Study On The Bioactivity, Culture Condition And Molecular Characterization Of Four Bacillus Thuringiensis Strains

Abstract: Bacillus thuringiensis is now the most sucessfully used bio-insecticide. As does not cause contamination and be safe to untargetted beings, it is an ideal substitute for chemical pesticide which have caused serious enviromental problems. But there are some problems that threaten the use of Bt, of which the most serious one is that some species have become resistant to it. Since the first reported field population of Plutella xyllostella (Linnaeus) become less sensitive to Bt, the number has reached above 150 now though most of it do happen in lab. To solve this problem, find new strains from the environment is a feasible method. In the past few years, our lab has separated stains YJ-1999 and YJ-2000 from the field, and another two stiains CAJ-2 and CAJ-3 were provided kindly by doctor Cai-Jun. In this thesis, we studied the toxity, culture condition and molecular characteration of these four strains.Using leaf-dip method, we tested the toxity of these four Bt strians to the 1st instar Bombyx mori, 3rd instar Prodenia litura (Fabricius) and Spodoptera exigwa(Hubner). The result indicate that all of them are toxic to Prodenia litura (Fabricius) and Spodoptera exigwa(Hubner), of them the most toxic to these two kinds of pests are YJ-2000 and CAJ-2 respectively, their LC50 are 57.3044 and 5.5418 mg/L. It is interesting that YJ-2000 seems to be safe to B.mori.The effects of pH, carbon sources, nitrogen sources and metal elements on the biomass andICPs production by these four strains were studied. Though do have many differences,, there are also. some similarities as below. (1) The growth and ICPs production of them are different;(2) All ofthem gain the highest biomass and ICPs production at pH 6.5;(3) Add certain concentration ofMg²⁺ favored the production of biomass and ICPs by the four strains.Finally we studied the molecular characteration of this four strains. The results indicates they are different in protein composition, insecticial genotype and protease genotype. Polymerase chain reactions (PCRs) revealed the presence of vip-like sequences in strains CAJ-2 and CAJ-3, cry2-like sequences in strains YJ-1999 and CAJ-2. The complete vip gene in strains CAJ-2 and CAJ-3 were sequenced and showed identities of 100 and above 99.9% to known Vip3A gene. The complete cry2gene in strains YJ-1999 and caj-2 were alse sequenced and showed identities of above 99.9% toknown Cry2Aa^ Cry2Ab gene respectively.