Efficacy of bacillus subtilis metabolites dissolved in propylene glycol qn bean anthracnose and coffee berry disease

Abstract

Work was done on formulation of crude antibiotic metabolites produced by Bacillus spp. isolates coded CAS and CA10 obtained from Dr.E.W.Mutitu, Department of Crop Protection,University of Nairobi where they had been preserved over 2 years in sterile soil at room temperature. Their efficacy in controlling plant pathogens of economic importance was determined. The coded isolates had been characterised and found to conform to Bacillus subtilis species both in physiological and morphological characteristics. They had also been proven to be antagonistic to various plant pathogens. Invitro studies on growth of C. kahawae, C. lindemuthianum and Alternaria sesami showed strong inhibition by the retrieved antagonistic Bacillus isolates CAS and CA 10. Fusarium oxysporum f.sp phaseoli was found to be less sensitive to the same isolates. Bacillus isolate CAS exhibited greater inhibitory activity on all the fungal pathogenic isolates tested as compared to isolate CA 10 when tested under the same conditions. The recovery of antibiotic metabolites from isolates CA 1a and CAS grown in shaken liquid medium containing soymeal, glucose, glycerol, yeast and mineral salts was 4% and 2.4%, respectively. However, the antibiotic metabolites from isolate CAS were found to be more inhibitory to the test fungal pathogens than those of isolate CA 1a. The culture filtrate containing the antibiotic metabolites from isolates CAS and CA 1a took 12-24 hrs to dry in a freeze drier at -11 aOc. The final product was chocolate (deep) brown tar-like material from isolate CAS and brown incase of isolate CA 1O. The two products were highly hygroscopic and dissolved easily in water. The antifungal activity of both products was retained even after freeze drying. The freeze-dried antibiotic metabolites dissolved easily in propylene glycol, which was used for formulation without significant sedimentation. The solubility was better with isolate CA5 antibiotic metabolites (1 gm I 6 ml) than isolate CA10 (1 gm I 8 ml). The formulated solution concentrate showed antifungal activity on C. kahawae and C. lindemuthianum in vitro. The antibiotic solution concentrate of Bacillus isolates CA10 and CA5, diluted at 1:3 (antibiotic: water), inhibited sporulation of C. kahawae on coffee twigs by 84.5% and 89.7%, respectively. The percentage sporulation inhibition from both isolates was comparable to 84.5% achieved with a broad-spectrum protectant fungicide Dithane M45 80 % WP. Greenhouse experiments with the formulated antibiotics reduced bean anthracnose severity and delayed symptom development. On the 5th week, isolate CA5, isolate CA10 and Dithane M45 gave a mean disease score of 2.6, 2.9 and 1.2 respectively compared to 6.2 recorded in the control. At higher concentrations the antibiotics were found to be phytotoxic to treated bean plants. The formulated antibiotics effectively controlled coffee berry disease on coffee seedlings without showing any phytotoxic effects. The mean disease score of coffee seedlings treated with isolate CA5, isolate CA10 and Dithane M45 was 2.4, 2.8 and 2.0 respectively compared to 3.8 recorded in the control. Using the filter paper disc bioassay method, the freeze dried culture filtrate of Bacillus isolates CA5 and CA10 were found to lose antifungal activity after storing them in the refrigerator at 4°c for one year. No inhibition zones were detected with Col/etotrichum kahawae, C. lindemuthianum, Alternaria sesami and Fusarium oxysporum f.sp. phaseoli the test fungal pathogens.