Isolation And Identification Of Bacillus Thuringiensis And Research Of High Toxicity Strains

Bacillus thuringiensis （Bt） is a ubiquitous gram-positive bacterium. Its activity is attributed largely to the insecticidal crystal proteins （Insecticidal Crystal Proteins, ICPs） which are also called as delta-endotoxin. Isolation, identification and evaluation of new B. thuringiensis strains were performed in this work. The results were as the following:1. 75 new Bt isolates were screened from 367 soil samples collected from different districts in China by sodium acetate-antibiotics selection. The isolated Bt rate was up 12.27%. Different shapes of parasporal crystals such as bipyramidal, spherical, spindle and anomalous shapes were observed in 75 Bt isolates.2. The insecticidal gene-types of the 75 Bt isolates were identified by PCR-RFLP. The results showed that there were 18 gene-types in 75 Bt isolates, which were cry1, cry1I, cry2, cry3, cry4, cry5, cry6, cry7, cry8, cry8N, cry9, cry13-14, cry18, cry22, cry26-28, cry34-35, cyt2 and vip3A. The cry2 gene was the most abundant, which account for 43.24%. The proportion of cry1I gene was 36.49%. Twelve isolates harbored unidentified genes.3. The spore-crystal mixtures from L-2, L-10, L-13, L-17, L-22, SC-39 and SC-40 isolates all showed high toxicity against the larvaes of Athetis lepigone. These strains simultaneously contained cry1Ac, cry2Ac, cry1I and vip3A genes. However, the cry1Ac gene was main responsible to insecticidal activity of Bt against A. lepigone larvaes.4. The Bt YX-1 strain showed broad spectrum insecticidal activities to Grapholita molesta, Adoxophyes orana, Phalera flavescens, Cydia pomonella, Hyphantria cunea, Helicoverpa armigera, Prodenia litura, Spodoptera exigua, Ostrinia furnacalis, Plutella xylostella. The toxicities of Bt YX-1 strain to these insect larvaes all were higher than that of Bt HD-1. Bt YX-1 had bipyramidal shape of crystals. SDS-PAGE analysis indicated that Bt YX-1 produced 130 kDаand 60 kDаprotein bands. PCR-RFLP analysis revealed that there were cry1Ac, cry2Ac, cry1I, cry34-35 and vip3Aa genes in Bt YX-1. The best medium formula of Bt YX-1 was: defatted soy flour 32g/L, defatted peanut flour 30g/L, fishmeal 16g/L, peptone 6g/L, cornmeal 14g/L, yeasts 6g/L, MgSO₄·7H₂O 0.2g/L, KH₂PO₄ 1g/L, CaCO₃ 0.5g/L, FeSO₄·7H₂O 0.06g/L.5. The Bt L-27 strain had spherical crystal. SDS-PAGE analysis showed that 130kDa and 60kDa protein band was heavy. PCR-RFLP analysis revealed that Bt L-27 contained cry8, cry8N, cry22 and cry26-28 genes. One cry8 gene from Bt L-27 was cloned. Sequencing analysis indicated that the cry8 gene contained 3525 nucleotides and showed 99% similarity to compare with cry8 （GenBank Accession number: AY551093.1）. The cry8 gene was submitted into GenBank, and Accession number was JQ389477. This cry gene was named as cry8Fa3 by B. thuringiensis Delta Endotoxin Nomenclature Committee.