Formação de uma coleção de fragmentos isolados de bactérias fitopatogênicas quarentenárias e não quarentenárias regulamentadas para fins diagnósticos

Abstract

The correct detection and identification of the causative agent of disease is an essential factor to prevent its introduction into new areas of cultivation, as well as in the adoption of effective control practices. For this purpose, several molecular biology techniques have been used in detection procedures in order to detect and characterize the pathogen quickly, as the enzymatic amplification directed by DNA primers or the polymerase chain reaction - PCR, widely used to detect phytobacteria. However, to minimize the possibility of performance failures and to ensure a reasonable safety margin in the technique, it´s necessary to have the means to validate the methodology employed. In this technique validation used to diagnose phytobacteria, pathogens positive controls are necessary so that does not occur false negative reports arising from problems in the diagnosis methodology performance, which would cause future problems to the producer, to the diagnosis laboratory and to the national agriculture. In the case of quarantine pests, there is a risk of introducing bacteria through the unintentional dispersal in regions where they do not exist if bacterial species viable cells were held and employed as a positive control. Assembling a positive controls library made up from these quarantine bacteria cloned fragments and stored DNA would eliminate the epidemic risk in new areas. Therefore, this work aims: assemble a positive controls library for quarantine and non-quarantine regulated bacteria to employ in research and diagnostic reports. With specific primers employed in the PCR methodology, thus obtaining amplified interesting genomic fragments, which were purified, cloned and stored, it was assembled a positive controls library for the following phytobacteria: Pantoea stewartii subsp. stewartii, Candidatus Liberibacter americanus, Candidatus Liberibacter asiaticus, Xanthomonas campestris pv. viticola, Xanthomonas citri subsp. citri, Xanthomonas axonopodis pv. phaseoli, Xanthomonas campestris pv. campestris, Curtobacterium flaccumfasciens pv. flaccumfasciens, Pseudomonas syringae pv. tabaci, Pseudomonas syringae pv. tomato, Clavibacter michiganensis subsp. michiganensis and Pseudomonas syringae pv. syringae.