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      Aislamiento y caracterización molecular, ribotipificación, de cepas nativas de Bacillus thuringiensis aisladas en suelos y larvas muertas de Hylesia metabus en la región nororiental de Venezuela Translated title: Isolation and molecular characterization, ribotyping, of Bacillus thuringiensis native strains isolated from soils and Hylesia metabus death larvae at northeast region of Venezuela

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          Abstract

          Bacillus thuringiensis es una bacteria esporogénica grampositiva del grupo B. cereus, caracterizada por la producción de δ-endotoxinas codificadas por una familia de genes cry, mayoritariamente plasmídicos con actividad patógena específica contra larvas de insectos. Esta propiedad ha sido usada en la formulación de productos comerciales para control de insectos plaga como Hylesia metabus “palometa peluda” que afecta a los pobladores de la región nororiental de Venezuela. En el Centro Venezolano de Colecciones de Microorganismos se ha iniciado la construcción de una colección de cepas nativas de B. thuringiensis, a partir de muestras de suelos y larvas muertas de H. metabus colectadas en zonas afectadas, que pudiera ser utilizada en la elaboración local de bioinsecticidas para control de ésta y otras plagas. El empleo de condiciones selectivas de crecimiento, pruebas bioquímicas complementarias y presencia de inclusiones parasporales, seguido de la caracterización molecular empleando la técnica de ribotipificación automatizada, permitió la identificación de 11 cepas nativas de B. thuringiensis. La variedad de ribotipos encontrados refleja la heterogeneidad genética de B. thuringiensis en la muestra examinada

          Translated abstract

          Bacillus thuringiensis is a Gram positive spore-forming bacteria of the B. cereus group, that produces δ-endotoxins, encoded mainly by cry genes, with pathogenic specific activity against some insect larvae. This property has been used in products applied to the control of Hylesia metabus, a pest known as “palometa peluda” that affects living population in the northeast region of Venezuela. In the Venezuelan Center for Culture Collections we have initiated the construction of a collection of B. thuringiensis native strains isolated from soils and H. metabus death larvae that could be applied in pest control. The use of selective growth conditions, biochemical tests and detection of parasporal inclusions, followed by ribotyping methods, allowed the identification of 11 B. thuringiensis native strains. Variety of ribotypes shows the genetic heterogeneity of B. thuringiensis identified

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          Worldwide Abundance and Distribution of Bacillus thuringiensis Isolates.

          We found the insect control agent Bacillus thuringiensis to be a ubiquitous soil microorganism. Using acetate selection to screen soil samples, we isolated B. thuringiensis in 785 of 1,115 soil samples. These samples were obtained in the United States and 29 other countries. A total of 48% of the B. thuringiensis isolates (8,916 isolates) fit the biochemical description of known varieties, while 52% represented undescribed B. thuringiensis types. Over 60% (1,052 isolates) of the isolates tested for toxicity were toxic to insects in the orders Lepidoptera or Diptera. Soil samples were collected from various habitats, including those habitats with different numbers of insects. The current presence of insects did not predict the presence of B. thuringiensis in a particular soil sample. B. thuringiensis was most abundant in samples from Asia.
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            Characterization of cry genes in a Mexican Bacillus thuringiensis strain collection.

            Mexico is located in a transition zone between the Nearctic and Neotropical biogeographical regions and contains a rich and unique biodiversity. A total of 496 Bacillus thuringiensis strains were isolated from 503 soil samples collected from the five macroregions of the country. The characterization of the strain collection provided useful information on the ecological patterns of distribution of B. thuringiensis and opportunities for the selection of strains to develop novel bioinsecticidal products. The analysis of the strains was based on multiplex PCR with novel general and specific primers that could detect the cry1, cry3, cry5, cry7, cry8, cry9, cry11, cry12, cry13, cry14, cry21, and cyt genes. The proteins belonging to the Cry1 and Cry9 groups are toxic for lepidopteran insects. The Cry3, Cry7, and Cry8 proteins are active against coleopteran insects. The Cry5, Cry12, Cry13, and Cry14 proteins are nematocidal. The Cry11, Cry21, and Cyt proteins are toxic for dipteran insects. Six pairs of general primers are used in this method. Strains for which unique PCR product profiles were obtained with the general primers were further characterized by additional PCRs with specific primers. Strains containing cry1 genes were the most abundant in our collection (49.5%). Thirty-three different cry1-type profiles were identified. B. thuringiensis strains harboring cry3 genes represented 21.5% of the strains, and 7.9% of the strains contained cry11 and cyt genes. cry7, cry8, and cry9 genes were found in 0.6, 2.4, and 2.6% of the strains, respectively. No strains carrying cry5, cry12, cry13, cry14, or cry21 genes were found. Finally, 14% of the strains did not give any PCR product and did not react with any polyclonal antisera. Our results indicate the presence of strains that may harbor potentially novel Cry proteins as well as strains with combinations of less frequently observed cry genes.
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              Use of 16S rRNA, 23S rRNA, and gyrB gene sequence analysis to determine phylogenetic relationships of Bacillus cereus group microorganisms.

              In order to determine if variations in rRNA sequence could be used for discrimination of the members of the Bacillus cereus group, we analyzed 183 16S rRNA and 74 23S rRNA sequences for all species in the B. cereus group. We also analyzed 30 gyrB sequences for B. cereus group strains with published 16S rRNA sequences. Our findings indicated that the three most common species of the B. cereus group, B. cereus, Bacillus thuringiensis, and Bacillus mycoides, were each heterogeneous in all three gene sequences, while all analyzed strains of Bacillus anthracis were found to be homogeneous. Based on analysis of 16S and 23S rRNA sequence variations, the microorganisms within the B. cereus group were divided into seven subgroups, Anthracis, Cereus A and B, Thuringiensis A and B, and Mycoides A and B, and these seven subgroups were further organized into two distinct clusters. This classification of the B. cereus group conflicts with current taxonomic groupings, which are based on phenotypic traits. The presence of B. cereus strains in six of the seven subgroups and the presence of B. thuringiensis strains in three of the subgroups do not support the proposed unification of B. cereus and B. thuringiensis into one species. Analysis of the available phenotypic data for the strains included in this study revealed phenotypic traits that may be characteristic of several of the subgroups. Finally, our results demonstrated that rRNA and gyrB sequences may be used for discriminating B. anthracis from other microorganisms in the B. cereus group.
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                Author and article information

                Journal
                rsvm
                Revista de la Sociedad Venezolana de Microbiología
                Rev. Soc. Ven. Microbiol.
                Organo Oficial de la Sociedad Venezolana de Microbiología. (Caracas, DF, Venezuela )
                1315-2556
                December 2010
                : 30
                : 2
                : 90-96
                Affiliations
                [02] orgnameUniversidad Central de Venezuela orgdiv1Instituto de Biología Experimental orgdiv2Laboratorio de Procesos Fermentativos
                [01] orgnameCentro Venezolano de Colecciones de Microorganismos
                [03] Caracas orgnameAcademia de Ciencias Físicas, Matemáticas y Naturales Venezuela
                Article
                S1315-25562010000200003 S1315-2556(10)03000203
                e6f3d3ca-c74e-4c38-a4ee-538cae57aca2

                http://creativecommons.org/licenses/by/4.0/

                History
                : 30 June 2010
                : 20 October 2010
                Page count
                Figures: 0, Tables: 0, Equations: 0, References: 32, Pages: 7
                Product

                SciELO Venezuela

                Categories
                Artículos Originales

                biocontrol,B. thuringiensis,H. metabus,ribotyping,ribotipificación,control biológico

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