Micro-organismos colonizadores de nódulos de Vigna spp. cultivadas em solos de Caatinga

Abstract

The study of the diversity of micro-organisms native to the Brazilian semi-arid region can be promising, both for knowledge of biodiversity and for selecting bacterial strains efficient in nitrogen fixation and the biocontrol of phytopathogens, improving the productivity of cowpea in the region. The study aimed to genetically characterize endophytic microorganisms from nodules of Vigna spp., cultivated in soils of Caatinga of Semi-arid, to evaluate the potential of bacteria in N fixation and in the biocontrol of phytopathogens in cowpea. Initially, a collection of 646 bacteria and 23 endophytic fungi from nodules of three species of Vigna was assembled: V. mungo; V. radiata, and V. unguiculata (BRS Pujante, BRS Novaera and V. unguiculata subsp. sesquipedalis) cultivated in five areas of preserved Caatinga in the states of Pernambuco, Piauí, and Bahia. The first study developed and validated a method for the simultaneous amplification of the nodC and nifH genes in duplex PCR reactions. The genetic diversity of the rhizobial isolates was assessed by the Amplified Ribosomal DNA Restriction Analysis (ARDRA), then 78 were selected for sequencing the 16S rRNA gene. 41 isolates had the nodC, recA, dnaK and gyrB genes sequenced. The symbiotic efficiency of the 41 isolates was assessed in two potted plant experiments. The first assay was conducted under gnotobiotic conditions, and the second in pots with non-sterile soil, in a nursery. The second study genetically characterized the collection of endophytic fungi, analyzing the profiles of the microsatellite marker (GTG) 5, and sequencing the ITS1-5.8S-ITS2 region. The pathogenicity of 18 fungi in the germination of cowpea seeds was evaluated in two assays, one in Petri dishes and the other in pots under gnotobiotic conditions. The biocontrol potential of the bacteria was evaluated in a pot experiment with a sterile substrate, by the co-inoculation of pathogenic fungi and bacteria isolated from the nodules of the same origin of plant and soil. A third study evaluated the genetic variability of a collection assembled with rhizobia isolated from nodules of cowpea cultivated in soils in areas of preserved Caatinga in the states of Ceará and Rio Grande do Norte. The genetic characterization was carried out through ARDRA and analysis of the BOX PCR profiles, enabling the selection of 26 isolates that had the 16S rRNA gene sequenced. Among these, 15 bacteria had the constitutive genes recA, dnaK and gyrB sequenced and proceeded to evaluate the symbiotic potential in assay with vessels under gnotobiotic conditions. This study showed that soils in the Semi-Arid are sources of diverse bacteria and belong to different taxonomic groups, with species possibly not yet described. Rhizobia of the genera were identified in the bacterial collection: Bradyrhizobium, Rhizobium, Agrobacterium, Ensifer, Mesorhizobium, Microvirga, and Paraburkholderia; in addition to non-nodulants: Bacillus spp., Enterobacter spp., Pseudomonas spp.. The endophytic fungi belonging to the genera: Nigrospora, Fusarium, Macrophomina, Aspergillus, Cladosporium. The last four with pathogenic strains in the germination of cowpea seeds. The collections show bacteria that stood out in the symbiotic association with cowpea, showing efficiency in N2-fixation and the biocontrol of pathogenic fungi, being selected for future field trials.