Activity of insecticidal Bacillus thuringiensis var. israelensis strains stored by various methods

Microbiological method of insecticidal pests control is an alternative to chemical pesticides. Insect control agents are based on different microorganisms, which should be stably effective against target pest organism. There are different origins of industrial strains including isolation from natural objects, screening of collections, selection of existing strains, genetic engineering etc. but in all cases beneficial features of the strains should be preserved. In this article, the problems of preserving beneficial features of insecticidal bacteria Bacillus thuringiensis var. israelensis (BTH14) are discussed. This strain is effective as the pest control agent against larvae of mosquitoes, midges and rice and champignons mosquitoes. Different methods and time of storage of various BTH14 strains are shown: 266/2 on meat-peptone agar (MPA) and in corpse of mosquitoes for one year, in sodium chloride crystals for one and a half year; 71 on MPA without reseeding for one year and for two years with reseeding every six months; 87a by cryopreservation for ten years; 87, 404, 19/43 as lyophilized bacteria for 28 years; 7-1/23, 71/82, 19/1 in sodium chloride crystals for 27 years. Culture of BTH14 strain was grown on MPA slants at 28-30 °C for 5-7 days until reaching the complete formation of spores and endotoxin crystals. Microscopic analysis was carried out with aniline black dye. Morphological analysis of colonies was performed with colony-purified BTH14. When BtH14 was stored by the lyophilization method, the spore culture in a tube on a slant MPA was washed with 5 ml of a 20 % NaCl solution. Then 0.5 ml of the resulting suspension with a titer of 107-108 CFU/ml was added with a Pasteur pipette into glass ampoules, covered with a sterile swab, then sterile stopper and frozen in a cold bath at a temperature of -22 °C for 1 hour, dried at -45 °C for 23 hours, sealed under vacuum over a gas burner and stored in a refrigerator at 3-5 °C. When using the BtH14 storage method in NaCl crystals, 5 ml of 0.9 % saline was added to a tube with spore culture on slant MPA, resuspended, and 0.5 ml of suspension was added to sterile tubes, covered with cotton-gauze stoppers and stored at room temperature. When BtH14 was stored by cryopreservation, the spore culture of BtH14 was suspended in meat-peptone broth (MBP) with 10 % glycerol. The resulting suspensions (200 μl each) were poured into cryovials and stored at -80 °C. The BtH14 titer and larvicidal activity for Aedes aegypti mosquitoes were measured once or twice per year. The results showed that the culture of the 266/2 strain after a year of storage in the corpses of mosquitoes Culex pipiens molestus or on MPA dissociated with the formation of 0.8 and 1.6 % of the IV S form morphotype which lost activity against A. aegypti mosquitoes. The titer of the spores and the larvicidity of the 71 strain were at the initial level after one year of storage on MPA in tubes with paraffinized plugs when reseeded every 6 months. These indicators decreased, respectively, by 12 and 16 % in a year and by 25-27 % after 2 years of storage. Cryopreservation of the 87a strain provided stability of titer and larvicidal activity after 10 years. Thus, the initial titer and larvicidal activity expressed as LC50 for A. aegypti mosquitoes were 2.74×109 CFU/ml and 0.178×10-3 %, respectively. After 6 and 10 years, they corresponded to the following indicators: 2.82×109 CFU/ml and 0.19×10-3 %; 2.72×109 CFU/ml and 0.18×10-3 %. The BtH14 strains 7-1/23, 71/82, and 19/1 were stored in NaCl crystals. After 27 years of storage, their titers and LC50 for A. aegypti mosquitoes varied within the range of 3.12×109-3.52×109 CFU/ml and 0.135×10-3<