The Screening Of High Toxicity Bacillus Thuringiensis Isolates And Solid State Fermentation

Bacillus thuringiensis （Bt） is currently the most widely used and in-depth study of biocontrol microorganisms. There are still shortcomings in the application process of Bt preparations such as narrow spectrum insecticide, poor resistance, and the high cost in combating which limit its further expanding in application. In order to obtain natural isolates to applicate in the practice, two wild strain of Bt including Bt CYZ-4 and Bt S-19 strains were screened by molecular identification methods in the lab in pre-screening study.After genotype alignment, they are predicted to be wild strains with broad insecticidal spectrum. In this study, we use bioassay system to study the virulence of two strains against the seven kinds of common pests and health pests and the resilience against adverse environmental conditions such as UV and high temperature meanwhile the Bt CYZ-4 strains which has a broad spectrum insecticide, strong resistance was sereened.To reduce production costs, low-cost solid state fermentation technology of the strains has been systematically studied. exploration Experiment of the synergy of Bt and chemical agents was conducted. We get the following main results:1. The two strains have broad insecticidal spectrum, but the different genotype showed differences pest activity.The bioassay results showed that, LC₅₀ of Bt CYZ-4 strains against Mythimna separata of newly hatched larvae 48h is 6.6×10⁶ cells/mL, against Helicoverpa armigera, Spodoptera exigua and Culex pipiens paliens of newly hatched larvae and Hyphantria cunea （Drury）, Phalera flavescens, Clostera anachoreta instar larvae 72h is 4.7×10⁶ cells/mL, 2.25×10⁵cells/mL, and 2.0×05 cells/mL and 2.9×10 6 cells /mL, 6.6×10⁷ cells/mL, 1.7×10⁶ cells/mL; LC₅₀ of Bt S-19 strains against the above pests, followed by 3.1×10⁷ cells/mL, 9.0×10⁶ cells/mL, 4.1×10⁴ cells/mL, 3.5×10⁴ cells/mL, and 1.8×10⁵cells/mL , 1.7×10⁶cells/mL, low to Clostera anachoreta.That means the two strains are broad-spectrum insecticide strains. The activity of Bt CYZ-4 strains against Mythimna separata, Helicoverpa armigera, Clostera anachoreta and Phalera flavescens is higher than Bt the S-19 strains, while the performance of the fall webworm, beet armyworm, and Culex pipiens, trend is the opposite. The two strains showed strong insecticidal activity to the fall webworm, cotton bollworm, beet armyworm and pale Culex.2 Resistance of Bt CYZ-4 strain is better than that of Bt S-19.Effects of temperature, UV treatment of the two strains at different times of changes in the activity of fall webworm larvae at 30℃, 33℃, 37℃, 42℃temperature gradient processing and 30min, 60 min, 120 min, 180 min, 240min and 360 min processing time. The results showed that the activity of the two bacteria under high temperature treatment was a downward trend with increasing temperature and treatment time. treated at 42℃for 240min. The activity of the former to 60%, the latter only the remaining 43.3% treated at 42℃for 360min.Description of Bt CYZ-4 anti-high temperature significantly higher than that of Bt S-19; Insecticidal activity of Bt was significantly reduced with the extension of exposure UV time. The same trend was demonstrated for UV-treatment between two isolates, Of Bt CYZ-4 application potential for UV resistance was significantly higher than Bt S-19; so, the Bt CYZ-4 strain resistance is better than of Bt S-19.3. The formula including corn starch or corn core were screened as Bt CYZ-4 strain by solid state fermentation,and with higher fermentation productsReference to the proportion of LB medium C source, N source and mineral substances, the higher products fermented formula of corn starch were screened. the the percentage of substances was corn starch 1%, nitrogen A 0.5%, nitrogen B 1%, ratio of 0.25% yeast extract, K₂HPO₄ 0.075%, MgSO₄ 0.035%, CaCO₃0.05%. with pH=8.4, 250mL flask fermentation at 30℃for 6d, the spore concentration can reach 2.96×10⁹cells/mL spores. Meanwhile the amount of the fermented formula of corn cob was corn cob 7.2g, 3g nitrogen source A, nitrogen source B, 3.2g, distilled water, 23.3g, K₂HPO₄, 0.075% MgSO₄, 0.05%, CaCO₃ 0.035%, pH=8.4, 250mL flask fermentation at 30℃for 6d, the spore concentration can reach 1.6×10¹⁰ / g.4. The synergism ofβ- cypermethrin and Bacillus thuringiensis against Hyphantria cuneaDue to deleterious effects of chemical pesticides on the environment and non target organisms, some efforts have been made to reduce the systematic application of pesticides and to replace them with biological control methods against fall webworm （Hyphantria cunea （Drury） ） in China in recent years. In this study, the synergetic effect betweenβ- cypermethrin, which was extensively applied in practice against the pest, and Bacillus thuringiensis, an worldwide bio-pestcide, was researched to the pest by bioassay through Bt pre-treatment or mixing together in the lab. The LC₅₀ ofβ- cypermethrin against 2nd larvae of H. cunea （Drury）, pre-treated by Bt or not, were 0.7625mg/L、1.2811mg/L in 48h respectively. While the LT₅₀ of treatment or CK were 9.23h、30.67h at the concentration of 2.25 mg/L ofβ- cypermethrin. These results demonstrated that there were synergized action between Bt andβ- cypermethrin by Bt pre-treatment. Whenβ-cypermethrin mixed together with Bt, the Co-toxicity coefficient （CTC） was 122.5 as the concentration ofβ- cypermethrin and Bt S19 was 6 mg/L and 1×10⁶cell/mL respectively, However, the mixture showed some antagonism as the concentration ofβ-cypermethrin increased. All these experiments indicated that Bt pre-treatment beforeβ-cypermethrin application was more efficiency than the mixture ofβ-cypermethrin and Bt against H. cunea （Drury）.