Microsymbionts of forage peanut under different soil and climate conditions belong to a specific group of Bradyrhizobium strains

Abstract

The use of mixed pastures of grasses with forage peanut (Arachis pintoi) is growing and offers promising results. This leguminous forage plant has a stoloniferous growth habit and tolerates more intense defoliation and shading. However, there are few studies on its symbiosis with nitrogen (N2) fixing bacteria and no studies on genetic identification of its native microsymbionts. The aim of this study was to evaluate phenotypic and genetic diversity and the symbiotic efficiency of bacterial strains isolated from nodules of forage peanut intercropped with grasses in pasture areas. Root nodules were collected from the Belomonte cultivar in Itabela, Bahia, and BRS Mandobi cultivar in Lavras, Minas Gerais, Brazil. The areas of sample collection had contrasting physical and chemical characteristics. Samples of 10 plants per area and 5 nodules per plant were taken. The strains isolated from the nodules were analyzed for culture characteristics (growth period and change in pH of the culture medium), their 16S rRNA, gyrB and nifH genes were partially sequenced, and authentication and analysis of symbiotic efficiency in forage peanut plants were carried out. Both the authentication and symbiotic efficiency experiments were carried out in a greenhouse under axenic conditions, using A. pintoi cv. BRS Mandobi in a completely randomized design with four replications. Authentication was performed in long neck bottles (500 mL) filled with nutrient solution, and symbiotic efficiency was performed in polypropylene seedling plug pots containing a mixture of sand and vermiculite with a nutrient solution. A total of 74 bacterial strains were obtained from isolation of the forage peanut nodules, 40 from Itabela, and 34 from Lavras. Partial sequencing of the 16S rRNA gene was obtained for 48 strains, 30 from Itabela, and 18 from Lavras. The strains isolated from nodules of forage peanut in pasture areas of Itabela, were identified in the following genera: Bradyrhizobium (21), Rhizobium (5), Burkholderia (2), Kocuria (1), and Paenibacillus (1). The strains coming from soils of Lavras, were identified in the following genera: Bradyrhizobium (13), Rhizobium (2), Burkholderia (1), Mucilaginibacter (1), and Enterobacter (1). In the authentication experiment, 17 strains from Itabela, and 13 strains from Lavras, exhibited nodulation. From these authenticated strains, 18 were identified by 16S rRNA sequencing as belonging to the Bradyrhizobium genus. Of these strains, 11 from the two areas exhibited high symbiotic efficiency in A. pintoi, and their results were better than those of the strains currently approved as inoculants for this crop. The non-nodulating strains are probably nodule endophytes. Sequencing of the gyrB housekeeping gene, performed in 13 strains, confirms phylogenetic groups of potential new species among the strains isolated from A. pintoi, which include strains able to nodulate this species, belonging to a specific phylogenetic group of Bradyrhizobium. Sequences of nifH gene of efficient strains belonged to a group different from that formed by non-nodulating strains.