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In this study, the effects of plant genotype, soil type and nutrient use efficiency on the composition of different bacterial communities associated with rice roots were investigated. Thus, total bacteria, Alpha- and Betaproteobacteria, Pseudomonas and Actinobacteria were studied using PCR, followed by denaturing gradient gel electrophoresis (PCR-DGGE). Rice genotype determined, to a large extent, the composition of the different bacterial communities across cultivars. Several cultivars belonging to Oryza sativa ssp. indica tended to select similar bacterial communities, whereas those belonging to subspecies japonica and aromatica selected ones with divergent community structures. An effect of soil type was pronounced for the Actinobacteria communities, while a small effect of 'improved' and 'traditional' plants was noted for all communities analyzed. A few dominant bands in PCR-DGGE, affiliated with Rhizobium radiobacter, Dickeya zeae, Mycobacterium bolletii and with members of the Rhizobiales, Rhodospirillaceae and Paenibacillaceae, were spread across cultivars. In contrast, a majority of bands (e.g. affiliated with Enterobacter cloacae or Burkholderia kururiensis) was only present in particular cultivars or was erratically distributed among rice replicates. These findings suggested that both bacterial adaptation and plant genotype contribute to the shaping of the dynamic bacterial communities associated with roots of rice plants.

The bias of widely used degenerate nifH-specific primer sets was first tested using denaturing gradient gel electrophoresis (DGGE), and their application for profiling of complex communities assessed for roots of Oryza longistaminata. When primers (P) with mismatches at nondegenerate positions were used on genomic DNA of Azotobacter vinelandii, which harbors three single divergent nifH genes, template-to-product ratios were highly skewed. In contrast, we obtained no evidence for a large PCR bias when we used highly degenerate primers with no mismatches (Z). Similar results were obtained for reverse transcription (RT)-PCR amplifications from root RNA from O. longistaminata grown at the river bed of the Okavango, where Z-primers detected a more complex nifH pool, corroborating that the P-primers are quite biased in the nifH sequences they amplify. In microcosms of O. longistaminata grown in the phytotron in the presence or absence of constant low nitrogen input (25 kg NH4NO3 ha(-1) year(-1)), roots of nitrogen-treated plants showed similar, slightly higher levels of nifH-mRNA. However, nitrogen treatment had a strong effect on the composition and diversity of expressed nifH pools that shifted towards methylotroph-related nitrogenases. Thus the active population of diazotrophs was not resistant towards low rates of nitrogen input and decreased significantly in richness, as also observed for plant species richness in grasslands by others.

Colophospermum mopane is an indigenous legume tree that grows in Southern Africa and is one of the predominant trees of the woodland vegetation. In order to increase knowledge about its ecology, especially how C. mopane thrives in the nitrogen-poor soils of the region, we analyzed the root-associated bacteria to assess the active diazotrophic diversity and total microbial diversity by culture-dependent and independent techniques. Root nodules were not detected but in some samples the lateral roots showed an outgrowth-like protuberance, that were not likely to have functions related to legume root nodules. The bacterial isolates recovered were related to Actinobacteria, Firmicutes and Proteobacteria. The total microbial diversity was dominated by Actinobacteria-related phylotypes, while the active diazotrophic diversity showed that the majority of the sequences were related to the order Rhizobiales but also to Spirochaetes, Firmicutes, Bacteroidetes and Deltaproteobacteria. Several isolates showed characteristics of plant growth-promoting bacteria. These findings increase the spectrum of possible phylotypes that can be found in legume trees that are typically nodulated by Alpha- and Betaproteobacteria, and reveal for the first time a surprising diversity of nitrogen-fixing bacteria active in legume tree roots.

Marama bean (Tylosema esculentum) is an indigenous non-nodulating legume to the arid agro-ecological parts of Southern Africa. It is a staple food for the Khoisan and Bantu people from these areas. It is intriguing how it is able to synthesize the high-protein content in the seeds since its natural habitat is nitrogen deficient. The aim of the study was to determine the presence of seed transmittable bacterial endophytes that may have growth promoting effects, which may be particularly important for the harsh conditions. Marama bean seeds were surface sterilized and gnotobiotically grown to 2 weeks old seedlings. From surface-sterilized shoots and roots, 123 distinct bacterial isolates were cultured using three media, and identified by BOX-PCR fingerprinting and sequence analyses of the 16S rRNA and nifH genes. Phylogenetic analyses of 73 putative endophytes assigned them to bacterial species from 14 genera including Proteobacteria (Rhizobium, Massilia, Kosakonia, Pseudorhodoferax, Caulobacter, Pantoea, Sphingomonas, Burkholderia, Methylobacterium), Firmicutes (Bacillus), Actinobacteria (Curtobacterium, Microbacterium) and Bacteroidetes (Mucilaginibacter, Chitinophaga). Screening for plant growth-promoting activities revealed that the isolates showed production of IAA, ACC deaminase, siderophores, endoglucanase, protease, AHLs and capacities to solubilize phosphate and fix nitrogen. This is the first report that marama bean seeds may harbor endophytes that can be cultivated from seedlings; in this community of bacteria, physiological characteristics that are potentially plant growth promoting are widespread.

In this study, the effects of plant genotype, soil type and nutrient use efficiency on the composition of different bacterial communities associated with rice roots were investigated. Thus, total bacteria, Alpha- and Betaproteobacteria, Pseudomonas and Actinobacteria were studied using PCR, followed by denaturing gradient gel electrophoresis (PCR-DGGE). Rice genotype determined, to a large extent, the composition of the different bacterial communities across cultivars. Several cultivars belonging to Oryza sativa ssp. indica tended to select similar bacterial communities, whereas those belonging to subspecies japonica and aromatica selected ones with divergent community structures. An effect of soil type was pronounced for the Actinobacteria communities, while a small effect of 'improved' and 'traditional' plants was noted for all communities analyzed. A few dominant bands in PCR-DGGE, affiliated with Rhizobium radiobacter, Dickeya zeae, Mycobacterium bolletii and with members of the Rhizobiales, Rhodospirillaceae and Paenibacillaceae, were spread across cultivars. In contrast, a majority of bands (e.g. affiliated with Enterobacter cloacae or Burkholderia kururiensis) was only present in particular cultivars or was erratically distributed among rice replicates. These findings suggested that both bacterial adaptation and plant genotype contribute to the shaping of the dynamic bacterial communities associated with roots of rice plants.

The bias of widely used degenerate nifH-specific primer sets was first tested using denaturing gradient gel electrophoresis (DGGE), and their application for profiling of complex communities assessed for roots of Oryza longistaminata. When primers (P) with mismatches at nondegenerate positions were used on genomic DNA of Azotobacter vinelandii, which harbors three single divergent nifH genes, template-to-product ratios were highly skewed. In contrast, we obtained no evidence for a large PCR bias when we used highly degenerate primers with no mismatches (Z). Similar results were obtained for reverse transcription (RT)-PCR amplifications from root RNA from O. longistaminata grown at the river bed of the Okavango, where Z-primers detected a more complex nifH pool, corroborating that the P-primers are quite biased in the nifH sequences they amplify. In microcosms of O. longistaminata grown in the phytotron in the presence or absence of constant low nitrogen input (25 kg NH4NO3 ha(-1) year(-1)), roots of nitrogen-treated plants showed similar, slightly higher levels of nifH-mRNA. However, nitrogen treatment had a strong effect on the composition and diversity of expressed nifH pools that shifted towards methylotroph-related nitrogenases. Thus the active population of diazotrophs was not resistant towards low rates of nitrogen input and decreased significantly in richness, as also observed for plant species richness in grasslands by others.