PCR-based approach for detection of novel Bacillus thuringiensis cry genes

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 #cryI# gene, is described in this article. (Résumé d'auteur

Distribution of cry1, cry2 and cry5 genes within Bacillus thuringiensis isolates from Spain

Using a PCR-based approach, a collection of 223 isolares of Bacillits tburingiensis from Spain was screened for the presence of cry genes belonging to three families. Genes from the cryI, cryII and cryV families were found in 54%, 42%, and 66% of the isolates, respectively. Only 23% of the isolates did not show the presence of any of the genes tested. Frequencies of these genes were compared in isolates from soil samples and from samples of cereal stores and mills, being this higher in the latter. Specific primers were used to detect cryIA(a), cryIA(b), cryIA(c), cryIA(d), cryIA(e), cryIB, cryIc, cryID, cryIE, cryIF and cryIG genes. Within the cryl family, the most frequent gene was cryIA(c) (62%), followed by cryIA(a), cryID, cryIC and cryIA(b) (49, 43, 35, and 34%, respectively). A high frequency of joint occurrence was observed for cryIC and cryID; the latter was present in 93% of the isolates containing cryIC. A random sample of 97 isolates was tested for toxicity against the insect pests Plutella xylostella and Spodoptera exigua. Among the isolates showing toxicity, the most common gene combination was cryIA-cryIC-cryID-cryII-cryV. Although in most cases toxicity could be related to gene content, in some others toxicity was unexpected according to the results obtained by PCR. We found no apparent relationship between gene content in our isolates and the serovar to which they belong. (Résumé d'auteur

Caracterização de novos isolados de Bacillus thuringiensis para o controle de importantes insetos-praga da agricultura

A bactéria Bacillus thuringiensis Berliner produz um corpo de inclusão paraesporal (cristal) de natureza proteica, formado durante a esporulação, que atua de forma eficiente no controle de insetos-praga de culturas economicamente importantes. Esse cristal é constituído de proteínas Cry, que são codificadas pelos genes cry; um isolado pode ser caracterizado pelo conteúdo de genes cry que apresenta. Visando caracterizar novos isolados no combate de insetos-praga pertencentes às ordens Lepidoptera e Coleoptera, 76 isolados bacterianos foram analisados molecularmente e tiveram seu potencial de controle avaliado por meio de bioensaios com larvas de Spodoptera frugiperda (J.E. Smith), Sphenophorus levis Vaurie e Tenebrio molitor Linnaeus. As análises moleculares indicaram 11 isolados (14,5% da coleção), contendo genes lepidóptero-específicos e 17 (22,37%) com genes coleóptero-específicos. As análises de patogenicidade revelaram dois isolados com alto potencial de controle para lagartas de S. frugiperda, um para larvas de S. levis e seis prejudiciais ao desenvolvimento das larvas de T. molitor. Esses isolados de B. thuringiensis podem ser promissores no controle biológico das referidas pragas