Isolation of soil actinomycetes, characterization and screening of their antibiotics against economically important plant pathogens
Muiru, W M
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Forty one actinomycete isolates were isolated from soils collected in three different sites namely cultivated land, pasture land and cattle shed. The cattle shed had the highest number of actinomycetes with 19 followed by cultivated land and pastureland with 13 and 9 respectively. Thirty-five of these actinomycetes produced antibiotics as shown by formation of inhibition zones in solid medium. Pythium sp was used as the primary assay pathogen. Nine actinomycete isolates that had a high level of antagonistic activity against Pythium sp were also found to inhibit the growth of Fusarium oxysporum fsp phaseoli, Alternaria sesami and Colletotrichum kahawae in agar medium. From the nine actinomycete isolates, 3 were found to be strongly antagonistic to Pythium sp and these were coded I4P, 28P and CS35. The three isolates produced antibiotics in soybean meal medium with 2% glucose when incubated for 7 days at 120 revolutions per minute at room temperature (22±2°C). Mean inhibitory diameters of 2.02 em, l.94 em and l.76 cm were produced in Pythium cultures by the isolates 14P, 28P and CS35 respectively when the culture filtrates were produced at the above incubation conditions. Antibiotics from isolates CS35 and 28P were found to be thermolabile from a temperature of 80°C and above, whereas the antibiotic from isolate 14P was found to be thermolabile from a temperature of 70°C and above. The antibiotics from isolates 28P and CS35 stored at 4°C, room temperature (22±2°C) and 40°C retained activity for the entire duration (5 months) tested. The antibiotics from isolate 14P stored at 40°C lost activity after 140 days of storage xviii but the antibiotics stored at room temperature (22±2°C) and at 4°C retained activity throughout the five months period. Bioautography results from culture filtrates of isolates 14P and CS35 showed one clear zone indicating the presence of only one antibiotic active against Pythium sp whereas culture filtrates of isolate 28P showed two clear zones indicating the presence of two antibiotics active against Pythium sp. Low pH (below 4.0) and high pH (above 10.0) were found to destabilize the antibiotics. The mean inhibitory diameters produced by culture filtrates from isolates 14P, 28P and CS35 subjected to pH 2.0 was 0.83, ern 0.8 cm and 0.4 cm respectively. These represented a percentage reduction of 61.4, 57.89 and 78.38 respectively compared to the mean inhibitory diameters obtained at pH 7.0. Mean inhibitory diameters of 1.5 cm, 1.52 em and 1.47 em were observed for the three isolates respectively when the culture filtrates were subjected to pH 4.0 and these represented a percentage reduction of 30.23, 20 and 20.5 respectively compared to the mean inhibitory diameters obtained at pH 7.0. Concentration of the culture filtrates of isolate 28P by partial purification and freeze drying enhanced activity by 10.53% and 20.53% respectively but concentration of culture filtrate from isolate CS35 through the same processes enhanced activity by 2.7% and 25.95% respectively. The antibiotics from the 3 isolates were found to reduce disease incidence, disease severity and the spread of the late blight of tomatoes in the greenhouse. The higher concentrations (double concentration and one and a half concentration) of the culture filtrates produced better performance in terms of low disease incidence compared to the low (half strength and XIX quarter strength) concentrations. A percentage disease incidence of27.6, 23.63 and 25.74 were observed when culture filtrates from isolates 14P, 28P and CS35 respectively were used at normal concentration. These represented a percentage reduction in disease incidence of 19.86,31.39 and 25.29 respectively compared to the control. Disease incidence of21.03%, 15.01% was observed when the culture filtrates from isolates 28P and CS35 respectively were concentrated two times. These represented a percentage reduction in disease incidence of38.94 and 50.42 respectively compared to the control. Phytotoxicity was not observed even at the concentrated levels. The performance of the above antibiotics was not as effective as the standard chemical used (Dithane M45) which suppressed the disease completely.