Utilização de diferentes técnicas para monitorar a colonização e estabelecimento de bactérias diazotróficas endofíticas em plantas de cana-de-açúcar

Abstract

The objective of this study was to evaluate the establishment of the diazotrophic bacteria BR 11504, BR 11335, BR 11115, BR 11281 and BR 11366 inoculated in sugarcane plants grown in greenhouse and in production regions of São Paulo and Rio Grande do Sul States. The sites of colonization and infection of these diazotrophs bacteria were identified by mapping shoots of sugarcane plants, in order to check which part of the plant has the largest population of bacteria from the inoculum. Diazotrophic bacteria were detected in all regions of the plants analyzed by the Most Probable Number (MPN) method, including the leaves. The mapping of diazotrophic bacteria community did not differ in the regions of the node and internode of the plant. The establishment of diazotrophic bacteria inoculated in sugarcane plants cultivated in the Brazilian states of São Paulo and Rio Grande do Sul was evaluated by MPN techniques and real-time PCR (qPCR). No statistical difference was observed in the population of endophytic diazotrophic bacteria among the control and the inoculated plants. The qPCR technique showed highest values in the quantification of diazotrophic bacteria when compared with the counting dependent of cultivation technique NMP. The real-time PCR showed to be quite accurate and sensitive in the detection and quantification of diazotrophic bacteria, and it can be used in sugarcane plants cultivated in field conditions. The population of diazotrophic bacteria varied during the crop cycle. The diversity of diazotrophic bacteria presented in the sugarcane varieties was studied using the technique of "nested PCR" with primers for the nifH gene. The establishment confirmation of the inoculated bacteria in the tissues of sugarcane plants was performed by using marked strains for antibiotic resistance in sugarcane varieties grown in greenhouse conditions.