Bacillus thuringiensis strains from natural sources in the Leningrad region: isolation and identification

Recently crystal-forming bacilli of thuringiensis group are considered the main microbial producers of insecticides. For these bacilli the high adaptability is characteristic leading to wide distribution of these anaerobic spore-forming bacteria in nature. The same Bacillus thuringiensis subspecies and variants were isolated on different continents regardless the presence and prevalence or absence of the host insects of this entomopathogen. In different countries and regions the researchers are searching for new B. thuringiensis isolates. In the paper the data are represented on B. thuringiensis isolation from natural substrates in the territory of Leningrad province. A total of 24 samples of soil, litter, water, silt, sick and died insects have been collected. The samples were cultivated on fish agar. Among more than 3,000 colonies, 62 ones with specific morphology were found. By microscopy with black aniline dye a total of 12 isolates of 62 isolates tested were found out to form both spores and differently shaped crystals of the endotoxin. The microorganisms were selected with regard to entomocidal and larvicidal activity and identified using H. De Barjac, A.A. Bonnefoi (1968) and O. Lysenko (1985) schemes. The investigation made it possible to classify isolates as B. thuringiensis of Н1 (var. thuringiensis, isolates №№ 12, 20, 40, 41), Н3а3в (var. kurstaki, isolates №№ 15, 29, 49) and Н14 (var. israelensis, isolates №№ 14, 25, 33, 38, 44) serovars. With regard to biological properties (production of acetyl methyl carbonate, lecithinase, pigment, b-exotoxin; pellicle in broth culture; sucrose, mannose, cellobiose, salicin fermentation; starch degradation; proteolytic activity) these isolates are close to standard strains. Isolates are characterized by high productivity, entomocidal and larvicidal activity and can be used as producers of biologicals against insects and larvae. In the isolates of BtH1, BtH3a3b and BtH14 serovars the titers varied as 2.42×109-2.78×109; 1.85×109-2.15×109 and 2.65×109-3.28×109 CFU/ml, respectively. The activity against Leptinotarsa decemlineata Say larvae in isolates №№ 12, 41 of the BtH1 serovar was the same as in standard strain BtH1 with LD50 at 0.19 %. Entomocidal activity of the isolates №№ 15, 29 and 49 of the BtH3a3b serovar expressed as LD50 for Ephestia kuehniella of the 2nd instar was 0.88; 0.82 and 0.92 %, respectively, while in the standard strain BtH3a3b the LD50 was 0.86 %. In the isolates №№ 33, 44 of the BtH14 serovar the titer was the same as in the standard strain, and the activity was even higher compared to the standard. In the isolates №№ 33, 44 the LD50 for the 4th instar Aedes aegypti larvae was 0.17×10-3 and 0.16×10-3 %, respectively, when in standard strain BtH14 it was 0.18×10-3 %. Thus, a total of 12 of the isolates which have been identified as B. thuringiensis are close to the type isolates on their biological characteristics and promising as producers of biologics with insecticidal action.