Isolation And Molecular Identification Of Bacillus Thuringiensis Against Plutella Xylostella

29 Bacillus thuringiensis isolates preserving in this lab which were toxic to Lepidopteran pests were bioassayed against Plutella xylostella 3rd larvae. Isolate XF50 showed the most toxicity to Plutella xylostella, and it was fully identified by bioassays, identification of cultivating and growth characteristics, analysis of plasmid, SDS-PAGE pattern, PCR identification and PCR-RFLP.The purpose of this paper was to find out new Bt resource for Plutella xylostella controlling and lay a basis for it's further development. The results were as follows.1. Bioassays of Bacillus thuringiensis isolatesThe results showed that 5 isolates had better acticity than other isolates at diluting the fermentation liquor 1,000 times with the mortality over 65% after 3d. Among them, XF50 demanstrated the best activity against Plutella xylostella. Comparing with standard strain HD73 and engineering bacteria WG-001, XF50 was much more toxic to Plutella xylostella and Spodoptera exigua, with the LC50 values as high as 2.08×106cfu/ml and 11.80×107cfu/ml respectively.2. Cultivating and growth characteristics identificationCultivating and growth characteristics of XF50 were similar to standard strain HD73. Both of them could produce rhombic crystal proteins which were almost as large as the nutrition somatic cells; the growth curve of XF50 was almost the same with standerd strain HD73. The lag phase was 2 h. Expoenetial phase was from 2 to 16 h, followed by the stationary phase. After 32 h it went into decline phase; Both of XF50 and HD73 formed circular colonies cultured in different mediums. Colony surface appeared smoothy or plicate, without producing soluble pigment; the isolate XF50 physio-biochemical characteristics were exactly the same with HD73. They could not hydrolyze gelatin. Indole test presented negative. But they could hydrolyze starch, lecithin and urea. V.P. test presented negative. And both of them could not break down D-glucose, sucrose and D- xylitol. Physio-biochemical characteristics of XF50 were similar to kurstaki subspecies. 3. Molecular identificationPlasmid pattern and protein pattern of XF50 were different from engineering bacteria WG-001 and standared strain HD73. Strain XF50 and WG-001 could both express two large plasmids which were almost 70Kb and 23Kb. XF50 could express 50Kb,6.7Kb,4.2Kb,3.8Kb,2.3Kb and 2.0Kb plasmids besides 70Kb and 23Kb. SDS-PAGE showed that XF50 could express at least two proteins,135KDa and 65KDa. Gene-type analysis by PCR indicated that XF50 contained abundant cry1-type genes like cry1Aa, crylAb, crylAc, cry1B, cry1I besides a cry2-type gene. The result was confirmed by PCR-RFLP.In summary, Bt isolate XF50 contained abundant cry genes and had high insecticidal activities. It was deserved to be further studied in the future. This new Bt isolate gained from this paper could be used as engineering bacteria because of its abundent cry-type genes.