Variation of plastid and mitochondrial DNAs in the genus Hedysarum

Summary Plastid and mitochondrial DNAs from Hedysarum species of the western Mediterranean basin, H. spinosissimum ssp eu-spinosissimum, H. spinosissimum ssp capitatum, H. carnosum, H. coronarium and H. flexuosum, were compared by restriction endonuclease fragment analysis. ctDNA fragment patterns for ssp eu-spinosissimum and ssp capitatum were indistinguishable in different enzyme digests. An identical ctDNA variation was found in Hpa II digests with two Sardinian populations of ssp capitatum. Each of the two subspecies was characterized by specific mt DNA patterns with Pst I, Bam HI, Sma I and EcoRI. No variation was detected in populations of different geographical origins for a given subspecies. H. carnosum, H. coronarium and H. flexuosum generated specific ct and mt DNA patterns. Comparison of mitochondrial fragments indicated: — a strong homology between the two subspecies, — a closer homology among the three other diploids, each being closer to the other two than to H. spinosissimum subspecies — as was also the case for the plastid genomes.     

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.     

Diversity of Rhizobia isolated from various Hedysarum species

Cultural and physiological properties, serology, plasmid profiles and infective traits were determined for 23 strains of rhizobia isolated from various Hedysarum species: H. coronarium (common name: sulla) (16), H. carnosum (1), H. alpinum (3), H. mackenzii (2) and H. pallens (1) from Portugal, Spain, Tunisia, Alaska and Israel. Strains isolated from H. alpinum, H. mackenzii and H. pallens have slow growth rates on yeast-extract mannitol medium and were unable to nodulate H. coronarium plants, whereas the latter were effectively nodulated by all sixteen fast growing strains from sulla. Regardless of the country of origin all H. coronarium strains fell into one serogroup and were not serologically related with strains of other Hedysarum species. The RAPD (random amplified polymorphic DNA) fingerprinting method which was carried out on five H. coronarium and three H. alpinum strains allowed distinction to be made among serologically related rhizobia. No particular plasmid profile pattern was observed in relation to the host or geographical origin of the strains.     

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.     

Genotypic and symbiotic diversity studies of rhizobia nodulating Acacia saligna in Tunisia reveal two novel symbiovars within the Rhizobium leguminosarum complex and Bradyrhizobium

Acacia saligna is an invasive alien species that has the ability to establish symbiotic relationships with rhizobia. In the present study, genotypic and symbiotic diversity of native rhizobia associated with A. saligna in Tunisia were studied. A total of 100 bacterial strains were selected and three different ribotypes were identified based on rrs PCR-RFLP analysis. Sequence analyses of rrs and four housekeeping genes (recA, atpD, gyrB and glnII) assigned 30 isolates to four putative new lineages and a single strain to Sinorhizobium meliloti. Thirteen slow-growing isolates representing the most dominant IGS (intergenic spacer) profile clustered distinctly from known rhizobia species within Bradyrhizobium with the closest related species being Bradyrhizobium shewense and Bradyrhizobium niftali, which had 95.17% and 95.1% sequence identity, respectively. Two slow-growing isolates, 1AS28L and 5AS6L, had B. frederekii as their closest species with a sequence identity of 95.2%, an indication that these strains could constitute a new lineage. Strains 1AS14I, 1AS12I and 6AS6 clustered distinctly from known rhizobia species but within the Rhizobium leguminosarum complex (Rlc) with the most closely related species being Rhizobium indicum with 96.3% sequence identity. Similarly, the remaining 11 strains showed 96.9 % and 97.2% similarity values with R. changzhiense and R. indicum, respectively. Based on nodC and nodA phylogenies and cross inoculation tests, these 14 strains of Rlc species clearly diverged from strains of Sinorhizobium and Rlc symbiovars, and formed a new symbiovar for which the name sv. “salignae” is proposed. Bacterial strains isolated in this study that were taxonomically assigned to Bradyrhizobium harbored different symbiotic genes and the data suggested a new symbiovar, for which sv. “cyanophyllae” is proposed. Isolates formed effective nodules on A. saligna.     

Genetic diversity of fast-growing rhizobia that nodulate soybean ( Glycine max L. Merr)

The fast-growing Rhizobium sp. strain NGR234, isolated from Papua New Guinea, and 13 strains of Sinorhizobium fredii, isolated from China and Vietnam, were fingerprinted by means of RAPD, REP, ERIC and ARDRA. ERIC, REP and RAPD markers revealed a considerable genetic diversity among fast-growing rhizobia. Chinese isolates showed higher levels of diversity than those strains isolated from Vietnam. ARDRA analysis revealed three different genotypes among fast-growing rhizobia that nodulate soybean, even though all belonged to a subcluster that included Sinorhizobium saheli and Sinorhizobium meliloti. Among S. fredii rhizobia, two strains, SMH13 and HH303, might be representatives of other species of nitrogen-fixing organisms. Although restriction analysis of the nifD–nifK intergenic DNA fragment confirmed the unique nature of Rhizobium sp. strain NGR234, several similarities between Rhizobium sp. strain NGR234 and S. fredii USDA257, the ARDRA analysis and the full sequence of the 16S rDNA confirmed that NGR234 is a S. fredii strain. In addition, ARDRA analysis and the full sequence of the 16S rDNA suggested that two strains of rhizobia might be representatives of other species of rhizobia.     

Molecular diversity,effectiveness and competitiveness of indigenous rhizobial population infecting mungbean <Emphasis Type="Italic">Vigna radiata</Emphasis> (L. Wilczek) under semi-arid conditions

Nodules from mungbean crop raised for the first time at Ram Dhan Singh (RDS) farm of Chaudhary Charan Singh (CCS) Haryana Agricultural University, Hisar were collected from 17 different locations. Twenty-five mungbean rhizobia were isolated and authenticated by plant infection test. DNA of all these rhizobia was extracted purified and amplified using enterobacterial repetitive intergenic consensus (ERIC) primers. All the mungbean rhizobial isolates were clustered into 4 groups at 65% of similarity and were further divided into 17 subclusters at 80% of similarity. All the 4 types of rhizobia were not present at any of the location and group 2 or 4 rhizobia were invariably present. Efficacy of these rhizobia in terms of nodulation, nitrogen uptake and chlorophyll a fluorescence was determined under pot culture conditions. Strain MB 307 showed maximum nitrogen uptake of 31.9 mg N plant⁻¹ followed by strain MB 1205, MB 1206(2), MB 308, MB 1524 and strain MB 1521 was found to be the least efficient in terms of N 2 fixation. Nodule occupancy by different rhizobia ranged from 5.5 to 40.3%. Most of the strains belonging to the 2nd group which clustered maximum number of strains were comparatively better competitors and formed 19.5–40.3% of the nodules and were also effective. Isolate MB 307, the most efficient strain, was found to have nodule occupancy of 31.5%. Such type of predominant, efficient and better competitor strains should be selected for enhancing nodule competitiveness.     

Serological grouping of indigenous Bradyrhizobium sp. (Arachis) isolated from various soils of Thailand

ELISA and antibody adsorption tests were applied to determine the minimal somatic antigen constitution of 243 strains of Bradyrhizobium sp. (Arachis) using 12 antisera. The 243 indigenous bradyrhizobial isolates were from 15 sites in four regions of Thailand. A total of 29 serogroups were identified. Most (80%) of the isolates tested had at least one heat-stable antigen in common with strain 280A, forming a so-called 280A serocluster. At 11 of 15 sites tested, 53 to 100% of the isolates fell into one or two predominant serogroups. The serological properties of the indigenous bradyrhizobia were not related to the cropping history of the cultivated fields from which they were isolated.Contribution from the Agricultural Research Organization, The Volcani Center, Bet Dagan, Israel; No. 3608-E, 1992 series.     

Diversity of rhizobia nodulating wild shrubs of Sicily and some neighbouring islands

Legume shrubs have great potential for rehabilitation of semi-arid degraded soils in Mediterranean ecosystems as they establish mutualistic symbiosis with N-fixing rhizobia. Eighty-eight symbiotic rhizobia were isolated from seven wild legume shrubs native of Sicily (Southern Italy) and grouped in operational taxonomic units (OTU) by analysis of the ribosomal internal transcribed spacer (ITS) polymorphism. Partial sequencing of 16S rRNA gene of representative isolates of each OTU revealed that most Genisteae symbionts are related to Bradyrhizobium canariense, B. japonicum and B. elkanii. Teline monspessulana was the only Genistea nodulated by Mesorhizobium strains, and Anagyris foetida (Thermopsideae) was promiscuosly nodulated by Rhizobium, Mesorhizobium, Agrobacterium and Bradyrhizobium strains. Analysis of the nodulation gene nodA assigned most Mediterranean Genisteae bradyrhizobia to clade II but also to clades IV, I and III, which included, so far, sequences of (sub)tropical and Australian isolates. The high diversity and low host specificity observed in most wild legumes isolates suggest that preferential associations may establish in the field depending on differences in the benefits conferred to the host and on competition ability. Once identified, these beneficial symbiosis can be exploited for rehabilitation of arid, low productive and human-impacted soils of the Mediterranean countries.     

Ectomycorrhizal symbiosis enhanced the efficiency of inoculation with two Bradyrhizobium strains and Acacia holosericea growth

Abstract Two strains of Bradyrhizobium sp., Aust 13C and Aust 11C, were dually or singly inoculated with an ectomycorrhizal fungus, Pisolithus albus to assess the interactions between ectomycorrhizal symbiosis and the nodulation process in glasshouse conditions. Sequencing of strains Aust 13C and Aust 11C confirmed their previous placement in the genus Bradyrhizobium. After 4 months culture, the ectomycorrhizal symbiosis promoted plant growth and the nodulation process of both Bradyrhizobium strains, singly or dually inoculated. PCR/RFLP analysis of the nodules randomly collected in each treatment with Aust 13C and/or Aust 11C: (1) showed that all the nodules exhibited the same patterns as those of the Bradyrhizobium strains, and (2) did not detect contaminant rhizobia. When both Bradyrhizobium isolates were inoculated together, but without P. albus IR100, Aust 11C was recorded in 13% of the treated nodules compared to 87% for Aust 13C, whereas Aust 11C and Aust 13C were represented in 20 and 80% of the treated nodules, respectively, in the ectomycorrhizal treatment. Therefore Aust 13C had a high competitive ability and a great persistence in soil. The presence of the fungus did not significantly influence the frequencies of each Bradyrhizobium sp. root nodules. Although the mechanisms remain unknown, these results showed that the ectomycorrhizal and biological nitrogen-fixing symbioses were very dependent on each other. From a practical point of view, the role of ectomycorrhizal symbiosis is of great importance to N₂ fixation and, consequently, these kinds of symbiosis must be associated in any controlled inoculation.     

Novel Endophytes of Rice form a Taxonomically Distinct Subgroup of Serratia marcescens

Six endophytic strains isolated from surface-sterilized rice roots and stems of different rice varieties grown in the Philippines were characterized. They were analyzed by physiological and biochemical tests, SDS-PAGE of whole-cell protein patterns, DNA-DNA hybridization and 16S rDNA sequencing. SDS-PAGE of whole-cell patterns showed that the six isolates fell into two subgroups which were similar but not identical in protein patterns to S. marcescens. The phylogenetic analysis of 16S rDNA sequences of two representative strains IRBG 500 and IRBG 501 indicated that they were closely related to S. marcescens(more than 99% identity). Physiological and biochemical tests corroborated that the isolates were highly related to each other and to S. marcescens. In cluster analysis, all six isolates were clustered together at 93% similarity level and grouped closely with Serratia marcescens at 86% similarity level. DNA-DNA hybridization studies revealed that the isolates shared high similarity levels with S. marcescens(≥86% DNA-DNA binding), indicating they belong to the same species. However, the isolates differed in several biochemical characteristics from the type strain. They produce urease and utilize urea and L(+) sorbose as a substrate, which is different from all known Serratia reference strains. These results suggest that the six endophytic isolates represent a novel, non-pigmented subgroup of S. marcescens.     

Characterization of Bradyrhizobium species isolated from root nodules of Cytisus villosus grown in Morocco

From a total of 73 bacterial strains isolated from root nodules of Cytisus villosus grown in soils of the central-western region of the Moroccan Rif, 68 strains clustered into 19 repetitive extragenic palindromic (REP)-polymerase chain reaction (PCR) groups. The nearly complete 16S rRNA gene sequence from each strain showed they were closely related to members of the genus Bradyrhizobium of the Alphaproteobacteria, but affiliation at the species level was not clear. Sequencing of the housekeeping genes glnII and recA, and their concatenated phylogenetic analysis showed that 11 out of the 19 strains belong to Bradyrhizobium canariense and that another three strains were Bradyrhizobium japonicum. The remaining five strains represented new lineages within the genus Bradyrhizobium since they were not identified with any previously described species. Sequencing of the symbiotic nodC and nifH genes from each bradyrhizobial strain revealed they were all similar to those of the strains included in biovar genistearum.     

Phylogenetic diversity of rhizobia associated with horsegram [Macrotyloma uniflorum (Lam.) Verdc.] grown in South India based on glnII, recA and 16S-23S intergenic sequence analyses

Horsegram [Macrotyloma uniflorum (Lam.) Verdc.) is an important grain legume and fodder crop in India. Information on root nodule endosymbionts of this legume in India is limited. In the present study, 69 isolates from naturally occurring root nodules of horsegram collected from two agro-eco-climatic regions of South India was analyzed by generation rate, acid/alkali reaction on YMA medium, restriction fragment length polymorphism analysis of 16S-23S rDNA intergenic spacer region (IGS), and sequence analyses of IGS and housekeeping genes glnII and recA. Based on the rDNA IGS RFLP by means of three restriction enzymes rhizobia were grouped in five clusters (I–V). By sequence analysis of 16S-23S rDNA IGS identified genotypes of horsegram rhizobia were distributed into five divergent lineages of Bradyrhizobium genus which comprised (I) the IGS type IV rhizobia and valid species B. yuanmingense, (II) the strains of IGS type I and Bradyrhizobium sp. ORS 3257 isolated from Vigna sp., (III) the strains of the IGS type II and Bradyrhizobium sp. CIRADAc12 from Acacia sp., (IV) the IGS type V strains and Bradyrhizobium sp. genospecies IV, and (V) comprising genetically distinct IGS type III strains which probably represent an uncharacterized new genomic species. Nearly, 87% of indigenous horsegram isolates (IGS types I, II, III, and V) could not be related to any other species within the genus Bradyrhizobium. Phylogeny based on housekeeping glnII and recA genes confirmed those results found by the analysis of the IGS sequence. All the isolated rhizobia nodulated Macrotyloma sp. and Vigna spp., and only some of them formed nodules on Arachis hypogeae. The isolates within each IGS type varied in their ability to fix nitrogen. Selection for high symbiotic effective strains could reward horsegram production in poor soils of South India where this legume is largely cultivated.     

Siderophore production byBradyrhizobium spp. strains nodulating groundnut

EighteenBradyrhizobium spp. strains, fourRhizobium spp. strains and oneAzorhizobium caulinodans strain were grown under Fe limitation and assayed for siderophore production. It was further assessed if Fe accumulation in two groundnut cultivars was influenced by inoculant strain or nitrate fertilisation. Growth ofBradyrhizobium spp. strains nodulating groundnut was slow with mean generation times from 11–24 h. All strains, except MAR 967, showed a reduced growth rate when deprived of Fe; none of the strains showed starvation at 1 M Fe. In the CAS (chrome azurol S)-agar assay, all strains, which formed colonies, produced siderophores as visualised by orange halos around the colonies on blue plates.Bradyrhizobium strains produced much smaller halos than the referenceRhizobium meliloti strain. In the CAS-supernatant assay, all strains, except MAR 967, gave positive responses (measured as absorbance at 630 nm) when supernatants of Fe-depleted cultures were assayed with CAS-indicator complex in comparison with Fe-supplemented cultures. Responses of all fourRhizobium spp. strains were large, while responses of allBradyrhizobium strains, exceptB. japonicum MAR 1491 (USDA 110), were small and mostly insignificant. A small response, i.e. a low Fe-scavenging ability, implies either the production of small quantities of siderophores or the production of low affinity siderophores. Among theBradyrhizobium strains, MAR 1574 and MAR 1587 gave the largest responses taken over the two assays. Fe accumulation in groundnut cultivar Falcon was seven times larger than in cultivar Natal Common. No correlation was found between the quantity of nodule tissue and Fe accumulation, making it unlikely that bacteroids are involved in Fe acquisition by groundnuts. Nitrate-fertilised plants accumulated significantly more Fe, suggesting involvement of nitrate reductase in Fe assimilation in groundnut. The two most successful Fe-scavengingBradyrhizobium spp. strains were also the most effective in nodulating groundnut, the reverse also being true. Strain MAR 967, with the lowest Fe requirement, produced the largest nodule dry weight. These data indicate that improved Fe scavenging properties and/or reduced Fe requirement improve rhizospheric growth and with that nodulation effectiveness.     

Wild peanut Arachis duranensis are nodulated by diverse and novel Bradyrhizobium species in acid soils

Aiming at learning the microsymbionts of Arachis duranensis, a diploid ancestor of cultivated peanut, genetic and symbiotic characterization of 32 isolates from root nodules of this plant grown in its new habitat Guangzhou was performed. Based upon the phylogeny of 16S rRNA, atpD and recA genes, diverse bacteria belonging to Bradyrhizobium yuanmingense, Bradyrhizobium elkanii, Bradyrhizobium iriomotense and four new lineages of Bradyrhizobium (19 isolates), Rhizobium/Agrobacterium (9 isolates), Herbaspirillum (2 isolates) and Burkholderia (2 isolates) were defined. In the nodulation test on peanut, only the bradyrhizobial strains were able to induce effective nodules. Phylogeny of nodC divided the Bradyrhizobium isolates into four lineages corresponding to the grouping results in phylogenetic analysis of housekeeping genes, suggesting that this symbiosis gene was mainly maintained by vertical gene transfer. These results demonstrate that A. duranensis is a promiscuous host preferred the Bradyrhizobium species with different symbiotic gene background as microsymbionts, and that it might have selected some native rhizobia, especially the novel lineages Bradyrhizobium sp. I and sp. II, in its new habitat Guangzhou. These findings formed a basis for further study on adaptation and evolution of symbiosis between the introduced legumes and the indigenous rhizobia.     

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.     

Signal molecules in the peanut–bradyrhizobia interaction

Main nodulation signal molecules in the peanut–bradyrhizobia interaction were examined. Flavonoids exuded by Arachis hypogaea L. cultivar Tegua were genistein, daidzein and chrysin, the latest being released in lower quantities. Thin layer chromatography analysis from genistein-induced bacterial cultures of three peanut bradyrhizobia resulted in an identical Nod factor pattern, suggesting low variability in genes involved in the synthesis of these molecules. Structural study of Nod factor by mass spectrometry and NMR analysis revealed that it shares a variety of substituents with the broad-host-range Rhizobium sp. NGR234 and Bradyrhizobium spp. Nodulation assays in legumes nodulated by these rhizobia demonstrated differences between them and the three peanut bradyrhizobia. The three isolates were classified as Bradyrhizobium sp. Their fixation gene nifD and the common nodulation genes nodD and nodA were also analyzed. Accession numbers: AY427207, EF202193, EF158295 (16S rRNA gene of strains NLH25, NOD31 and NDEHE, respectively); DQ295199, DQ295200, DQ295201 (Partial nifD gene sequences of strains NLH25, NOD31 and NDEHE, respectively).     

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.     

Fitness variation among host species and the paradox of ineffective rhizobia

Legumes can preferentially select beneficial rhizobial symbionts and sanction ineffective strains that fail to fix nitrogen. Yet paradoxically, rhizobial populations vary from highly beneficial to ineffective in natural and agricultural soils. Classic models of symbiosis focus on the single dimension of symbiont cost‐benefit to sympatric hosts, but fail to explain the widespread persistence of ineffective rhizobia. Here, we test a novel framework predicting that spatio‐temporal and community dynamics can maintain ineffective strains in rhizobial populations. We used clonal and multistrain inoculations and quantitative culturing to investigate the relative fitness of four focal Bradyrhizobium strains varying from effective to ineffective on Acmispon strigosus. We found that an ineffective Bradyrhizobium strain can be sanctioned by its native A. strigosus host across the host's range, forming fewer and smaller nodules compared to beneficial strains. But the same ineffective Bradyrhizobium strain exhibits a nearly opposite pattern on the broadly sympatric host Acmispon wrangelianus, forming large nodules in both clonal and multistrain inoculations. These data suggest that community‐level effects could favour the persistence of ineffective rhizobia and contribute to variation in symbiotic nitrogen fixation.