Organic Name Reactions for the students and aspirants of Chemistry12th.pptx
Faba bean root rot defense final.ppt
1. IMPORTANCE, VARIABILITY IN THE PATHOGEN AND
ALTERNATIVE MEASURES FOR THE MANAGEMENT OF FABA
BEAN ROOT ROT (FUSARIUM SOLANI) IN THE HIGHLANDS
OF NORTHEASTERN ETHIOPIA
Eshetu Belete
2. INTRODUCTION
2
Faba bean (Vicia faba L.)
• Most important pulse crops grown in the
highlands of Ethiopia.
• Ranks first among highland pulses in area
coverage and grain production
• Considered as one of the center of
secondary diversity
Roles
• Food, animal feed, improving soil fertility,
good source of income and export items.
• But the current average yield is low
• 1.5 tons/ha (CSA, 2010).
3. Introduction cont…
The production is constrained by several biotic
and abiotic factors.
Fungal diseases are the major factors affecting
the production and productivity as well as the
quality of the crop.
Economically important diseases: -
3
Constraints
Chocolate spot
(Botrytis fabae),
Rust
(Uromyces
vicae fabae)
Black root rot
(Fusarium solani)
4. 4
Highlands of Ethiopia, faba bean root rot (F.
solani) is an important disease.
Black clay soils for which water logging is a
problem.
Major production problem in the area today.
Yield loss - 45% was estimated (PPRC, 1996).
Favorable conditions-complete crop failure.
Farmers substitute this crop with other
legumes, mainly grass pea (Lathyrus
sativus).
Introduction cont…
5. F. solani is commonly a part of the root rot
complex and causes seedling blight and
root rot.
Black discoloration observed on main root
and laterals, start at the soil level or a
little above the ground.
Yellowing of leaves starts from the lowest
part of the plant and the infected plants do
not produced pods.
Finally the infected plant dies.
5
Symptoms
Introduction cont…
6. Factors that reduce root growth increase
susceptibility to Fusarium root rot.
Vertisols it holds a lot of water that favor
epidemic development (Dereje and Tesfaye,
1994).
The pathogen is very persistent in soil as
clamydospores and capable of surviving in
infested fields for long period.
Can also survive as mycelium or spores.
6
Disease development
Introduction cont…
7. Agronomic practice:- cultural practice.
Resistant varieties:- most important and cost-effective
But the durability are not uniform across locations.
B/c of pathogenic variability in F. solani
Hence, determination of genetic diversity in
Fusarium spp. is important for appropriate
management.
Biological Control (BC)-
Ecologically friendly, safe and specific
Used individual or as an IDM component.
Limited attempts on BC of faba bean root rot in
Ethiopia (Tesfaye, 1999; Temesgen, 2003).
None of them has found practical applications.
7
Introduction cont…
Control
8. In spite of the serious yield losses caused by
root rot to faba bean production, there has
been no detailed studies on the distribution and
pathogen variability in Ethiopia.
Investigation on pathogen variability within the
pathogen population is important
Such information is crucial for epidemiological
studies leading to development of effective
disease management strategies.
8
Introduction cont…
9. OBJECTIVES
To study the distribution and importance of the
root rot disease, and characterize pathogen
variability and develop alternative management
options to reduce the negative impact of the
disease in northeastern Ethiopia.
9
10. I. Associations of biophysical factors with faba bean root
rot (Fusarium solani) epidemics in the northeastern
highlands of Ethiopia. 2013. Crop Protection, 52: 39-46.
II. Morphological and pathogenic variability of Fusarium
solani populations affecting faba bean in the highland
of northeast Ethiopia. (Submitted).
III. Antagonistic Effects of Native Bacillus isolates against
root rot (Fusarium solani) on faba bean (Submitted).
IV. Evaluation of local isolates of Trichoderma spp. against
root rot (Fusarium solani) on faba bean (under
preparations). 10
Manuscripts
11. PAPER I
Associations of biophysical factors with faba bean
root rot (Fusarium solani) epidemics in the
northeastern highlands of Ethiopia
11
12. To assess the distribution of faba bean root rot in the
major faba bean-growing districts of the northeast
highlands of Ethiopia in relation to biophysical
factors.
12
Objective
14. Sampling and disease assessments
A total of 240 farmers fields’ were assessed
during 2010 and 2011 main cropping seasons.
Disease incidence as percentage of plants
showing faba bean root rot symptoms in
averages of the five quadrats were taken for
each field, and
Disease severity was estimated from
randomly selected ten faba bean plants as
the area of root tissue damaged using a 0-5
rating scale.
14
Material and Methods Cont…
15. Disease severity scores were converted into
percentage severity index (PSI)
Other biophysical factors : -
● preceding crops ● land preparation,
● cropping system ● plant density
● elevation ● sowing time
● extent of weeding ● soil type
● crop season
15
Material and Methods Cont…
16. The associations of faba bean root rot with
biophysical variables were analyzed by logistic
regression model (Yuen, 2006; Fininsa and
Yuen, 2001) using SAS procedure of the
GENMOD (SAS, 2004).
Logistic regression model allows evaluating the
importance of multiple independent variables
that affect the response variable (McCullagha
and Nelder, 1989)
16
Data analysis
17. Distribution, incidence and severity of faba
bean root rot
Fusarium solani was shown to be the most
common and frequently isolated (73%)
pathogen from infected roots of faba bean
Others : F. oxysporium, R. bataticola, R.
solani, Alternaria spp., D. dipsaci and
unidentified fungi.
All faba bean fields surveyed in the two
cropping seasons were infested by root rot
and the prevalence was 100%.
17
D. dipsaci
Result
18. 18
Variable Variable
class
DI (%) PSI
2010 2011 Mean 2010 2011 Mean
District Delanta 44.6 30.3 37.4 40.1 23.4 31.8
Jama 47.1 23.8 35.5 41.3 19.8 30.6
Worillu 56.0 27.9 42.0 39.6 23.9 31.8
Altitude ≤2650 51.3 24.5 37.3 41.3 20.9 30.7
>2650 47.4 30.8 39.3 39.4 24.1 32.1
Soil type Vertisol 49.7 28.4 39.3 40.4 22.9 31.8
Non-verti 47.4 23.9 35.1 40.1 20.8 29.9
Land
prepapara
Flat 51.9 29.7 41.6 40.8 23.7 32.8
Furrow 41.1 22.8 30.5 38.8 19.9 27.9
Sowing
time
June 57.0 24.8 43.5 41.7 21.0 33.0
July 45.3 28.2 36.2 39.6 22.8 30.7
Table 1. Incidence and Percent severity index (mean ) of faba bean root rot
for different independent variables.
19. 19
Variable Variable
class
DI (%) PSI
2010 2011 Mean 2010 2011 Mean
Cropping
system
Sole 48.8 27.4 38.6 40.5 22.4 31.9
Mixed 54.1 27.3 36.3 37.3 22.5 27.4
Plant
density
High 50.1 24.6 42.2 39.4 20.1 33.4
Normal 48.7 28.8 37.5 39.0 23.2 30.1
Low 49.2 26.2 37.1 42.8 22.1 32.0
Weed
managt.
Poor 51.7 29.0 40.6 41.5 24.3 33.1
Inter. 47.5 26.1 36.4 39.3 21.3 30.0
Good 47.1 26.2 36.9 39.6 20.8 30.4
Preceding
crop
Tef 51.3 28.0 38.6 40.2 23.3 30.9
Wheat 48.4 27.0 38.2 40.4 21.9 31.6
Table 1. Cont…
20. Association of faba bean root rot with biophysical factors
Independent
Variables df
Incidence, LRT PSI, LRT
VEF VEL VEF VEL
DR Pr >2 DR Pr >2 DR Pr >2 DR Pr >2
Districts 2 78.9 0.0001 37.55 0.0001 3.78 0.15 1.86 0.39
Altitude 1 0.53 0.46 0.08 0.78 0.10 0.75 0.49 0.48
Soil type 1 44.6 0.0001 119.4 0.0001 6.30 0.012 17.55 0.0001
Land prep. 1 198.0 0.0001 170.7 0.0001 30.35 0.0001 26.5 0.0001
Cropping syst. 1 0.54 0.46 0.79 0.37 2.00 0.16 1.37 0.24
Sowing time 1 15.08 0.0001 8.42 0.004 0.00 0.99 1.17 0.28
Density 2 2.57 0.27 0.92 0.63 7.25 0.026 15.45 0.0004
Weed mangt. 2 10.45 0.005 10.06 0.006 31.47 0.0001 30.64 0.0001
Preceding crop 1 0.07 0.78 0.07 0.78 0.02 0.88 0.02 0.88
Season 1 1230.6 0.0001 851.4 0.0001 904.2 0.0001 655.1 0.0001
Table 2. Logistic regression model of faba bean root rot incidence and PSI
and likelihood ratio test on independent variables in two cropping seasons
The independent variables of soil type, land preparation, weed
management and cropping season were tested in both reduced
multiple-variable model.
21. Added
Variables
Residua
devianc df
LRT
Variable
class
Estimate
Log SE
Odd
ratio
DR Pr >2
Intercept 2534.0 0 -1.455 0.08 0.23
Soil type 1179.3 1 117.48 0.0001 Vertosol 0.41 0.037 1.51
Non-
vertisol
0 1
Land
preparati
981.2 1 168.27 0.0001 Flat sowing 0.45 0.045 1.57
Furrow 0 1
Sowing
time
965.6 1 8.41 0.0037 June 0 1
July -0.12 0.039 0.89
Weed
managet.
938.9 2 8.53 0.0005 Good -0.117 0.04 0.89
Intermediat -0.085 0.035 0.92
Poor 0 1
Season 1303.3 1 830.54 0.0001 2010 0.89 0.031 2.43
2011 0 1
Table 3. Analysis of deviance, natural logarithms of odds ratio and
standard error of percent root rot incidence in two cropping seasons
Low disease incidence was associated with non-vertisols, furrow
sowing, late planting, good weed management and the 2011
cropping season
22. Added
Variables
Residua
devianc df
LRT
Variable
class
Estimate
Log SE
Odd
ratio
DR Pr >2
Intercept 1392.6 0 -1.359 0.079 0.257
Soil type 478.2 1 17.43 0.0001 Vertosol 0.163 0.039 1.177
Non-
vertisol
0 1
Land
preparati
447.8 1 26.35 0.0001 Flat sowing 0.182 0.035 1.199
Furrow 0 1
Weed
managet.
403.5 2 22.81 0.0005 Good -1.99 0.04 0.136
Intermediat -1.68 0.036 0.196
Poor 0 1
Season 488.4 1 640.3 0.0001 2010 0.822 0.03 2.275
2011 0 1
Table 4. Analysis of deviance, natural logarithms of odds ratio and
standard error of percent root rot severity in two cropping seasons
Low PSI was associated with non-vertisols, furrow sowing, good
weed management and the 2011 cropping season
Both root rot incidence and PSI were highly influenced by the soil
type, land preparation, weed management and season.
23. Root rot was widely distributed and a major
problem in the area today.
The incidence and severity of the disease varied
among the soil type, land preparation, weed
management practice and cropping season.
Logistic regression analyses indicated that
vertisols, flat sowing, high weed infestation and
high soil moisture were associated with the root
rot incidence and severity and had significant
contributions to the development of epidemics.
23
Discussion
24. PAPER II
24
Morphological and pathogenic variability of
Fusarium solani populations affecting faba bean in
the highland of northeast Ethiopia
25. To determine morphological, cultural and pathogenic
variability of F. solani causing root rot of faba bean
and its cross infectivity on cool-season food legumes.
25
Objective
26. Fungal Isolations
Infected roots of faba bean plants were collected
from three major faba bean growing districts.
Segments of root portion of plants showing lesions
characteristics to F. solani infections were
disinfected and cultured on PDA.
Developing Fusarium colonies were sub-cultured on
PDA and pure cultures were maintained on PDA
slants at 4oC.
Colonies were sub-cultured on PDA and CLA and
isolation of F. solani were identified based on
morphological characteristics (Leslie and
Summerell 2006).
26
Material and Methods
27. Cultural and morphological characteristics
Macroscopic and microscopic characteristics were
studied following the procedures described by
Lesilie and Summerell (2006).
Aerial mycelial texture, colony shape, obverse and
reverse colony color, radial growth and colony
margins were recorded (Hosen et al., 2010).
Radial growth rates of each isolate were estimated
from colony diameter measurement (mm).
Condial size (length and width), appearance of
chlamydospores and condial septations were
determined.
27
28. Pathogenic variability
The isolates of F. solani was tested in the
greenhouse using five released faba bean
cultivars namely
Degaga (R878-3),
Kussie (KUSE2-27-33),
Mesay (74TA12050x74TA236),
Selalle (Selale Kasim 91-13) and
Wayu (Wayu 89-5).
28
29. Cross infectivity
Cross infectivity tests of F. solani isolates
were carried out by isolating representative
F. solani isolates from faba bean, field pea,
lentil, grass pea and chickpea plants.
Each isolate was examined for their
pathogenicity in a growth chamber.
29
30. Data analyses
Using General Linear Modeling (GLM) procedure of
SAS® System for Windows Version 9.1 software (SAS
2004).
Severity ratings were normalized before analysis
using square root transformation.
Treatment means were separated using LSD test at
5% level of significance
Cluster analysis was used to determine the
relationship of the isolates based on cultural and
morphological characteristics and their virulence
on faba bean cultivars (Forina et al., 2002;
Mahmoud et al., 2007).
30
31. Cultural characteristics
Isolate Colony color Colony
texture
Colony
shape
Colony
margin
Surface Reverse
PPRC White White Fluffy RWOS Regular
DV036 White L. Blue Fluffy RWOS Regular
DV037 White White Velvet Irreg. Irregular
DV038 L. pinkish Pinkish Fluffy RWOS Regular
DV043 White L. pinkish Fluffy RWS Regular
DV045 White White Velvet Irreg. Irregular
JV001 L. Pinkish White Velvet RWOS Regular
JV009 White Pinkish Effuse RWOS Regular
JV010A White White Fluffy RWS Regular
JV010B White White Fluffy RWOS Irregular
JV011 White L. pinkish Velvet RWOS Regular
31
Table. 1 Cultural characteristic of isolates of F. solani 10 days
after incubation on PDA agar.
Results
32. Isolate Colony color Colony
texture
Colony
shape
Colony
margin
Surface Reverse
WV016A White L. Pinkish Fluffy RWOS Regular
WV016B White White Velvet RWOS Regular
WV018 White L. Pinkish Fluffy RWOS Regular
WV019 White L. Pinkish Fluffy RWOS Regular
WV022 White White Velvet RWS Regular
WV023 White Pinkish Fluffy RWOS Regular
WV024 White Pinkish Fluffy RWS Regular
WV026 White L. Pinkish Fluffy RWOS Regular
WV027 Pinkish Pinkish Effuse RWS Regular
WV029 White L. Pinkish Velvet RWOS Regular
32
Table. 1 cont…
The cultural characteristics of F. solani isolates showed
variation in colony color, texture, shape and margin.
33. Isolate
Colony diameter
4 days 5 days 6 days 7 days
PPRC 39.8±0.03d-g 54.3±0.03e-h 70.0±0.00c-e 84.2±0.03c-g
DV036 37.7±0.03gh 51.8±0.08g-i 64.8±0.03fg 79.3±0.06k
DV037 38.0±0.15f-h 56.3±0.06c-e 73.0±0.13bc 82.2±0.18g-i
DV038 38.5±0.18e-h 51.3±0.38hi 66.5±0.38d-f 80.5±0.43h-j
DV043 41.2±0.08b-e 55.0±0.05d-g 69.8±0.03c-e 83.7±0.10d-g
DV045 43.2±0.12a-c 57.8±0.14b-d 72.7±0.17bc 82.5±0.10f-h
JV001 43.7±0.16ab 58.7±0.15a-c 73.8±0.21bc 86.8±0.17b
JV009 43.3±0.03ab 59.7±0.03ab 76.7±0.10ab 89.5±0.09a
JV010A 37.2±0.11g-I 51.7±0.03hi 66.2±0.08d-f 79.7±0.08ij
JV010B 34.7±0.49i 46.7±0.66j 61.0±0.89g 79.2±0.21k
JV011 45.0±0.05a 61.3±0.10a 79.2±0.06a 90.0±0.03a 33
Table 2. Mean colony diameter (mm) of F. solani isolates during
seven day incubation period on PDA.
34. Isolate
Colony diameter
4 days 5 days 6 days 7 days
WV016A 41.8±0.03b-d 57.3±0.03b-e 73.2±0.08bc 85.8±0.08b-d
WV016B 41.7±0.03b-d 57.0±0.05b-e 73.3±0.06bc 86.5±0.00bc
WV018 42.3±0.03a-c 56.7±0.06b-e 71.3±0.06c 84.7±0.03b-f
WV019 37.5±0.17gh 52.0±0.15g-i 66.0±0.18ef 80.3±0.19h-j
WV022 40.5±0.17c-f 55.8±0.08c-f 71.5±0.15c 85.2±0.12b-e
WV023 42.5±0.00a-d 57.0±0.00b-e 72.7±0.03bc 82.0±0.05g-i
WV024 37.5±0.10gh 51.8±0.08g-i 66.3±0.15d-f 80.2±0.12h-j
WV026 36.2±0.33hi 50.0±0.35i 64.7±0.39fg 79.3±0.27k
WV027 38.0±0.15f-h 53.0±0.18f-i 70.2±0.10cd 83.2±0.07e-g
WV029 42.7±0.06a-c 57.3±0.08b-e 71.8±0.08c 84.2±0.08c-g
Mean 40.1 54.9 70.2 83.3
Lsd (5%) 0.271 0.332 0.4108 0.2517
34
Table. 2 cont…
Significant differences (P≤ 0.05) were observed among F.
solani isolates for their mycelial growth during different
days of incubation
35. Morphological characteristics
Macroconidia - predominantly 3-septae
but there were conidia with 2-4
septae.
The isolates also showed variability in
the size of the macroconidia.
The width and length was 4.7~5.8 µm
and 24.2~31.4 µm, respectively.
Microconidia - were abundant, mono or
bi-cellular, oval in shape and formed
on very long monophialides.
Chlamidospores - produced abundantly,
mostly single and rarely in pair. 35
38. Isolates Faba bean cultivar
Degaga Kuse Mesay Selalle Wayu
WV016A 0.0(0.7)d 16.0(4.1)a-d 0.0(0.7)f 25.0(5.0)a 18.0(4.3)a-c
WV016B 21.7(4.7)a 15.0(3.9)b-d 13.3(3.6)c-e 0.0(0.7)e 16.7(4.1)a-c
WV018 0.0(0.7)d 21.7(4.7)ab 25.0(5.0)a 0.0(0.7)e 13.3(3.7)bc
WV019 0.0(0.7)d 0.0(0.7)f 15.0(3.8)b-d 0.0(0.7)e 0.0(0.7)d
WV022 0.0(0.7)d 0.0(0.7)f 10.0(3.2)de 18.3(4.3)a-c 0.0(0.7)d
WV023 23.3(4.8)a 11.7(3.5)c-e 0.0(0.7)f 16.7(4.1)a-c 13.7(3.6)c
WV024 13.0(3.7)bc 18.0(4.3)a-c 0.0(0.7)f 11.0(3.3)cd 20.0(4.5)ab
WV026 18.3(4.2)ab 0.0(0.7)f 21.7(4.7)ab 20.0(4.5)ab 0.0(0.7)d
WV027 0.0(0.7)d 6.7(2.5)e 0.0(0.7)f 11.7(3.1)b-d 0.0(0.7)d
WV029 11.7(3.5)bc 15.0(3.8)b-d 0.0(0.7)f 11.7(3.1)b-d 0.0(0.7)d
Lsd 0.9141 1.0719 0.9653 1.1654 0.8715
Mean 7.54(2.25) 10.70(2.94) 6.59(2.11) 10.52(2.84) 6.84(2.11)
38
Table 4. cont…
The cultivar isolate interaction suggested that F. solani
population showed variation in disease reactions
depending on the type of isolates.
The differences in root rot infection between isolates
were not the same for each cultivar, that is, cultivars
responded differently to the isolates.
39. 39
WV016B
JV011
WV024
WV023
DV043
WV018
WV016A
JV001
WV026
DV038
WV029
JV010A
DV045
DV037
WV022
JV010B
WV027
DV036
JV009
WV019
PPRC01
0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0 1.1 1.2 1.3 1.4
I
II
III
IV
V
Average distance between clusters
Cluster analysis showed that the isolates were grouped into
five different clusters.
Fig 2. Dendrogram showing relationship among F. solani isolates
based on pathogenicity on five faba bean cultivars.
Weak
virulence
Strong
virulence
On isolate-cultivar interaction,
the mean percentage severity
index of the isolates ranged from
2 to 13%, indicating that isolates
varied considerably in their
pathogenicity.
40. Cross infectivity
The isolates of F. solani from faba bean and field
pea showed various degree of pathogenicity on all
tested cool-season food legumes.
In contrast, the pathogens isolated from chickpea,
lentil and grasspea did not infect faba bean and
field pea.
Isolates of F. solani from lentil and grasspea were
pathogenic on lentil and grasspea, while isolate
from chickpea did not infect the other pulses.
40
41. F. solani isolates showed high level of cultural,
morphological and pathogenic variability.
The existence of variation among F. solani
isolates in the area might be attributed due to
the difference in farming practices and env’tal
conditions.
Moreover, the occurrence of the broad genetic
diversity of faba bean in the country might hosts
diverse isolates of F. solani population.
41
Discussion
42. However, characterization of the pathogen
population based on morphological and
pathogenicity are not adequate enough to properly
distinguish different isolates of Fusarium pathogen.
Molecular techniques could overcome these
limitations and will characterize the genetic
variability of fungal population more precisely.
Information regarding the variability of F. solani
from this study will serve as baseline information on
the future prospects of breeding for root rot
resistance in faba bean in the highlands of Ethiopia.
42
Discussion cont …
43. 43
Paper III
Antagonistic effects of native Bacillus isolates against
root rot (Fusarium solani) on faba bean
Objective
To identify native Bacillus spp. and evaluate their
antagonistic effect in managing root rot on F. solani
pathogen.
44. Isolation
Isolations of antagonistic bacteria from health
faba bean roots and rhizosphere soil were done
using dilution technique (Watesman, 1922).
One ml of serially diluted suspension from both
soil and root dilutions were pipetted into King’ B
medium and Nutrient agar (NA) media.
Isolates of bacteria colonies were picked for
antagonism studies.
44
Material and Methods
45. Isolation cont…
All Gram +ve, rod-shaped, catalase +ve, growing
at 45˚C and endospore forming bacteria were
identified as Bacillus isolates and were maintained
at 4˚C on slants of NA for further studies (Schaad
et al., 2001). .
45
46. Testing in vitro antagonistic activity
Dual culture
Isolates of Bacillus spp. were tested for their
antagonist effects against F. solani.
Cultures were observed daily and data on growth
inhibition zone and colony diameter were
recorded.
The radii of the fungal colony towards and away
from the antagonistic colony were measured
Percent growth inhibition was calculated.
Spore Germination
The effect of culture filtrate of Bacillus isolates
on conidia germination of F. solani was evaluated
and spore germination percent were recorded. 46
47. Green house experiment
Antagonist effect of Bacillus isolates were tested
on faba bean root rot development on faba bean
seedlings.
Root rot severity was scored on the roots and
hypocotyls using 0-4 rating scale (McFadden et al.,
1989)
Percentage of root rot suppression was calculated
(Villajuan-Abgona et al., 1996).
47
48. Data analyses
Using General Linear Modeling (GLM) procedure of
SAS® System for Windows Version 9.1 software (SAS
2004).
Severity ratings were normalized before analysis
using square root transformation.
Treatment means were separated using LSD test at
5% level of significance
48
49. In vitro antagonistic activity
All isolates of the antagonists reduced mycelial
growth of the pathogen.
Significant differences (P≤0.05) were observed
among isolates on inhibiting the mycelial
growth and the inhibition effects of the
pathogen (Table 1)
49
Results
51. 51
Fig1. In vitro antagonistic effect of selected Bacillus isolates
against F. solani using in dual culture method.
The antagonist Bacillus isolates was able to inhibit
mycelia growth and clear inhibition zone were
formed between antagonists and pathogen.
52. 52
Culture filtrates of Bacillus isolates significantly
(P≤0.05) reduced spore germination of F. solani.
As the culture filtrate concentration increase, the level
of germinated spores was reduced.
The germination of untreated (control) spores was
about 83.4%, while at 100% culture filtrate
concentration the lowest level of germination (9.9%),
with 88% reduction over the control.
53. Greenhouse experiment
Application of Bacillus antagonists as seed treatment
significantly (P ≤ 0.05) reduced root rot infection and
enhanced the height and biomass of bean seedlings as
in comparison to F. solani alone inoculated bean
seedlings (Table 3).
53
A B
A B
Bacillus treated (A) and untreated (B)
faba bean seedlings .
55. Antibiosis produced by the bacteria suppressed and
restricted the growth of the pathogen.
Bacillus isolates able to reduce spore germination of
F. solani pathogen at lower culture filtrate conce.
Effectiveness at low concentration is the indication
of the ideal characteristics of ideal antagonists.
Better overall growth of faba bean plants indicates
the efficacy of the antagonists in controlling faba
bean root rot.
Therefore, the selected antagonists suggests that it
is a promising biocontrol agent against the pathogen.
55
Discussion
56. 56
Paper IV
Evaluation of local isolates of Trichoderma spp. against root rot
(Fusarium solani) on faba bean
To identify local Trichoderma spp. and evaluate their
antagonistic effect in managing root rot on F. solani
pathogen.
Objective
57. Isolation
Isolations of Trichoderma spp. from rhizosphere soil
were done using dilution technique (Watesman,
1922).
One ml of serially diluted suspension plated on PDA.
Isolates of Trichoderma colonies were picked for
antagonism studies.
Trichoderma isolates were identified based on their
conidial morphology, color and texture, and growth
characteristics (Kubicek and Harman, 2002)
57
Material and Methods
58. Testing in vitro antagonistic activity
Trichoderma spp. were tested for their antagonist
effects using dual culture method (Same as Exp. 3)
Green house experiment
Same as Exp. 3
Data analyses
Same as Exp. 3
58
59. In vitro test
Dual culture tests clearly showed that all the
isolated Trichoderma spp. significantly (P≤ 0.05)
inhibited the radial growth of F. solani (Table 2)
59
Results
60. Isolate Inhibition (mm) Inhibition (%) T. radius F. radius
TS004 19.3±0.1a 40.2±0.1f 49.7±0.1b-d 32.3±0.1a
TS007 13.3±0.1c 55.1±0.2d 52.3±0.2ab 29.7±0.1b-d
TS010 12.7±0.2cd 60.5±5.1b-d 54.0±0.1a 32.0±0.2ab
TS015 20.7±0.1a 33.9±2.7g 50.3±0.2bc 31.3±0.2a-c
TS018 16.7±0.2b 47.4±5.0e 50.0±0.2b-d 31.7±0.1a-c
TS019 11.3±0.2d-f 61.4±4.4a-c 52.3±0.2ab 29.3±0.1c-e
TS022 12.3±0.2c-e 60.7±3.8b-d 50.0±0.1b-d 31.3±0.1a-c
TS025A 10.7±0.1ef 65.9±1.2ab 51.7±0.2ab 31.3±0.1a-c
TS027 19.3±0.1a 34.8±1.7fg 47.0±0.2de 29.6±0.2c-e
TS030 19.3±0.1a 37.0±2.8fg 45.7±0.5e 30.7±0.0a-c
TS032 20.0±0.0a 34.8±1.3fg 47.0±0.3de 30.7±.01a-c
TS036 10.3±0.1f 67.0±1.6a 49.6±0.1b-d 31.3±.01a-c
TS037 16.0±0.1b 47.2±3.5e 49.7±0.1b-c 30.3±0.1a-c
TS041A 17.0±0.2b 40.1±5.9f 48.0±0.3c-e 28.4±0.3de
TS047 12.0±0.1c-f 60.4±3.2b-d 51.3±0.2ab 30.3±0.2a-c
TS050 11.3±0.1d-f 63.0±0.5a-c 49.3±0.2c-d 30.7±0.1a-c
TS058 12.3±0.1c-e 58.6±5.8cd 49.3±0.1b-d 30.0±0.1a-c
TS064 19.6±0.1a 34.3±2.0fg 49.7±0.1b-d 30.0±0.0a-c
TS090 20.0±0.0a 35.9±5.1fg 49.3±0.1b-d 31.2±0.3a-c
CV% 7.05 7.11 3.89 5.3
60
Table 2. Antagonistic activity of Trichoderma isolates towards F. solani
Nine Trichoderma isolates could significantly inhibit the
colony growth of F. solani with the highest mycelial
growth inhibition percent, with more than 50% inhibition.
61. 61
Fig.1. In vitro antagonistic effect of selected Trichoderma isolates against
F. solani using in dual culture method after four (top) and five (bottom)
days inoculation.
The antagonists inhibited the mycelial growth of F. solani
but could not overgrow the pathogen until 3 to 4 days.
However, five days later the Trichoderma were overgrew
the pathogen and occupied the medium.
62. Greenhouse experiment
Trichoderma antagonists as seed
treatment significantly (P ≤ 0.05)
reduced root rot infection and enhanced
the height and biomass of bean seedlings
as compared to F. solani alone
inoculated bean seedlings (Table 4).
62
A B
A
B
Trichoderma treated (A) and untreated (B)
faba bean seedlings .
A
B
64. Trichoderma isolates grow much faster and inhibited
the growth of F. solani pathogen, thus competing
efficiently for space and nutrients.
So that competition for limiting nutrients results in
biological control of fungal pathogens.
Subsequent profuse sporulation of Trichoderma and its
ability to over grow the pathogenmay indicate its
ability to directly parasitize the pathogen.
Better overall growth of faba bean plants indicates the
efficacy of the antagonists in controlling faba bean
root rot.
64
Discussion
65. Root rot is widely distributed and is a major problem
in most faba bean-growing areas of the NEH of
Ethiopia.
Vertisols, flat sowing, high weed infestation and high
soil moisture were associated with the development
of root rot epidemics.
Substantial pathogenic variability exists among
isolates of F. solani in the area.
Information regarding the variability of F. solani will
serve as baseline information on the future prospects
of breeding for root rot resistance in faba bean. 65
CONCLUSION
66. Development and use of resistant varieties require
exploration and a through understanding of the
variability of the pathogen.
Use of indigenous Bacillus and Trichoderma
species as a biopesticide in the control of F. solani
of faba bean is a promising natural biopesticide
and used as one of component of integrated
disease management strategy.
66
CONCLUSION CONT…
67. Root rot disease management measures that are
suitable for adoption by the small-scale farmers
are important.
Use of weed management and proper drainage
practice can reduce the risk of faba bean root rot.
Breeding for resistance to faba bean root rot
should be given high priority and should be
supported with good agronomic management
practices that do not favor disease epidemics.
67
RECOMMENDATION
68. Further investigation is required to develop
differential host varieties and reveal the full extent
of pathogenic variability across the country.
Still there is a need that the more aggressive strain
should be employed in cultivar screening. Developing
resistance genotypes using virulent isolates, together
with a mixture of isolates, in order to test the
disease interactions and select for faba bean
genotypes.
The relationship of stem nematodes to the root rot
of faba bean needs to be investigated in relation to
its role in root rot.
68
RECOMMENDATION CONT…
69. Biological control research should be directed
towards mass multiplication, formulation and mode
of delivery of these effective antagonistic isolates.
Field studies should be undertaken to confirm the
effectiveness of the antagonistic isolates under
natural conditions in the field as components of
integrated disease management.
69
RECOMMENDATION CONT…
70. My advisors Dr Amare A. and Dr Seid A.
HU, School of Plant Sc. – facilitating courses and issues
WU and MOE – providing study leave and budget support
My family – moral supports, love and encouragement.
All staffs and postgraduate students – kind and
friendliness
Plant pathology lab. Staffs- Haimanot, Wogayeh, Kidest,
Marta, Adiss and Yegilla- technical support.
70
ACKNOWLEDGEMENT
