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Evaluate for potential biocontrol agent by assessing isolates for
The effect of Rhizobium isolates on the development of radial mycelium of
In dual culture, 27 rhizobium isolates inhibited the radial growth of
Beneficial traits strongly assist the efficiency of candidate antagonists for desired biocontrol, emphasizing the value of concerted mechanisms of action. The result indicated the possible use of Rhizobial isolates as an alternative means of BRR management but further study is needed to verify actual use in agricultural production.
In Ethiopia, grain crops are grown annually on approximately 12.5 million hectares of land, of these, 1.5 million hectares is covered by pulses out of which 443,074.68 hectares is dedicated to Faba bean with annual production of about 8,389,438.97 quintals [
In spite of their huge importance, the productivity of Faba bean in Ethiopia remains far below the crop’s potential greater than 3 ton/ha. Production of Faba bean has been constrained by several biotic and abiotic factors [
Root rot is among the major production constraints limiting the yield of Faba bean in many countries of the world especially where poor nutrient supply and overly wet soil conditions prevail [
Numerous efforts have been made to manage BRR in Ethiopia as such using host plant resistance, cultural practice and synthetic chemicals [
Many species of
In spite of the existence of rhizobia in faba bean rhizosphere, there is little information on rhizobia antagonists against black root rot. Therefore, the major aims of the current study were to determine
The study was conducted at Jimma University College of Agriculture and Veterinary Medicine (JUCAVM), Jimma, Ethiopia. Laboratory activities were conducted in Plant Pathology Laboratory. Pot experiments were conducted in JUCAVM which is located at 7042 N Latitude 36057 E Longitude and at altitude 1710 m. a.s.l. The maximum and minimum temperature of the area is 26.8 and 11.8 0c, respectively, with relative humidity of 91% and the mean rainfall of 1500 mm per annum [
Fourteen (14) rhizobium isolates were obtained from the culture collection of Holeta Agricultural Research Centre, Microbial Biotechnology Laboratory. The other twenty seven (27) isolates were obtained from Jimma University, Department of Biology, Applied Microbiology Laboratory (Table
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JU2(4) | Faba bean | Mena | Jimma | JU3(1) | Faba bean | Mena | Jimma |
JU-E(1) | Haricot bean | Mena | Jimma | Ho-4-1SG | Faba bean | S. Gonder | Holeta |
JU1(2) | Faba bean | Mena | Jimma | JU47(2) | Faba bean | Mena | Jimma |
JU1(3) | Faba bean | Mena | Jimma | JU2(2) | Faba bean | Mena | Jimma |
JU8(1) | Pea | Mena | Jimma | JU2(3) | Faba bean | Mena | Jimma |
JU1(5) | Faba bean | Mena | Jimma | JU-Y(1) | Haricot b | Yebu | Jimma |
Ho-1-1NG | Faba bean | N. Gonder | Holeta | JU3(2) | Faba bean | Mena | Jimma |
Ho-1018 | Faba bean | N. Gonder | Holeta | JU8(2) | Faba bean | Mena | Jimma |
Ho-3WG | Faba bean | W. Gonder | Holeta | JU8(3) | Faba bean | Mena | Jimma |
JU26(1) | Faba bean | Mena | Jimma | JU-D(1) | Haricot b | Dedo | Jimma |
JU3(1) | Faba bean | Mena | Jimma | Ho-3-1SG | Faba bean | S. Gonder | Holeta |
JU15(2) | Faba bean | Mena | Jimma | JU5(2) | Faba bean | Mena | Jimma |
JU-S(1) | Haricot b | Seribo | Jimma | JU21(4) | Faba bean | Mena | Jimma |
JU11(1) | Faba bean | Mena | Jimma | JU-Sr(1) | Haricot b | Seribo | Jimma |
JU-G(2) | Haricot b | Ginibbe | Jimma | JU26(2) | Faba bean | Mena | Jimma |
Ho-2ST | Faba bean | S. Tigria | Holeta | Ho-7EG | Faba bean | E. Gonder | Holeta |
Ho-1WG | Faba bean | W. Gonder | Holeta | Ho-1035 | Faba bean | N. Gonder | Holeta |
JU3(3) | Faba bean | Mena | Jimma | Ho-2-1ST | Faba bean | S. Tigria | Holeta |
Ho-5EG | Faba bean | E. Gonder | Holeta | Ho-2-1WG | Faba bean | W. Gonder | Holeta |
Ho-2EG | Faba bean | E. Gonder | Holeta | ||||
Ho-1EG | Faba bean | E. Gonder | Holeta | ||||
JU1(4) | Faba bean | Mena | Jimma |
The inhibitory effect of the
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Where, C is the radial growth of fungus in control,and T is the radial growth of fungus in dual culture after 7 days of incubation.
The rhizobial isolates showing antagonistic activities against mycelium growth were further studied with different biochemical tests to determine the probable mechanisms of antimicrobial activities of the isolates.
To test for chitinolytic activity of isolates were spot inoculated individually on medium containing fine powdered chitin [
Proteolytic activity was assessed on skimmed milk agar [
Lipolytic activity was assessed on Yeast Extract Mannitol Agar medium containing 1% Tween 80R. Single bacterial isolate was spot inoculated onto solidified plates of medium. The uninoculated plates served as control. The presence of lipase activity was indicated by insoluble olic acid forming halos around the bacterial colonies [
Hydrogen cyanide production was detected as described by [
Indole Acetic Acid (IAA) production was detected as described by Gordon and Weber [
Isolates were tested for phosphate solubilizing ability on Pikovskaya medium [
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One of our Fusarium isolate (JUFS1) was obtained from the purified culture slant in test tube and maintained on PDA medium to produce conidia. The culture of fungal pathogen was multiplied by cultivating on Potato Dextrose Agar (PDA) medium in Petri plates (9 cm) at 25±2 oC for 15 days in an incubator. The colonies were harvested by scraping the surface with spatula and homogenized with 200 ml of sterile distilled water and filtered through a cheese cloth to make a fungal suspension for inoculation. The spores 1 x 106 / mL were counted by haemocytometr [
Antagonistic effect of
All plants were harvested from each pot at full bloom flowering stage (45 days). Excess soil was removed from the roots by placing the root mass on a sieve and washing with running tap water. Cleaned plants were separated into nodules, root (everything below the first node except nodules) and shoots and the following parameter were assessed. Disease incidence was evaluated after 45 days of planting. The number of plants exhibiting root rot symptoms and percent disease incidence were estimated according the equation by Reddy
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To determine disease severity, individual plant root tissues assessed according to a 1-9 scale proposed by Abawi and Pastor-Corrales [
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The percentage of disease reduction (DR %) was calculated according to Edginton
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where Dc is disease on the control plants that treated with only pathogen and Dt is disease on the treated with antagonist and pathogen.
Shoot, Root, root nodulation and also flower and leaf number were recorded. In order to measure dry weight of nodules, roots and shoots from each plant was placed in paper bags, dried at 70oC for 72 h in a hot oven, and weighed [
The data were subjected to analysis of variance (ANOVA) using the General Linear Modeling (GLM) procedure of SAS-9.2 software [
A total of 41
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JU2(4) | Faba bean | 50.0f | JU1(4) | Faba bean | 36.2ji |
JU-E(1) | Haricot bean | 29.4lmn | JU13(1) | Faba bean | 24.5op |
JU1(2) | Faba bean | 56.9e | Ho-4-1SG | Faba bean | 35.2jk |
JU1(3) | Faba bean | 44.1g | JU47(2) | Faba bean | 26.4opn |
JU8(1) | Pea | 25.4op | JU2(2) | Faba bean | 23.5qp |
JU1(5) | Faba bean | 14.7r | JU2(3) | Faba bean | 30.3lm |
Ho-1-1NG | Faba bean | 0.0s | JU-Y(1) | Haricot bean | 61.7cbd |
Ho-1018 | Faba bean | 27.4omn | JU3(2) | Faba bean | 32.3lk |
Ho-3WG | Faba bean | 62.7cbd | JU8(2) | Faba bean | 24.5op |
JU15(2) | Faba bean | 64.7b | JU8(3) | Faba bean | 59.8ed |
JU3(1) | Faba bean | 41.1hg | JU-D(1) | Haricot bean | 60.7cd |
JU26(1) | Faba bean | 70.5a | Ho-3-1SG | Faba bean | 41.1hg |
JU S(1) | Haricot bean | 29.4lmn | JU5(2) | Faba bean | 29.4lmn |
JU11(1) | Faba bean | 39.2hi | JU21(4) | Faba bean | 39.2hi |
JU-G(2) | Haricot bean | 59.8ed | JU-Sr(1) | Haricot bean | 20.5q |
Ho-2ST | Faba bean | 0.0s | JU26(2) | Faba bean | 20.5q |
Ho-1WG | Faba bean | 63.7cb | Ho-7EG | Faba bean | 35.2jk |
JU3(3) | Faba bean | 24.5op | Ho-1035 | Faba bean | 20.5q |
Ho-5EG | Faba bean | 11.7r | Ho-2-1ST | Faba bean | 11.7r |
Ho-2EG | Faba bean | 20.5q | Ho-2-1WG | Faba bean | 26.4opn |
Ho-1EG | Faba bean | 32.3lk | Control | - | 0.0s |
CV (%) | 2.98 |
There were significant differences (
The seven (25.9%) isolates that showed remarkable inhibitory effect against
In this investigation, twelve (44.4%) out of those that showed inhibitory effect against
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JU-D(1) | + | + | - | - | + | 0.1 | 1.08h |
Ho-1 WG | + | + | - | - | + | 1.3 | 1.90a |
JU-Y(1) | - | + | + | - | - | 0.2 | 1.12fhg |
JU47(2) | + | + | - | - | + | 0.2 | 1.11hg |
JU8(3) | - | + | - | - | + | - | - |
JU1(2) | - | + | - | - | + | 0.3 | 1.08h |
JU-E(1) | - | - | - | - | - | - | - |
JU-S(1) | + | - | - | - | + | - | - |
Ho-2-1WG | - | - | - | - | + | 0.5 | 1.37c |
JU11(1) | - | - | + | - | + | 0.3 | 1.18feg |
JU4-1SG | - | - | - | + | + | - | - |
JU-G(2) | - | + | - | - | + | 0.2 | 1.20fe |
JU1(3) | - | - | - | + | + | - | - |
JU3(2) | + | + | - | - | + | - | - |
JU3(1) | - | - | - | - | + | 0.4 | 1.67b |
Ho-3-1SG | - | - | - | + | + | 0.2 | 1.09h |
JU21(4) | - | + | - | - | - | 0.7 | 1.23de |
Ho-3WG | - | - | + | - | + | - | - |
JU5(2) | - | - | - | + | + | - | - |
JU26(1) | + | + | - | - | + | 0.3 | 1.32dc |
JU1(4) | - | - | - | + | + | - | - |
JU2(3) | - | - | - | + | + | 0.5 | 1.38c |
JU8(1) | - | + | - | - | + | 0.7 | 1.88a |
Ho-1EG | - | - | - | + | + | - | - |
JU2(4) | - | - | + | - | + | - | - |
JU15(2) | + | + | - | - | + | 0.2 | 1.17fheg |
Ho-1018 | - | - | - | + | + | - | - |
Control | - | - | - | - | - | 0.0 | 0.00i |
The
In this study, 4(14.8%) of those that showed inhibitory effect against
Color of the picrate/ Na2CO3-impregnated paper strips changed from yellow (control) to light brown, brown or reddish brown as an indication of the strength of the cyanide produced (Fig.
A growing body of evidence showed that the inhibitory effect of
Most of the
Over 50% of the total
The SI of the potential P solubilising rhizobial isolates differed significantly (p< 0.05) and ranged from 1.0 to 1.9. The bacterial strain Ho-1WG showed the largest SI (1.9 cm) of solubilization, followed by JU8(1) and JU3(1). Phosphate solubilizing rhizobacteria convert insoluble phosphates into soluble monobasic form for plant uptake [
The results showed that there was significant (p< 0.05) interaction effect among types of treatment used and time of application of the isolates in reduction of disease incidence (Table
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JU26(1) | Before | (50)45.0b±0.0 | 50.0 | (55.5)48.1cd±0.0 | 37.5 |
At time | (50)45.0b±0.0 | 50.0 | (61.1)51.4bc±0.0 | 27.7 | |
After | (50)45.0b±0.0 | 50.0 | (64.8)53.6b±1.90 | 27.0 | |
JU15(2) | Before | (25)30.0c±0.0 | 75.0 | (38.8)38.5e±0.00 | 56.3 |
At time | (25)30.0c±0.0 | 75.0 | (51.8)46.0d ±1.8 | 41.6 | |
After | (41)40.0b±8.6 | 59.0 | (55.5)48.1cd±0.0 | 37.5 | |
Ho-1WG | Before | (25)30.0c±0.0 | 75.0 | (31.4)34.0f±2.00 | 64.6 |
At time | (25)30.0c±0.0 | 75.0 | (38.5)38.5e ±0.0 | 56.6 | |
After | (50)45.0b±0.0 | 50.0 | (57.3)49.2cd±1.8 | 35.4 | |
Combination | Before | (0)1.4d ±0.0 | 100.0 | (23.7)29.1g±1.8 | 73.3 |
At time | (0)1.4d ±0.0 | 100.0 | (31.2)33.9f ±2.3 | 64.8 | |
After | (25)30.0c±0.0 | 75.0 | (35.1)36.3ef±1.9 | 60.4 | |
Control (diseased) | Before | (100)88.6a±0.0 | 0 | (88.8)70.4a±0.00 | 0 |
Mean | (35.85)35.5±0.6 | 64.1 | 48.73(44.4)±1.04 | 44.8 | |
CV (%) | 5.3 | 2.8 |
Application of combination of
All of the
The present study showed that
Combined application of plant growth promoting rhizobacteria has significantly lowered
The reduction of root rot incidence and severity obtained in
Treatment | Time of Application | Nodulation | |
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Number | Dry Weight | ||
JU26(1) | Before | 0.97g ± 0.0 | 0.04g ± 0.0 |
At time | 0.0g ± 0.0 | 0.0g ± 0.0 | |
After | 0.0g ± 0.0 | 0.0g ± 0.0 | |
JU15(2) | Before | 4.43f ± 0.0 | 0.02ef ± 0.0 |
At time | 1.0g ± 0.0 | 0.01fg ± 0.0 | |
After | 0.0g ± 0.0 | 0.0g ± 0.0 | |
Ho-1WG | Before | 26.42a ± 0.9 | 0.15b ±0.0 |
At time | 24.3b ± 0.2 | 0.02de± 0.0 | |
After | 16.3d± 0.1 | 0.01fg ± 0.0 | |
Combination | Before | 25.33b ± 0.5 | 0.17a ± 0.0 |
At time | 22.3c ± 0.5 | 0.1c ± 0.0 | |
After | 14.4e ± 0.3 | 0.03d ± 0.0 | |
Health (un-inoculated) | 0.0g± 0.0 | 0.0g± 0.0 | |
Control (diseased) | 0.0g± 0.0 | 0.0g± 0.0 |
Timing of
The roots of uninoculated and those infected with
Before and at time of pathogen inoculation of Ho-1WG, combination and JU15(2) were significantly (p<0.05) increased nodule dry weight compared to the control (diseased) and health (uninoculated). The of suspension isolate JU26(1) was not significantly increased nodule dry weight in either of time of application. Application of isolate combination resulted in the highest (0.17 g/plant) nodule dry weight when applied before followed by at the same time of pathogen inoculation.
Results reported herein indicated that
IAA synthesizing rhizobia have been found to nodulate more intensely than IAA negative mutants [
Over all, significant results have been shown in before application followed by at time of pathogen inoculation. The result of this study corresponds with work done by Yaqub
Shoot height of control plant (infected with
Shoot dry weight of control plant infected with
Means with the same letter are not significantly different at
Root length was significantly lower in plants infected with
Treatment | Time of Application | Growth Parameter | |||||
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Shoot | Root | Flower | Leaf | ||||
Height | Dry wt. | Length | Dry wt. | Number | Number | ||
JU26(1) | Before | 38.2cd ± 0.6 | 1.8ef± 0.0 | 28.6bcd±1.1 | 1.0e ± 0.0 | 14.6cd ±0.5 | 30.6cd ± 0.2 |
At time | 35.8de ± 1.5 | 1.4gh± 0.0 | 25.3e±0.5 | 0.8f ± 0.0 | 9.6e ± 0.5 | 28.9def± 0.7 | |
After | 34.3de± 1.8 | 1.0h ± 0.0 | 22.3fg±0.5 | 0.6gh ± 0.0 | 6.6f ±0.5 | 27.3efg± 0.2 | |
JU15(2) | Before | 37.0cde± 0.6 | 2.0e± 0.0 | 29.6bc±0.5 | 1.0e ± 0.0 | 17.0b ± 1.0 | 29.4de± 0.1 |
At time | 35.8de ± 1.5 | 1.3gh± 0.0 | 29.6bc±0.5 | 1.0e ± 0.0 | 13.6d ± 0.5 | 28.0ef ± 0.6 | |
After | 35.0de ± 1.3 | 1.1gh± 0.0 | 21.0g±1.0 | 0.7g ± 0.0 | 13.0d ±1.0 | 25.2gh ± 0.4 | |
Ho-1WG | Before | 40.6bc ± 1.8 | 2.9cd ± 0.0 | 31.0b±1.0 | 1.5c ± 0.0 | 20.3a ±1.1 | 33.9ab± 0.9 |
At time | 36.4de±0.3 | 2.6d± 0.0 | 29.3bc±1.1 | 1.2d ± 0.0 | 14.6cd ±0.5 | 32.3bc ± 1.1 | |
After | 36.8cde± 2.5 | 1.5fg ± 0.5 | 26.0de±1.0 | 0.9ef ± 0.0 | 14.3cd ± 0.5 | 29.0def± 0.1 | |
Combination | Before | 48.0a± 0.7 | 3.6a± 0.1 | 35.6a±1.1 | 2.7a ± 0.0 | 20.6a ±0.5 | 36.0a ± 0.7 |
At time | 42.8b± 1.0 | 3.3ab ± 0.0 | 27.3cde±1.1 | 2.5b ± 0.0 | 15.6bc ±1.1 | 32.0bc ± 1.0 | |
After | 37.5cde±1.0 | 3.1bc ± 0.0 | 24.6ef±0.5 | 0.7gh ±0.0 | 14.3cd ±0.5 | 27.0fg ± 0.9 | |
Health (un-inoculated) | 34.0e ± 0.0 | 1.5fg ± 0.0 | 27.0cde±1.0 | 1.2d ± 0.0 | 7.3f ±0.2 | 24.6h ± 0.7 | |
Control (diseased) | 25.2f±1.2 | 1.0h± 0.0 | 17.3h± 0.5 | 0.5h ± 0.0 | 4.3g ±0.2 | 20.6i ± 0.7 |
Root dry weight was significantly lower in plants infected with
No significant (p>0.05) difference was observed between combination and Ho-1WG application on flower number of Faba bean plants. The combination of isolates produced the highest (36.0/plant) leaf number in response to before inoculation followed by at time of pathogen inoculation. After inoculation of JU15(2) produced the lowest (25.2/plant) leaf number followed by at the same time.
The Faba bean plants remained stunted and showed poor growth response in the absence of
The findings suggest that incorporation of
Inoculation of combinations of
The average disease reduction for mixtures was 45.1% compared to 29.2% for individual strains. In addition to disease reduction strain mixtures increased biomatter production and yield compared to individual strains [
It was suggested that these multiples of beneficial traits strongly assist the efficiency of candidate antagonists for desired biocontrol methods, emphasizing the great value of concerted mechanisms of action. It has been strongly suggested that the main success of biocontrol agents is largely attributable to their multifunctional characteristics [
The result of study clearly demonstrated that rhizobial isolates exhibited inhibition of the radial growth of
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Sand | % | 8.00 |
Silt | % | 18.00 |
Clay | % | 74.00 |
Texture class | - | Clay |
pH (1:2.5 soil: water) | - | 5.29 |
E. C. (1:2.5 soil: water) | ds/m | 0.07 |
Organic matter | % | 3.83 |
Organic carbon | % | 2.22 |
Total nitrogen | % | 0.19 |
Available phosphorus | ppm | 3.41 |
Not applicable.
The authors declare no conflict of interest, financial or otherwise.
The research is financed by Jimma University and Humbo Woreda Administration. Thanks for Microbial Biotechnology Unit, Holeta Agricultural Research Center.