Distribution and Genetic Diversity of Genes from Brazilian Bacillus thuringiensis Strains Toxic to Agricultural Insect Pests Revealed by Real-Time PCR.

Bacillus thuringiensis is a Gram-positive aerobic bacterium and the most used biopesticide worldwide. Given the importance of B. thuringiensis strain characterization for the development of new bioinsecticides or transgenic events and the identification and classification of new B. thuringiensis genes and strains to understand its distribution and diversity, this work is aimed at creating a gene identification system based on qPCR reactions utilizing core B. thuringiensis genes cry1, cry2, cry3, cry4, cry5, app6, cry7, cry8, cry9, cry10, cry11, vpb1, vpa2, vip3, cyt1, and cyt2 for the characterization of 257 strains of B. thuringiensis. This system was based on the Invertebrate Bacteria Collection from Embrapa Genetic Resources and Biotechnology and analyzed (a) the degree of correlation between the distribution of these strains and the origin of the substrate from which the strain was isolated and (b) between its distribution and geoclimatic conditions. This study made it possible to observe that the cry1, cry2, and vip3A/B genes occur homogeneously in the Brazilian territory, and some genes are found in specific regions. The biggest reservoir of variability is within B. thuringiensis strains in each region, and it is suggested that both geoclimatic conditions and regional crops interfere with the genetic diversity of the B. thuringiensis strains present in the region, and B. thuringiensis strains can constantly exchange genetic information.

Biocontrol potential of bacteria belonging to the Bacillus subtilis group against pests and diseases of agricultural interest through genome exploration.

The usage of microorganisms as biocontrol agents and biofertilizers has been recommended and recognized as an ecologically correct alternative to maintaining the productivity and safety of crops. Thus, the objectives of this work were to characterize twelve strains belonging to Invertebrate Bacteria Collection of Embrapa Genetic Resources and Biotechnology by molecular, morphological, and biochemical methods and to evaluate the pathogenicity of these strains against pests and diseases of agricultural interest. The morphological characteristic of the strains was performed according to the principles of Bergy's Manual of Systematic Bacteriology. The genomes of the 12 strains were sequenced in Macrogen, Inc. (Seoul, Korea) using the HiSeq2000 and GS-FLX Plus high-performance platforms. In the determination of antibiotic sensibility profiles, disc-diffusion methods (Cefar Diagnótica Ltda) were adopted©. Selective bioassays were carried out with insects of the Lepidoptera (Spodoptera frugiperda, Helicoverpa armigera, and Chrysodeixis includens), Coleoptera (Anthonomus grandis), Diptera (Aedes aegypti) and Hemiptera (Euschistus heros) orders, and with the nematode Caenorhabditis elegans. In addition, the antagonistic action of the phytopathogens Fusarium oxysporum f. sp. vasinfectum and Sclerotinia sclerotiorum against the strains under study, and in vitro assays of phosphate solubilization were also performed. Sequencing of the complete genome of the 12 strains determined that all of them belonged to the Bacillus subtilis sensu lato group. In the strains genome were detected genic clusters responsible for encoding secondary metabolites such as surfactin, iturin, fengycins/plipastatin, bacillomycin, bacillisin, and siderophores. Due to the production of these compounds, there was a survival reduction of the Lepidoptera order insects and a reduction in the phytopathogens mycelial growth. These results show that the species of group B. subtilis s.l. can become promising microbiological alternatives to pest and disease control.