Comparison of Paenibacillus azotofixans Strains Isolated from Rhizoplane, Rhizosphere, and Non-Root-Associated Soil from Maize Planted in Two Different Brazilian Soils

Abstract

ABSTRACT Paenibacillus azotofixans is a nitrogen-fixing bacterium often found in soil and in the rhizospheres of different grasses. In this study, two Brazilian clay soils were planted with cross-hybrid maize (BR-201) and four stages of plant growth were analyzed to characterize the P. azotofixans populations present in the rhizoplanes, rhizospheres, and non-root-associated soils (herein called nonrhizospheres). A total of 106 strains were isolated and identified as P. azotofixans with an API 50CH kit, by classical biochemical tests, and via the use of specific primers based on the 16S rRNA gene in PCRs. To compare the isolated strains, phenotypic characteristics were determined and three different probes were used in hybridization experiments: two nif probes and one probe comprising a 0.58-kb fragment cloned from the P. azotofixans C3L4 genome. These results were used to construct a dendrogram, in which two main clusters could be observed. One cluster contained exclusively strains from Várzea soil, and the other contained the majority of strains from Cerrado soil. The 60 strains from Várzea soil and the 46 strains from Cerrado soil were further analyzed with REP and BOX primers, respectively. Based on the patterns obtained, it was possible to identify 21 different groups among strains from Várzea soil and 4 different groups among strains from Cerrado soil. These different patterns were tested by multivariate analysis of variance, and differences in the populations of P. azotofixans during the four stages of plant growth were demonstrated. Moreover, strains isolated from the rhizoplanes, rhizospheres, and nonrhizospheres of maize planted in Cerrado and Várzea soils were shown to be statistically different; the diversity of P. azotofixans strains was affected by the soil type.