RESUMO
The novel strains of Bacillus thuringiensis PM9 and NA69, isolated from soil samples in Spain, were classified and characterized in terms of their crystal proteins, plasmid profile, cry genes content, and their toxicological properties against several species of Lepidoptera, Coleoptera, and Diptera. Both strains share morphological and biochemical characteristics with previously described B. thuringiensis strains, although their unique H antigens identify them as two new serotypes. Two new serovar names, B. thuringiensis serovar balearica (H serotype 48) and B. thuringiensis serovar navarrensis (H serotype 50) are proposed for the type strains PM9 and NA69, respectively.
Assuntos
Bacillus thuringiensis/classificação , Bacillus thuringiensis/fisiologia , Toxinas Bacterianas , Insetos/fisiologia , Controle Biológico de Vetores , Animais , Antígenos de Bactérias/análise , Bacillus thuringiensis/genética , Bacillus thuringiensis/isolamento & purificação , Toxinas de Bacillus thuringiensis , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/imunologia , Técnicas de Tipagem Bacteriana , Eletroforese em Gel de Poliacrilamida , Endotoxinas/química , Endotoxinas/genética , Endotoxinas/imunologia , Proteínas Hemolisinas , Immunoblotting , Corpos de Inclusão , Plasmídeos/genética , Reação em Cadeia da Polimerase , Sorotipagem , Microbiologia do SoloRESUMO
The type strain Bacillus thuringiensis var. bolivia (serotype H63), isolated from the Bolivian high valleys, has been characterized at different levels. Its parasporal crystal has an unusual shape and it is composed of a protein of 155 kDa which shows two bands of 75 and 80 kDa after activation. Analysis by PCR shows the presence of cry1 genes, and amplification with specific primers gave products for cry1 E, cry1 D, cry4 A and cry4 B with sizes different to those expected. Immunoblotting tests showed positive reaction for Cry1 E, Cry3 A, Cry4 A and Cry11 A crystal proteins. The plasmid pattern revealed two large and two small plasmids. Toxicity tests were performed against 14 insects and a slight toxicity was found against Plutella xylotella and Trichoplusia ni.
Assuntos
Bacillus thuringiensis/classificação , Bacillus thuringiensis/isolamento & purificação , Besouros/microbiologia , Animais , Bacillus thuringiensis/ultraestrutura , Toxinas de Bacillus thuringiensis , Proteínas de Bactérias/genética , Proteínas de Bactérias/toxicidade , Toxinas Bacterianas/genética , Toxinas Bacterianas/toxicidade , Western Blotting , Bolívia , Eletroforese em Gel de Poliacrilamida , Endotoxinas/genética , Endotoxinas/toxicidade , Proteínas Hemolisinas , Larva/microbiologia , Microscopia Eletrônica de Varredura , Plasmídeos/genéticaRESUMO
Bacillus thuringiensis was isolated from 301 out of 1,005 samples collected in Spain from agricultural and non-cultivated soils, dust from stored products, and dead insects. Based on the production of parasporal crystals, 1,401 isolates were identified as B. thuringiensis after examining 11,982 B. thuringiensis-like colonies. We found a greater presence of B. thuringiensis in dust from grain storages than in other habitats. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the spore-crystal mixtures revealed diverse populations of B. thuringiensis which were differentiated in at least 92 distinct protein profiles. Serological identification also showed great diversity among the Spanish isolates which were distributed among 38 of the 58 known serovars. The most frequently found serovars were aizawai, kurstaki, konkukian, morrisoni, and thuringiensis, which together represented more than 50% of the serotyped isolates. In preliminary toxicity assays, a number of isolates were found to show significant insecticidal activity against the lepidopterans Heliothis armigera (76.1% of the assayed isolates), Spodoptera exigua (50.5%), and Plutella xylostella (19.7%). Thirty five isolates were toxic to both H. armigera and S. exigua, and eight were toxic to S. exigua and P. xylostella. Four and one isolates were toxic to the coleopterans Leptinotarsa decemlineata and Colaspidema atrum, respectively, and three to the dipteran Tipula oleracea. The electrophoretic pattern and serovar of most of the isolates with toxic activity were consistent with those reported in the literature, although other isolates revealed unusual protein profiles, were assigned to new H serovars, or were included in H serovars not previously reported within such pathotypes.
Assuntos
Bacillus thuringiensis/isolamento & purificação , Microbiologia Ambiental , Insetos/efeitos dos fármacos , Animais , Bacillus thuringiensis/classificação , Bacillus thuringiensis/metabolismo , Toxinas Bacterianas/farmacologia , Produtos Agrícolas , Poeira , Ecologia , Eletroforese em Gel de Poliacrilamida , Insetos/microbiologia , Controle Biológico de Vetores , Sorotipagem , EspanhaRESUMO
A two-step strategy, named exclusive PCR or E-PCR, has been developed to overcome the main limitation of PCR, which is the detection of already-known sequences only. This strategy allows the ability to detect and further clone and sequence genes for which no specific primers are available and in which a variable region exists between two conserved regions. This approach has been applied to Bacillus thuringiensis cryI genes by the use of mixtures of degenerate and specific primers recognizing well-known sequences. The first step allows the accurate identification of already-characterized cryI genes by the use of three primers. During the second step, the same sets of primers are used to exclude known sequences and to positively detect cryI genes unrecognized by any specific primer. The method, as well as its application to detect, clone, and sequence a novel cryIB gene, is described in this article.
