Soybeans inoculated with root zone soils of Canadian native legumes harbour diverse and novel Bradyrhizobium spp. that possess agricultural potential

An assessment was made of the evolutionary relationships of soybean nodulating bacteria associated with legumes native to eastern Canada to identify potential new sources of soybean inoculant strains.Short season soybeans were used to selectively trap bacteria from root zone soils of four native legume species. Screening of more than 800 bacterial isolates from soybean root nodules by analysis of recA gene sequences followed by analyses of selected genotypes using six core and two symbiosis (nodC and nifH) gene sequences permitted identification of diverse taxa that included eight novel and four named Bradyrhizobium species as well as lineages attributed to the genera Afipia and Tardiphaga.Plant tests showed that symbionts related to four named species as well as a novel Bradyrhizobium lineage were highly efficient with regard to nitrogen fixation on soybeans relative to an inoculant strain.A new symbiovar (sv. septentrionalis) is proposed based on a group of four novel Bradyrhizobium spp. that possess distinctive nodC and nifH gene sequences and symbiotic characteristics.Evidence is provided for horizontal transfer of sv. septentrionalis symbiosis genes between novel Bradyrhizobium spp., a process that rendered recipient bacteria ineffective on soybeans.Diverse lineages of non-symbiotic and symbiotic Bradyrhizobium spp. co-occured within monophyletic clusters in a phylogenetic tree of concatenated core genes, suggesting that loss and/or gain of symbiosis genes has occurred in the evolutionary history of the bacterial genus.Our data suggest that symbiont populations associated with legumes native to eastern Canada harbour elite strains of Bradyrhizobium for soybean inoculation.     

Plasmids impact on rhizobia-legumes symbiosis in diverse environments

Rhizobia are a well-known group of soil bacteria that establish symbiotic relationship with leguminous plants, fix atmospheric nitrogen, and improve soil fertility. To fulfill multiple duties in soil, rhizobia are elaborated with a large and complex multipartite genome composed of several replicons. The genetic material is divided among various replicons, in a way to cope with, and satisfy the diverse functions of rhizobia. In addition to the main chromosome, which is carrying the essential (core) genes required for sustaining cell life, the rhizobia genomes contain several extra-chromosomal plasmids, carrying the nonessential (accessory) genes. Occasionally, some mega-plasmids, denoted as secondary chromosomes or chromids, carry some essential (core) genes. Furthermore, specific accessory gene sequences (the symbiotic chromosomal islands) are incorporated in the main chromosome of some rhizobia species in Bradyrhizobium and Mesorhizobium genera. Plasmids in rhizobia are of variable sizes. All of the plasmids in a Rhizobium cell constitute about 30–50% of the genome. Rhizobia plasmids have specific characters such as miscellaneous genes, independent replication system, self-transmissibility, and instability. The plasmids regulate several cellular metabolic functions and enable the host rhizobia to survive in diverse habitats and even under stress conditions. Symbiotic plasmids in rhizobia are receiving increased attention because of their significance in the symbiotic nitrogen fixation process. They carry the symbiotic (nod, nif and fix) genes, and some non-symbiotic genes. Symbiotic plasmids are conjugally-transferred by the aid of the non-symbiotic, self-transmissible plasmids, and hence, brings about major changes in the symbiotic interactions and host specificity of rhizobia. Besides, the rhizobia cells harbor one or more accessory, non-symbiotic plasmids, carrying genes regulating various metabolic functions, rhizosphere colonization, and nodulation competitiveness. The entire rhizobia-plasmid pool interacting in harmony and provides rhizobia with substantial abilities to fulfill their complex symbiotic and non-symbiotic functions in variable environments. The above concepts are extensively reviewed and fairly discussed.     

Soil acidity in relation to groundnut-Bradyrhizobium symbiotic performance

Effects of soil acidity on groundnut-Bradyrhizobium symbiotic performance were studied in a potted, sandy soil in a glasshouse in Zimbabwe. The soil was limed to soil-pH levels of 5.0 and 6.5. Soil acidity negatively affected plant development, measured as leaf area and plant dry weight, while nodulation was enhanced. This acidity-enhanced nodulation was most evident when nodulation was caused by the indigenousBradyrhizobium population. Effects of soil acidity differed between groundnut cultivars andBradyrhizobium spp. strains, the former having greater importance. TwoArachis hypogaea L. Spanish-type cultivars, Falcon and Plover, performed equally well at neutral soil pH, but Falcon was more acid tolerant. Comparison of the symbiotic performance in neutral versus acid soil of twoBradyrhizobium spp. strains, MAR 411 (3G4b20) and MAR 1510 (CB 756), showed that MAR 411 performed superiorly in neutral soil, but MAR 1510 in acid soil. The indigenousBradyrhizobium population was more effective than was inoculation with strains MAR 411 or MAR 1510. Comparison of twelveBradyrhizobium spp. strains for their symbiotic performance in acid soil showed that some strains were totally ineffective under acidity stress (MAR 253, MAR 967 and MAR 1506), while others performed well.Bradyrhizobium spp. strain MAR 1576 (32 H1) ranked highest for nitrogen accumulation, plant dry weight and leaf area, with strains MAR 1555 (TAL 11) and MAR 1510 following closely. Nitrate fertilisation of groundnut plants led to soil alkalinisation, while nitrogen fixation resulted in soil acidification. Soil acidity in combination with soil sterilisation gave rise to symptoms associated with Al and Mn toxicity.     

Formation of nodular structures on the non-legumes Brassica napus,B. campestris,B. juncea and Arabidopsis thaliana with Bradyrhizobium and Rhizobium isolated from Parasponia spp. or legumes grown in tropical soils

Strains of Bradyrhizobium formed nodule-like structures on Arabidopsis and species of Brassica in pots with sandvermiculite and in glass tubes on a nitrogen-free mineral salts agar. Broad-host-range Rhizobium strains NGR234 from Lablab purpureus and NGR76 from Phaseolus vulgaris formed similar nodule-like structures on Brassica spp. The size of these structures on plants in pots were large, often reaching 10 mm in diameter.The frequency of inoculated Brassica plants in pots with nodule-like structures was 25–50%, depending on the inoculum strain. The inheritable nature of factors involved in the formation of the nodule-like structures was demonstrated when the structures occurred on 100% of inoculated B. napus seedlings derived from plants with the nodule-like structures.Nodule-like structures occurred without, but not with, the application of a cellulase-pectolyase-PEG treatment to the roots. Attempts to isolate Bradyrhizobium or Rhizobium from the nodule-like structures failed. Internal infection of these structures could not be detected using either the light or electron microscope. The inoculum strains of root-nodule bacteria were detected in high numbers in the rhizosphere of plants 5 months after inoculation. On agar plates bacterial colonies could be seen, with undiminished growth, over the surface of the agar extending to the root surface. However, ground root tissue of Brassica was toxic to Bradyrhizobium strains. This suggested that Bradyrhizobium strains would not survive after infecting the roots of Brassica spp. Nitrogen fixation was associated with high rhizosphere populations of Azospirillum and not with Bradyrhizobium induced nodule structures of Brassica spp.     

Biological nitrogen fixation: Investments,expectations and actual contributions to agriculture

Inputs of biologically fixed N into agricultural systems may be derived from symbiotic relationships involving legumes and Rhizobium spp., partnerships between plants and Frankia spp. or cyanobacteria, or from non-symbiotic associations between free-living diazotrophs and plant roots. It is assumed that these N₂-fixing systems will satisfy a large portion of their own N requirements from atmospheric N₂, and that additional fixed N will be contributed to soil reserves for the benefit of other crops or forage species. This paper reviews the actual levels of N₂ fixation attained by legume and non-legume associations and assesses their role as a source of N in tropical and sub-tropical agriculture. We discuss factors influencing N₂ fixation and identify possible strategies for improving the amount of N₂ fixed.     

Breeding for better symbiosis

The present review gives a critical assessment of the literature dealing with symbiosis between rhizobia and legumes and between AM fungi and most plants. Associative N₂ fixation (even though strictly speaking not a symbiotic relationship) does have some characteristics of symbiosis due to mutualistic dependence and usefulness of the relationship, and is therefore covered in this review. Nodulation in the rhizobia–legume symbiosis may be limited by an insufficient amount of the nod-gene inducers released from seed and/or roots. However, there is genotypic variation in the germplasm of legume species in all components of the signalling pathway, suggesting a prospect for improving nodulation by selecting and/or transforming legume genotypes for increased exudation of flavonoids and other signalling compounds. Deciphering chromosomal location as well as cloning nod, nif and other genes important in nodulation and N₂ fixation will allow manipulation of the presence and expression of these genes to enhance the symbiotic relationship. Increased efficacy of symbiotic N₂ fixation can be achieved by selecting not only the best host genotypes but by selecting the best combination of host genotype and nodule bacteria. As flavonoids exuded by legume seedlings may not only be nod-gene inducers, but also stimulants for hyphal growth of the AM fungi, selecting and/or transforming plants to increase exudation of these flavonoids may result in a double benefit for mycorrhizal legumes. Mutants unable to sustain mycorrhizal colonisation are instrumental in understanding the colonisation process, which may ultimately pay off in breeding for the more effective symbiosis. In conclusion, targeted efforts to breed genotypes for improved N₂ fixation and mycorrhizal symbiosis will bring benefits in increased yields of crops under a wide range of environmental conditions and will contribute toward sustainability of agricultural ecosystems in which soil-plant-microbe interactions will be better exploited.     

Symbiotic properties of a chimeric Nod-independent photosynthetic Bradyrhizobium strain obtained by conjugative transfer of a symbiotic plasmid

The lateral transfer of symbiotic genes converting a predisposed soil bacteria into a legume symbiont has occurred repeatedly and independently during the evolution of rhizobia. We experimented the transfer of a symbiotic plasmid between Bradyrhizobium strains. The originality of the DOA9 donor is that it harbours a symbiotic mega-plasmid (pDOA9) containing nod, nif and T3SS genes while the ORS278 recipient has the unique property of inducing nodules on some Aeschynomene species in the absence of Nod factors (NFs). We observed that the chimeric strain ORS278-pDOA9* lost its ability to develop a functional symbiosis with Aeschynomene. indica and Aeschynomene evenia. The mutation of rhcN and nodB led to partial restoration of nodule efficiency, indicating that T3SS effectors and NFs block the establishment of the NF-independent symbiosis. Conversely, ORS278-pDOA9* strain acquired the ability to form nodules on Crotalaria juncea and Macroptillium artropurpureum but not on NF-dependent Aeschynomene (A. afraspera and A. americana), suggesting that the ORS278 strain also harbours incompatible factors that block the interaction with these species. These data indicate that the symbiotic properties of a chimeric rhizobia cannot be anticipated due to new combination of symbiotic and non-symbiotic determinants that may interfere during the interaction with the host plant.     

Expression and evolution of functionally distinct haemoglobin genes in plants

Haemoglobin genes have been found in a number of plant species, but the number of genes known has been too small to allow effective evolutionary inferences. We present nine new non-symbiotic haemoglobin sequences from a range of plants, including class 1 haemoglobins from cotton, Citrus and tomato, class 2 haemoglobins from cotton, tomato, sugar beet and canola and two haemoglobins from the non-vascular plants, Marchantia polymorpha (a liverwort) and Physcomitrella patens (a moss). Our molecular phylogenetic analysis of all currently known non-symbiotic haemoglobin genes and a selection of symbiotic haemoglobins have confirmed the existence of two distinct classes of haemoglobin genes in the dicots. It is likely that all dicots have both class 1 and class 2 non-symbiotic haemoglobin genes whereas in monocots we have detected only class 1 genes. The symbiotic haemoglobins from legumes and Casuarina are related to the class 2 non-symbiotic haemoglobins, whilst the symbiotic haemoglobin from Parasponia groups with the class 1 non-symbiotic genes. Probably, there have been two independent recruitments of symbiotic haemoglobins. Although the functions of the two non-symbiotic haemoglobins remain unknown, their patterns of expression within plants suggest different functions. We examined the expression in transgenic plants of the two non-symbiotic haemoglobins from Arabidopsis using promoter fusions to a GUS reporter gene. The Arabidopsis GLB1 and GLB2 genes are likely to be functionally distinct. The class 2 haemoglobin gene (GLB2) is expressed in the roots, leaves and inflorescence and can be induced in young plants by cytokinin treatment in contrast to the class 1 gene (GLB1) which is active in germinating seedlings and can be induced by hypoxia and increased sucrose supply, but not by cytokinin treatment.     

Vigna mungo, V. radiata and V. unguiculata plants sampled in different agronomical–ecological–climatic regions of India are nodulated by Bradyrhizobium yuanmingense

Vigna mungo, Vigna radiata and Vigna unguiculata are important legume crops cultivated in India, but little is known about the genetic resources in native rhizobia that nodulate these species. To identify these bacteria, a core collection of 76 slow-growing isolates was built from root nodules of V. mungo, V. radiata and V. unguiculata plants grown at different sites within three agro-ecological-climatic regions of India. The genetic diversity of the bacterial collection was assessed by restriction fragment length polymorphism (RFLP) analysis of PCR-amplified DNA fragments of the 16S–23S rDNA intergenic spacer (IGS) region, and the symbiotic genes nifH and nodC. One rDNA IGS type grouped 91% of isolates, but more diversity was found at the symbiotic loci (17 symbiotic genotypes). Overall, no host plant specificity was shown, the three host plant species sharing common bradyrhizobial genotypes that represented 62% of the collection. Similarly, the predominant genotypes were found at most sampling sites and in all agro-ecological-climatic regions. Phylogenies inferred from IGS sequencing and multi-locus sequence analysis of the dnaK, glnII and recA genes indicated that all isolates but one were clustered with the Bradyrhizobium yuanmingense species. The nifH phylogeny also grouped the different nif haplotypes within a cluster including B. yuanmingense, except for one infrequent nif haplotype which formed a new lineage within the Bradyrhizobium genus. These results may reflect a long history of co-evolution between B. yuanmingense and Vigna spp. in India, while intra-species polymorphism detected in the symbiotic loci may be linked with the long history of diversification of B. yuanmingense coinciding with that of its host legumes.     

Heavy metal tolerant Metalliresistens boonkerdii gen. nov., sp. nov., a new genus in the family Bradyrhizobiaceae isolated from soil in Thailand

Bacterial strains from inoculated soybean field soil in Thailand were directly isolated using Bradyrhizobium japonicum selective medium (BJSM), on the basis of Zn²⁺ and Co²⁺ resistance of B. japonicum and B. elkanii. The isolates were classified into symbiotic and non-symbiotic groups by inoculation assays and Southern hybridization of nod and nif genes. In this study, a nearly full-length 16S rRNA gene sequence showed that the non-symbiotic isolates were more closely related to members of Rhodopseudomonas and to a number of uncultured bacterial clones than to members of Bradyrhizobium. Therefore, a polyphasic study was performed to determine the taxonomic positions of four representatives of the non-symbiotic isolates. Multilocus phylogenetic analysis of individual genes and a combination of the 16S rRNA and three housekeeping genes (atpD, recA and glnII) supported the placement of the non-symbiotic isolates in a different genus. The ability of heavy metal resistance in conjunction with phenotypic analyses, including cellular fatty acid content and biochemical characteristics, showed that the non-symbiotic isolates were differentiated from the other related genera in the family Bradyrhizobiaceae. Therefore, the non-symbiotic isolates represented a novel genus and species, for which the name Metalliresistens boonkerdii gen. nov., sp. nov. is proposed. The type strain is NS23 (= NBRC 106595^T = BCC 40155^T).     

Alleviation of water stress effects in cowpea by Bradyrhizobium spp. inoculation

Experiments were carried out to investigate the effects of different degrees of water stress on cowpea in the presence and absence of Bradyrhizobium spp. inoculation and to evaluate physiological responses to stress. The soil used was Yellow Latosol, pH 6.3 and the crop used was cowpea (Vigna unguiculata (L.) Walp.) cv. ‘IPA 204’. Stress was applied continuously by the control of matric potential (ψ _m ) through a porous cup. The lowered soil ψ _m had a direct effect on the N2 fixation, but the strains Bradyrhizobium introduced by inoculation in the cowpea plants were superior to the indigenous strain demonstrating the importance of inoculation in the stressed plants. At the more negative ψ _m plants inoculated with the strains EI 6 formed associations of greater symbiotic efficiency which helped the cowpea plants to withstand drought stress better than the strain BR 2001 and the uninoculated control. The leghaemoglobin concentration was not inhibited in the drought-stressed plants at ψ _m -70 kPa when inoculated with the strain EI 6, which confered a differential degree of drought resistance in plants. The ψ _w declined in the stressed plants reaching values of -1.0 MPa which was sufficient to cause disturbance in nodulation and biomass production. This revised version was published online in June 2006 with corrections to the Cover Date.     

Investigation into the ability of Gluconacetobacter sacchari to live as an endophyte in sugarcane

The relatively low numbers and sporadic pattern of incidence of the acetic acid bacterium Gluconacetobacter sacchari with the pink sugarcane mealybug (PSMB) Saccharicoccus sacchariCockerell (Homoptera: Pseudococcidae) over time and from different sugarcane-growing regions do not indicate that Glac. sacchari is a significant commensal of the PSMB, as has been previously proposed. This study was conducted to investigate the hypothesis that Glac. sacchari is, like its closest relative Glac. diazotrophicus, an endophyte of sugarcane (Saccharum officinarum L.). In this study, bothGlac. sacchari and Glac. diazotrophicus were isolated from internal sugarcane tissue, although the detection of both species was sporadic in all sugarcane-growing regions of Queensland tested. To confirm the ability of Glac. sacchari to live endophytically, an experiment was conducted in which the roots of micropropagated sugarcane plantlets were inoculated with Glac. sacchari, and the plantlets were subsequently examined for the presence of the bacterium in the stem cells. Pure cultures of Glac. sacchari were grown from homogenized surface sterilized sugarcane stems inoculated withGlac. sacchari.Electron microscopy was used to provide further conclusive evidence that Glac. sacchari lives as an endophyte in sugarcane. Scanning electron microscopy of (SEM) sugarcane plantlet stems revealed rod-shaped cells of Glac. sacchari within a transverse section of the plantlet stem cells. The numbers of bacterial cells inside the plant cell indicated a successful infection and colonization of the plant tissue. Using transmission electron microscopy, (TEM) bacterial cells were more difficult to find, due to their spatial separation. In our study, bacteria were mostly found singularly, or in groups of up to four cells inside intercellular spaces, although bacterial cells were occasionally found inside other cells.     

Genetic diversity and distribution of Bradyrhizobium and Azorhizobium strains associated with the herb legume Zornia glochidiata sampled from across Senegal

Herb legumes have great potential for rehabilitation of semi-arid degraded soils in Sahelian ecosystems as they establish mutualistic symbiosis with N₂-fixing rhizobia. A phylogenetic analysis was performed for 78 root nodule bacteria associated with the common Sahelian herb legume Zornia glochidiata Reichb ex DC in Senegal. Based on ITS (rDNA16S-23S) and recA sequences, these strains were shown to belong to the two genera Bradyrhizobium and Azorhizobium. Strains of this latter, although frequent, formed small and ineffective nodules and suggested a parasitism rather than a symbiotic association. A potential negative effect of Azorhizobium on Zornia growth was tested for when inoculated alone or in association with a Bradyrhizobium strain. Bradyrhizobium isolates were distributed in four groups. Groups A and B were two sister clades in a larger monophyletic group also including Bradyrhizobium liaoningense, Bradyrhizobium yuanmingense, and Bradyrhizobium japonicum. Strains of cluster D fell in a sister clade of the photosynthetic Bradyrhizobium sp. group, including ORS278, whereas group C appeared to be divergent from all known Bradyrhizobium clusters. Amplified fragment length polymorphism (AFLP) clustering was congruent with ITS and recA phylogenies, but displayed much more variability. However, within the main Bradyrhizobium clades, no obvious relationship could be detected between clustering and geographical origin of the strains. Each sub-cluster included strains sampled from different locations. Conversely, Azorhizobium strains showed a tendency in the phylogeny to group together according to the site of sampling. The predominance of ineffective Azorhizobium strains in the nodules of Zornia roots, the large Bradyrhizobium genetic diversity and the geographical genetic diversity pattern are explored.     

Identification and genetic diversity of Fusarium sacchari associated with pokkah boeng disease of sugarcane in Northeast Brazil

Isolates from the Fusarium fujikuroi species complex, mainly F. sacchari, have been reported to be the causal agents of pokkah boeng in sugarcane in Brazil. However, inadequate information was available on the occurrence and genetic diversity of F. sacchari in Northeast Brazil, which is a limiting factor on management. Thus, isolates of F. subglutinans sensu lato from sugarcane plants with symptoms of pokkah boeng were evaluated using the sexual cross-fertility to determine species. All the isolates produced black perithecia when they were crossed with the test isolates of F. sacchari. Three weeks after the crossing, the formation of fertile ascospores cirri was observed. Thirty-four isolates were self-sterile hermaphrodites, while 21 were fertile only as males. Five isolates were homothallic. The effective size [N_e(f)] of the population as a function of the frequency of hermaphrodites and female sterile strains was 95.5%. The F. sacchari isolates were separated into four genetic groups independent of geographic location. The mean of polymorphism among all populations was 79%, and the average unbiased genetic diversity (uh) was considered moderate (0.31). This study in addition to confirming that F. sacchari as the main species associated with pokkah boeng in sugarcane in Northeast Brazil, reveals the relationship of mating type and genetic diversity of F. sacchari. The unrestricted gene flow between regions is probably the best explanation for the low geographic correlation. This knowledge will help in the adoption of management measures with fungicides or resistant cultivars.     

In vitro generation of somaclonal variant plants of sugarcane for tolerance to Fusarium sacchari

Key message

A combination of in vitro culture and mutagenesis using ethyl methanesulfonate (EMS) followed by culture filtrate-mediated selection produced variant sugarcane plants tolerant and resistant to Fusarium sacchari.

Abstract

Eldana saccharina is a destructive pest of the sugarcane crop in South Africa. Fusarium sacchari PNG40 (a fungal strain harmful to E. saccharina) has the potential to be an endophytic biological control agent of the stalk borer. However, the fungus causes Fusarium stalk rot in sugarcane. In the current study, sugarcane plants tolerant and resistant to F. sacchari PNG40 were produced by exposing embryogenic calli to the chemical mutagen ethyl methanesulfonate (EMS), followed by in vitro selection during somatic embryogenesis and plantlet regeneration on media containing F. sacchari culture filtrates (CF). The incorporation of 100 ppm CF in the culture media at the embryo maturation stage, at germination, or at both, resulted in callus necrosis and consequent reduced plantlet yield. Subsequent trimming of the roots of regenerated plants and their exposure to 1,500 ppm CF served as a further selection treatment. Plants produced from EMS-treated calli displayed improved root re-growth in the presence of CF pressure compared with those from non-treated calli. The tolerance of CF-selected plants was confirmed in greenhouse tests by inoculation with F. sacchari PNG40, re-isolation of Fusarium spp. from undamaged tissue of asymptomatic plants and establishment of the identity of fungal isolates as PNG40 using molecular analysis. The restriction of PNG40 presence to the inoculation lesion in some plants suggested their resistance to the fungus. Genotypes exhibiting symptomless endophytic colonization by PNG40 were identified and will be utilised for testing biological control strategies against E. saccharina.     

Biocontrol of fungal root rot diseases of crop plants by the use of rhizobia and bradyrhizobia

Twenty-oneRhizobium andBradyrhizobium strains were testedin vitro against the mycelial growth of three pathogenic fungi on solid and liquid media. All tested rhizobia and bradyrhizobia significantly suppressed the growth of the three soil-borne root-infecting fungi (Fusarium solani, Macrophominia phasolina andRhizoctonia solani) either in the absence or presence of iron. This indicates that the siderophore played a minor role in the biocontrol potential ofRhizobium andBradyrhizobium against pathogenic fungi. Pot experiments revealed that the numbers of propagules causing disease after 4 weeks of planting varied with species and host plant. The three most activeRhizobium andBradyrhizobium strains (R. leguminosarum bv.phaseoli TAL 182,B. japonicum TAL 377 andBradyrhizobium sp. (lupin) WPBS 3211 D) tested under greenhouse conditions for their ability to protect one leguminous (soybean) and two non-leguminous (sunflower and okra) seedlings from root rot caused byFusarium solani, Macrophominia phaseolina andRhizoctonia solani provided significant suppression of disease severity compared with nonbacterized control in both leguminous and non-leguminous seedlings.Bradyrhizobium sp. (lupin) WPBS 3211 D provided the lowest degree of resistance against all the tested pathogens with all host plants. *** DIRECT SUPPORT *** A00EN058 00013     

Rhizobium phaseoli: A molecular genetics view

We have used molecular genetics techniques to analyze the structural and functional organization of genetic information ofRhizobium phaseoli, the symbiont of the common bean plantPhaseolus vulgaris. As in otherRhizobium species, the genome consists of the chromosome and plasmids of high molecular weight. Symbiotic determinants, nitrogen fixation genes as well as nodulation genes, are localized on a single replicon, the symbiotic (sym) plasmid. Thesym plasmid of differentR. phaseoli strains was transferred to anAgrobacterium tumefaciens strain cured of its native plasmids. In all cases, Agrobacterium transconjugants able to nodulate bean plants were obtained. Some of the transconjugants had the capacity to elicit an effective symbiosis. The genome ofR. phaseoli is complex, containing a large amount of reiterated DNA sequences. In mostR. pahseoli strains one of such reiterated DNA families corresponds to the nitrogenase structural genes (nif genes). A functional analysis of these genes suggested that the presence of reiteratednif genesis is related to the capacity of fixing atmospheric nitrogen in the symbiotic state. The presence of several repeated sequences in the genome might provide sites for recombination, resulting in genomic rearrangements. By analyzing direct descendants of a single cell in the laboratory, evidence of frequent genomic rearrangements inR. phaseoli was found. We propose that genomic rearrangements constitute the molecular basis of the frequent variability and loss of symbiotic properties in different Rhizobium strains.     

Phylogenetic perspectives of nitrogen-fixing actinobacteria

It was assumed for a long time that the ability to catalyze atmospheric nitrogen (diazotrophy) has a narrow distribution among actinobacteria being limited to the genus Frankia. Recently, the number of nitrogen fixation (nifH) genes identified in other non-Frankia actinobacteria has dramatically increased and has opened investigation on the origin and emergence of diazotrophy among actinobacteria. During the last decade, Mycobacterium flavum, Corynebacterium autotrophicum and a fluorescent Arthrobacter sp. have been reported to have nitrogenase activity, but these studies have not been further verified. Additional reports of nitrogen fixation by Agromyces, Microbacterium, Corynebacterium and Micromonospora isolated from root nodules of leguminous and actinorhizal plants have increased. For several actinobacteria, nitrogen fixation was demonstrated by the ability to grow on nitrogen-free medium, acetylene reduction activity, ¹⁵N isotope dilution analysis and identification of a nifH gene via PCR amplification. Moreover, the analyses of draft genome sequences of actinobacteria including Slackia exigua, Rothia mucilaginosa and Gordonibacter pamelaeae have also revealed the presence of nifH-like sequences. Whether these nifH sequences are associated with effective nitrogen fixation in these actinobacteria taxa has not yet been demonstrated. These genes may be vertically or horizontally transferred and be silent sequences. These ideas merit further investigation. This minireview presents a phylogenetic comparison of nitrogen fixation gene (nifH) with the aim of elucidating the processes underlying the evolutionary history of this catalytic ability among actinobacteria.     

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.