Isolation, Identification And Development Of Highly Potent Bt Strains Of Plutella Xylostella

The environmentally friendly insecticide crystal proteins produced by Bacillus thuringiensis are of great scientific interest because of their potency and specificity to a wide range of insect pests. Because of their highly specific toxicity, Bt toxins offer tremendous benefits for insect pest management. Now the Bt products are the most widely used as biopesticides in agriculture for pest management. Also Bt Cry toxins genes have been engineered into crop plants for decade years. The wide application of Bt formulations and Bt transgenic plants brought out the threat of Bt resistance in insect of field populations. It was reported that some insects have been confirmed to show resitance to Bt in field, but the diamondback moth (DBM, Plutella xylostella) is still the only insect having resistance to Bt formulations in the open field up to now.The major objectives of this present study were to scree B. thuringiensis strains toxic to resistant diamondback moth and to understand the resistance mechanism of P. xylostella, which will provide a theoretical basis in the resistance management in the open field. In this paper, the B. thuringiensis strains toxic to diamondback moths which were selected by Bt toxin were identified and the biological characteristics and cry-type genes characterization of these strains were carried out. The results were as follows.(1) The population dynamics of P. xylostella in the Heilongjiang area were investigated in2009and2010. Over the same period, the toxicity of11insecticides introduced to control the diamondback moth was evaluated. Due to different weather conditions the occurrence of P. xylostella in2010was10days later than that in2009. There was the same general trend in this two years. P. xylostella first appeared in the first ten-day period of May, different peaks of abundance occurred in June to July, P. xylostella disappeared in the later August. Toxicities of the11insecticides were tested using the leaf dipping bioassay method. The results indicate that Fipronil was the most toxic with an LD50value of0.58mg/ml. The analysis of drug tolerance showed that P. xylostella had the last drug tolerance to B. thuringiensis though that have low resistance to B. thuringiensis, which means B. thuringiensis resistant was more hard than others in P. xylostella.(2) Two resistant diamondback moth populations were achieved by the selection with Bt and Cry1Ac toxin. At last The SH-R population has resistance ratios of1655folds to Bt and the SZ-R population has resistance ratios of over1500folds compared with SS population.(3) Differential expression of genes from the resistant and sensitive P. xylostella strains to Bt toxin were studied through AFLP technique.12polymorphic primer combinations between the two parents were found from65AFLP primer combinations.32different bands were amplified. By checking the polymorphism between the resistant and sensitive P. xylostella strains to Bt toxin, a dominant marker, Eaaa/Mcta-776bp, is found. The AFLP maker was retrieved、sequenced、designed a pair of suitable primer and transformed a SCAR maker.3resistant strains and a sensitive strain of P. xylostella were tested by the SCAR maker, the result showed that the gene-specific fragment was likely related to the reistance of P. xylostella to Bt.(4) Insecticidal activity of ten strains of Bacillus thuringiensis isolated from Plutella xylostella larvae showing disease symptoms in Harbin against P. xylostella larvae were characterized. All strains demonstrated sensitive larval mortality above80%and the strain DBW902showed the highest insecticidal activity with LC50of13.99mg/L at48h. The9B. thuringiensis isolates were moderate to high insecticidal activity against resistant P. xylostella. DBW903was the most toxic with an LD50value of43.85mg/ml to the SH-R population and DBW93was the most toxic with an LD50value of49.33mg/ml to the SZ-R population. The biochemical characteristics were determined. The biochemical characteristics of the strains, DBW904, DBW93and DBW962had homology to B. thuringiensis subsp. Shandongiensis, but not the other strains.(5) This study used the specific primer on the basis of the cry1gene part to identify the10B. thuringiensises isolates in the test, the results showed that all Bts contain Cry1gene part. Then the specific primer for the full-length cry1gene was designed to identify the10B. thuringiensises isolates, the results showed that8Bts contain full-length cry1gene, which were no difference to each other and were homologous to cry1Ac in Genebank. The specific primer for the full-length cry2gene was designed to identify the10B.thuringiensises isolates, the results showed that8Bts contain full-length cry2gene, which were homologous to cry1Aa or cry2Ab gene in Genebank.(6) The optimum culture conditions of B.thuringiensis DBW902which was a good one in all isolates moderate to high insecticidal activity against sensitive resistant P. xylostella were determined with shaking flask cultures. Results showed that the suitable culture conditions were as follows:seed age10h, inoculum quantity2%, initial pH6.5-7.5, shaking speed200r/min, temperature30℃and culture time48h. Under these conditions, viable cells and viable spores counts were2.99×109CFU/ml and8.88×108CFU/ml respectively, and crystal protein level was20.0mg/ml.(7) On this basis, a series of Bt powder of B.thuringiensis DBW902with different concentration gradient mineral filler have been prepared. The results indicate that permutite had strong protection on Bt powder when the spores were detected in the series of Bt powder and the permutite made Bt.DBW902the high insecticidal activity against P. xylostella in the concentration of1.0%to3.0%.