In vitro screening of bacillus strains for antagonism against selected phytopathogenic microorganisms and their potential use in biocontrol

Abstract

A total of 137 endospore-forming bacteria isolated and identified as members of the genus Bacillus in a previous study were tested for viability by subculturing onto fresh nutrient agar (NA) plates from slants in tubes stored at 4°C. Out of this number, 83 isolates were found to be viable. Preliminary screening of the isolates against four fungal pathogens of agricultural importance, namely: Colletotrichum lindemuthianum, C. kahawae, Fusarium oxysporum fsp. phaseoli and Alternaria solani in vitro showed that 23 of the viable isolates were antagonistic against at least one of the phytopathogens. Bacillus isolate 66 showed antagonism against all the four pathogens with a considerable % reduction in their growth on artificial medium. It reduced the colony size of Colletotrichum kahawae by 69.3%, C. lindemuthianum by 57.8%, Fusarium oxysporum f sp. phasoli by 53% and Alternaria solani by 64.9%, which were the highest recorded. The isolate produced appreciable amounts of antibiotics in liquid media containing soybean meal and mannitol. Its culture filtrate exhibited stronger antibiotic activity against C. kahawae compared to the other three phtytopathogens, an indication that it was more susceptible to the antibiotics pro~uced by the Bacillus strain. This was demonstrated in the mean inhibition ,diameters produced by dried II-mm-diameter paper , discs immersed in the culture filtrate of the antagonistic Bacillus strain. Inhibition diameters of 25.4 mm, 20.94 mm, 22.97 mm and 16.72 mm were recorded on plates seeded with C. kahawae, C. lindemuthianum, A. solani and F. oxysporum fsp. phaseoli respectively. Paper chromatography combined with bioautography of -the isolate's culture filtrate produced two continuous zones of inhibition against f. lindemuthianum, an indication that the culture filtrate contained two active compounds." The culture filtrate was found to be stable within a wide range of pH levels between 3 and 11 with the perceived optimal pH level being pH 6. Tests on stability of culture filtrate after subjecting them to different temperatures for a period of 15 minutes showed little variation except when subjected to autoclaving temperatures of 121°C, which resulted in loss of activity of the antibiotic. The activity of antibiotics obtained from the isolate was retained for a period of 3 months when stored at the two storage conditions tested: at refrigeration (4°C) and at room temperature (22 ± 2°C). Mean zones of inhibition produced at day 0 and the 120th day of storage against Colletotrichum lindemuthianum were not significantly different at (p S 0.05) when the culture filtrate was stored under refrigeration. At room temperature, in spite of possible differences in incubation conditions, mean inhibition zones were as well not statistically different. Partial purification of the antibiotic culture filtrate was done by adsorbing onto activated charcoal and later eluting with a solution of 80% acetone in water. This procedure enhanced the activity of the filtrate. This was reflected by a 17.73% increase in size of the clear zones of inhibition produced against Colletotrichum lindemuthianum when the partially purified culture filtrate was used. Biochemical tests confirmed that isolate 96 was Bacillus licheniformis as had earlier been identified in a previous study. In vivo tests in the greenhouse showed that the antibiotic culture filtrate significantly delayed and suppressed (p S 0.01) the development of bean anthracnose caused by C. lindemuthianum on bean leaves. The culture filtrate Compared favourably with Benlate 50%WP, a systemic fungicide used in the control 6.£ bean anthracnose. Benlate and Bacillus Isolate 66-culture filtrate (double concentration) gave mean disease scores of 0.9 and 1.24 respectively compared to 3.72 recorded in the control. Phytotoxicity was observed on bean plants treated with. the Bacillus culture filtrate though to tolerable levels.