Unveiling remarkable bacterial diversity trapped by cowpea (Vigna unguiculata) nodules inoculated with soils from indigenous lands in Central-Western Brazil

Abstract

Cowpea (Vigna unguiculata) is recognized as a promiscuous legume in its symbiotic relationships with rhizobia, capable of forming associations with a wide range of bacterial species. Our study focused on assessing the diversity of bacterial strains present in cowpea nodules when inoculated with soils from six indigenous lands of Mato Grosso do Sul state, Central-Western Brazil, comprising the Cerrado and the Pantanal biomes, which are known for their rich diversity. The DNA profiles (BOX-PCR) of 89 strains indicated great genetic diversity, with 20 groups and 23 strains occupying single positions, and all strains grouped at a final similarity level of only 25%. Further characterization using 16S rRNA gene sequencing revealed a diverse array of bacterial genera associated with the cowpea nodules. The strains (number in parenthesis) were classified into ten genera: Agrobacterium (47), Ancylobacter (2), Burkholderia (12), Ensifer (1), Enterobacter (1), Mesorhizobium (1), Microbacterium (1), Paraburkholderia (1), Rhizobium (22), and Stenotrophomonas (1), split into four different classes. Notably, only Ensifer, Mesorhizobium, Rhizobium, and Paraburkholderia are classified as rhizobia. Phylogenetic analysis was conducted based on the classes of the identified genera and the type strains of the closest species. Our integrated analyses, combining phenotypic, genotypic, and phylogenetic approaches, highlighted the significant promiscuity of cowpea in associating with a diverse array of bacteria within nodules, showcasing the Brazilian soils as a hotspot of bacterial diversity.

Keywords: 16S rRNA; BOX-PCR; Biological nitrogen fixation; Diversity; Rhizobia.