Characterization and determination of lethal dose (LDSO)of metabolites from two soil Bacilli and evaluation of their If efficacy in the control of bean anthracnose in the greenhouse.

Abstract

Two Bacillus isolates coded as CA5 and CAIO were retrieved from sterile loam soils where they had been preserved. They were tested against four fungal pathogens of agricultural importance namely Fusarium sp Colletotrichum lindemuthianum, Helminthosporium nodulosum and Alternaria sesami in vitro. Bothisolates CA5 and CA10 were shown to be antagonistic to each of the above pathogens, however CA5 was more active than CA10. Growth of Colletotrichum lindemuthinum was reduced by 45% and 47% by CA10 and CA5 respectively. The two Bacillus isolates produced appreciable amounts of antibiotics in liquid medium containing soybean meal and glucose. Culture filtrate from isolate CA5 showed stronger antibiotic activity against all the four fungal pathogens when compared to isolate CA10. This was demonstrated using Colletotrichum lindemuthianum where CA5 produced a mean inhibition zone of 27mm compared to 20mm by CA10. Paper chromatography combined with bioautography for both isolates CA5 and CA10 produced one continuos zone of inhibition against Fusarium spp indicating that each isolate contained one active compound against Fusarium spp. The culture filtrate from isolate CA10 was found to be stable on a wide range of pH levels between 4 and 10 as opposed to CA5 that was stable between pH 4 and pH 8. Tests on temperature stability showed that CA5 was more heat stable than CA10. Activity was demonstrated by CA5 after autoc1aving at 1210C but CA10 was inactivated at temperatures above 1000C. The antibiotics from the two isolates retained activity when stored for 120 days at room temperature (220C) and in the refrigerator at 40C. Using storage temperature of 40C the inhibition zones produced at 120th day by CA5 and CAI0 on Colletotrichum lindemuthianum were 27.3 mm and 20.1mm respectively while those at room temperature were 27.lmm and 20.6 mm respectively. The zones were not significantly different at (p S 0.05). Partial purification of the two antibiotics were done by adsorbing them onto activated charcoal and later eluting with a solution of 80% acetone in water. The partial purification enhanced the activity of the filtrates. This was reflected by the increase in the size of the clear zones produced. For partially purified antibiotic from CA5 the inhibition zone against Colletotrichum lindemuthianum was 11% more than that of crude filtrate and 20% more with CA10 antibiotics. In vivo tests in the greenhouse using the two antibiotics showed that they significantly delayed and suppressed (pS5%) the development of bean anthracnose caused by C lindemuthianum on bean leaves sprayed with culture filtrates. Isolate CA5 compared favorably with benlate 50% W.P, a systemic fungicide used in the control of bean anthracnose. Both benlate and isolate CA5 gave a mean disease score of 2.3 and 2.5 respectively compared to 7.3 recorded in the control. The culture filtrates however, were found to be a little phytotoxic. Determination of the lethal dose (LD50) to the mice of the two culture filtrates after freeze drying was done using three weeks old male albino mice that weighed between 20g and 40g body weight. Injecting the reconstituted culture filtrates intraperitoneally the LD50 of CA5 and CAI0 antibiotics were determined and found to be 2.35 and 2.93g/Kg body weight respectively. According to the World Health Organisation (WHO) classification of toxicity and according to Loomis (1974) any substance with LD50 of lrng/Kg or less is graded as extremely toxic and products giving LD50 of 5g/Kg are graded as relatively harmless. The two culture filtrates from CA5 and CAI0 can be classified therefore, as slightly toxic and thus safe for agricultural use.