Research On Diversity Of Species And Molecular Genetic Of Biocontrol Bacillus Strains

Aflatoxin is the most toxic and cancer caused natural substance and it seriously threat to human health. Aflatoxin is difficult to remove and can infect milk, meat and food crops, so it seriously plagued the development of food industry. Peanuts are one of the major oil crops, but peanuts prone to mildew, so this harms the safety of peanut food and cooking oil and troubles peanut planting and peanut exports.In this research we filter biocontrol bacterial strains against aflatoxin from groundnut, and we chose 52 strains of them to do species identification. In the conventional species identification we found that all the strains grew well in the culture conditions of 35℃,medium with neutral pH value,sterile air. The colony morphology can be divide to 5 classes: the typical wrinkle-like colony, the typical wax-drop like and so on. In the microscopic examination after Gram staining and spore staining, we found that 37 strains are spore Gram-positive bacilli and 15 strains are Gram-negative bacilli witch do not produce spores. And we measured the size of each bacterial strains. Finally we conclude that the 37 spore Gram-positive bacilli strains may belong to Bacillus, Paenibacillus and so on.The 37 spore Gram-positive strains were chosen to detect their ability of controlling the growth of Aspergillus parasiticus and producing of NA. We found 20 strains witch can completely control the producing of NA, and 10 strains witch can inhibit the growth of Aspergillus parasiticus more than 80%. By combining analysis, we found 7 strains with best biocontrol effect(completely control the producing of NA and inhibit the growth of Aspergillus parasiticus more than 85%), and several strains witch can completely control the NA and have different activity in controlling the growth of Aspergillus parasiticus.We did 16S rDNA molecular identification to the 37 biocontrol strains ,and constructed phylogenetic trees. After combining analysis of them, we found that all the 37 biocontrol strains belong to Bacillus, the most of them are Bacillus subtilis, and others are 5 Bacillus cereus strains, 3 Bacillus amyloliquefaciens strains, 2 Bacillus thuringiensis strains, and 1 Bacillus licheniformis strain.We did 16S-23S IGS PCR-RFLP to the 37 biocontrol bacillus. We used 7 kinds of restriction enzymes, and they are Hinf I,Hae III,Rsa I,Cfo I(Hha I),Dde I,Tru9 I,Nde II(Mbo I). The clustering trees have significant diversity and regular. After analysis we amended the result of 16S rDNA molecular identification and got the result of molecular genetic diversity and cluster groups. We found that different strains in the same species bacillus have good molecular genetic diversity. A cluster group with 2 Bacillus cereus strains and 2 Bacillus thuringiensis strains separated at the similarity level of 65%, the major of the remaining strains are Bacillus subtilis strains, and they have good molecular genetic diversity.