RAPD Delineation of Native Bacillus spp. and Analyses of Their Biocontrol Effect on Tomato Fusarium Wilt
Ajilogba, Caroline Fadeke
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Tomato (Lycopersicon esculentum mill) is a very important vegetable crop and easily susceptible to fungal diseases such as Fusarium wilt which is caused by Fusarium solani. The use of biocontrol can be used to ameliorate this challenge. Eleven Bacillus isolates that were obtained from plant rhizosphere were characterized using Random Amplified Polymorpruc DNA Polymerase Chain Reaction (RAPD-PCR). Four out of the eleven were used as antagonists in dual culture of the Bacillus and F. solani in vitro and in vivo. In vitro analysis also included hydrogen cyanide production, phosphate solubilization and indole acetic acid production whle in vivo analysis included measurement of growth parameters and using EDX 720 for analysis of mineral/elemental composition of tomato. In vitro levels of inhibition of F. solani growth revealed that Bacillus amyloliquefaciens inhibited the growth of F. solani the most by 95.20% and was significantly different (p=0.05) from Bacillus subtilis, Bacillus pumilus and Bacillus cereus having 82.10%, 70.46% and 55.70% inhibition respectively. They were all significantly different (p=0.05) from the control which had I 00% growth. In vivo inhibition of F. solani growth was measured as percent disease control and disease incidence. Percent disease control in vivo was slightly different from in vitro and varied from 62.50% in both B. pumilus and B. subtilis; 75% in B. amyloliquefaciens to 81.25% in B. cereus which exhibited the highest disease incidence. Disease incidence ranged from 18.75% in B. cereus, 25% in B. amyloliquefaciens to 37.50% in both B. pumilus and B. subtilis. This shows that the four Bacillus isolates were effective in disease control and reducing disease incidence. Treatment with B. cereus enhanced plant growth the most in terms of shoot and root length and root and shoot wet and dry mass while treatment with B. amyloliquefaciens enhanced plant growth the most in terms of number of leaves, branches and fruits. The tomatoes from the different plants treated with the antagonists were also analyzed for their mineral composition. The mineral elements common to all treatments are Potassium (K), Calcium (Ca) and Iron (Fe). The analysis showed that in all the treatments except the control, the mineral that was mostly abundant in the tomato was potassium (K). B. amyloliquefaciens had the highest percentage of 86.46% with cps/f.lA of 16.33 while control had the lowest of 22.93% and cps/uA of 8.81. Calcium (Ca) content of B. cereus was the highest both in percentage and cps/uA while the control had the lowest concentration of Ca. There was significant difference in the concentration of Iron (Fe) in the treatments compared to the control with the concentration of B. pumilus as the highest having 0.88% and 0.70 cps/uA. None of the antagonist produced hydrogen cyanide but all of them showed readings of absorbance with and without tryptophan compared to the control. Treatment with B. amyloliquefaciens and B. cereus had the highest readings of absorbance with tryptophan while B. cereus had the highest without tryptophan. Only B. amyloliquefaciens showed clear zones around the streaked isolates in the test for phosphate solubilization. Three primers, A9B7, OPH 19 and S4 were used to depict the diversity of the eleven Bacillus isolates and they produced a total of 76 bands. From the dendrograms drawn, only B. pumilus exhibited clear-cut difference compared to the other Bacillus isolates. The relatedness of B. amyloliquefaciens and B. subtilis was emphasized by primer A9B7 even though B. cereus was in a different cluster but not distant. Primer OPH 19 revealed that B. amyloliquefaciens is closer to B. cereus while B. subtilis is in a not-distant cluster. In the same vain, primer S4 revealed that B. amyloliquefaciens is closely related to B. pumilus while B. cereus and B. subtilis are closely related. It was observed from this study that all randomly selected Bacillus isolates used, antagonised the growth of F. solani in vitro and in vivo. Despite the fact that B. amyloliquefaciens antagonised growth by the highest percentage in vitro, B. cereus, reduced disease incidence the most in vivo. This indicated that sometimes there is no correlation between in vitro antagonism and in vivo disease control. Further research studies can be done to provide information on this non-correlation and also how to clone best expressed potentials from each isolate into one organism.