planting time lost their importance when
entered into reduced variable model.

33
Table 5. Independent variables used in logistic regression analysis and likelihood ratio
statistics for eleven variables entered first and last into a model.
Independent Type 1 analysis a Type 3 analysis b
variable df DC PR>X2 DC PR>X2
PA 10 1819.0c 0.000 413.37 0.000
Crop Variety 1 11.59 0.001 4.23 0.04
Crop density 1 2.22 0.136 0.25 0.614
Planting time 1 6.25 0.012 0.86 0.353
Weed condition 2 0.41 0.815 3.56 0.169
Altitude 1 6.75 0.009 10.91 0.001
Crop history 2 6.81 0.033 8.29 0.016
Field size 1 0.15 0.699 0.00 0.998
Land preparation 1 0.01 0.924 0.01 0.923
Growth stage 1 0.03 0.865 0.31 0.580
Slope 1 2.73 0.098 2.73 0.098
Dependent Variable=Disease severity index
Model=Peasant association, Crop Variety, crop density, planting time, Weed condition,
Altitude, Crop history, Field size, land preparation, Growth stage and Slope.
df, degrees of freedom; DC, deviance change; PR, probability of a chi square value
exceeding the deviance; LRT, likelihood ratio test.
a
Type 1 analysis =variable entered first in to the model.
b
Type 3 analysis = variable entered last in to the model.

The results for analysis of deviation for variable and variable class are presented in Table 5.
The independent variables such as peasant associations, crop variety, crop density, altitude,
weed managements and slope were tested in a reduced multiple variable models. The
deviation analysis of these variables in a reduced multiple variable models showed the
significance of their association with disease severity.

The parameter estimates, standard error and odds ratio presented in Table 6 indicates:
probability of lowest severity (≤ 45) was highly associated with the mid-altitude PAs (≤ 2200
m.a.s.l) (Dali, Sengetsi, Kechi and Gozoshasho), good weed management practices and use of
improved varieties. High chocolate spot severity had high probability of association to high-
altitude (> 2200 m.a.s.l) (Bale, Waka, Botori and Mari) PAs, none weeded fields and use of

34
local varieties. The severity was greater in none weeded fields than weeded ones. All other
variables such as crop density, field size, and slope of farms, planting time, previous crop and
ploughing frequency did not show significance association on the severity of chocolate spot.

Table 6. Analysis of deviance, natural logarithms of odds ratio and standard error of the
selected independent variables in a reduced model analyzing chocolate spot severity.
b d
Independent LRS Variable Estimate Odds ratio
a 2 c SE
variable df RD DC PR>x class
Intercept 0 0.083 - - - 0.11 0.37 1.11
Crop variety 1 4.41 12.88 0.036 improved -0.34 0.16 0.71
Local 0* 0* 1
Crop density 1 0.3 2.47 0.584 <55plants /m2 -0.11 0.20 0.90
>55 plants /m2 0* 0* 1
Altitude 1 14.31 10.96 0.000 <2200m -0.61 0.16 0.54
>2200m 0* 0* 1
Weed mg’t 2 3.38 0.34 0.07 Weeded -0.019 0.15 1.33
0.79 0.37 Intermediate -0.466 0.16 0.87
Not weeded 0* 0* 1
Cropping history 2 8.82 9.67 0.003 Cereals 1.07 0.36 2.91
11.53 1.156 0.001 Legumes 1.16 0.34 3.18
Fallow 0* 0* 1
Slope 1 3.09 3.09 0.079 >8% -0.20 0.11 0.82
<8% 0* 0* 1
PA 10 4.662 35.12 0.000 Tulema 0.15 0.22 1.16
Dali -0.28 0.26 0.75
Sengetsi -0.09 0.26 0.91
Waka 0.02 0.24 1.02
Mari -0.07 0.21 0.93
Gozoshasho -0.45 0.39 1.57
Medhanalem -0.22 0.21 0.80
Botori 0.02 0.22 1.02
Kechi -0.19 0.35 1.21
Gessachere 0.03 0.30 1.03
Bale 0* 0* 1
2
df, degrees of freedom; DC, the changes in deviance; PR>x ,probability of a chi square
value exceeding the deviance; SE, standard error of the estimate; *Reference group.
a
Independent variables added in to the reduced model; RD = residual deviance(Unexplained
variations after fitting the model); bLikelihood ratio statistics.
c
Estimates from the model with the independent variables added in to a reduced model.
d
Exponentiating the estimates.

35
 4.3. Chocolate Spot Epidemics and Progression

4.3.1. Disease onset and symptoms observation

At both at Tocha and Mari, chocolate spot was first observed on the local cultivar in spreader
rows around the end of July at about second to third leaf emergence and it was recorded on
the experimental plots on first to second week of August, 2011. The disease was observed on
the improved varieties at 20 days after planting (DAP) at these two experimental sites, while
it occurred at later ( after 27 DAP) at Turi.

After the establishment on all experimental plots, it spreads rapidly within a crop and at 4 - 5
days of infection spores formed on infected tissue and initiated secondary spread of the
disease.

Figure 3. Early infection of chocolate spot on faba bean varieties, 30 Days after planting.

36
Chocolate symptoms are varied, and range from small discrete red-brown lesions (1 - 3 mm)
on the leaves to complete blackening of the entire plant. Leaves are the main part of the plant
affected at early stage of growth (Figure 3), under high altitude ranges (Tocha and Mari); it
also spreads to stems, flowers, pods and seeds (Figure 4B-E). Two stages of the disease are
frequently recognized. First, a non-aggressive phase, when discrete reddish-brown spots over
the leaves and stems, and then an aggressive phase, when spots darken in color and coalesce
to form larger grey-brown target spots that may eventually cover the entire plant. On stems,
dark brown streaks may become noticeable on the badly diseased plants (Figure 4E). Infection
of stems can lead to lodging. Indeed, infected flowers and pods may abort (Figure 4C).

Note: A =Leave areas die leading to defoliation; B and D = Pods and Seeds – discolorations;
C= Abnormal leaf fall and abortion of infected flowers and pods; E=Sclerotia of Botrytis
fabae on faba bean stems

Figure 4. Aggressive stages of chocolate spot symptoms or signs on local cultivar.

37
When the crop was infected at early growth stage (before flowering), aggressive development
of infection late in the season can cause the poor pod set, the crop to lodge and drop of leaves
in the vicinity of the plant and whole plant death was observed. Also the secondary branches
may severely attacked and becomes stunted and weak (not vigor) on plots of certain varieties
such as Local, Mesay, Kuse and Moti.

4.3.2. Analysis of variance for location

The results of analysis of variance for disease reaction and yield related traits at each location
showed highly significant variations among faba bean varieties .These are an indication that
the responses of the varieties differ across location for chocolate spot infections and yield
related traits under consideration (Appendices Table 3 - 10).

4.3.3. Chocolate spot progress at test locations

The chocolate spot severity indices recorded at 35, 42, 49, 56, 63, 70, 77, 84, 91 and 98 days
after planting revealed significant (P < 0.01) effects of the tested varieties at all assessment
dates after disease appearance on experimental plots across location except for Mari at 35
DAP; and Turi at 35 and 42 DAP confirming differences in disease reaction among the
varieties (Appendix Table 3). Maximum chocolate spot severity, up to 46.3 % was recorded
on the local cultivar plots at Tocha while a lower chocolate spot severity of 23 % was
recorded on Degaga at Turi 98 DAP (Table 7).

At Tocha, after 63 DAP onward, the chocolate spot severity was varied on CS20DK, Degaga,
Tesfa and Bulga-70 and began to show significantly lower levels up to 98 DAP. Severity
levels in plots of the Local cultivar, Kuse, Hachalu, Gebelcho were statistically at far with that
of the CS20DK, Degaga, Bulga-70 and Tesfa during all the successive assessments of the
disease onward 63 DAP (Figure 5).

38
 CS20DK

45 Degaga
Tesfa
40
Bulga-70
Disease severity index (%)

35 Kasa

30 Nc-58
Mesay
25
Moti
20 Walki

15 Tumsa
Gebelcho
10
Kuse
5 Hachalu
35 42 49 56 63 70 77 84 91 98
Local
Days after planting

Figure 5. Comparison of disease progress curves for epidemics of chocolate spot on faba bean
varieties at Tocha, 2011 growing season.

At Mari, severity levels in plots of the variety CS20DK, Degaga, Bulga-70, Tesfa and Kasa
began to show significantly lower levels of disease severity after 56 to 83 DAP compared
with severity levels in the plots of local, Hachalu Gebelcho, Kuse, Moti, Mesay, NC-58,
Tumsa and Walki during all the successive assessments of the disease. But after 84 DAP
variety Bulga-70 showed significant increment of severity as those varieties of high infection
rate while Kasa decreases as 98 DAP. In contrast, Disease severity was constantly lowered on
CS20Dk, Degaga and Tesfa (Figure 6).

39
 45 CS20DK
Degaga
40 Tesfa
Bulga-70
Disease severity index (%)

35 Kasa
Walki
30
Kuse
Gebelcho
25
Hachalu
Nc-58
20
Mesay
Moti
15
Tumsa
Local
10
35 42 49 56 63 70 77 84 91 98
Days after plantig

Figure 6. Comparison of disease progress curves for epidemics of chocolate spot faba bean
varieties at Mari, 2011 growing season.

Also at Turi, the varieties showed different levels of chocolate spot severity starting from the
4th disease assessment (at 56 DAP).The final levels of chocolate spot severity were
significantly different (P < 0.05) among varieties but they were lower by comparison to the
severity levels at Mari and Tocha (Figure 7).

40
 30 Degaga

28 CS20DK

26 Hachalu
Disease severity index (%)

Gebelcho
24
Tumsa
22
Walki
20
Mesay
18
Moti
16
Tesfa
14
Kasa
12 Kuse
10 Local
35 42 49 56 63 70 77 84 91 98
Bulga-70
Days after planting
Nc-58

Figure 7. Comparison of disease progress curves for epidemics of chocolate spot on faba bean
varieties at Turi, 2011 growing season.

Mean disease severity index at 98 DAP ranged from 24.1 to 46.3% in experimental plots of
both Tocha and Mari in comparison with 23.1 to 28.7 % in Turi. According to terminal DSI
values, the five faba bean varieties namely Bulga-70, CS20DK, Degaga, Kasa and Tesfa
showed the moderately resistance to chocolate spot at Tocha and Mari where high disease
pressure was observed. But at Turi the maximum chocolate spot severity was 28.7 % recorded
from NC-58 and it was not significantly different from other varieties except from the
CS20DK and Degaga. All the tested varieties showed moderately resistance to chocolate spot
at Turi and rated low disease severity (Table 7).

41
Table 7. Grouping of 14 faba bean varieties into reaction groups at three test locations,
2011growing season.

Variety Tocha Mari Turi

e c bc
Bulga-70 32.8 31.5 24.1
f d c
CS20DK 24.1 24.1 23.1
f d c
Degaga 25.5 24.7 23.0
bc b bc
Gebelcho 42.6 41.7 25.0
ab b bc
Hachalu 44.4 41.7 25.0
d c ab
Kasa 38.9 33.3 27.0
bc b a-c
Kuse 42.6 42.0 26.0
a a bc
Local 46.3 45.0 25.0
d ab bc
Mesay 38.9 43 25.0
cd ab bc
Moti 39.8 44.0 25.0
d b a
Nc-58 38.9 42.0 28.7
e d a-c
Tesfa 33.3 25.0 26.0
cd a bc
Tumsa 39.8 45.0 25.0
d b bc
Walki 38.9 42.0 25.0
Mean 37.6 37.4 25.2
SD (+) 6.45 7.8 1.7
CV (%) 2.8 2.9 4.5
p-value <0.0001 <0.0001 <0.0001
Similar letter(s) down the columns are not significantly different (P < 0.05)
DSI =disease severity index assessed at 98 days after planting.
The cut-off values for moderately resistant ranging between >15.0 - 40.0, and moderately
susceptible ranging between >40.0 - 60.0 as expressed by Abo-Hegazy et al. (2012)

42
 4.3.4. Area under the disease progress curve (AUDPC)

Areas under the disease progress curve (AUDPC) for chocolate spot were significantly
different (P < 0.01) among the varieties tested in all locations (Appendix Table 4). The
highest AUDPC was observed at Tocha and Mari experimental plots (1289 to 2387 % - days),
on CS20DK and local cultivar respectively, compared with at Turi (1195 to 1539 % - days on
CS20Dk or Degaga and NC-58, respectively (Figure 8 - 10).

At Tocha, according to their mean AUDPC values, tested varietis were categorized into five
groups, the first group were local and Hachalu, the second group were Kuse and Gebelcho;
the third group were Tumsa,Walki, Moti, Nc-58,Mesay and Kasa ;the fourth group were
Bulga-70 and Tesfa; and the last with least AUDPC was Degaga and CS20DK (Figure 9).

2600
a
2400 ab
bc
2200 c
AUDPC (%-days)

d d d
2000 d
d d
1800
e e
1600
f
1400 f

1200

1000
70

lu
ki
a

sa
i

ho
sfa

l
e
ay
a
DK

58

ot

ca
ag

us
as

ha
al

m
a-

elc
es

M
Te

Lo
c-
eg

K
W

Tu

ac
20

lg

M
N

eb
D

Bu

H
CS

Varieties

Figure 8. Classification of 14 varieties of faba bean according to AUDPC values determined

at Tocha, 2011 growing season.

Bars with similar letters are not significantly different (P < 0.05).

43
At Mari, based on their mean AUDPC values tested varieties were classified into four groups,
the first group were Local and Tumsa, Moti, and Mesay, the second group were Nc-
58,Hachalu,Gebelcho, Kuse ,Walki and Kasa ; the third group was Bulga-70;the fourth group
and the last with least AUDPC were Tesfa ,Degaga and CS20DK (Figure 9).

2200 ab a
a-c
a-c
a-d a-d
2000 cd cd cd
AUDPC (%-days)

d
1800
e
1600

f
1400 f
f

1200

1000
lu
70

ki
a

sa
i
ho
Bu a

l
e

ay
a

58
DK

ot

ca
ag

sf

us
as

ha
al

m
a-

elc

es

M
Te

Lo
c-
eg

K
W

Tu
ac
20

lg

M
N
eb
D

H
CS

Varieties

Figure 9. Classification of 14 varieties of faba bean according to AUDPC values determined

at Mari, 2011 growing season.

Bars with similar letters are not significantly different (P < 0.05).

Also at Turi, highly significant (P < 0.01) difference with AUDPC values were realized
(Appendix Table 4) but the least AUDPC values were obtained compared with other test
locations. Among varieties Nc-58, Bulga-70 and local cultivar had higher AUDPC that of
Degaga and CS20DK. Others categorized as similar trends with each other (Figure 10).

44
 1600
a

1500 bc

bc
b-d
AUDPC(%-days)

1400
c-e
c-f
e-g d-g d-g
1300 e-g
e-g
fg
g g
1200

1100

1000

70
lu

ki
a

sa

i
ho

l
sfa

e
ay

a
DK

58
ot

ca
ag

us
as
ha

al
m

a-
elc

es
M

Te

Lo

c-
eg

K
W
Tu
ac
20

lg
M

N
eb
D

Bu
H
CS

Varieties

Figure 10. Classification of 14 varieties of faba bean according to AUDPC values determined
at Turi, 2011 growing season.

Bars with similar letters are not significantly different (P < 0.05).

4.3.5. Chocolate spot infection rate

Disease progress data were fitted to logistic and Gompertz growth models. Based on
coefficients of determination (R2), standard errors of the estimate, and examination of
residuals, the logistic model was found to provide the best fit and, therefore, this model was
used to quantify disease progression to make compressions among the fourteen varieties.

ANOVA for apparent infection rates (IR) were significantly different (p < 0.05) for tested
varieties for each of the test locations (Appenidix Table 5). The mean IR ranging from 0.118
to 0.207 disease units per day. Apparently low rate of chocolate spot infection was quantified
for Degaga (0.118 disease units per day) at Tocha, CS20Dk, Degaga and Tesfa (0.110, 0.118
and 0.104 disease units per day at Mari, respectively) and CS20Dk and Degaga (0.118 disease
units per day) at Turi compared with other varieties within each location. The acceptable

45
regression equations with coefficient of determinations (R 2) ranging from 73.8 to 97.3% were
produced when chocolate spot severity was regressed over time in days after planting for all
plots. The other varieties did not affect the infection rate of chocolate spot significantly within
location. The rates of infection on those plots were ranged from 0.122 to 0.196 disease units
per day, at Turi and Mari, respectively (Table 8).

Table 8. Infection rates of chocolate spot on 14 faba bean varieties at three locations in
Dawuro zone, 2011 growing season.
Variety Tocha Mari Turi
2 2 2
IR S I R IR S I R IR S I R
Bulga-70 0.151b 0.47 -2.11 93.8 0.131
bc
0.32 -1.82 87.6 0.122
def
0.39 -2.22 92.1
Cs-20Dk 0.181ab 0.64 -2.62 73.8 0.110
c
0.34 -2.18 87.6 0.118
f
0.37 -2.31 93.1
c c f
Degaga 0.118 0.39 -2.07 83.9 0.118 0.37 -2.18 87.6 0.118 0.37 -2.31 93.6
Gebelcho 0.185a 0.57 -1.97 96.3 0.207
a
0.64 -2.25 97.1 0.124
c-f
0.39 -2.30 94.7
a a ef
Hachalu 0.192 0.6 -1.91 94.8 0.195 0.60 -2.13 96.7 0.122 0.38 -2.32 93.1
a ab a
Kasa 0.189 0.58 -2.20 96.6 0.170 0.55 -2.11 86.6 0.139 0.44 -2.30 93.4
ab a a-c
Kuse 0.167 0.52 -1.81 95.4 0.175 0.62 -2.25 97.3 0.134 0.42 -2.23 92.8
ab a c-f
Local 0.167 0.51 -1.69 96.0 0.192 0.59 -2.00 96.7 0.123 0.39 -2.14 91.2
ab ab a-e
Messay 0.181 0.56 -2.11 96.4 0.170 0.59 -2.06 96.6 0.131 0.41 -2.30 93.2
a a a-d
Moti 0.186 0.58 -2.13 95.9 0.186 0.57 -1.98 96.5 0.133 0.41 -2.31 94.7
a a a-e
Nc-58 0.189 0.58 -2.20 96.6 0.196 0.61 -2.13 96.5 0.131 0.41 -2.09 91.8
a c ab
Tesfa 0.187 0.43 -2.25 84.9 0.104 0.34 -2.06 87.1 0.136 0.42 -2.31 95.5
ab a b-f
Tumsa 0.171 0.52 -1.98 96.8 0.191 0.59 -2.00 97.1 0.126 0.39 -2.31 95.0
ab a c-f
Walki 0.165 0.52 -1.96 92.9 0.178 0.63 -2.27 97.3 0.124 0.39 -2.27 94.9
Mean 0.173 0.166 0.127
CV 10.7 15.3 5.15
SD 0.025 0.034 0.009
p-value 0.0031 <0.0001 0.0113
Means in a column with the same letter are not significantly different (P < 0.05).
IR =infection rate; S= standard error of infection rate; I=intercept; R 2 =Coefficient of
determination for the logistic model (%).
Intercept and infection progress rate represent the equation of the line.

46
 4.4. Yield and Yield Components

4.4.1. Number of pods per plant (NPP) and number of seeds per pod (NSP)

Analysis of variance showed highly significant difference (p < 0.01) for NPP among varieties
at three locations but significant difference for NSP (p < 0.05) was realized only at Turi
(Appendix Table 6 and 7).

The mean values of number of pods per plant and number of seeds per pod are presented in
Table 10. Mean values ranged from 5.6 for (Gebelcho) to 17 for (Degaga) at Tocha; 8.6
(Hachalu) to 15.7 (NC-58) at Mari; and 9 (Gebelcho and Hachalu) to 15.7 (NC-58) at Turi.
Nine varieties namely Walki, Tesfa, Nc-58, Messay, Kuse, Kasa, Degaga, CS20Dk and Bulga-
70 had high NPP than the average at each location (Table 9).

Among the varieties for SNP the highest SNP is 4 at Turi for Bulga-70, Hachalu and Tumsa
while the lowest is 2.7 for varieties Walki at Tocha and Gebelcho at Mari and Turi. Four
varieties namely Bulga-70, Hachalu, Tumsa and Moti had high NSP than the average at Turi
(Table 9).

47
Table 9. Mean values of number of pods per plant and number of seeds per pod of faba bean
varieties tested at three locations in Dawuro zone, 2011 cropping season.
Number of pods per plant Number of Seeds per Pod
Variety
Tocha Mari Turi Tocha Mari Turi
b-d ab ab a a
Bulga-70 13.0 14.0 14.0 3.3 3.3 4.0 a
cd abc a-c a a
CS20Dk 12.0 12.7 12.7 3.3 3.0 3.0ab
a ab a-c a a
Degaga 17.0 13.7 12.7 3.3 4.0 3.0 ab
h de d a a
Gebelcho 5.7 9.0 9.0 3.0 2.7 2.7b
f-h e d a a
Hachalu 8.3 8.7 9.0 4.0 3.3 4.0a
ab a ab a a
Kasa 15.3 14.7 14.0 3.0 3.0 3.0ab
b-d a-d a-d a a
Kuse 13.0 12.3 12.3 3.0 3.0 3.0ab
efg b-e b-d a a
Local 9.0 10.7 11.0 3.3 3.0 3.0ab
a-c ab ab a a
Messay 14.3 13.7 13.7 3.3 3.0 3.0ab
f-h cde cd a a
Moti 8.0 10.0 10.0 3.7 3.3 3.3ab
a-c a a a a
Nc-58 15.0 15.7 15.7 3.3 3.3 3.0ab
a-c a-d a-d a a
Tesfa 14.3 12.3 12.3 3.0 3.0 3.7ab
d-f cde cd a a
Tumsa 10.7 9.3 9.3 3.0 3.3 4.0a
gh a a a a
Walki 7.0 15.0 14.7 2.7 3.0 3.0ab
Mean 11.62 11.46 12.16 3.24 3.17 3.26
SD(±) 3.57 2.48 2.36 0.48 0.49 0.54
CV (%) 9.2 9.29 9.9 11.95 14.93 11.95
p-value <0.0001 <0.0001 <0.0001 0.1174 0.26 0.0011
Means in a column with the same letter are not significantly different (P < 0.05).

48
4.4.2. Seed Yield (SYD) and Hundred Seed Weight (HSW)

The analysis of variance displayed significant differences among varieties for SYD (P <
0.01) (Appendix Table 8) and hundred seed HSW (P < 0.01) (Appendix Table 9). Mean
values of seed yield and hundred seed weight are presented in Table 10.

At Tocha, SYD ranged from 2100 kg/ha (CS20Dk) to1305 kg/ha (Mesay); at Mari1327 kg/ha
(Degaga) to 533 kg/ha (local) and at Turi 1221 kg/ha (Degaga) to 697 kg/ha (local). All
varieties produced 1305 kg/ha and above at Tocha and only Degaga yielded more than 1305
kg/ha at Mari. But, at Turi all varieties yielded less than 1305 kg/ha (Table 11). CS20DK
(2100 kg/ha), NC-58 (2027 kg/ha), Moti (1973 kg/ha) and Degaga (1910 kg/ha) were highest
seed yield performer at Tocha where as Local cultivar (1873 kg/ha), Bulga-70(1500 kg/ha)
and Kasa (1520kg/ha) are low yielders. Similarly, at Mari high SYD (kg/ha) of 1327,880 and
870 was obtained from Degaga, CS20Dk and NC-58 respectively. Variety Degaga (1221
kg/ha) was high yield performed at Turi where as local cultivar (697 kg/ha), Tumsa (860
kg/ha) and Walki (873 kg/ha) produced low yield.

The mean for HSW ranged 99.1g (Hachalu) at Tocha to 28.6g (Kuse) at Turi (Table 10). At
Tocha the Varity Hachalu (99.1g), Moti (91.3g), Gebelcho (86.3g) and Tumsa (86.3g) were
produced heavier seeds where as Kuse (38.6g), Local (38.6g) and Bulga-70 (44.1g) produced
small seeds. Similarly at Mari heavier seeds were recorded from Hachalu (95.6g), Moti
(83.7), Tumsa (73.7g), Gebelcho (72.7g) and Walki (65.3g) where as lighter seeds was
obtained from variety Kuse (35.7g), Local (36.3g), Tesfa (38.3g) and Bulga-70 (39.3g). Also
at Turi; Moti (84g), Hachalu (82.8g) and Gebelcho (77.1g) were produced heavier seeds and
Kuse (28.6g), Local (44.5g) and Tesfa (39.6g) produce smaller seeds.

49
Table 10. Mean values of seed yield (kg/ha) and hundred seed weight (g) of faba bean
varieties, 2011cropping season.

Seed yield (kg/ha) Hundred Seed weight (g)

Variety
Tocha Mari Turi Tocha Mari Turi
g bc bc gh g gh
Bulga-70 1500 813 963 44.1 39.3 42.3
a b ab ef e f
CS20Dk 2100 880 1043 55.3 57.7 49.0
b-d a a d e e
Degaga 1910 1327 1221 62.4 58.7 53.7
c-e bc bc b cd b
Gebelcho 1810 803 1013 86.3 71.2 77.1
b-e bc bc a a a
Hachalu 1890 840 937 99.1 95.7 82.8
g bc bc de e c
Kasa 1520 813 1027 58.5 54.0 72.3
fg bc bc h g i
Kuse 1570 863 977 38.6 35.7 28.6
b-e d d h g h
Local 1873 533 697 38.6 36.3 40.8
h c ab fg f g
Messay 1305 737 1050 50.7 47.2 44.5
a-c bc bc b b a
Moti 1973 760 970 91.3 83.7 84.0
ab b bc d-f ef f
Nc-58 2027 870 993 56.7 53.3 48.0
c-e bc bc g g g
Tesfa 1840 790 903 46.7 38.3 39.6
de bc cd b c d
Tumsa 1773 833 860 84.5 73.7 66.1
ef bc b-d c d c
Walki 1727 820 873 44.1 65.3 69.8
Mean 1772.74 834.5 996.24 63.27 57.9 57.04
SD (±) 225 165.3 125.05 19.78 18.06 17.55
CV (%) 3.8 5.08 6.13 3.35 3.53 1.77
p-value <0.0001 <0.0001 <0.0001 <0.0001 <0.0001 <0.0001
Means in a column with the same letter are not significantly different (P < 0.05).

50
 4.4.2. Performance of tested faba bean varieties across location

The analysis of variance for yield and yield components was combined after Bartlett’s test for
homogeneity of error variance. The test identified the error variance as homogeneous for only
number of seeds per pod at Tocha and Mari (Appendix Tables 6 - 9).

However, combined analysis of variance was recommended to be done even if the test is
found significant by excluding the data of locations where coefficient of variation for the trait
analyzed is greater than twenty (Gomez and Gomez, 1984). Therefore, in the present study,
the coefficients of variation at all locations were less than twenty for all traits. The analysis
showed that the effects due to all components namely variety, location, and variety - location
interaction were highly significant (p < 0.01) for yield and related traits except for pod
number per plant for location effect, and seeds per pod for interaction effect (Appendix
Table10) . This indicated the presence of variability among the varieties and locations and
emphasized consideration of variety - location interaction when selecting faba bean varieties
for production.

The mean SYD of the varieties ranged between 1486 kg/ha (Degaga) to 845kg/ha (Local).
High yielding varieties were Degaga (1486), CS20Dk (1341kg/ha), Nc-58 (1297 kg/ha) and
Moti (1234 kg/ha); while Local (845kg/ha), Bulga-70 (1092 kg/ha) and Kasa (1120) were low
yielding varieties (Table 11). When locations were compared, the highest SYD (1772.7 kg/ha)
was obtained at Tocha which is followed by Turi (966.2 kg/ha), while Mari (834.5 kg/ha) was
poor yielding (Table 12).

There was significant (p < 0.05) variation for HSW across locations. High value of HSW was
recorded for the varieties Hachalu (92.5g), Moti (86.3g), Gebelcho (78.2g), Tumsa (74.8g)
and Walki (69.4g) which had large seed size. Kuse had low (34.3g) HSW (Table 11). When
locations were compared, the highest SYD (1772.7 kg/ha) was obtained at Tocha which is
followed by Turi (966.2 kg/ha), while Mari (834.5 kg/ha) was poor yielding (Table 12).

51
Table 11. Combined mean values for chocolate spot severity and yield related traits of tested
faba bean varieties.

Variety NPP SNP SYD (kg/ha) HSW(g)

a-c ab f
Bulga-70 13.7 3.6 1092 41.9j
CS20DK 12.4c 3.1a-c 1341b 54.0h
Degaga 14.4ab 3.4a-c 1486a 58.2g
Gebelcho 7.9e 2.8c 1209c-e 78.2c
Hachalu 8.7de 3.8a 1222c-e 92.5a
Kasa 14.7ab 3.0bc 1120f 61.6f
Kuse 12.6c 3.0bc 1137ef 34.3l
Local 10.2d 3.1 a-c 845g 38.6k
Mesay 13.9a-c 3.1 a-c 1220c-e 47.5i
Moti 9.3de 3.4 a-c 1234cd 86.3b
Nc-58 15.4a 3.2 a-c 1297bc 52.7h
cb a-c d-f
Tesfa 13.0 3.2 1178 41.5jk
Tumsa 9.8d 3.4 a-c 1156d-f 74.8d
Walki 12.2c 2.9bc 1140ef 69.4e
Mean 12.02 3.2 1191 59.4
SD (±) 2.9 0.50 451.8 18.6
CV (%) 9.4 13.4 4.55 3.2
p-value <0.0001 <0.0001 <0.0001 <0.0001
Means in a column with the same letter are not significantly different (P < 0.05).
NPP=number of pods per plant, NSP=number of seed per pod, SYD=seed yield in kg per
hectare, HSW=hundred seed weight in gram.

52
Table 12. Combined mean values for yield and yield components of the tested faba bean
varieties across locations.

location NPP SNP SYD (kg/ha) HSW (g)

Tocha 11.6b 3.2a 1772.7a 63.3a
Mari 12.3a 3.2a 834.5c 57.9b
Turi 12.2ab 3.3a 966.2b 57.0b
Mean 12.0 3.2 1191.0 59.4
SD(+) 2.8 0.5 451.8 18.5
CV (%) 9.4 13.4 4.55 3.2
p-value <0.001 <0.0001 <0.0001 <0.0001
Means with in a column with the same letter are not significantly different (P < 0.05).
NPP=number of pods plant-1, NSP=number of seeds per pod, SYD=seed yield in kilogram
per hectare, HSW=hundred seed weight in gram.

4.5. Relation of Chocolate Spot to Faba Bean Yield Components

There was variation between epidemiological measurements and yield and yield related
components in their associations. But all three epidemiological measurements (final disease
severity, area under the disease progress curves and infection rate) were significantely (p <
0.05) associated with grain yield at all test locations (Appendix Table 12). Chocolate spot
severity was significantly associated (p < 0.05) at Tocha and Mari and negatively related to
seed yield of faba bean starting from the first date until the last date of the assessment (98
DAP). Disease severity from 35 to 56 DAP at Tocha experimental site and from 35 to 98
DAP in Mari experimental site was negatively correlated and highly significant with seed
yield (p < 0.01) having correlation values ranging from -0.592 to -0.361. But in Turi disease
severity was negatively correlated and not significant (p < 0.05) except at 84 DAP with
correlation value -0.335 (Table 13).

53
Table 13. Coefficient of correlation between seed yield and chocolate spot severity for
assessment dates, 2011 growing season.
Tocha Mari Turi
DAP
r P-value r P-value r P-value
35 -0.453 0.0026 -0.394 0.010 _ _
42 -0.592 < 0.0001 -0.524 0.000 -0.149 0.347
49 -0.505 0.0006 -0.418 0.006 -0.262 0.094
56 -0.491 0.001 -0.477 0.001 -0.261 0.095
63 -0.361 0.0189 -0.493 0.001 -0.295 0.058
70 -0.324 0.0364 -0.563 0.000 -0.245 0.118
77 -0.369 0.0163 -0.504 0.001 -0.268 0.086
84 -0.362 0.0185 -0.499 0.001 -0.335 0.030
91 -0.386 0.0116 -0.474 0.002 -0.166 0.293
98 -0.439 0.0037 -0.528 0.000 -0.153 0.333
DAP =days after planting; r =Coefficient of correlation

54
The seed weight correlation with disease severity followed a different trend in the
experimental plots, with the exception of Tocha and Mari locations (Table 14).

Table 14. Coefficient of correlation between 100 seed weight and severity of chocolate spot
for assessment dates, 2011 growing season.

Tocha Mari Turi

DAP
r p-value r p-value r p-value
35 -0.353 0.02 -0.166 0.29 _ _
42 -0.262 0.09 -0.212 0.18 0.285 0.07
49 -0.454 0.001 -0.171 0.28 0.404 0.01
56 -0.502 0.001 -0.190 0.23 0.305 0.05
63 -0.527 0.001 -0.134 0.40 0.377 0.01
70 -0.577 <0.0001 -0.304 0.05 0.369 0.02
77 -0.536 0.001 -0.298 0.06 0.293 0.06
84 -0.502 0.001 -0.272 0.08 0.255 0.10
91 -0.519 0.001 -0.272 0.08 0.352 0.02
98 -0.512 0.001 -0.327 0.04 0.293 0.06
DAP =days after planting; r =Co-efficient of correlation

The linear regression of AUDPC better described the relationships between faba bean pod
number and seed yield with disease severity in Tocha and Mari compared to severity values of
each assessment dates . The relationship described by the model accounted 22.4 to 64.8% for
pod number per plant and 21.7 - 60.8 % for seed yield of the variance. The estimated slope of
the regression line obtained were -0.805 and -2.1298 , -0.6402 and -0.3267, and 1.0809 and -
1.3279, for pod number per plant and seed yield (kg/ha) at Tocha, Mari , and Turi,
respectively (Figure 11A and B).

55
Figure 11A. Linear regression relating AUDPC with pod numbers per plant of faba bean
under three locations, 2011cropping seasons.

56
Figure 11B. Linear regression relating chocolate spot AUDPC with seed yield of faba bean
under Tocha, Mari and Turi experimental sites, 2011cropping seasons.

4.6. Morphological and Cultural Characterization

All Botrytis isolates examined in this study, size of conidia had a maximum length of 11 to 14
μm and mean width of 7 to 10μm (mean 12.5 × 7.8 µm). No Significant differences were
observed in the frequency of isolates with specific morphological or cultural traits in the high-
altitude locations compared to the mid-altitude parts of the study area. But there was
significant difference observed in the frequency of isolates among PAs. This was the case
with colony color and colony growth rate. Where 48% of the isolates were grayish white, 27%
gray and 25% white; and while 45% of isolates were fast-growing, 39% moderate growth
rate, and 16 % had a slow growth rate compared among all isolates (Table 15).

57
Table 15. Morphological characteristics B. fabae isolates from faba bean fields in Dawuro zone, southwest Ethiopia (n=44).

Colony color Colony shape Colony texture Growth rate

PA W GW G Un Ir Sr Ap Fcu Flco S M F
Bale 2 1 1 2 2 0 1 2 1 2 2 0
Dali 2 2 0 2 2 0 0 2 2 0 2 2
Sengeti 2 0 2 0 3 1 0 2 2 1 1 2
Waka 2 0 2 1 2 1 0 2 2 0 2 2
Mari 2 1 1 1 3 0 0 4 0 1 3 0
Gozoshasho 0 2 2 0 4 0 0 2 2 1 3 0
Medanalem 1 3 0 1 3 0 0 2 2 0 3 1
Botori 0 3 1 2 1 1 0 1 3 0 0 4
Kechi 0 4 0 1 3 0 1 1 2 2 0 2
Gessachare 0 3 1 2 2 0 0 2 2 0 0 4
Tulema 0 2 2 2 2 0 0 2 2 0 1 3
Total 11 21 12 14 27 3 2 22 20 7 17 20
X2=35.94,df=2,p<0.05 X2=17.7,df=2,ns X2=14.5,df=2,ns X2=38.7,df=2,p<0.01
PA=peasant association, W=white, GW=grayish white, G=gray, U= uniform, Ir=irregular,
Sr = Sectoring, AP = Appressed, Fcu =Flocculose, Flco= Floccose, S=Slow, M=medium, F=fast, X2= chisquare, df =degree of
freedom, ns=non- significant difference in frequency of isolates.

58
 4.7. Aggressiveness of the Botrytis fabae isolates

In the greenhouse test, 12 (27%) isolates produced the highest leaf necrosis 87.04 - 92.2% of
DSI and lowest 2.61 days of incubation period and grouped as more aggressive, while 28 (64
%) of the isolates induced leaf necrosis of 45. 7% to 62.4% and 1.5 to 1.78 incubation period
belonged to moderately aggressive. The rest four 4 (9 %) isolates induced 1 to 12 % necrosis
and longest 4 to 6.5 days of incubation period and grouped as less aggressive (Table 16).
Esera and Mareka districts had the highest frequency of more aggressive isolates while
isolates from Tocha and Loma districts were moderately aggressive without or weakly
aggressive isolates. Mostly, isolates obtained from Bale, Waka and Mari had a higher
frequency of more aggressive isolates than others. These three PAs accounts 75 % of more
aggressive isolates.

Table 16. Aggressiveness of B. fabae isolates against faba bean under greenhouse conditions.

Number of isolates with aggressiveness group

District PA
More Moderate Less
Essera Bale 3 1 -
Dali 2 1 1
Sengetsi - 2 2
Mareka Waka 3 1 -
Mari 3 1 -
Gozoshasho 1 2 1
Tocha Medhanalem - 4 -
Botori - 4 -
Kechi - 4 -
Loma Gessachere - 4 -
Tulema - 4 -
Total (%) 12 (27) 28 (64) 4(9)
PA = Peasant association, 2= Aggressive was determined using the1 – 3 rating scale of
ICARDA (1986). Less = DS ≤ 3; Medium = 3 < DS < 7; more = DS ≥ 7

59
 5. DISCUSSION

This study has provided information on the distribution and epidemics of chocolate spot on
different faba bean varieties in production areas, and characteristics of B. fabae isolates of
Dawuro zone, southwest Ethiopia. Chocolate spot is a damaging disease of faba bean
throughout production areas; during 2011 growing season, it was found widely distributed in
both surveyed faba bean fields and experimental plots with variations in their severity. The
highest mean chocolate spot severity ranged from 45.8 to 69.4 % at Waka, Bale, Tulema,
Mari, Medhanalem, Botori, Gessachere and Kechi and the lowest ranged from 33.4 to 44.2 %
in Dali, Gozoshasho and Sengetsi peasant associations (PAs). Generally, disease severity was
relatively higher above 2200 m.a.s.l by 6.4 – 34.6 % than mid altitudes (< 2200m) (Figure 2).
In the 2011 cropping season, the weather conditions were more favorable for disease
development at high altitude parts. These situations were attributed to the higher rainfall and
lower temperature, where the rainfall is more than sufficient for crop growth, whereas at the
mid-altitudes there is a scarcity of rainfall and relatively high temperature (Appendix Table
2). On average, the temperature ranged from 14.9 to 21.6 0C and 27 rainy days per month for
these particular locations during the growing season. This suggests that moist and mild
climate is more favorable for chocolate spot development and epidemics. This fact
extensively studied by others, demonstrating that several cycles of infection may take place in
a short period of time, particularly when mild temperature giving rise to extended necroses
that can frequently cause defoliation and even death of whole plants (Assefa and Gorfu, 1985,
Harrison, 1988; Villegas-Fernandez et al., 2010; Fernandez-Aparicio et al., 2011).

Farmers interviewed for their seed source, most farmers (86 %) were used local cultivar
maintained from previous seasons, which favored the occurrence of chocolate spot. It has
been reported by Entwistle (1990) that the inoculum levels will inevitably be within the crop,
or debris from a previous crop in the vicinity. Also it had been observed that higher severity
of infected plants with chocolate spot symptoms was recorded in densely populated fields
than in sparsely populated fields. Although the data was not significantly different, it might be
due to secondary infections in the fields. It has been reported that in a dense population of
faba bean plants, the severity increases due to more plant to-plant spread of the Botrytis fabae
inoculum. According to Khalil et al. (2011), plant density is affecting phonological

60
development, source sink relationship and assimilates partitioning of faba bean. Dense plant
density can cause less light penetration in the crop canopy, reduce photosynthetic efficiency
and may lead to chocolate spot epiphytotics.

In the same way, chocolate spot severity was observed more in unweeded fields where there
was competition for soil nutrients, spacing and moisture and as a result, the faba bean plants
were weak and more prone to the disease due to reduction in plant vigor. Again outbreaks of
chocolate spot are highly dependent on microclimate within crops, such as reduced
temperature and increased moisture (wet) condition that favoring the disease development.

There was a strong association established between the severity of chocolate spot and PAs
located at high altitude ranges, where farmers experienced continues faba bean production
year after year. This continued growing may increases the inoculum level and could result in
rapid intensification of chocolate spot. The inoculum may carry-over from one season to the
next on infected faba bean seed, stubble and volunteer plants. Hawthorne (2004) mentioned
that, continuous growing of faba bean leads to accumulation of the chocolate spot sclerotia in
the soil that increases occurrence of chocolate spot. A similar association had been reported
for faba bean cercospora leaf spot and other disease (Vereijssen et al., 2006; Kimber, 2011).
Therefore, if the level of initial inoculums is high and conditions are favorable for primary
infection, disease may be severe even when few secondary cycles occur (Paul and Munkvold,
2005). Our experiments conducted in the field was also suited at previous fields of faba bean
confirmed that chocolate spot infested from debris remaining on the soil surface as a
predominant factor in the availability of B. fabae inoculum to infect faba bean planted in
succeeding seasons.

Faba bean fields with fallow- cropped had lower chocolate spot severity. This might be due to
the low inocula effect of the fallowed fields and the reduced source of inoculum during the
fallow periods. Harrison (1988) reported the significant role of the amount and quality of
inoculum delivered to the crop canopy as well as the time of arrival of inoculum in relation to
the stage of the crop development on the increase of the disease.

61
In the field experiment, the epidemics of chocolate spot were successful as all plants were
infected with occasional hot – spots simulating the action of a natural outbreak. Furthermore,
Investigations of the effect of environmental conditions on the severity of chocolate spot also
provided a greater understanding of the dynamics of disease. Differences in disease onset and
development with earlier and higher disease spread at Tocha and Mari could be explained by
lower daily average temperature associated with prolonged precipitation favorable to
chocolate spot, leading to rapid expansion of lesions on infected faba bean leaves and
premature defoliation. For instance, in August, where early chocolate symptoms observed,
26 days having rain presented with an average temperature below 20 0C. There was late
appearance of the disease at Turi that related to unfavorable weather conditions particularly
the absences of precipitation in mid July till early August. Vandenberg (2006); Villegas-
Fernandez et al. (2009) and El-Sayed et al. (2011) mentioned that, chocolate spot infection
and development is favored by mild and moist conditions. Dry conditions are known to
hamper the initiation and development of chocolate spot epidemics since leaf wetness is
needed for successful infection and disease establishment (Bretag and Raynes, 2004).

Based on their final disease severity indices (DSI), great difference was observed as different
varieties planted at three locations, the higher percentage on local cultivar, the lowest on
CS20DK rated at Tocha and Mari locations (Table 8). This indicate that the disease
development and spread of the pathogen was higher on the plants sown at high altitude
ranges, where the mean temperature of growing season was still low 17.4 0C, than on the
plants sown at Turi, where the temperature increased up to 24 0C. This result supports our
survey results in that why chocolate spot is favored at high altitude ranges rather than mid
altitude. In warm conditions chocolate spot is typically important later in the season during
flowering and after canopy closure (Table 13) where significant (p < 0.05) and negatively
correlated results of DSI with seed yield at 83 DAP.

However, during longer periods of wet weather the fungus thrives and the leaf is killed (RHS,
2012). The most important damage usually occurs when plants are flowering as that is when
the environmental conditions are often more conducive to disease development, decaying
flower petals are available for growth of the fungus and yield reductions may be serious due
to spread of the pathogen from the flowers into the developing pods (Stoddard et al., 2010).

62
Harrison (1981) also suggested that, at any stage of plant growth, the rate of aggressive lesion
progress was linear and proportional to the optimum temperature between 6 to 20 0C, and
neither light intensity nor a film of free water affected lesion development. Tocha and Mari
locations had extended rainfall and were favorable for chocolate spot development. Hanounik
(1979) and Dereje et al. (1994) reported prolonged rainfall is conducive for chocolate spot
development leading to complete crop loss.

The linearization of disease progress curve was essential to determine epidemic speed, to
project future disease and to estimate initial disease. However, disease progress curves
indicated that epidemics on tested varieties progressed slowly at early growth stage, and then
increased rapidly mostly at Tocha and Mari 56 DAP onwards until late assessment date, after
which epidemics slowed as fewer healthy plants remained to be infected. The chocolate spot
progress curves for these two locations are generally attained typical sigmoid shape except for
the moderately resistant plots of few varieties (Figure 5 and 6). At Turi, the chocolate spot
growth curves did not attain the sigmoid shape due to the reduced disease development
(Figure 7). The chocolate spot development revealed that growth curves vary depending on
the resistance level of the variety and environmental factors. The difference in shape and
magnitude of chocolate spot progress curves at Tocha, Mari and Turi indicate the effect of
environmental factors on disease progress and final disease development as stated by
Campbell and Madden (1990). Epidemics that progressed in this fashion are best described by
a logistic (or similar) growth function (Campbell and Madden, 1990) and have been termed as
compound interest or polycyclic diseases (Van der Plank, 1963).

In describing the rate of chocolate spot infection, Logistic model was superior to Gompertz
model. The coefficient of determination (R2) was higher for Logistic model consistently for
all assessment dates except for the terminal chocolate spot assessment in which Gompertz
model was slightly better than Logistic model. This indicates that chocolate spot infection rate
is apparently related to the logarithm of the ratio of the amount of diseased and healthy tissues
present as described by Campbell and Madden (1990). However, chocolate spot progress was
not significant for the weekly assessed plots of the tested varieties apparently except for
Degaga (0.118 disease units per day) at Tocha, CS20Dk, Degaga and Tesfa (0.110, 0.118 and
0.104 disease units per day at Mari, respectively) and CS20Dk and Degaga (0.118 disease

63
units per day) at Turi where retarded infection rate compared with other varieties. The other
varieties did not affect significantly the infection rate of chocolate spot within test locations
(Table 6). Nevertheless, infected faba bean plants with in each plot were contributing the new
infected plants (Van der Plank, 1963). To lessen the epidemics of that increase logistically, it
is important to reduce the rate of plant to plant spread of the pathogen within the plot, thereby
allowing more time for plants to set high number of fruiting nodes. El-Bramawy et al. (2010)
recommended that, use of appropriate agricultural practices for agronomic characters
improvement and for enhancing the resistance to foliar diseases, which could be used to
minimize the harmful effects of fungal pathogens and to increase the grain production. The
moderately resistant faba bean varieties, for instance CS20DK, Degaga and Tesfa, which
reduced the apparent infection rate of chocolate spot in this study by 13 - 50% compared to
other tested varieties, may also provided a substantial benefit in terms of faba bean yield.

Host resistance to chocolate spot pathogen is another important factor that could slow
epidemic. The tested varieties were significantly different (p < 0.01) for their reaction to the
disease without expressing the extreme reactions (Table 7). For instance Mesay, Tumsa, NC-
58 and Walki which recorded moderately resistant (MR) at Tocha and Turi turned to be
moderately susceptible (MS) under Mari conditions. Local cultivar, Gebelcho, Hachalu and
Kuse both at Tocha and Mari, and Mesay, Moti, NC-58, Tumsa and Walki at Mari rated to be
MS to chocolate spot having the highest degree of infection. Other faba bean varieties
recorded as moderately resistant (MR) to chocolate spot infection at all locations. Neither
highly resistant nor highly susceptible varieties were rated by the disease severity.

Area under disease progress curve (AUDPC) is a resistance parameter calculated from the
percentages of the leaf area affected. Calculation of the AUDPC as a measure of quantitative
disease resistance entails repeated disease assessments at different times during the epidemic.
The analysis of variance for AUDPC revealed highly significant differences (p < 0.01) among
the tested varieties. Five varieties such as Bulga-70, Cs-20-Dk, Degaga, Kasa and Tesfa were
significantly less affected by chocolate spot even at Tocha and Mari where the chocolate spot
infection was higher. Moreover, AUDPC values were greater in MS varieties than in MR
varieties (Figure 9 and 10). This indicates MS varieties rated higher AUDPC values were not
limited disease spread as effectively as did MR varieties (Figure 5 and 6) and also this may

64
reflect the incompetent of those varieties to slow down the disease at different phases of its
progression. In general, the higher the AUDPC, the more susceptible is the variety. The loss
of active leaf area results in less photosynthetic available region during the flowering and or
pod setting stage which eventually results in producing less productive pods per plant. This
reduction may eventually contribute to the overall yield losses. The advantage of using
AUDPC was that it was simple to calculate and uses multiple evaluations which are often
based on assumptions about the distribution of the data points (Campbell and Madden, 1990).

The use of AUDPC values rather than a single score of severity at a specific stage may prove
the importance under fluctuating climatic conditions influencing the development of the
disease. In addition, the stage after natural infection of the disease may coincide with a
different seasonal period under different environmental conditions. The variability observed
in this experiment showed that faba bean varieties possess remarkable genetic diversity for
chocolate spot resistance. Similarly, Abo-Hegazy et al. (2012) found that thirteen faba bean
varieties varied in their reactions to Botrytis fabae infection between indoor and outdoor
experiments. Moreover, similar results were obtained by ICARDA (2006) in the field
conditions for performance of 17 Iraqi faba bean genotypes in relation to infection with
chocolate spot and other foliar diseases. Therefore, the present study and those of others show
that faba bean varieties vary in their resistance to chocolate spot disease. In a recent report,
Gebrehiwot (2011) indicated that 38 faba bean genotypes differed significantly in their
reactions to Botrytis fabae isolates studied in the laboratory.

There were also the highly significant differences (P < 0.01) of the variety × location
interaction indicate that fluctuation of varieties in their responses to the different
environments (Appenidex Table 10). There was tremendous change in yield and related traits
of the varieties across locations. Regarding the mean number of pods per plant (NPP) (Table
11) shows big ranges over the three locations 5.7 to 17. Degaga (17) both at Tocha and Mari
produced greatest number of pods /plant, followed by Nc-58 (15.6), and Walki (14.6) at Turi.
Nine varieties namely Walki, Tesfa, Nc-58, Messay, Kuse, Kasa, Degaga, Cs-20Dk and
Bulga-70 had high NPP than the average (12.02) at each location (Table 9). These varieties
also showed highly significant differences (P < 0.01) in grain yield performance (Appendix
Table 8). Based on the means obtained over the three locations, it is obvious that the seed

65
yield (SYD) range 1486 (Degaga) – 845 kg/ha (local), and with respect to hundred seed
weight (HSW), it was ranged from 34.3 - 92.5g with mean of 57.04g (Table 11).

The most commonly used varieties in the area were local cultivar which produced low yields
of 845 kg/ha. Hence, the result clearly showed that high yielding varieties such as Degaga
(1486), Cs-20Dk (1341kg/ha), Nc-58 (1297 kg/ha) and Moti (1234 kg/ha) could be best
substitutes the local cultivar and other tested varieties and can be introduced in seed
production and distribution. When locations were compared, the highest SYD (1772.7 kg/ha)
was obtained at Tocha which is followed by Turi (966.2 kg/ha), while Mari (834.5 kg/ha) was
poor yielding (Table 10). This is due to soil physico - chemical property differences at the
experimental sites (Appendix Table 1B). According to Richardson and Horsham (2008) soil
pH had shown a strong relationship with disease severity. Acidic soil (below pH 5.8) had
significantly greater disease incidence caused by B. fabae than plants in basic soil (above pH
6.7). High value of HSW was recorded for the varieties Hachalu (92.5g), Moti (86.3g),
Gebelcho (78.2g), Tumsa (74.8g) and Walki (69.4g) which had large seed size. Kuse had low
(34.3g) HSW. This may be attributed to the long growing period of the crop at Tocha and
Mari while lower rain shower especially late in the growing season at Turi (Appendix Table
2).

Correlations were found between all epidemiological measurements and yield and yield
related traits used to quantify the effect of chocolate spot epidemics in the field (Appendix
table 12). For this purpose, chocolate spot severity indices of the growing season and AUDPC
values considered the simplest method. These represent the summation of host – environment
interactions over the course of a growing season (Vander plank, 1984).

Chocolate spot severity was negatively related to seed yield of faba bean starting from the
first date of disease assessment (35 DAP) until the last day of the assessment (98 DAP) except
for Turi where no disease appeared at early time of assessments. Disease severity from 35 to
56 DAP at Tocha experimental site and from 35 to 98 DAP in Mari experimental site were
negatively correlated and highly significant with seed yield (p <0.01) having correlation
values ranging from -0.592 to -0.361. But in Turi disease severity was negatively correlated
and not significant (p < 0.05) except at 84 DAP with correlation value -0.335 (Table 13).

66
Also the AUDPC value showed varied negative correlation with pod number per plant and
seed yield , at the Mari and Tocha experimental plots, and it is more in Mari (r = -0.847 to -
0.593, 0.05) than in Tocha (r = -0.761, P < 0.01) while at Turi negative and non significant
correlation value was showed for seed yield (Figure 11A and B).

The linear regression of AUDPC better described the relationships between faba bean pod
number per plant and seed yield with disease severity in Tocha and Mari compared to severity
values of each assessment dates . The relationship described by the model accounted 22.4 to
64.8% for pod number per plant and 21.7 - 60.8 % for seed yield of the variance. The
estimated slope of the regression line obtained were -0.805 and -2.1298, -0.6402 and -0.3267,
and 1.0809 and -1.3279, for pod number per plant and seed yield at Tocha, Mari, and Turi,
respectively. The estimates showed that for each unit increase in percent of chocolate spot
AUDPC, there was a seed yield reduction 2.13, 0.33, and 1.32 kg/ha in Tocha, Mari and Turi,
respectively (Figure 11B). This indicates that chocolate spot affected faba bean yield by
flower abortion and plant damage. When compared to Tocha and Mari, chocolate spot
epidemics were sufficiently delayed in Turi to allow the plants to set more productive pods. In
fact, in Turi the mean seed weight was less for tested varieties than in other test locations, a
reversal of the ranking of varieties for pod number per plant where chocolate spot was rated
higher. These Turi data support the view that chocolate spot is mainly responsible for the
yield loss at Tocha and Mari by early defoliation of flowers consequently low pod set. This
kind of disease relation has also been seen in chocolate spot disease (Sahile et al., 2010)
where Severity and AUDPC were inversely correlated with faba bean grain yield and some
other foliar diseases such as leaf anthracnose (Colletotrichum graminicola) on alfalfa crop
(Thomas et al. 1996), and spot blotch (Bipolaris sorokiniana) (Joshi et al. 2002).

Atotal of fourty four Botrytis isolates were isolated and identified from spotted leaves, pods,
and stems and of faba bean plants, a result that was consistent with others reports (Mazen,
2004; Eisa et al., 2006; Elwakil et al., 2009). All were found to be B. fabae and none fitted the
morphological description of B. cinerea. In pathogenicity tests, representative isolates caused
typical chocolate spot symptoms and were re-isolated from infected leaves, indicating that B.
fabae is the causal agent of chocolate spot in Dawuro faba bean production areas. This finding
supports (Sahile et al., 2012) the situation of the disease in Northern Ethiopia. Also there were

67
significant differences in the frequency of isolates. This was the case with colony color and
colony growth rate (Table 15), where 48 % of the isolates were grayish white, 27 % gray and
25 % white while 45 % of isolates were fast-growing, 39 % moderate growth rate, and 16 %
had a slow growth rate compared among all isolates collected from faba bean fields.
Similarly, Dereje (1996) reported that there was morphological variability among isolates of
B. fabae. On the other hand, variations in the size of conidia from different isolates of Botrytis
sp. were not uncommon (Mirzaei et al, 2007; Pandel et al., 2010). Although colonies of B.
cinerea and B. fabae on artificial media can be difficult to distinguish, possibly accounting for
the apparent confusion between the species, the two species can always be distinguished by
measuring their conidia (Harrison, 1988).

Also there was a wide variation of B. fabae isolates collected from Dawuro zone in their level
of aggressiveness. The most virulent isolates, 27 % of the total, produced a greater leaf
necrosis and with a shorter incubation period than less virulent isolate (Table 16). This
indicates that more virulent isolate was able to invade and colonize host tissue more rapidly
and resulted in more leaf damage. Similarly, Setti et al. (2009) and Muiru et al. (2010)
reported that the most virulent isolates had a shorter incubation period with a larger lesion size
and disease severity. Sakr et al. (2009) also showed that isolates having shorter latent periods
and greater spore production are considered to be more virulent than isolates having longer
latent periods and lesser spore production.

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 6. SUMMARY AND CONCLUSIONS

Faba bean is one of the major pulse crops produced in Ethiopia that serve as a multi-purpose
crop in the socioeconomic life of farming communities mainly grown as a valuable source of
protein and energy for both human food and animal feed . The yield of the crop is affected
by fungal diseases like chocolate spot (B. fabae), cultural practices, their genetic make-up and
the growing environmental conditions existing in the study area.

Chocolate spot is a damaging disease of faba bean throughout production areas and it was
found widely distributed in both surveyed farmers fields and experimental plots of faba bean
with variation in their severity. The mean chocolate spot severity ranged from 24.1% at Turi
experimental plots to 69.4 % at Waka on farmers field. The higher mean chocolate spot
severity frequently observed at high altitude ranges (6.4 – 34.6 %) than mid altitude ranges.

The association of independent variables such as peasant association, crop variety, planting
time, altitude, and crop history was significantly associated with chocolate spot severity when
entered into the logistic regression model as a single variable. However, when all variables
entered last into the regression model, peasant association, altitude and crop history were the
most important variable in its association with severity. Also it had been observed that higher
severity of infected plants with chocolate spot in densely populated and unweeded fields than
in sparsely populated and weeded fields.

The earliness of chocolate spot onset under field conditions could be explained by lower daily
average temperature associated with prolonged precipitation at high altitude ranges (>2200m).
At this suituations disease progresses up the canopy, with the spots rapidly expanding into
large patches, which blight the leaves. Plants defoliate and lose flowers and pods. Flowers and
pods may also develop lesions. Stems can become reddish-brown and weakened, aggressive
development of stem infection late in the season can cause the crop to lodge. Whereas
chocolate spot is likely to be yield limiting in later growth stages especially after canopy
closure and after flowering in mid altitude ranges.

69
Disease progress curves indicated that epidemics on tested varieties progressed slowly at early
growth stage, and then increased rapidly mostly at Tocha and Mari 56 DAP onwards until late
assessment date, after which epidemics slowed as fewer healthy plants remained to be
infected.

The tested varieties were significantly different for their reaction to chocolate spot without
expressing the extreme reactions; they rated either moderate resistant (MR) or moderately
susceptible (MS). It also showed the highly significant difference in terms of AUDPC.
Moreover, AUDPC values were greater in MS than in MR varieties. This reflects the
incompetent of MS varieties to slow down the disease at different phases of its progression.
Five varieties such as Bulga-70, CS20Dk, Degaga, Kasa and Tesfa rated low AUDPC and
less affected by chocolate spot and rated MR under all [Link] varieties reduced the
apparent infection rate of chocolate spot by 13 - 50 % compared to others and also provided a
substantial benefit in terms of faba bean yield.

Over all tested locations varieties such as CS20DK, Nc-58 and Moti were the top yielding
varieties and best substitutes the exsisting local cultivar which can be preferable in cultivation
and seed production.

Chocolate spot severity for all assessment dates was negatively related to seed yield of faba
bean. But highly significant correlation were obtained 35 to 56 DAP at Tocha and for all
assessment dates at Mari experimental sites having correlation values ranging from -0.592 to -
0.361. But at Turi significant correlation was obtained later at 83 DAP with correlation value -
0.335. Also the AUDPC value showed varied negative correlations at the Mari and Tocha
experimental plots, and it is more in Mari (r =-0.847 & -0.593) than in Tocha (r = -0.761 & -
0.555) for pod number per plant and seed yield, respectively. At Turi negative and non
significant correlation value was showed only for seed yield.

The linear regression of AUDPC between faba bean pod number per plant and seed yield
accounted 22.4 to 64.8% and 21.7 - 60.8 %, respectively. The estimated slope of the
regression line obtained were -0.805 to 1.0809 for pod number per plant and -0.3267 to

70
1.3279 for seed yield. When compared to Tocha and Mari, chocolate spot epidemics were
sufficiently delayed in Turi.

Based on morphological characteristics of conidia, all isolates examined were found to be B.

fabae and none fitted the morphological description of B. cinerea. A significant difference
was observed in the color and growth rate of the colony. There was also a wide variation
among of B. fabae isolates in their level of aggressiveness and incubation period.

In conclusion, chocolate spot is important disease in faba bean production areas of southwest
Ethiopia, and can influenced by cultural practices, growing situations and genetic makeup of
the crop. However, field evaluations at existing agro-ecologies and disease tolerance of
released varieties were best meets growers interest which helps for seed production and
cultivation. The insight of this finding also help to design research objectives to overcome
rejection of varieties developed by researchers alone, enhances the acceptance of varieties and
reduces costs associated with variety development. In most cases improved varieties will
perform better if accompanied by recommended cultural practices.

Even though, the tested varieties were not immune to the chocolate spot; but have much
higher levels of tolerance than local cultivar. Therefore, an integrated approach is the key to
successful management of chocolate spot in faba bean but attention to other management
practices can reduce disease pressure and yield loss:
 Use the varieties with the highest level of tolerance to chocolate spot in respective
production area like CS20DK, Degaga, Tesfa, Bulga-70 and Kasa.
 Farm hygiene and crop separation – preferably, the current crop should be sown
more than 500 m from faba bean fields in the previous year. This will isolate it
from sources of infection for fungal diseases. Volunteer faba bean plants appearing
in late season can help to carry over diseases and should be eradicated.
 Seed should be sourced from the ‘cleanest’ crops. Old, frosted or damaged seed
may have reduced germination and reduced vigor.
 Make faba bean part of a cropping system with cereals; crop rotation not more
than 1 faba bean crop in 4 years.

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  Fallow management - Soils should be well structured, clay loam or heavier in
texture, and pH 5.2 to 8; treating the acidic soils with lime is also important.
 Intercropping with cereals to reducing chocolate spot severity by suppressive
effects of a combination of host biomass reduction, altering microclimate and
physical barriers to spore dispersal.
 Delayed sowing and wide row spacing to keep the canopy open longer- preferably
for varieties having higher vegetative growth characters and wider canopy like
Hachalu, Gebelcho, Walki, Tumsa, and Moti.
 Plan to harvest as early as possible to minimize disease infection on seed.
 Use of foliar fungicides like Chlorothalonil or mancozeb could be preferable as a
foliar spray with added benefit of controlling other foliar diseases, when
significant infection could be occurred at early growth stages. To achieve the
cheapest fungicide management program, faba bean growers must also observe
other aspects of the crop establishment package. That is, selecting a farm that is
remote from where faba bean were grown in the previous year, both on their own
and their neighbours' properties.

Critical issues for future research are tracking changes in population structure of key
pathogens and also validation and up-scaling of proven integrated disease management
strategies, working with different varieties released for the same agro-ecologies in
Ethiopia. Climatic/season variability will affect the importance of diseases; therefore
model-based decision support system research needs special attention. The current faba
bean varieties are rated moderately resistant to moderately susceptible to chocolate spot
and not immune. Therefore, development of new varieties that can be the substitute for
them should be considered.

72
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8. APPENDIXES

85
Appendix Table [Link] features of study areas in four districts of Dawuro zone.

District PA1 Altitude (m.a.s.l)2 Latitude 2 Longitude2

Essera Bale 2407-2543 7062'151"N 37091'733"E
Dali 2125-2132 7017'240"N 37020'365"E
Sengetsi 1982-2106 7008'940"N 37003'551"E
Mareka Waka 2304-2474 7005'140"N 37016'096"E
Mari 2445-2489 7002'185"N 37005'870"E
Gozoshasho 1805-1912 7005'528"N 37001'113"E
Tocha Medhanalem 2666-2782 7005'528"N 37002'158"E
Botori 2354-2411 7052'940"N 37006'421"E
Kechi 1995 -2135 7006'147"N 37011'696"E
Loma Gessachere 2015-2104 7012'055"N 37014'713"E
Tulema 2243-2478 7017'940"N 37009'521"E
1
PA=peasant association; 2 geographic locations =Measured using GPS

Appendix Table 1B. The results of soil analysis at the experimental sites.

*Soil samples collected from top 20cm depth & analyzed at Hawasa soil testing center.

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Appendix Table 2. Monthly average minimum and maximum temperature ( 0C) and total
rainfall (mm) of the experimental period, 2011 growing season.

Month 1 2
Tocha and Mari Turi
Air temperature (0c) Air temperature (0c)
TRF RD TRF RD
Min. Max. Av. Min. Max. Av.
July 8.3 18.5 11.4 158 30 14 22.4 17.95 150 21
August 8.1 17.8 12. 5 279 28 14 23.9 19.8 218 18
September 15.5 21.5 16.5 174 30 15 23.4 19.3 104 21
October 17.2 22.3 17.75 108 27 18 24.2 20.2 109 16
November 19.1 24.8 20.5 129 24 19 25.6 22.4 72.8 17
December 21.2 24.9 21 116 21 22 24.7 23.4 106 12
Mean 14.9 21.6 17.4 160 27 17 24 20.5 127 18
TRF= monthly total rain fall (mm), RD = number of rainy days per month,
1 and 2
Recorded at Loma1 and Tercha meteorological stations.

Appendix Table 3. ANOVA table for the disease severity index (DSI) of each assessment
dates under field conditions.

Disease Assessment days after planting

Location Source
df 35 42 49 56 63 70 77 84 91 98
** ** ** ** ** ** ** ** **
Tocha Variety 13 17.9** 25.9 34.5 37.1 27.7 60.2 72 100.5 128.4 128.8
ns ns ns ns ns ns ns ns ns ns
Block 2 0.8 1.1 7.6 3.7 0.2 3.0 4.5 0.75 0.5 2.31
Residue 26 1.2 2.2 3.2 2.7 1.2 1.8 2.28 1.15 0.3 1.12
* ** * ** ** ** ** ** ** **
Mari Variety 13 6.6 9.4 5.9 8.6 33.8 57.6 113.9 153.2 182.4 189.9
ns ns ns ns ns ns ns ns ns ns
Block 2 1.7 0.6 3.5 2.2 6.7 6.7 1.7 0.3 2.1 0.21
Residue 26 1.9 1.2 1.9 1.9 4.1 3.12 2.3 1.3 2.4 1.15
** ** ** ** ** ** **
Turi Variety 13 0 0.9* 5.8** 7.4 10.7 12.9 8.9 6.7 9.3 6.2
ns ns ns ns ns ns ns ns
Block 2 0 0.6 3.9* 1.7 2.9 2.3 1.3 0.2 0.7 1.0
Residue 26 0 1.2 0.9 1.2 1.6 1.56 1.5 0.9 1.1 1.3
a Values are mean squares. * Significant at P < 0.05; ** significant at P<0.01; ns, not
significant at 5%

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Appendix Table 4. ANOVA table for AUDPC of fourteen faba bean varieties, 2011 cropping
season.

Location Source DF SS MS F Value Pr > F

Tocha Model 15 3590403 239360 114.59 <.0001
Variety 13 3580186 275399 131.85 <.0001
Block 2 10216 5108 2.45 0.1064
Error 26 54308 2089
Total 41 3644711
R2=0.99 CV=2.4
Mari Model 15 3682229 245482 54.39 <.0001
Variety 13 3671312 282409 62.57 <.0001
Block 2 10917 5458 1.21 0.3146
Error 26 117345 4513
Total 41 3799574
R2= 0.97 CV=3.65
Turi Model 15 423674.2 28244.9 22.94 <.0001
Variety 13 417689 32129.9 26.09 <.0001
Block 2 5985.2 2992.6 2.43 0.1078
Error 26 32016.8 1231.4
Total 41 455691
R2= 0.93 CV=2.66

Appendix Table 5. ANOVA table for infection rate of fourteen faba bean varieties, 2011
cropping season.

Location Source DF SS MS F Value Pr > F

Tocha Model 15 0.016 0.0011 3.13 0.0053
Variety 13 0.0156 0.0012 3.52 0.0031
Block 2 0.0004 0.0002 0.58 0.5661
Error 26 0.0089 0.0003
Total 41 0.0249
R2= 0.64 CV= 10.66
Mari Model 15 0.04829 0.00322 5 0.0002
Variety 13 0.04772 0.00367 5.7 <.0001
Block 2 0.00057 0.00028 0.44 0.6493
Error 26 0.01673 0.00064
Total 41 0.06502
R2= 0.74 CV=15.28
Turi Model 15 0.0021 0.00014 2.57 0.0168
Variety 13 0.002 0.00016 2.84 0.0113
Block 2 0.0001 0.00004 0.81 0.4568
Error 26 0.0014 0.00006
Total 41 0.0035
R2=0.60 CV= 5.8

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Appendix Table 6. ANOVA table for pod number per plant of fourteen faba bean varieties,
2011 cropping season.

Location Source DF SS MS F Value Pr > F

Tocha Model 15 496.81 33.12 31.78 <0.0001
Variety 13 483.9 37.22 35.72 <0.0001
Block 2 12.9 6.45 6.19 0.0063
Error 26 27.1 1.04
Total 41 523.9
R2=0.95 CV=8.79
Mari Model 15 218.36 14.56 11.21 <0.0001
Variety 13 215.45 16.57 12.76 <0.0001
Block 2 2.9 1.45 1.12 0.342
Error 26 33.76 1.3
Total 41 252.12
R2= 0.87 CV=9.29
Turi Model 15 190.12 12.67 8.74 <0.0001
Variety 13 186.5 14.35 9.89 <0.0001
Block 2 3.62 1.81 1.25 0.3039
Error 26 37.71 1.45
Total 41 227.83
2
R =0.83 CV=9.9
Appendix Table 7. ANOVA table for seed number per pod of fourteen faba bean varieties,
2011 cropping season.

Location Source DF SS MS F Value Pr > F

Tocha Model 15 4.6 0.308 1.6 0.1418
Variety 13 4.3 0.33 1.71 0.1174
Block 2 0.3 0.167 0.87 0.4321
Error 26 5 0.192
Total 41 9.62
R2= 0.48 CV=13.54
Mari Model 15 4 0.27 1.2 0.33
Variety 13 3.8 0.29 1.32 0.26
Block 2 0.2 0.1 0.43 0.66
Error 26 5.8 0.22
Total 41 9.8
R2= 0.41 CV=14.93
Turi Model 15 8.167 0.544 3.58 0.0021
Variety 13 8.119 0.625 4.11 0.0011
Block 2 0.048 0.024 0.16 0.8558
Error 26 3.952 0.152
Total 41 12.119
R2= 0.67 CV=11.95

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Appendix Table 8. ANOVA table for seed yield (kg/ha) of fourteen faba bean varieties, 2011
cropping season.

Location Source DF SS MS F Value Pr > F

Tocha Model 15 1983892.3 132259 36.58 <0.0001
Variety 13 1982126.8 152471 42.16 <0.0001
Block 2 1765.5 883 0.24 0.7852
Error 26 94017.9 3616
Total 41 2077910.1
R2= 0.95.5 CV=3.4
Mari Model 15 1072940.5 71529.4 39.82 <0.0001
Variety 13 1068907.1 82223.6 45.78 <0.0001
Block 2 4033.3 2016.7 1.12 0.3406
Error 26 46700 1796.2
Total 41 1119640.5
R2= 0.96 CV=5.08
Turi Model 15 549946 36663.1 10.45 <0.0001
Variety 13 545540 41964.6 11.97 <0.0001
Block 2 4406 2203 0.63 0.5415
Error 26 91183 3507.1
Total 41 641130
Appendix Table 9. ANOVA table for hundred seed weight (g) of fourteen faba bean varieties,
2011 cropping season.

Location Source DF SS MS F Value Pr > F

Tocha Model 15 15895.8 1059.7 195.12 <0.0001
Variety 13 15867.7 1220.6 224.75 <0.0001
Block 2 28 14 2.58 0.0949
Error 26 141.2 5.4
Total 41 16037
R2=0.99 CV=3.68
Mari Model 15 13264.9 884.3 212.12 <0.0001
Variety 13 13241.2 1018.5 244.32 <0.0001
Block 2 23.7 11.8 2.84 0.0766
Error 26 108.4 4.17
Total 41 13373.3
R2=0.99 CV=3.53
Turi Model 15 12602.6 840.2 823.7 <0.0001
Variety 13 12538.9 964.5 945.62 <0.0001
Block 2 63.6 31.8 31.2 <0.0001
Error 26 26.5 1
Total 41 12629.1
R2= 0.99 CV=1.77

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Appendix Table10. ANOVA table for combined yield and related parameters, 2011 cropping
season.

NPP Model 43 909.79 21.2 16.65 <0.0001

VAR 13 684.63 52.7 41.5 <0.0001
LOC 2 10.1 5.1 3.98 0.0224
BLK 2 13.83 6.9 5.44 0.006
VAR*LOC 26 201.22 7.74 6.09 <0.0001
Error 82 104.17 1.27
Total 125 1013.97
R2= 0.90 CV=9.4
YLD (kg) Model 43 25270348 587682 200.1 <0.0001
VAR 13 2395025 184232.7 62.7 <0.0001
LOC 2 21672517 10836259 3689.3 <0.0001
BLK 2 1256.33 628 0.21 0.8079
VAR *LOC 26 1201549 46213 15.7 <0.0001
Error 82 240850 2937
Total 125 25511198
R2=0.99 CV= 4.55
HSW Model 43 42716 993.4 283.4 <0.0001
VAR 13 39809.2 3062.3 873.7 <0.0001
LOC 2 963.8 481.9 137.5 <0.0001
BLK 2 104.1 52 14.8 <0.0001
VAR*LOC 26 1838.6 70.71 20.2 <0.0001
Error 82 287 3.5
Total 125 43003
R2= 0.99 CV=3.2

Appendix Table 11. ANOVA table for Mean severity index of chocolate spot at eleven
peasant asociations (PA) of farmers’ field, 2011 cropping season.

Source DF SS MS F Value Pr > F

Model 10 2705 271 8.43 <0.0001
PA 10 2705 271 8.43 <0.0001
Error 33 1059 32.1
Total 43 3764
R2=0.72 CV=10.8

91
Appendix Table 12. Pearson correlation coefficients between epidemiological measurements
and yield and yield components at three locations, 2011 growing season.

Location Parameter AUDPC IR NPP NSP HSW YLD

DSI 0.87** 0.37* -0.51** -0.01 0.23 -0.44**
AUDPC 0.28 -0.46** 0.05 0.32 -0.26
IR -0.22 0.01 0.2 0.18
Tocha
NPP 0.03 -0.55 0.09
NSP 0.22 0.23
HSW 0.44**
DSI 0.97** 0.82** -0.33* -0.17 0.28 -0.53**
AUDPC 0.78** -0.29 -0.13 0.28 -0.55**
IR -0.23 -0.29 0.36* -0.35*
Mari
NPP -0.04 -0.50** 0.22
NSP 0.16 -0.45**
HSW 0.15
DSI 0.61** 0.64** 0.29 -0.04 -0.03 -0.15
AUDPC 0.24 0.46* 0.03 -0.45 -0.26
IR 0.11 -0.04 -0.03 -0.03
Turi
NPP -0.36* -0.42** 0.24
NSP 0.08 -0.28
HSW 0.05
* Significant at P < 0.05; ** highly significant at P<0.01
DSI=Disease severity index at last assessment date; AUDPC=Area under disease progress
curve for growing season; IR=chocolate spot infection rate; NPP=Number of pods per plant;
NSP=Number of seeds per plant; HSW=Hundred seed weight; SYD=Seed yield

92
Apendex plate1. Isolation and identification of Botrytis fabae isolates.

93
 Less
M
After 2 days of
inoculation After 8 da

Appendix plate 2. Pathogenicity of Botrytis fabae isolates on var. CS20DK under green house
conditions.

94