Avaliação da população de duas espécies diazotróficas associativas em tecidos de braquiária e milho utilizando PCR quantitativa

Abstract

Diazotrophic bacteria, from Azospirillum and Herbaspirillum genus, in association with grasses, such as maize and brachiaria, can promote benefits for the plant, such as biological nitrogen fixation (BNF). Quantitative PCR technique (qPCR) is effective in monitoring the endophytic bacterial population, however its applicability at strainspecific level needs further study. This study aimed to select oligonucleotides (primers) for quantification of strains populations of A. brasilense in roots and stems of brachiaria (Brachiaria spp.) and H. seropedicae in root of maize (Zea mays) by strain-specific qPCR. The primers designed were evaluated by conventional PCR for specificity for strain and species. Primers sensitivity and efficiency were determined by qPCR reactions. The quantification by strain-specific qPCR of the A. brasilense and H. seropedicae associated with brachiaria and maize tissues were made by using two different standard curves. The results of quantitative PCR using the selected primers were compared to the microdrop count technique. The strain A. brasilense Sp245 was quantified from DNA extracted from stems and root tissues of inoculated and noninoculated brachiaria cultivars grown under field conditions. The bacterial population of the A. brasilense Sp245 and H. seropedicae ZAE94 strains was quantified by maize root genomic DNA from inoculated and non-inoculated plants. Maize plants were grown under greenhouse conditions with sterile substrate and different N doses (3 and 0.3 mM). The primer Sp245p10 was specific for Sp245 strain, and the primers ZAEF1R1 and ZAEF2R2 were specific for the ZAE94 strain. Quantifications by qPCR of Sp245 and ZAE94 strains presented similar results to micro drop count. In the field-grow brachiaria experiment, the number of Sp245 strain cells was higher in inoculated plants from Basilik and Piatã cultivars. In the greenhouse experiment, the N dose with 3 mM favored the endogenous population of Sp245 bacteria in non-inoculated maize plants, while the N dose did not affect the Sp245 and ZAE94 population in inoculated plants