Land plants interact with microbes primarily at roots. Despite the importance of root microbial communities for health and nutrient uptake, the current understanding of the complex plant-microbe interactions in the rhizosphere is still in its infancy. Roots provide different microhabitats at the soil-root interface: rhizosphere soil, rhizoplane, and endorhizosphere. We discuss technical aspects of their differentiation that are relevant for the functional analysis of their different microbiomes, and we assess PCR (polymerase chain reaction)-based methods to analyze plant-associated bacterial communities. Development of novel primers will allow a less biased and more quantitative view of these global hotspots of microbial activity. Based on comparison of microbiome data for the different root-soil compartments and on knowledge of bacterial functions, a three-step enrichment model for shifts in community structure from bulk soil toward roots is presented. To unravel how plants shape their microbiome, a major research field is likely to be the coupling of reductionist and molecular ecological approaches, particularly for specific plant genotypes and mutants, to clarify causal relationships in complex root communities.

Despite the relevance of complex root microbial communities for plant health, growth and productivity, the molecular basis of these plant-microbe interactions is not well understood. Verrucomicrobia are cosmopolitans in the rhizosphere, nevertheless their adaptations and functions are enigmatic since the proportion of cultured members is low. Here we report four cultivated Verrucomicrobia isolated from rice, putatively representing four novel species, and a novel subdivision. The aerobic strains were isolated from roots or rhizomes of Oryza sativa and O. longistaminata. Two of them are the first cultivated endophytes of Verrucomicrobia, as validated by confocal laser scanning microscopy inside rice roots after re-infection under sterile conditions. This extended known verrucomicrobial niche spaces. Two strains were promoting root growth of rice. Discovery of root compartment-specific Verrucomicrobia permitted an across-phylum comparison of the genomic conformance to life in soil, rhizoplane or inside roots. Genome-wide protein domain comparison with niche-specific reference bacteria from distant phyla revealed signature protein domains which differentiated lifestyles in these microhabitats. Our study enabled us to shed light into the dark microbial matter of root Verrucomicrobia, to define genetic drivers for niche adaptation of bacteria to plant roots, and provides cultured strains for revealing causal relationships in plant-microbe interactions by reductionist approaches.

Twenty one strains of symbiotic bacteria from root nodules of local races of cowpea (Vigna unguiculata), Bambara groundnut (Vigna subterranea) and peanuts (Arachis hypogaea) grown on subsistence farmers' fields in the Kavango region of Namibia, were previously characterized as a novel group within the genus Bradyrhizobium. To verify their taxonomic position, the strains were further analysed using a polyphasic approach. 16S rRNA gene sequences were most similar to Bradyrhizobium manausense BR 3351 T , with Bradyrhizobium ganzhouense RITF806 T being the most closely related type strain in the phylogenetic analysis, and Bradyrhizobium yuanmingense CCBAU 10071 T in the ITS sequence analysis. Phylogenetic analysis of concatenated glnII-recA-rpoB-dnaK placed the strains in a highly supported lineage distinct from species of the genus Bradyrhizobium with validly published names; they were most closely related to Bradyrhizobium subterraneum 58 2-1 T . The status of the species was validated by results of DNA-DNA hybridization. The combination of phenotypic characteristics from several tests, including carbon source utilization and antibiotic resistance, could be used to differentiate representative strains of species of the genus Bradyrhizobium with validly published names. Novel strain 7-2 T induced effective nodules on Vigna subterranea, Vigna unguiculata, Arachis hypogaea and on Lablab purpureus. The DNA G+C content of strain 7-2 T was 65.4 mol% (Tm). Based on the data presented, we conclude that these strains represent a novel species for which the name Bradyrhizobium vignae sp. nov. is proposed, with strain 7-2 T [LMG 28791 T , DSMZ 100297 T , NTCCM0018 T (Windhoek)] as the type strain.In the Kavango region of Namibia, agriculture is largely dominated by smallholder farms, without irrigation or agrochemical inputs. Local farmers are confronted with low yields and decreasing soil fertility (Pröpper et al., 2010). The intercropping of grain legumes is common practice with local cereals interspersed in an irregular pattern (Grönemeyer et al., 2013). Cowpea (Vigna unguiculata (L.) Walp., local name in Kavango: makunde) is the main grain legume grown by farmers, but also Bambara groundnut [Vigna subterranea (L.) Verdc., local name: nongomene ] and peanuts (Arachis hypogaea L.) are planted, albeit to a lesser extent (Grönemeyer et al., 2013). Inoculants adapted to the crops and the harsh environmental conditions (long dry seasons and high temperatures) may help to increase yields for local smallholders.In a previous study (Grönemeyer et al., 2014), populations of symbiotic bacteria from nodules of pulses collected in the Kavango region of Namibia and in the Angolan province Bié were sampled. Pure cultures of Bradyrhizobium were isolated and characterized. On the basis of multilocus sequence analysis (MLSA) of concatemers of three protein-coding genes (glnII-recA-rpoB) and intergenic spacer (ITS) sequences, several novel lineages within the genus Bradyrhizobium were suggested by phylogenetic analyses (Gröne-meyer et al....

Four strains of symbiotic bacteria from root nodules of hyacinth bean (Lablab purpureus (L.) Sweet) from Namibia were previously identified as a novel group within the genus Bradyrhizobium. To confirm their taxonomic status, these strains were further characterized by taking a polyphasic approach. The type strain possessed 16S rRNA gene sequences identical to Bradyrhizobium paxllaeri LMTR 21 and Bradyrhizobiumicense LMTR 13, the full-length sequences were identical to those retrieved from SAMN05230119 and SAMN05230120, respectively. However, the intergenic spacer sequences of the novel group showed identities of less than 93.1 % to described Bradyrhizobium species and were placed in a well-supported separate lineage in the phylogenetic tree. Phylogenetic analyses of six concatenated housekeeping genes, recA, glnII, gyrB, dnaK, atpD and rpoB, corroborated that the novel strains belonged to a lineage distinct from named species of the genus Bradyrhizobium, with highest sequence identities to Bradyrhizobiumjicamae and B. paxllaeri (below 93 %). The species status was validated by results of DNA-DNA hybridization and average nucleotide identity values of genome sequences. The combination of phenotypic characteristics from several tests, including carbon source utilization and antibiotic resistance, could be used to differentiate representative strains from recognized species of the genus Bradyrhizobium. Phylogenetic analysis of nodC and nifH genes placed the novel strains in a group with B. paxllaeri and B.lablabi. Novel strain 5-10 induces effective nodules on Lablab purpureus, Vigna subterranea, Vigna unguiculata and Arachis hypogaea. Based on our results, we conclude that our strains represent a novel species for which the name Bradyrhizobium namibiense sp. nov. is proposed, with type strain 5-10[LMG 28789, DSM 100300, NTCCM0017 (Windhoek)].

Pterocarpus angolensis, a leguminous tree native to the dry woodlands of Southern Africa, provides valuable timber, but is threatened by land conversion and overharvesting while showing limited natural regeneration. Nitrogen-fixing root nodule symbionts that could improve establishment of young seedlings have not yet been described. Therefore, we investigated the ability of P. angolensis to form nodules with a diverse range of rhizobia. In drought-prone areas under climate change with higher temperatures, inoculants that are heat-tolerant and adapted to these conditions are likely to be of advantage. Sources of bacterial isolates were roots of P. angolensis from nurseries in the Kavango region, other shrubs from this area growing near Pterocarpus such as Indigofera rautanenii, Desmodium barbatum, Chamaecrista sp., or shrubs from drought-prone areas in Namaqualand (Wiborgia monoptera, Leobordea digitata) or Kalahari (Indigofera alternans). Only slight protrusions were observed on P. angolensis roots, from which a non-nodulating Microbacterium sp. was isolated. Rhizobia that were isolated from nodules of other shrubs were affiliated to Bradyrhizobium ripae WR4T, Bradyrhizobium spp. (WR23/WR74/WR93/WR96), or Ensifer/Mesorhizobium (WR41/WR52). As many plant growth-promoting rhizobacteria (PGPR), nodule isolates produced siderophores and solubilized phosphate. Among them, only the Bradyrhizobium strains nodulated P. angolensis under controlled conditions in the laboratory. Isolates were further characterized by multilocus sequence analysis and were found to be distant from known Bradyrhizobium species. Among additional reference species tested for nodulation on P. angolensis, Bradyrhizobium vignae 7-2T and Bradyrhizobium namibiense 5-10T from the Kavango region of Namibia as well as Bradyrhizobium elkanii LMG6234T and Bradyrhizobium yuanmingense LMG21728T induced nitrogen-fixing nodules, while Bradyrhizobium diazoefficiens USDA110T and Bradyrhizobium tropiciagri SEMIA6148T did not. This suggests a broad microsymbiont range from Bradyrhizobium japonicum and B. elkanii lineages. Phylogenetic analysis of nodC genes indicated that nodulating bradyrhizobia did not belong to a specific symbiovar. Also, for I. rautanenii and Wiborgia, nodule isolates B. ripae WR4T or Mesorhizobium sp. WR52, respectively, were authenticated. Characterization of symbionts inducing effective root nodules in P. angolensis and other shrubs from Subsahara Africa (SSA) give insights in their symbiotic partners for the first time and might help in future to develop bioinoculants for young seedlings in nurseries, and for reforestation efforts in Southern Africa.