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.

Identification of cry genes in Bacillus thuringiensis by multiplex real-time PCR.

Bacillus thuringiensis is an important bacterium of the group Bacillus cereus sensu lato due to its insecticidal properties. This microorganism has high genetic variability and its strains produce different Cry toxins, known as δ-endotoxins, which are mainly responsible for its toxic effect on insects that are agricultural pests or vector human diseases. Each strain can express a variety of cry genes, out of a total of 789 cry genes described so far. The detection of these genes is very important to characterize strains, as they may indicate their toxic potential. Several methods have been used to characterize B. thuringiensis strains, but one of the most common techniques is Polymerase Chain Reaction (PCR) from primers that detect the presence of cry genes. This technique has been optimized to make real-time multiplex quantitative PCR (qPCR) assays faster, more efficient, and safer, because the presence of three genes can be detected in a single reaction. In this work, a multiplex assay was developed to identify the presence of genes from the cry1A, cry1C, and cry1F families whose respective toxins are present in both bioinsecticides, and commercial transgenic plants used to control caterpillars. Specific primers were designed to identify the families of the cited genes and the system was validated with samples that were sequenced by next-generation sequencing (NGS). The system was implemented and used to characterize 214 strains. Of these, eight were submitted to conventional PCR, and the results matched, again validating the system. Thus, the application of the proposed technique allows the reliable evaluation through this system to detect the presence of the genes of the families cry1A, cry1C, and cry1F in samples of B. thuringiensis.

Selection and characterization of Bacillus thuringiensis isolates with a high insecticidal activity against Spodoptera frugiperda (Lepidoptera: Noctuidae) / Seleção e caracterização de isolados de Bacillus thuringiensis com Alta atividade inseticida contra Spodoptera frugiperda (Lepidoptera: Noctuidae)

Spodoptera frugiperda (SMITH, 1797) (Lepidoptera: Noctuidae) affects diverse crops of great economic interest, for instance, it can cause severe yield losses in maize, rice and sorghum. In this study, a selection and characterization of Bacillus thuringiensis (BERLINER, 1911) isolates with a high insecticidal activity against S. frugiperda was performed. Fifty-two crystal-forming B. thuringiensis isolates that were identified from 3384 Bacillus-like colonies were examined and screened by PCR for the presence cry genes (cry1, cry1Aa, cry1Ab, cry1Ac, cry1D, cry2 and cry2Ab). Four isolates that showed high toxicity towards S. frugiperda were shown to harbor cry2 genes. The crystals were analyzed by electron microscopy and showed bipyramidal and cuboidal shapes. Furthermore, these four isolates had lethal concentration (LC50) values of 44.5 ng/cm2 (SUFT01), 74.0 ng/cm2 (SUFT02), 89.0 ng/cm2 (SUFT03) and 108 ng/cm2 (SUFT 04) to neonate S. frugiperda larvae. An ultrastructural analysis of midgut cells from S. frugiperda incubated with the SUFT01 spore-crystal complex showed disruptions in cellular integrity and in the microvilli of the midgut columnar cells. The isolates characterized in this work are good candidates for the control of S. frugiperda, and could be used for the formulation of new bioinsecticides.

Spodoptera frugiperda (SMITH, 1797) (Lepidoptera: Noctuidae) afeta diversas culturas de grande interesse econômico, por exemplo, pode causar severas perdas em milho, arroz e sorgo. Neste estudo, foi realizada uma seleção e caracterização de isolados de Bacillus thuringiensis (BERLINER, 1911) com elevada atividade inseticida contra S. frugiperda. Cinquenta e dois isolados formadores de cristal B. thuringiensis que foram identificados a partir de 3384 colônias foram examinados e testados por PCR para a presença dos genes cry (cry1, cry1Aa, cry1Ab, cry1Ac, cry1D, cry2 e cry2Ab). Quatro isolados que apresentaram alta toxicidade contra S. frugiperda foram mostrados para abrigar os genes cry2. Os cristais foram analisados por microscopia eletrônica e mostraram formas bipiramidais e cúbicas. Os valores da concentração letal (CL50) destes quatro isolados foram de 44,5 ng / cm2 (SUFT01), 74,0 ng / cm2 (SUFT02), 89,0 ng / cm2 (SUFT03) e 108 ng / cm2 (suft 04) para larvas recém-eclodidas de S. frugiperda. Uma análise ultra-estrutural das células do intestino médio de S. frugiperda incubadas com complexo esporo-cristal do isolado SUFT01 mostrou rupturas na integridade celular e microvilosidades das células cilíndricas do intestino médio. Neste estudo, o alto nível de atividade inseticida de isolados os torna excelentes candidatos para o controlo de S. frugiperda, e pode proporcionar alternativas no controle destas populações de pragas, bem como a formação de novos bioinsecticidas.

Evaluation of cytotoxicity, genotoxicity and hematotoxicity of the recombinant spore-crystal complexes Cry1Ia, Cry10Aa and Cry1Ba6 from Bacillus thuringiensis in Swiss mice.

The insecticidal properties of Cry-endotoxins from Bacillus thuringiensis (Bt) have long been used as spore-crystals in commercial spray formulations for insect control. Recently, some Bt-endotoxin genes have been cloned in many different plants. Toxicological evaluations of three spore-crystal endotoxins, BtCry1Ia, BtCry10Aa and BtCry1Ba6 from B. thuringiensis, were carried out on mice to understand their adverse effects on hematological systems and on genetic material. These three spore-crystals have shown toxic activity to the boll weevil, which is one of the most aggressive pests of the cotton crop. Cry1Ia, Cry10Aa and Cry1Ba6 did not increase the micronucleus frequency in the peripheral erythrocytes of mice and did not cause changes in the frequency of polychromatic erythrocytes. However, some hematologic disburbances were observed, specifically related to Cry1Ia and Cry1Ba6, respectively, for the erythroid and lymphoid lineage. Thus, although the profile of such adverse side effects can be related to their high level of exposure, which is not commonly found in the environment, results showed that these Bt spore-crystals were not harmless to mice, indicating that each spore-crystal endotoxin presents a characteristic profile of toxicity and might be investigated individually.

Midgut GPI-anchored proteins with alkaline phosphatase activity from the cotton boll weevil (Anthonomus grandis) are putative receptors for the Cry1B protein of Bacillus thuringiensis.

Cry toxins from Bacillus thuringiensis (Bt) are used for insect control. They interact with specific receptors located on the host cell surface and are activated by host proteases following receptor binding resulting in midgut epithelial cells lysis. In this work we had cloned, sequenced and expressed a cry1Ba toxin gene from the B thuringiensis S601 strain which was previously shown to be toxic to Anthonomus grandis, a cotton pest. The Cry1Ba6 protein expressed in an acrystaliferous B. thuringiensis strain was toxic to A. grandis in bioassays. The binding of Cry1Ba6 toxin to proteins located in the midgut brush border membrane of A. grandis was analyzed and we found that Cry1Ba6 binds to two proteins (62 and 65kDa) that showed alkaline phosphatase (ALP) activity. This work is the first report that shows the localization of Cry toxin receptors in the midgut cells of A. grandis.

Translocation and insecticidal activity of Bacillus thuringiensis living inside of plants.

The major biological pesticide for the control of insect infestations of crops, Bacillus thuringiensis was found to be present naturally within cotton plants from fields that had never been treated with commercial formulations of this bacterium. The ability of B. thuringiensis to colonize plants as an endophyte was further established by the introduction of a strain marked by production of green fluorescent protein (GFP). After inoculation of this preparation close to the roots of cotton and cabbage seedlings, GFP-marked bacteria could be re-isolated from all parts of the plant, having entered the roots and migrated through the xylem. Leaves taken from the treated plants were able to cause toxicity when fed to the Lepidoptera Spodoptera frugiperda (cotton) and Plutella xylostella (cabbage). These results open up new horizons for understanding the natural ecology and evolution of B. thuringiensis and use of B. thuringiensis in insect control.