Symbiotic Competence Research Articles

Molecular Analysis of Genes in Nostoc punctiforme Involved in Pilus Biogenesis and Plant Infection

Hormogonia are the infective agents in many cyanobacterium-plant symbioses. Pilus-like appendages are expressed on the hormogonium surface, and mutations in pil-like genes altered surface piliation and reduced symbiotic competency. This is the first molecular evidence that pilus biogenesis in a filamentous cyanobacterium requires a type IV pilus system.

Root and root hair mechanisms that confer symbiotic competence for nodulation in acidic soils within Medicago species: a holistic model

Three experiments were undertaken to investigate the mechanisms used by the annual medic species, Medicago murex (Murex medic) to achieve nodulation more rapidly in acidic soils than the perennial species Medicago sativa (lucerne). In experiment 1, numbers and locations of root hairs on the primary roots of medic and lucerne were determined from plants grown in soil of pH 4.3 and 7.0. Experiment 2 enumerated the numbers of Sinorhizobium medicae (rhizobia) associated with the roots of medic and lucerne when grown in an acidic soil. Experiment 3 used a GFP-marked strain of rhizobia to determine the localised distribution of rhizobia along various zones of the primary roots of medic and lucerne in soil of pH 4.3 and 7.0. When grown in an acidic soil, medic produced 60% more root hairs/mm root along the primary root axis compared with lucerne, from 7 days after sowing. In soil of low pH, medic also had higher numbers of rhizobia associated with its entire root system than lucerne, with about 103 cfu/cm root throughout the 24-day period of the experiment, compared with lucerne for which rhizobial numbers decreased from 102 to 11 cfu/cm root over the same time period. When the intensity of fluorescence emitted by a GFP-marked transconjugant of rhizobia was measured at localised zones along the primary root of medic and lucerne grown in the acidic soil, it was 1.8-fold higher from the roots of medic than lucerne at 7 days after sowing, indicating higher numbers of rhizobia along medic roots. Hence, the numbers of rhizobia associated with the entire root system, and those colonised on localised zones of the primary root of medic were both higher than that of lucerne. These fundamental differences between the 2 species, combined with the earlier finding that lucerne tends to acidify its rhizosphere more than medic, allowed us to construct conceptual models that attempt to explain the increased symbiotic competency of medic compared with lucerne, and the different nodulation responses between medic and lucerne at low pH.

Vibrio fischeri flagellin A is essential for normal motility and for symbiotic competence during initial squid light organ colonization.

The motile bacterium Vibrio fischeri is the specific bacterial symbiont of the Hawaiian squid Euprymna scolopes. Because motility is essential for initiating colonization, we have begun to identify stage-specific motility requirements by creating flagellar mutants that have symbiotic defects. V. fischeri has six flagellin genes that are uniquely arranged in two chromosomal loci, flaABCDE and flaF. With the exception of the flaA product, the predicted gene products are more similar to each other than to flagellins of other Vibrio species. Immunoblot analysis indicated that only five of the six predicted proteins were present in purified flagella, suggesting that one protein, FlaF, is unique with respect to either its regulation or its function. We created mutations in two genes, flaA and flaC. Compared to a flaC mutant, which has wild-type flagellation, a strain having a mutation in the flaA gene has fewer flagella per cell and exhibits a 60% decrease in its rate of migration in soft agar. During induction of light organ symbiosis, colonization by the flaA mutant is impaired, and this mutant is severely outcompeted when it is presented to the animal as a mixed inoculum with the wild-type strain. Furthermore, flaA mutant cells are preferentially expelled from the animal, suggesting either that FlaA plays a role in adhesion or that normal motility is an advantage for retention within the host. Taken together, these results show that the flagellum of V. fischeri is a complex structure consisting of multiple flagellin subunits, including FlaA, which is essential both for normal flagellation and for motility, as well as for effective symbiotic colonization.

Identification and use of actinomycetes for enhanced nodulation of soybean co-inoculated with Bradyrhizobium japonicum.

The utilization of actinomycetes as potential soybean (Glycine max (L.)) co-inoculants was evaluated. Soil samples from Carbondale and Belleville, Ill., were used to inoculate pre-germinated soybean plants to determine antibiotic sensitivity in the native Bradyrhizobium japonicum population. Sensitivity was in the order kanamycin > tetracycline > oxytetracycline > rifampicin > neomycin. Antagonism by five actinomycete cultures toward seven test strains of B. japonicum was also assessed. The ranking average inhibition (across all seven B. japonicum strains) by these actino mycetes was Streptomyces kanamyceticus = Streptomyces coeruleoprunus > Streptomyces rimosus > Streptomyces sp. > Amy colatopsis mediterranei. Ten antibiotic combinations were used to isolate antibiotic-resistant mutants of B. japonicum I-110 and 3I1B-110 via successive cycles of mutation. Eighty-one antibiotic-resistant strains were isolated and tested for symbiotic competency; nine of which were selected for further characterization in a greenhouse pot study. Few differences in nodule number were caused by these treatments. Nodule occupancy varied from 0% to 18.3% when antibiotic-resistant strains of B. japonicum were used as the sole inoculants. However, when three mutant strains of B. japonicum were co-inoculated with S. kanamyceticus, significant increases in nodule occupancy (up to 55%) occurred. Increases in shoot nitrogen composition (27.1%-40.9%) were also caused by co-inoculation with S. kanamyceticus.

Cloning and characterization of a second acid phosphatase from Sinorhizobium meliloti strain 104A14.

Sinorhizobium meliloti has two nonspecific periplasmic acid phosphatases. The NapD enzyme has been previously described, and a second acid phosphatase, NapE, is described in this report. NapE was partially purified from an S. meliloti napD mutant and characterized with respect to molecular mass and substrate range. As predicted from SDS-PAGE analysis, the subunit molecular mass of NapE is approximately 35.8 kDa and gel filtration experiments estimated the native molecular mass to be approximately 70 kDa, indicating that the active enzyme is a homodimer. NapE demonstrated significant activity with p-nitrophenyl phosphate, phenyl phosphate, and alpha-naphthyl-phosphate. The pH optimum was between 4.5 and 5.0. The gene encoding NapE was also sequenced and the inferred amino acid sequence from the predicted ORF was found to be 60% identical and 75% similar to that encoded by napD. An S. meliloti napE mutant was constructed and assessed for symbiotic competence. This mutant did not differ from the wild-type parent strain in nodulation and symbiotic efficiency.

An overview of the genome of Nostoc punctiforme, a multicellular, symbiotic cyanobacterium.

Nostoc punctiforme is a filamentous cyanobacterium with extensive phenotypic characteristics and a relatively large genome, approaching 10 Mb. The phenotypic characteristics include a photoautotrophic, diazotrophic mode of growth, but N. punctiforme is also facultatively heterotrophic; its vegetative cells have multiple developmental alternatives, including terminal differentiation into nitrogen-fixing heterocysts and transient differentiation into spore-like akinetes or motile filaments called hormogonia; and N. punctiforme has broad symbiotic competence with fungi and terrestrial plants, including bryophytes, gymnosperms and an angiosperm. The shotgun-sequencing phase of the N. punctiforme strain ATCC 29133 genome has been completed by the Joint Genome Institute. Annotation of an 8.9 Mb database yielded 7432 open reading frames, 45% of which encode proteins with known or probable known function and 29% of which are unique to N. punctiforme. Comparative analysis of the sequence indicates a genome that is highly plastic and in a state of flux, with numerous insertion sequences and multilocus repeats, as well as genes encoding transposases and DNA modification enzymes. The sequence also reveals the presence of genes encoding putative proteins that collectively define almost all characteristics of cyanobacteria as a group. N. punctiforme has an extensive potential to sense and respond to environmental signals as reflected by the presence of more than 400 genes encoding sensor protein kinases, response regulators and other transcriptional factors. The signal transduction systems and any of the large number of unique genes may play essential roles in the cell differentiation and symbiotic interaction properties of N. punctiforme.

Genealogy of legume-Rhizobium symbioses.

Accumulating evidence suggests that lateral transfer of nodulation capacity is an important driving force in symbiotic evolution. As a consequence, many distantly related soil bacteria have acquired the capacity to invade plants and fix nitrogen within them. In addition to these proteins required for bacteroid development and nitrogen fixation, core symbiotic competence seems to require flavonoids, NodD proteins, lipochitooligosaccharidic Nod-factors, extra-cellular polysaccharides, as well as various exported proteins. Plants respond to different levels and combinations of these substances in species specific ways. After contact has been initiated by flavonoids and NodD proteins, constant signal exchange fine-tunes these symbiotic demands, especially to overcome defence reactions.

The periplasmic, group III catalase of Vibrio fischeri is required for normal symbiotic competence and is induced both by oxidative stress and by approach to stationary phase.

The catalase gene, katA, of the sepiolid squid symbiont Vibrio fischeri has been cloned and sequenced. The predicted amino acid sequence of KatA has a high degree of similarity to the recently defined group III catalases, including those found in Haemophilus influenzae, Bacteroides fragilis, and Proteus mirabilis. Upstream of the predicted start codon of katA is a sequence that closely matches the consensus sequence for promoters regulated in Escherichia coli by the alternative sigma factor encoded by rpoS. Further, the level of expression of the cloned katA gene in an E. coli rpoS mutant is much lower than in wild-type E. coli. Catalase activity is induced three- to fourfold both as growing V. fischeri cells approach stationary phase and upon the addition of a small amount of hydrogen peroxide during logarithmic growth. The catalase activity was localized in the periplasm of wild-type V. fischeri cells, where its role could be to detoxify hydrogen peroxide coming from the external environment. No significant catalase activity could be detected in a katA null mutant strain, demonstrating that KatA is the predominately expressed catalase in V. fischeri and indicating that V. fischeri carries only a single catalase gene. The catalase mutant was defective in its ability to competitively colonize the light organs of juvenile squids in coinoculation experiments with the parent strain, suggesting that the catalase enzyme plays an important role in the symbiosis between V. fischeri and its squid host.

Host-derived amino acids support the proliferation of symbiotic bacteria.

Animals are typically colonized by diverse bacterial symbionts, many of which are commensal and, in numerous cases, even essential for their host's proper development and growth. In exchange, the host must supply a sufficient array and quantity of nutrients to support the proliferation and persistence of its microbial community. In this investigation, we have examined such a nutritional environment by determining the symbiotic competence of auxotrophic mutants of the bioluminescent bacterium Vibrio fischeri, and have demonstrated that the host squid Euprymna scolopes provides at least 9 aa to the growing culture of symbiotic V. fischeri present in its light-emitting organ. We also collected and analyzed the extracellular fluid from this organ, in which the symbionts reside, and confirmed that it contained significant amounts of amino acids. The combined results suggested that host-derived free amino acids, as well as peptides or proteins, are a source of the amino acids that support the growth of the symbionts. This work describes a technique to sample the symbionts and their surrounding environment without contamination by host tissue components and, in combination with molecular genetic studies, allows the characterization of the nutritional conditions that support a cooperative animal-bacterial symbiosis.

Biochemical and symbiotic properties of histidine‐requiring mutants of Rhizobium leguminosarum biovar trifolii

A perturbation of the histidine biosynthetic pathway in legume microsymbionts can abolish their symbiotic competence. Twenty-one histidine-requiring (His - ) mutants were isolated from berseem clover-nodulating, symbiotically-competent (Nod + , Fix + ) Rhizobium leguminosarum bv. 'trifolii' strain RTH 48 Sm r by N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) mutagenesis followed by enrichment. These mutants were analysed for their biochemical defect and the corresponding effect, if any, on their symbiotic abilities. Cross-feeding, supplementation and enzymatic studies identified three types of mutants. Group I mutants, His-2 and His-12, grew with histidine supplementation but not with the addition of either L-histidinul or L-histidinol phosphate to the medium; they lacked histidinol dehydrogenase (EC 1.1.1.23) activity and consequently formed only ineffective, or anon-fixing' nodules. Group 2 mutant, His-17, grew when supplemented with either L-histidinol or L-histidine, had low histidinol phosphate phosphatase (EC 3.1.3.15) activity (37% of wild-type), and consequently failed to nodulate berseem clover. Group 3, the remaining 18 mutants, grew when supplemented with L-histidinul phosphate, L-histidinul or histidine, and did not nodulate. Typically, reversion rates were between 10 -7 and 10 -8 , Defects in early steps of the pathway abolished nodulating ability, whereas lesions in the last step did not. The last step, however, as required for symbiotic nitrogen fixation. It is hypothesized that histidine may be supplied by the host in sufficient quantity for nodulation hy histidinol dehydrogenase mutants to occur whereas the amount provided in the nodule may be insufficient to support bacteroid development and nitrogen fixation.

Differentiation of Clover Rhizobium Isolated from Biosolids‐Amended Soils with Varying pH

AbstractMetal contamination may alter the diversity of microbes residing in soil. The genetic structure and phenotypic characteristics of clover Rhizobium isolated from contaminated and control soils were compared. Plant infection and symbiotic competence tests were used for phenotypic characterization. Variation across isolates in fingerprint patterns determined with primers for repetitive extragenic palindromic (REP) sequences and the polymerase chain reaction (PCR) were used for genetic characterization. Two phenotypic groups of effective and ineffective isolates were identified using the symbiotic effectiveness test. Soil pH was the primary factor influencing this phenotypic characteristic. Effective isolates were associated with higher soil pH and ineffective isolates were associated with lower soil pH regardless of soil metal content. The isolates were genetically diverse. The variation of isolates from the different soils overlapped, indicating that neither the heavy metals nor the low soil pH resulted in the selection of a single genotype. Isolates from the most heavily contaminated soils were more variable than isolates from control soils. Soil pH, and not heavy metal content, was important in the selection of rhizobia that formed ineffective N2‐fixing symbioses.

Construction and symbiotic competence of a luxA-deletion mutant of Vibrio fischeri

Bioluminescence by the squid Euprymna scolopes requires colonization of its light organ by the symbiotic luminous bacterium Vibrio fischeri. Investigation of the genetic determinants underlying bacterial symbiotic competence in this system has necessitated the continuing establishment and application of molecular genetic techniques in V. fischeri. We developed a procedure for the introduction of plasmid DNA into V. fischeri by electroporation, and isolated a mutant strain that overcame the apparent restriction barrier between V. fischeri and Escherichia coli. Using the technique of electroporation in combination with that of gene replacement, we constructed a non-luminous strain of V. fischeri (ΔluxA::erm). In addition, we used the transducing phage rp-1 for the first time to transfer a chromosomal antibiotic resistance marker to another strain of V. fischeri. The luxA mutant was able to colonize E. scolopes as quickly and to the same extent as wild type. This result suggested that, at least during the initial stages of colonization, luminescence per se is not an essential factor for the symbiotic infection.

Symbiotic competence, genetic diversity and plasmid profiles of Egyptian isolates of a Rhizobium species from Leucaena leucocephala (Lam.) Dewit

L.D. KUYKENDALL, D.M. SWELIM, F.M. HASHEM, S.M. ABDEL-WAHAB AND N.I. HEGAZI. 1996. There is considerable interest in improving nitrogen fixation in tropical legume trees to increase soil fertility, particularly in developing countries. To provide information needed for the development of improved strains, characterization of strains of Leucaena-nodulating Rhizobium was performed. Thirteen strains were isolated from root nodules of Leucaena leucocephala grown in different geographical regions in Egypt. Plasmid DNA profile groups, including identification of symbiosis-controlling plasmids, were defined. Symbiotic competence was determined in plant tests and some strains were perhaps more symbiotically proficient at fixing nitrogen with L. leucocephala than were reference strains. The genetic diversity of these strains was determined by RFLP analysis of total DNAs using, as probes, six arbitrarily selected cosmid clones from a gene library of strain TAL 1145, a well-characterized reference strain. There were four plasmid profile groups which did not correlate with either symbiotic competence or RFLP analysis. RFLP analysis, unlike plasmid profiles, permitted a determination that all 13 Egyptian strains were evidently homogeneous and quite distinct from previously studied Leucaena-nodulating Rhizobium as represented by reference strains.

Symbiotic ineffectiveness of trpCD deletion mutants of Bradyrhizobium japonicum

Genes responsible for tryptophan biosynthesis are thought to be essential for symbiotic competence of Bradyrhizobium japonicum. The B. japonicum trpCD region, cloned as a 2.8-kb HindIII- BamHI fragment, had an internal 1.2-kb SmaI fragment that was deleted and replaced in vitro with interposon omega carrying a gene specifying streptomycin and spectinomycin resistance. Site-directed marker exchange mutagenesis of the B. japonicum genome was performed in vivo and the construction of deletion mutants was confirmed by Southern blot analysis of the DNA of the exconjugants. Since the trpCD deletion mutants grew on indole they evidently were unaffected in expression of trpAB and we interpret these data as indicating that the organization of the genes for tryptophan biosynthesis in B. japonicum differs from that of enterics. The new Trp − deletion mutants lacked activities for the trpC- and trpD-encoded enzymes, indole glycerol phosphate synthase (EC 4.1.1.48) and phosphoribosyl anthranilate transferase (EC 2.4.2.18), respectively, but had a higher specific activity for the trpEG-encoded enzyme, anthranilate synthase (EC 4.1.3.27). The new trpCD deletion mutants failed to effectively nodulate soybean. Thus, we conclusively demonstrate the essential role of trpCD in symbiosis.

Symbiotic competence and genetic diversity of Rhizobium strains used as inoculants for alfalfa and berseem clover

To provide the necessary information for strain improvement and the development of competitive strains, characterization of some agronomically important inoculant Rhizobium strains specific for either alfalfa or berseem clover is an ongoing project in this laboratory. Plasmid DNA content and identification of symbiosis-controlling plasmids were previously determined. In this study, to determine their symbiotic competence and their apparent genetic diversity, plant tests and RFLP analysis of total DNAs were conducted. Symbiotic effectiveness varied significantly among the Rh. meliloti strains but not among the berseem clover symbionts. Only one strain, Rh. meliloti ARC 104, was symbiotically ineffective. RFLP analysis using a ribosomal RNA operon probe, with three different enzyme digests, produced four related groups which correlated with plasmid profiles. The use of a nifHD gene probe produced similar results which clustered the berseem clover microsymbionts but more clearly separated two distinct groups of Rh. meliloti. A cosmid clone, from a Rh. leguminosarum biovar trifolii T24 gene library, distinguished each strain. This new information will be useful in identifying and producing improved recombinant strains by plasmid transfer.

Variation of Clonal, Mesquite-Associated Rhizobial and Bradyrhizobial Populations from Surface and Deep Soils by Symbiotic Gene Region Restriction Fragment Length Polymorphism and Plasmid Profile Analysis

Genetic characteristics of 14 Rhizobium and 9 Bradyrhizobium mesquite (Prosopis glandulosa)-nodulating strains isolated from surface (0- to 0.5-m) and deep (4- to 6-m) rooting zones were determined in order to examine the hypothesis that surface- and deep-soil symbiont populations were related but had become genetically distinct during adaptation to contrasting soil conditions. To examine genetic diversity, Southern blots of PstI-digested genomic DNA were sequentially hybridized with the nodDABC region of Rhizobium meliloti, the Klebsiella pneumoniae nifHDK region encoding nitrogenase structural genes, and the chromosome-localized ndvB region of R. meliloti. Plasmid profile and host plant nodulation assays were also made. Isolates from mesquite nodulated beans and cowpeas but not alfalfa, clover, or soybeans. Mesquite was nodulated by diverse species of symbionts (R. meliloti, Rhizobium leguminosarum bv. phaseoli, and Parasponia bradyrhizobia). There were no differences within the groups of mesquite-associated rhizobia or bradyrhizobia in cross-inoculation response. The ndvB hybridization results showed the greatest genetic diversity among rhizobial strains. The pattern of ndvB-hybridizing fragments suggested that surface and deep strains were clonally related, but groups of related strains from each soil depth could be distinguished. Less variation was found with nifHDK and nodDABC probes. Large plasmids (>1,500 kb) were observed in all rhizobia and some bradyrhizobia. Profiles of plasmids of less than 1,000 kb were related to the soil depth and the genus of the symbiont. We suggest that interacting selection pressures for symbiotic competence and free-living survival, coupled with soil conditions that restrict genetic exchange between surface and deep-soil populations, led to the observed patterns of genetic diversity.

Characteristics of Hormogonia Formation by Symbiotic Nostoc spp. in Response to the Presence of Anthoceros punctatus or Its Extracellular Products

Nostocacean cyanobacteria typically produce gliding filaments termed hormogonia at a low frequency as part of their life cycle. We report here that all Nostoc spp. competent in establishing a symbiotic association with the hornwort Anthoceros punctatus formed hormogonial filaments at a high frequency in the presence of A. punctatus. The hormogonia-inducing activity was produced by A. punctatus under nitrogen-limited culture conditions. The hormogonia of the symbiotically competent Nostoc spp. were characterized as motile (gliding) filaments lacking heterocysts and with distinctly smaller cells than those of vegetative filaments; the small cells resulted from a continuation of cell division uncoupled from biomass increase. An essentially complete conversion of vegetative filaments to hormogonia occurred within 12 h of exposure of Nostoc sp. strain 7801 to A. punctatus growth-conditioned medium. Hormogonia formation was accompanied by loss of nitrogen fixation (acetylene reduction) and by decreases in photosynthetic CO(2) fixation and in vivo NH(4) assimilation of 30% and approximately 40%, respectively. The rates of acetylene reduction and CO(2) fixation returned to approximately the control rates within 72 to 96 h after hormogonia induction, as the cultures of Nostoc sp. strain 7801 differentiated heterocysts and reverted to the vegetative growth state. The relationship between hormogonia formation and symbiotic competence is discussed.

Role of Motility and Chemotaxis in Efficiency of Nodulation by Rhizobium meliloti

Spontaneous mutants of Rhizobium meliloti L5-30 defective in motility or chemotaxis were isolated and compared against the parent with respect to symbiotic competence. Each of the mutants was able to generate normal nodules on the host plant alfalfa (Medicago sativa), but had slightly delayed nodule formation, diminished nodulation in the initially susceptible region of the host root, and relatively low representation in nodules following co-inoculation with equal numbers of the parent. When inoculated in growth pouches with increasing dosages of the parental strain, the number of nodules formed in the initially susceptible region of the root increased sigmoidally, with an optimum concentration of about 10(5) to 10(6) bacteria/plant. The dose-response behavior of the nonmotile and nonchemotactic mutants was similar, but they required 10- to 30-fold higher concentrations of bacteria to generate the same number of nodules. The distribution frequencies of nodules at different positions along the primary root were very similar for the mutants and parent, indicating that reduced nodulation by the mutants in dose-response experiments probably reflects reduced efficiency of nodule initiation rather than developmentally delayed nodule initiation. The number of bacteria that firmly adsorbed to the host root surface during several hours of incubation was 5- to 20-fold greater for the parent than the mutants. The mutants were also somewhat less effective than their parent as competitors in root adsorption assays. It appears that motility and chemotaxis are quantitatively important traits that facilitate the initial contact and adsorption of symbiotic rhizobia to the host root surface, increase the efficiency of nodule initiation, and increase the rate of infection development.

Clustering of nitrogen fixation (nif) genes in Rhizobium meliloti.

A cloned 17.3-kilobase region of the Rhizobium meliloti genome with homology to the Klebsiella pneumoniae nitrogenase structural genes was studied. Limits on the extent of homology were determined. Transposon mutagenesis of this region of the genome verified that it contained functional nif genes, Some transposon insertions resulted in a defective symbiotic phenotype, whereas others had no noticeable effect on symbiotic competence. The relative position of insertions yielding these two phenotypic classes suggested that at least three distinct units of gene expression are present in this region. Hybridization of RNA from alfalfa root nodules and from vegetatively grown Rhizobium to this cloned DNA showed that at least 11.1 kilobases of the region was transcribed actively and that transcription was specific for the symbiotic state.

N 2-fixing competence of Rhizobium japonicum strains in soybean infected with tobacco ringspot virus

Soybean budblight and nodule infection by tobacco ringspot virus (TRSV) is a most severe, although not the most prevalent, soybean disease in the USA as it reduces growth, nodulation, N2-fixation, and yield. Infection of cv. “Tracy” soybean with tobacco ringspot virus (TRSV) had a severe effect upon the symbiotic competence of eight Rhizobium japonicum (Rj) strains in combination with this soybean. This effect was associated with a significant decrease in nodule (g fresh wt plant−1) and N2-fixation activity as expressed by the acetylene reduction assay. This was particularly evident with TRSV infection at the unifoliate developmental stage of plant growth as shown by a 63% decrease in nodulation and 62% decrease in acetylene reduction activity. However, with TRSV infection at the trifoliate stage there was no decrease in nodulation although acetylene reduction activity decreased 36%. The significantly higher N2-fixing competence of USDA Rj strains 110, 122, and 143 in healthy plants was associated with their greater nodulation. No such significant association was demonstrated with the other Rj strains tested. The re-isolation of the virus from nodules of plants inoculated with TRSV in either growth stage, and the kill of cowpea and limabean plants inoculated with extracts from TRSV-infected nodules, showed that the virus remained viable. Serotyping of somatic antigens of the Rj strains in extracts from TRSV-infected nodules from plants in the unifoliate or in the trifoliate inoculation showed that the virus within the nodule did not always suppress agglutinability. These results indicate that the inherent high N2-fixing competence of Rj strains 110, 122, and 143 in particular is consistent with their reported ability for hydrogenase-enhanced nitrogenase N2-fixation and that TRSV is a significant repressing factor through its effect on plant growth and the soybean-Rhizobium symbiosis.