Genetic and symbiotic diversity of nitrogen-fixing bacteria isolated from agricultural soils in the western Amazon by using cowpea as the trap plant

Abstract

Cowpea is a legume of great agronomic importance that establishes symbiotic relationships with nitrogen-fixing bacteria. However, little is known about the genetic and symbiotic diversity of these bacteria in distinct ecosystems. Our study evaluated the genetic diversity and symbiotic efficiencies of 119 bacterial strains isolated from agriculture soils in the western Amazon using cowpea as a trap plant. These strains were clustered into 11 cultural groups according to growth rate and pH. The 57 nonnodulating strains were predominantly fast growing and acidifying, indicating a high incidence of endophytic strains in the nodules. The other 62 strains, authenticated as nodulating bacteria, exhibited various symbiotic efficiencies, with 68% of strains promoting a significant increase in shoot dry matter of cowpea compared with the control with no inoculation and low levels of mineral nitrogen. Fifty genotypes with 70% similarity and 21 genotypes with 30% similarity were obtained through repetitive DNA sequence (BOX element)-based PCR (BOX-PCR) clustering. The 16S rRNA gene sequencing of strains representative of BOX-PCR clusters showed a predominance of bacteria from the genus Bradyrhizobium but with high species diversity. Rhizobium, Burkholderia, and Achromobacter species were also identified. These results support observations of cowpea promiscuity and demonstrate the high symbiotic and genetic diversity of rhizobia species in areas under cultivation in the western Amazon.

Fig 1

Distribution of the 119 strains isolated from soil under agricultural use into culture groups according to time (days) to appearance of isolated colonies and pH change of the medium: FA, fast growth, medium acidification; FN, fast growth, no alteration of medium pH; FAL, fast growth, medium alkalinization; FAN, fast growth, localized acidification, i.e., around the colonies; IA, intermediate growth, medium acidification; IN, intermediate growth, no alteration of medium pH; IAL, intermediate growth, medium alkalinization; IAN, intermediate growth, localized acidification, i.e., around the colonies; SA, slow growth and medium acidification; SN, slow growth, no alteration of medium pH; SAL, slow growth, medium alkalinization.

Fig 2

Distribution of 62 nodulating strains into groups by relative efficiency (RE %) according to the Scott-Knott test at 5% similarity. RE = (inoculated DMS/DMS with N) × 100, where inoculated DMS is the dry matter of shoots in the treatment with inoculation of respective strain and DMS with N is the dry matter of shoots in the control treatment with mineral nitrogen. The RE of each strain was the mean for three replicates, and each replicate had one plant.

Fig 3

Dendrogram showing the genetic similarity (based on BOX-PCR profiles) of bacterial strains that nodulated cowpea and of type and reference strains of known rhizobium species. These bacteria were isolated from soils under agricultural use in the western Amazon. Groups were obtained at 70% similarity. “∗” indicates isolates for which the 16S rRNA gene has been sequenced (see Table 1).

Fig 4

Phylogenetic relationships based on 16S rRNA sequences among strains isolated from cowpea nodules and strains representative of alpha- and betaproteobacteria. Phylogeny was determined by the neighbor-joining method. Bootstrap values were based on 1,000 trials.