Polymerase chain reaction denaturing gradient gel electrophoresis analysis of the N₂‐fixing bacterial diversity in soil under Acacia tortilis ssp. raddiana and Balanites aegyptiaca in the dryland part of Senegal

Abstract

Denaturing gradient gel electrophoresis (DGGE) of amplified nifH gene fragments was used to study the diazotrophic community of soil samples under Acacia tortilis ssp. raddiana (legume tree) and Balanites aegyptiaca (non-legume tree), two dominant plant species growing naturally in the dryland part of Senegal. Samples were taken along transects from the stem up to 10 m distance from it, at depths of 0-0.25 m and 0.25-0.50 m. Sampling was done in the dry season (25 June 1999) and in the rainy season (28 August 1999). The community structure and diversity of the bacterial groups from the different samples was analysed further using different techniques, such as statistical analysis and diversity index evaluation of the band patterns. Diazotrophic diversity was lower under B. aegyptiaca than under A. tortilis ssp. raddiana. Multidimensional scaling (MDS) analysis and ANOSIM tests showed a significant effect of the tree on the diazotroph assemblages. SIMPER analysis showed that the major elements responsible for the dissimilarity are a member of the genus Sinorhizobium, which is characteristic of the samples taken under A. tortilis ssp. raddiana and a member of the cluster Bradyrhizobium for the samples taken under B. aegyptiaca. Forty-four major bands were partially sequenced, yielding 33 different nifH sequences, which were used in phylogenetic reconstructions. Most sequences were affiliated with the alpha- beta- and gamma-proteobacteria. Five nifH sequences were identical to those of Pseudomonas stutzeri, and one sequence showed 100% similarity to that of Azotobacter vinelandii. Four bands were affiliated with the Cyanobacteria and a single one with the Firmicutes. For both trees, there were also clear differences between the samples taken in the dry and rainy seasons. Only for the samples taken under A. tortilis ssp. raddiana was a significant difference found between the two sampling depths.

Effects of Plant Biomass, Plant Diversity, and Water Content on Bacterial Communities in Soil Lysimeters: Implications for the Determinants of Bacterial Diversity

“…Several of the above-cited studies were found to have been limited by the resolution of the culture-independent methods used (clone libraries of limited coverage, ribosomal DNA fingerprinting using eubacterial primers) (23). Supporting this conclusion, a significant correlation between particular plants and soil bacterial populations could be detected when the diazotrophic community was analyzed separately and at high resolution by amplifying the specific functional gene nifH (13); Rhizobium spp. were found to be less diverse in pastures planted with soybeans (11).…”

“…Specific associations between particular plant species and soil bacterial populations have so far been documented for a few individual groups, like diazotrophic (13,24) or dissimilatory (24) nitrate-reducing bacteria. In the current study, however, 20 out of the 160 sequence types analyzed were found at increased abundance in lysimeters devoid of higher plant species.…”

Analysis of Diversity of Diazotrophic Bacteria Associated with the Rhizosphere of a Tropical Arbor, Melastoma malabathricum L.

“…As direct amplification failed, to increase sensitivity, the nifH gene fragments were retrieved by nested PCR. Demba Diallo et al (7) explained that direct amplification failed using PolF and AQER in the first PCR due to quite a low nifH copy number compared with the other nifH gene pools in the sample. Another possible explanation is contamination, by inhibiting substances (e.g., humic acids), of the DNA extracted from soil samples (38).…”

“…Although there have been many attempts to describe the diversity of nifH gene pools in environmental samples (6,7,9,12,19,30,31,33), few studies have simultaneously displayed both qualitative and quantitative differences in nifH gene composition (18,26,27,32). Phylogenetic trees have been frequently used to describe visually the microbial diversity in diverse environments.…”

“…Molecular biological techniques such as PCR-DGGE and RFLP have been used to compare differences in diazotrophic diversity, based on fingerprint patterns (7,8,19,31). These techniques are suitable for distinguishing existing microbial community structures and provide the advantage of high sample throughput.…”