Infection and Invasion of Roots by Symbiotic, Nitrogen-Fixing Rhizobia during Nodulation of Temperate Legumes

Bacteria belonging to the genera Rhizobium, Mesorhizobium, Sinorhizobium, Bradyrhizobium, and Azorhizobium (collectively referred to as rhizobia) grow in the soil as free-living organisms but can also live as nitrogen-fixing symbionts inside root nodule cells of legume plants. The interactions between several rhizobial species and their host plants have become models for this type of nitrogen-fixing symbiosis. Temperate legumes such as alfalfa, pea, and vetch form indeterminate nodules that arise from root inner and middle cortical cells and grow out from the root via a persistent meristem. During the formation of functional indeterminate nodules, symbiotic bacteria must gain access to the interior of the host root. To get from the outside to the inside, rhizobia grow and divide in tubules called infection threads, which are composite structures derived from the two symbiotic partners. This review focuses on symbiotic infection and invasion during the formation of indeterminate nodules. It summarizes root hair growth, how root hair growth is influenced by rhizobial signaling molecules, infection of root hairs, infection thread extension down root hairs, infection thread growth into root tissue, and the plant and bacterial contributions necessary for infection thread formation and growth. The review also summarizes recent advances concerning the growth dynamics of rhizobial populations in infection threads.     

Diversity of rhizobia and interactions among the host legumes and rhizobial genotypes in an agricultural-forestry ecosystem

To investigate the diversity of rhizobia and interactions among the host legumes and rhizobial genotypes in the same habitat, a total of 97 rhizobial strains isolated from nine legume species grown in an agricultural-forestry ecosystem were identified into seven genomic species and 12 symbiotic genotypes within the genera Bradyrhizobium, Mesorhizobium, Rhizobium and Sinorhizobium based upon analyses of genomic DNA regions and symbiotic genes. The results evidenced that the symbiotic genotypes of rhizobia were consistent with their hosts of origin; revealed that vertical transfer was the main mechanism in rhizobia to maintain the symbiotic genes but lateral transfer of symbiotic genes might have happened between the closely related rhizobial species; suggested the existence of co-distribution and co-evolution among the legume hosts and compatible rhizobia. All of these data demonstrated that the biogeography of rhizobia was a result of interactions among the host legumes, bacterial genomic backgrounds and environments.     

Denitrifying capacity of rhizobial strains of Argentine soils and herbicide sensitivity

Agrochemical application in soils is a matter of environmental concern, and among soil microorganisms, rhizobia and their action before different pesticides are interesting to study, due to their taxonomic and functional diversity. The objectives of the present work were to assess the capacity of rhizobial populations to use herbicides as source of nutrients, as well as their ability to reduce nitrates and / or denitrify. Eighty-one strains belonging to four populations of different genera of rhizobia (Rhizobium, Mesorhizobium, Ensifer and Bradyrhizobium) were assessed. The effect of glyphosate, 2,4-dichlorophenoxyacetic acid, and atrazine on growth of the strains, as well as the ability of the strains to act on herbicide transformation to reduce nitrate and denitrify, were evaluated. The genera studied showed different responses to pesticides. Bradyrhizobium had the greater capacity to utilize the herbicides and among the compounds evaluated, atrazine was the most used as a source of energy. To conclude, some Bradyrhizobium strains were able both to denitrify and to use the atrazine herbicide. The results obtained in this study increase expectations of the use of rhizobia as inoculants, causing changes at the agricultural and environmental level and allowing an appropriate management of agricultural soil fertilization, efficiency in nitrogen fixation and a faster biodegradation of pesticides in soil.     

Biodiversity of rhizobia isolated from a wide range of forest legumes in Brazil

Tropical forests have a high diversity of plant species; are they associated with a correspondingly rich microbial flora? We addressed this question by examining the symbiotic rhizobium bacteria that nodulate a diverse pool of forest legume species in Brazil. The 44 strains studied had been isolated from 29 legume tree species representing 13 tribes including all three subfamilies of the Leguminosae, and were chosen to represent major groups from a larger sample that had previously been characterized by SDS–PAGE of total proteins. Partial 16S rRNA gene sequence was determined, corresponding to positions 44–303 in the Escherichia coli sequence. Fifteen sequences were found, including six novel ones. However, all but one of them could be assigned to a genus because they grouped closely with sequences from previously described rhizobial species. Fast-growing strains had sequences similar to Rhizobium spp., Sinorhizobium spp. or Mesorhizobium spp., while the slow-growing strains had sequences similar to Bradyrhizobium spp. One strain with an intermediate growth rate had a unique sequence which indicated that the strain might belong to the genus Azorhizobium. Although the strains showed a variety of sequences, it was surprising that these strains isolated from taxonomically very diverse host plants in previously unexplored environments were mostly very similar to strains described previously, largely from agricultural systems.     

Diversity of sporadic symbionts and nonsymbiotic endophytic bacteria isolated from nodules of woody, shrub, and food legumes in Ethiopia

Fifty-five bacterial isolates were obtained from surface-sterilized nodules of woody and shrub legumes growing in Ethiopia: Crotalaria spp., Indigofera spp., and Erythrina brucei, and the food legumes soybean and common bean. Based on partial 16S rRNA gene sequence analysis, the majority of the isolates were identified as Gram-negative bacteria belonging to the genera Achromobacter, Agrobacterium, Burkholderia, Cronobacter, Enterobacter, Mesorhizobium, Novosphingobium, Pantoea, Pseudomonas, Rahnella, Rhizobium, Serratia, and Variovorax. Seven isolates were Gram-positive bacteria belonging to the genera Bacillus, Paenibacillus, Planomicrobium, and Rhodococcus. Amplified fragment length polymorphism (AFLP) fingerprinting showed that each strain was genetically distinct. According to phylogenetic analysis of recA, glnII, rpoB, and 16S rRNA gene sequences, Rhizobium, Mesorhizobium, and Agrobacterium were further classified into six different genospecies: Agrobacterium spp., Agrobacterium radiobacter, Rhizobium sp., Rhizobium phaseoli, Mesorhizobium sp., and putative new Rhizobium species. The strains from R. phaseoli, Rhizobium sp. IAR30, and Mesorhizobium sp. ERR6 induced nodules on their host plants. The other strains did not form nodules on their original host. Nine endophytic bacterial strains representing seven genera, Agrobacterium, Burkholderia, Paenibacillus, Pantoea, Pseudomonas, Rhizobium, and Serratia, were found to colonize nodules of Crotalaria incana and common bean on co-inoculation with symbiotic rhizobia. Four endophytic Rhizobium and two Agrobacterium strains had identical nifH gene sequences with symbiotic Rhizobium strains, suggesting horizontal gene transfer. Most symbiotic and nonsymbiotic endophytic bacteria showed plant growth-promoting properties in vitro, which indicate their potential role in the promotion of plant growth when colonizing plant roots and the rhizosphere.     

Unexpectedly Diverse Mesorhizobium Strains and Rhizobium leguminosarum Nodulate Native Legume Genera of New Zealand, while Introduced Legume Weeds Are Nodulated by Bradyrhizobium Species

The New Zealand native legume flora are represented by four genera, Sophora, Carmichaelia, Clianthus, and Montigena. The adventive flora of New Zealand contains several legume species introduced in the 19th century and now established as serious invasive weeds. Until now, nothing has been reported on the identification of the associated rhizobia of native or introduced legumes in New Zealand. The success of the introduced species may be due, at least in part, to the nature of their rhizobial symbioses. This study set out to address this issue by identifying rhizobial strains isolated from species of the four native legume genera and from the introduced weeds: Acacia spp. (wattles), Cytisus scoparius (broom), and Ulex europaeus (gorse). The identities of the isolates and their relationship to known rhizobia were established by comparative analysis of 16S ribosomal DNA, atpD, glnII, and recA gene sequences. Maximum-likelihood analysis of the resultant data partitioned the bacteria into three genera. Most isolates from native legumes aligned with the genus Mesorhizobium, either as members of named species or as putative novel species. The widespread distribution of strains from individual native legume genera across Mesorhizobium spp. contrasts with previous reports implying that bacterial species are specific to limited numbers of legume genera. In addition, four isolates were identified as Rhizobium leguminosarum. In contrast, all sequences from isolates from introduced weeds aligned with Bradyrhizobium species but formed clusters distinct from existing named species. These results show that native legume genera and these introduced legume genera do not have the same rhizobial populations.     

Lotus endemic to the Canary Islands are nodulated by diverse and novel rhizobial species and symbiotypes

Genetic and symbiotic characterization of 34 isolates from several Lotus species endemic to the Canary Islands showed extraordinary diversity, with bacteria belonging to different species of the genera Mesorhizobium (17 isolates), Sinorhizobium (12 isolates) and Rhizobium/Agrobacterium (5 isolates). In a previous report, we showed that the Sinorhizobium isolates mostly belonged to S. meliloti. Here, we focused on the remaining isolates. The Lotus mesorhizobial strains were distributed in the rrs tree within six poorly resolved branches. Partial sequences from atpD and recA genes produced much better resolved phylogenies that were, with some exceptions, congruent with the ribosomal phylogeny. Thus, up to six different mesorhizobial species were detected, which matched with or were sister species of M. ciceri, M. alhagi, M. plurifarium or M. caraganae, and two represented new lineages that did not correspond to any of the currently recognized species. Neither M. loti nor Bradyrhizobium sp. (Lotus), recognized as the typical Lotus-symbionts, were identified among the Canarian Lotus isolates, although their nodulation genes were closely related to M. loti. However, several subbranches of mesorhizobia nodulating Lotus spp. could be differentiated in a nodC tree, with the isolates from the islands distributed in two of them (A1 and A3). Subbranch A1 included reference strains of M. loti and a group of isolates with a host range compatible with biovar loti, whereas A3 represented a more divergent exclusive subbranch of isolates with a host range almost restricted to endemic Lotus and it could represent a new biovar among the Lotus rhizobia.     

Genetic diversity and biogeography of rhizobia associated with Caragana species in three ecological regions of China

Twenty-two genospecies belonging mainly to Mesorhizobium, and occasionally to Rhizobium and Bradyrhizobium, were defined among the 174 rhizobia strains isolated from Caragana species. Highly similar nodC genes were found in the sole Bradyrhizobium strain and among all the detected Mesorhizobium strains. A clear correlation between rhizobial genospecies and the eco-regions where they were isolated was found using homogeneity analysis. All these results demonstrated that Caragana species had stringently selected the rhizobia symbiotic genotype, but not the genomic background; lateral transfer of symbiotic genes from Mesorhizobium to Bradyrhizobium and among the Mesorhizobium species has happened in the Caragana rhizobia; and biogeography of Caragana rhizobia exists. Furthermore, a combined cluster analysis, based upon the patterns obtained from amplified 16S rRNA gene and 16S–23S intergenic spacer restriction analyses, BOX PCR and SDS-PAGE of proteins, was reported to be an efficient method to define the genospecies.     

Diversity and Biogeography of Rhizobia Isolated from Root Nodules of <Emphasis Type="Italic">Glycine max</Emphasis> Grown in Hebei Province,China

A total of 215 rhizobial strains were isolated and analyzed with 16S rRNA gene, 16S–23S intergenic spacer, housekeeping genes atpD, recA, and glnII, and symbiotic genes nifH and nodC to understand the genetic diversity of soybean rhizobia in Hebei province, China. All the strains except one were symbiotic bacteria classified into nine genospecies in the genera of Bradyrhizobium and Sinorhizobium. Surveys on the distribution of these rhizobia in different regions showed that Bradyrhizobium japonicum and Bradyrhizobium elkanii strains were found only in neutral to slightly alkaline soils whereas Bradyrhizobium yuanmingense, Bradyrhizobium liaoningense-related strains and strains of five Sinorhizobium genospecies were found in alkaline–saline soils. Correspondence and canonical correspondence analyses on the relationship of rhizobial distribution and their soil characteristics reveal that high soil pH, electrical conductivity, and potassium content favor distribution of the B. yuanmingense and the five Sinorhizobium species but inhibit B. japonicum and B. elkanii. High contents of available phosphorus and organic matters benefit Sinorhizobium fredii and B. liaoningense-related strains and inhibit the others groups mentioned above. The symbiotic gene (nifH and nodC) lineages among B. elkanii, B. japonicum, B. yuanmingense, and Sinorhizobium spp. were observed in the strains, signifying that vertical gene transfer was the main mechanism to maintain these genes in the soybean rhizobia. However, lateral transfer of symbiotic genes commonly in Sinorhizobium spp. and rarely in Bradyrhizobium spp. was also detected. These results showed the genetic diversity, the biogeography, and the soil determinant factors of soybean rhizobia in Hebei province of China.     

Diversity of endophytic bacteria associated with nodules of two indigenous legumes at different altitudes of the Qilian Mountains in China

A total of 201 endophytic root nodule-associated bacteria collected from two legumes indigenous to different Qilian Mountain altitudes (Hexi Corridor) were characterized through 16S rDNA polymerase chain reaction (PCR)-restriction fragment length polymorphism, 16S rRNA gene sequence analysis, and enterobacterial repetitive intergenic consensus-PCR clustering. The isolates phylogenetically belonged to 35 species in the Phyllobacterium, Ensifer, Rhizobium, Microvirga, Sphingomonas, Paracoccus, Mycobacterium, Paenibacillus, Cohnella, Sporosarcina, Bacillus, Staphylococcus, Brevibacterium, Xenophilus, Erwinia, Leclercia, Acinetobacter, and Pseudomonas genera. Phylogenetic nodA sequence analysis showed higher similarity to Sinorhizobium meliloti with strains related to the Rhizobium, Sinorhizobium, and Acinetobacter genera. Sequence analysis of the nifH gene revealed that the strains belonging to Xenophilus, Acinetobacter, Phyllobacterium, and Rhizobium had genes similar to those of Mesorhizobium and Sinorhizobium. The results indicated that horizontal gene transfer could have occurred between rhizobia and non-rhizobial endophytes. Canonical correspondence analysis revealed that altitude and host plant species contributed more to the bacterial endosymbiont separation than other ecological factors. This study provided valuable information on the interactions between symbiotic bacteria, non-symbiotic bacteria and their habitats, and thus provided knowledge on their genetic diversity and ecology.     

Phenotypic,genomic and phylogenetic characteristics of rhizobia isolated from root nodules of <Emphasis Type="Italic">Robinia pseudoacacia</Emphasis> (black locust) growing in Poland and Japan

Rhizobial strains, rescued from the root nodules of Robinia pseudoacacia growing in Japan and Poland, were characterized for the phenotypic properties, genomic diversity as well as phylogeny and compared with the reference strains representing different species and genera of nodule bacteria. They had a moderately slow growth rate, a low tolerance to antibiotics, a moderate resistance to NaCl and produced acid in yeast mannitol agar. Cluster analysis based on the phenotypic features divided all bacteria involved in this study into four phena, comprising: (1) Rhizobium sp. + Sinorhizobium sp., (2) Bradyrhizobium sp., (3) R. pseudoacacia microsymbionts + Mesorhizobium sp., and (4) Rhizobium galegae strains at similarity coefficient of 74%. R. pseudoacacia nodule isolates and Mesorhizobium species were placed on a single branch clearly distinct from other rhizobium genera lineages. Strains representing R. pseudoacacia microsymbionts shared 98–99% 16S rDNA sequence identity with Mesorhizobium species and in 16S rDNA phylogenetic tree all these bacteria formed common cluster. The rhizobia tested are genomically heterogeneous as indicated by the AFLP (Amplified Fragment Length Polymorphism) method. The bacteria studied exhibited high degree of specificity for nodulation. Nitrogenase structural genes in these strains were located on 771–961 kb megaplasmids. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Evolution of root nodule bacteria: Reconstruction of the speciation processes resulting from genomic rearrangements in a symbiotic system

The processes of speciation and macroevolution of root nodule bacteria (rhizobia), based on deep rearrangements of their genomes and occurring in the N₂-fixing symbiotic system, are reconstructed. At the first stage of rhizobial evolution, transformation of free-living diazotrophs (related to Rhodopseudomonas) to symbiotic N₂-fixers (Bradyrhizobium) occurred due to the acquisition of the fix gene system, which is responsible for providing nitrogenase with electrons and redox potentials, as well as for oxygen-dependent regulation of nitrogenase synthesis in planta, and then of the nod genes responsible for the synthesis of the lipo-chitooligosaccharide Nod factors, which induce root nodule development. The subsequent rearrangements of bacterial genomes included (1) increased volume of hereditary information supported by species, genera (pangenome), and individual strains; (2) transition from the unitary genome to a multicomponent one; and (3) enhanced levels of bacterial genetic plasticity and horizontal gene transfer, resulting in formation of new genera—of which Mesorhizobium, Rhizobium, and Sinorhizobium are the largest—and of over 100 species. Rhizobial evolution caused by development and diversification of the Nod factor-synthesizing systems may result in either relaxed host specificity range (transition of Bradyrhizobium from autotrophic to symbiotrophic carbon metabolism in interaction with a broad spectrum of legumes) or narrowed host specificity range (transition of Rhizobium and Sinorhizobium to “altruistic” interaction with legumes of the galegoid clade). Reconstruction of the evolutionary pathway from symbiotic N₂-fixers to their free-living ancestors makes it possible to initiate the studies based on up-to-date genome screening technologies and aimed at the issues of genetic integration of organisms into supraspecies complexes, ratios of the macro- and microevolutionary mechanisms, and development of cooperative adaptations based on altruistic interaction between the symbiotic partners.     

Polyphasic characterization of rhizobia isolated from Leucaena leucocephala from Panxi, China

Leucaena leucocephala was introduced into Panxi, Sichuan, China, in the 1980s and 1990s for afforestation and preventing water loss and soil erosion in this area. The co-introduction of rhizobial symbionts of introduced plants has drawn attention since they may influence local soil communities. We studied the phylogenetic position of the L. leucocephala isolates and assessed if the rhizobia were introduced together with the host to Panxi, Sichuan, China. The glnII and atpD genes of fifteen representative isolates were sequenced and analyzed, and applied multilocus sequence analyses in which the housekeeping genes recA, glnII and atpD were included. Furthermore, we estimated the within species diversity directly with 23S rDNA and IGS RFLP and indirectly through phenotypic analysis of forty L. leucocephala isolates. The isolates represented seven species and 38 diversified strains in the genera Ensifer, Mesorhizobium, Bradyrhizobium and Rhizobium. The within species diversity of the Ensifer isolates was large, proposing a potential to occupy novel niches. There was not conclusive evidence to show that any of the strains would have been co-introduced with L. leucocephala. On the contrary, we came to a conclusion that the possible introduction should not be inferred from sequence data alone.     

Root nodules of Genista germanica harbor Bradyrhizobium and Rhizobium bacteria exchanging nodC and nodZ genes

A collection of 18 previously unstudied strains isolated from root nodules of Genista germanica (German greenweed) grown in southeast Poland was evaluated for the level of genetic diversity using the BOX-PCR technique and the phylogenetic relationship based on both core (16S rRNA, dnaK, ftsA, glnII, gyrB, recA, rpoB) and nodulation (nodC and nodZ) gene sequences. Each of the 18 G. germanica root nodule isolates displayed unique BOX-PCR patterns, indicating their high level of genomic heterogeneity. Based on the comparative 16S rDNA sequence analysis, 12 isolates were affiliated to the Bradyrhizobium genus and the other strains were most similar to Rhizobium species. Phylogenetic analysis of the core gene sequences indicated that the studied Bradyrhizobium bacteria were most closely related to Bradyrhizobium japonicum, whereas Rhizobium isolates were most closely related to Rhizobium lusitanum and R. leguminosarum. The phylogenies of nodC and nodZ for the Rhizobium strains were incongruent with each other and with the phylogenies inferred from the core gene sequences. All Rhizobium nodZ gene sequences acquired in this study were grouped with the sequences of Bradyrhizobium strains. Some of the studied Rhizobium isolates were placed in the nodC phylogenetic tree together with reference Rhizobium species, while the others were closely related to Bradyrhizobium bacteria. The results provided evidence for horizontal transfer of nodulation genes between Bradyrhizobium and Rhizobium. However, the horizontal transfer of nod genes was not sufficient for Rhizobium strains to form nodules on G. germanica roots, suggesting that symbiotic genes have to be adapted to the bacterial genome.     

Forms of natural selection controlling the genomic evolution in nodule bacteria

The role of different forms of natural selection in the evolution of genomes in root nodule bacteria (rhizobia) is analyzed for the first time. In these nitrogen-fixing symbionts of leguminous plants, two types of genome organization are revealed: (i) unitary type, where over 95% of genetic information is encoded by chromosomes (5.3–5.5 Mb in Azorhizobium, 7.0–7.8 Mb in Mesorhizobium, 7.3–10.1 Mb in Bradyrhizobium); (ii) multipartite type, where up to 50% of genetic information is allocated to plasmids or chromids which may exceed 2 Mb in size and usually control the symbiotic properties (pSyms) in fast-growing rhizobia (Rhizobium, Sinorhizobium, Neorhizobium). Emergence of fast-growing species with narrow host ranges are correlated to the extension of extrachromosomal parts of genomes, including the increase in pSyms sizes (in Sinorhizobium). An important role in this evolution is implemented by diversifying selection since the genomic diversity evolved in rhizobia owing to symbiotic interactions with highly divergent legumes. However, analysis of polymorphism in nod genes (encoding synthesis of lipo-chitooligosaccharide signaling Nod factors) suggests that the impacts of diversifying selection are restricted to the bacterial divergence for host specificity and do not influence the overall genome organization. Since the extension of rhizobia genome diversity results from the horizontal sym gene transfer occurring with low frequencies, we suggest that this extension is due to the frequency-dependent selection anchoring the rare genotypes in bacterial populations. It is implemented during the rhizobia competition for nodulation encoded by the functionally diverse cmp genes. Their location in different parts of bacterial genomes may be considered as an important factor of their adaptive diversification implemented in the host-associated microbial communities.     

The Symbiotic Biofilm of Sinorhizobium fredii SMH12, Necessary for Successful Colonization and Symbiosis of Glycine max cv Osumi,Is Regulated by Quorum Sensing Systems and Inducing Flavonoids via NodD1

Bacterial surface components, especially exopolysaccharides, in combination with bacterial Quorum Sensing signals are crucial for the formation of biofilms in most species studied so far. Biofilm formation allows soil bacteria to colonize their surrounding habitat and survive common environmental stresses such as desiccation and nutrient limitation. This mode of life is often essential for survival in bacteria of the genera Mesorhizobium, Sinorhizobium, Bradyrhizobium, and Rhizobium. The role of biofilm formation in symbiosis has been investigated in detail for Sinorhizobium meliloti and Bradyrhizobium japonicum. However, for S. fredii this process has not been studied. In this work we have demonstrated that biofilm formation is crucial for an optimal root colonization and symbiosis between S. fredii SMH12 and Glycine max cv Osumi. In this bacterium, nod-gene inducing flavonoids and the NodD1 protein are required for the transition of the biofilm structure from monolayer to microcolony. Quorum Sensing systems are also required for the full development of both types of biofilms. In fact, both the nodD1 mutant and the lactonase strain (the lactonase enzyme prevents AHL accumulation) are defective in soybean root colonization. The impairment of the lactonase strain in its colonization ability leads to a decrease in the symbiotic parameters. Interestingly, NodD1 together with flavonoids activates certain quorum sensing systems implicit in the development of the symbiotic biofilm. Thus, S. fredii SMH12 by means of a unique key molecule, the flavonoid, efficiently forms biofilm, colonizes the legume roots and activates the synthesis of Nod factors, required for successfully symbiosis.     

Analysis of nifH Gene Pool Complexity in Soil and Litter at a Douglas Fir Forest Site in the Oregon Cascade Mountain Range

Nitrogen-fixing microbial populations in a Douglas fir forest on the western slope of the Oregon Cascade Mountain Range were analyzed. The complexity of the nifH gene pool (nifH is the marker gene which encodes nitrogenase reductase) was assessed by performing nested PCR with bulk DNA extracted from plant litter and soil. The restriction fragment length polymorphisms (RFLPs) of PCR products obtained from litter were reproducibly different than the RFLPs of PCR products obtained from the underlying soil. The characteristic differences were found during the entire sampling period between May and September. RFLP analyses of cloned nifH PCR products also revealed characteristic patterns for each sample type. Among 42 nifH clones obtained from a forest litter library nine different RFLP patterns were found, and among 64 nifH clones obtained from forest soil libraries 13 different patterns were found. Only two of the patterns were found in both the litter and the soil, indicating that there were major differences between the nitrogen-fixing microbial populations. A sequence analysis of clones representing the 20 distinct patterns revealed that 19 of the patterns had a proteobacterial origin. All of the nifH sequences obtained from the Douglas fir forest litter localized in a distinct phylogenetic cluster characterized by the nifH sequences of members of the genera Rhizobium, Sinorhizobium, and Azospirillum. The nifH sequences obtained from soil were found in two additional clusters, one characterized by sequences of members of the genera Bradyrhizobium, Azorhizobium, Herbaspirillum, and Thiobacillus and the other, represented by a single nifH clone, located between the gram-positive bacteria and the cyanobacteria. Our results revealed the distinctness of the nitrogen-fixing microbial populations in litter and soil in a Douglas fir forest; the differences may be related to special requirements for degradation and mineralization processes in the plant litter.     

Phenotypic and genotypic characterisation of root nodule bacteria nodulating Millettia pinnata (L.) Panigrahi, a biodiesel tree

Aims

Milletia pinnata is a leguminous tropical tree that produces seed oil suitable for biodiesel and is targeted to be planted on marginal land associated with nitrogen poor soil. This study aimed to identify effective rhizobia species for M. pinnata.

Methods

Soil samples were collected from M. pinnata grown in Kununurra, Australia. Rhizobia were trapped, characterised and sequenced for 16S rRNA, atpD, dnaK and recA genes.

Results

Forty isolates tolerated pH 7 – 9, temperatures 29 – 37 °C, salinity below 1 % NaCl, and had optimal growth on mannitol, arabinose or glutamate as a single carbon source, a few grew on sucrose and none grew on lactose. Inoculation of isolates increased shoot dry weight of M. pinnata’s seedlings in nitrogen minus media. Slow-growing isolates were closely related to Bradyrhizobium yuanmingense, Bradyrhizobium sp. DOA10, Bradyrhizobium sp. ORS305 and B. liaoningense LMG 18230^T. The fast-growing isolates related to Rhizobium sp. 8211, R. miluonense CCBAU 41251^T, R miluonense CC-B-L1, Rhizobium sp. CCBAU 51330 and Rhizobium sp. 43015

Conclusions

Millettia pinnata was effectively nodulated by slow-growing isolates related to Bradyrhizobium yuanmingense, Bradyrhizobium sp. DOA10 Bradyrhizobium sp. ORS305, B. liaoningense LMG 18230^T and fast-growing isolates related Rhizobium sp. 8211, R. miluonense, Rhizobium sp. CCBAU 51330 and Rhizobium sp. 43015     

Molecular diversity and phylogeny of rhizobia associated with Lablab purpureus (Linn.) grown in Southern China

As an introduced plant, Lablab purpureus serves as a vegetable, herbal medicine, forage and green manure in China. In order to investigate the diversity of rhizobia associated with this plant, a total of 49 rhizobial strains isolated from ten provinces of Southern China were analyzed in the present study with restriction fragment length polymorphism and/or sequence analyses of housekeeping genes (16S rRNA, IGS, atpD, glnII and recA) and symbiotic genes (nifH and nodC). The results defined the L. purpureus rhizobia as 24 IGS-types within 15 rrs-IGS clusters or genomic species belonging to Bradyrhizobium, Rhizobium, Ensifer (synonym of Sinorhizobium) and Mesorhizobium. Bradyrhizobium spp. (81.6%) were the most abundant isolates, half of which were B. elkanii. Most of these rhizobia induced nodules on L. purpureus, but symbiotic genes were only amplified from the Bradyrhizobium and Rhizobium leguminosarum strains. The nodC and nifH phylogenetic trees defined five lineages corresponding to B. yuanmingense, B. japonicum, B. elkanii, B. jicamae and R. leguminosarum. The coherence of housekeeping and symbiotic gene phylogenies demonstrated that the symbiotic genes of the Lablab rhizobia were maintained mainly through vertical transfer. However, a putative lateral transfer of symbiotic genes was found in the B. liaoningense strain. The results in the present study clearly revealed that L. purpureus was a promiscuous host that formed nodules with diverse rhizobia, mainly Bradyrhizobium species, harboring different symbiotic genes.     

Coexistence of Burkholderia, Cupriavidus, and Rhizobium sp. Nodule Bacteria on two Mimosa spp. in Costa Rica

rRNA gene sequencing and PCR assays indicated that 215 isolates of root nodule bacteria from two Mimosa species at three sites in Costa Rica belonged to the genera Burkholderia, Cupriavidus, and Rhizobium. This is the first report of Cupriavidus sp. nodule symbionts for Mimosa populations within their native geographic range in the neotropics. Burkholderia spp. predominated among samples from Mimosa pigra (86% of isolates), while there was a more even distribution of Cupriavidus, Burkholderia, and Rhizobium spp. on Mimosa pudica (38, 37, and 25% of isolates, respectively). All Cupriavidus and Burkholderia genotypes tested formed root nodules and fixed nitrogen on both M. pigra and M. pudica, and sequencing of rRNA genes in strains reisolated from nodules verified identity with inoculant strains. Inoculation tests further indicated that both Cupriavidus and Burkholderia spp. resulted in significantly higher plant growth and nodule nitrogenase activity (as measured by acetylene reduction assays) relative to plant performance with strains of Rhizobium. Given the prevalence of Burkholderia and Cupriavidus spp. on these Mimosa legumes and the widespread distribution of these plants both within and outside the neotropics, it is likely that both β-proteobacterial genera are more ubiquitous as root nodule symbionts than previously believed.