Strain USDA Research Articles

Competitiveness of native Bradyrhizobium japonicum strains in two different soil types

Interstrain competitiveness is a key factor affecting the performance of rhizobium inoculant. In the present study five native strains of Bradyrhizobium japonicum, namely SSF 4, SSF 5, SSF 6, SSF 7 and SSF 8, were assessed for their competitiveness in nodulating soybean using serological methods. The strains were inoculated individually or with the type strain USDA 110 at a 1:1 ratio. Nodule occupancy determined by immunofluorescence and dot immunoblot assay revealed that under in vitro conditions SSF 8 is more competitive than USDA 110 whereas the others were less competitive. The competitive ability of these strains was also estimated in pot culture in the field. In red soil both SSF 8 and USDA 110 were equally competitive whereas in black soil SSF 8 competed better than USDA 110 and produced more nodules. In a black soil field trial using a randomized block design, USDA 110 or SSF 8, when inoculated alone, occupied the majority of the nodules and enhanced nodule dry weight and shoot biomass. SSF 8 was more competitive when the strains were co-inoculated.

Structural characterization of the K antigens from Rhizobium fredii USDA257: evidence for a common structural motif, with strain-specific variation, in the capsular polysaccharides of Rhizobium spp.

Rhizobium fredii participates in a nitrogen-fixing symbiosis with soybeans, in a strain-cultivar-specific interaction, and past studies have shown that the cell surface and extracellular polysaccharides of rhizobia function in the infection process that leads to symbiosis. The structural analysis of the capsular polysaccharides (K antigens) from strain USDA257 was performed in this study. The K antigens were extracted from cultured cells with hot phenol-water and purified by size exclusion chromatography. We isolated two structurally distinct K antigens, both containing a high proportion of 3-deoxy-D-manno-2-octulosonic acid (Kdo). The polysaccharides were characterized by matrix-assisted laser desorption ionization-time-of-flight-mass spectrometry, nuclear magnetic resonance spectrometry, and gas chromatography-mass spectrometry analyses. The primary polysaccharide, which constituted about 60% of the K-antigen preparation, consisted of repeating units of mannose (Man) and Kdo, [-->)3-beta-D-Manp-(1-->5)-beta-D-Kdop-(2-->], and a second polysaccharide consisted of 2-O-MeMan and Kdo, [-->)3-beta-D-2-O-MeManp-(1-->5)-beta-D-Kdop-(2-->]. These structures are similar to yet distinct from those of other strains of R. fredii and R. meliloti, and this finding provides further evidence that the K antigens of rhizobia are strain-specific antigens which are produced within a conserved motif.

Effects of common bean (Phaseolus vulgaris L.) cultivar and rhizobium strain on plant growth, seed yield and nitrogen content

The common bean is generally regarded as a relatively inefficient fixer of nitrogen. Information on the relative importance of host cultivar and rhizobium strain would be useful in guiding a breeding program to improve nitrogen fixation in common bean. Seventeen cultivars of common bean (15 of Meso-American and 2 of Andean origin, differing in nodulation potential, maturity, growth habit and market class) were investigated for symbiotic compatibility with 10 genetically diverse strains of bean rhizobia. In a greenhouse (27/22 °C), five sets of 170 pots were replicated over time: three sets were harvested after 30 d for determination of acetylene reduction activity, plant dry weights, and nitrogen content; two replications were grown to maturity for determination of seed dry weight and nitrogen content. There were highly significant effects of both strain and cultivar on most measured characteristics, but there was no interaction between cultivar and strain. Italian Barlotti produced the greatest plant weight after 30 d, but its final seed yield did not differ significantly from that of the highest yielding cv. BAT271. The strain TAL182 was associated with the greatest plant weight at 30 d, but the final seed yield associated with it was less than that of the most productive strain USDA9001. Strain USDA 2667 was characterized as type I by determination of its 16S rRNA gene nucleotide sequence. The nodules of plants in symbiosis with strains of type IIB had high levels (8.1%) of nitrogen and high levels (227 µmol g−1 h−1) of acetylene reduction activity (ARR). Nodules occupied by strains of type IIA and the type I USDA2667 had intermediate levels of nitrogen (6.9%) and ARR (166 µmol g−1 h−1), while nodules occupied by type 1 strains had low levels of nitrogen (5.2%) and ARR (144 µmol g−1 h−1). Screening of lines, parents and other breeding materials for improved N-fixation may be achieved with any effective strain since no host × strain interaction was detected. Key words: Common bean, Phaseolus vulgaris, nodulation, nitrogen fixation, rhizobium strains, cultivar × strain interaction

Sulphation of Rhizobium sp. NGR234 Nod factors is dependent on noeE, a new host-specificity gene.

Rhizobia secrete specific lipo-chitooligosaccharide signals (LCOs) called Nod factors that are required for infection and nodulation of legumes. In Rhizobium sp. NGR234, the reducing N-acetyl-D-glucosamine of LCOs is substituted at C6 with 2-O-methyl-L-fucose which can be acetylated or sulphated. We identified a flavonoid-inducible locus on the symbiotic plasmid pNGR234a that contains a new nodulation gene, noeE, which is required for the sulphation of NGR234 Nod factors (NodNGR). noeE was identified by conjugation into the closely related Rhizobium fredii strain USDA257, which produces fucosylated but non-sulphated Nod factors (NodUSDA). R. fredii transconjugants producing sulphated LCOs acquire the capacity to nodulate Calopogonium caeruleum. Furthermore, mutation of noeE (NGRdelta noeE) abolishes the production of sulphated LCOs and prevents nodulation of Pachyrhizus tuberosus. The sulphotransferase activity linked to NoeE is specific for fucose. In contrast, the sulphotransferase NodH of Rhizobium meliloti seems to be less specific than NoeE, because its introduction into NGRdelta noeE leads to the production of a mixture of LCOs that are sulphated on C6 of the reducing terminus and sulphated on the 2-O-methylfucose residue. Together, these findings show that noeE is a host-specificity gene which probably encodes a fucose-specific sulphotransferase.

Characterization of the Overlapping Promoters of nolB and nolW, Two Soybean Cultivar Specificity Genes from Rhizobium fredii Strain USDA257

The transcripts of nolW and nolB, two divergently oriented cultivar specificity genes of Rhizobium fredii strain USDA257, are known to be initiated 14 bp apart from promoters that face one another. We show here that expression of nolB is dependent both on induction with flavonoid signals and on the regulatory gene, nodD1. Expression of nolW is constitutive and independent of flavonoids and nodD1. Normal expression of nolB is retained with a promoter that extends only 61 bp upstream of the transcript start site, but it is lost if an additional 24 bp are removed. Substantial expression of nolW is retained with a promoter that contains only 34 bp of DNA upstream from the transcript initiation site. The dual control region for the two genes is thus only about 109 bp in length.

Survival of Bradyrhizobium sp. (Arachis) on fungicide-treated peanut seed in relationship to plant growth and yield

Survival and viability of Bradyrhizobium inoculant on fungicide-treated peanut seed and the resulting effects on nitrogen fixation, plant growth and seed yield were determined. Vitavax and Benomyl had the most and least lethal actions against Bradyrhizobium strains grown on YEM medium containing a fungicide, respectively, while Thiram and Captan effects were intermediate. Survival of Bradyrhizobium USDA 3384 and USDA 3456, as single strain peat inoculants, on peanut (Arachis hypogaea L. var. Florunner) seeds treated with Benomyl or Vitavax at the rate of 3g/kg seed was also examined. Both fungicides inhibited the growth and affected the survival of strain USDA 3384 on peanut seed. Vitavax killed the inoculant in 9 h. In contrast, USDA 3456 resisted both fungicides, and survived for up to 72h. Nodule formation on greenhouse-grown plants inoculated with USDA 3384 was inhibited by all fungicides. Shoot dry weight and plant nitrogen content significantly decreased as compared to controls. Fungicides, except Vitavax, had a slight effect on nodulation and plant growth when USDA 3456 was used as inoculant. The agronomic importance of fungicide-inoculant interaction was examined in field experiments conducted in Egypt in soil free of peanut-nodulating Bradyrhizobium, where seeds were treated with a combination of two fungicides and a single strain peat inoculant of either USDA 3384 or USDA 3456. All fungicides decreased nodulation, nitrogen fixation, plant growth and seed yield, especially with USDA 3384 as inoculant. Fungicides inhibited viability and survival of Bradyrhizobium on peanut seeds which decreased nodule formation leading to reduced peanut seed yield.

Short communication: Screening of native and foreign Bradyrhizobium japonicum strains for high N2 fixation in soybean

Four local rhizobia isolates selected after two screening experiments and five USDA Bradyrhizobium japonicum strains were estimated for N2 fixation in soybean using the (15)N isotope dilution technique. Strain USDA 110 was superior to the local isolates in nodulation and N2 fixation when inoculated onto soybean cv TGX 1497-ID in a Nigerian soil and could therefore be used as an inoculant for enhanced N2 fixation in soybean in Nigeria.

Rhizobium fredii synthesizes an array of lipooligosaccharides, including a novel compound with glucose inserted into the backbone of the molecule

Flavonoid cues from the plant host cause symbiotic, nitrogen-fixing rhizobia to synthesize lipochitooligosaccharides (LCOs), which act as return signals to initiate the nodulation process. Rhizobium fredii strain USDA257 is known to produce four LCOs, all substituted with vaccenic acid (C 18:1). We show here that a mutant strain can replace vaccenic acid with a C 16:0 side chain, and that strain USDA191 synthesizes additional LCOs that differ from one another in the length and unsaturation of their fatty acyl substituents. USDA191 and 257DH4 both produce a novel LCO with glucose substituted for N- acetyl- d - glucosamine in the backbone of the molecule.

Inheritance of Host‐Controlled Restriction of Nodulation by Bradyrhizobium japonicum Strain USDA 110

The use of soybean [Glycine max (L.) Merr.] genotypes which specifically restrict nodulation by competitive indigenous Bradyrhizobium populations has been proposed as an approach to favor increased nodulation by more effective, inoculant‐quality strains. Soybean plant introduction (PI) PI 417566 restricts nodulation by Bradyrhizobium japonicum (Kirchner, Jordan) strains in serogroup 110. In this study, the interaction of PI 417566 with strains in serogroup 110 is being used as a model system to determine whether host‐controlled nodulation restriction can be used to alter the competitiveness of indigenous bradyrhizobia. The objectives of this study were to determine the type of inheritance and the number of genes in PI 417566 which condition restriction of nodulation by strain USDA 110 and whether this gene(s) could function to prevent nodulation of serogroup 110 strains under field conditions. Inheritance of host‐controlled nodulation restriction was determined by crossing P1417566 with the nonrestricting cultivars Agassiz and Evans. The nodulation phenotype of 126 F2 plants from Agassiz × PI 417566 and 44 F2 plants from Evans × PI 417566 was assessed in growth chamber studies. In the former cross, 98 nodulating and 28 non‐nodulating plants, and in the latter, 32 nodulating and 12 non‐nodulating plants were observed. In both cases, a Chi‐squared test indicated a good fit to a 3:1 segregation ratio, indicating that a single recessive gene conditions restricted nodulation in P1 417566. A 2‐yr field study utilizing F2:4 and F2:5 progeny from Agassiz × PI 417566 and F2:4 progeny from Evans × PI 417566 demonstrated that the lines had the ability to restrict nodulation by serogroup 110 strains under field conditions and that restriction occurred even in the presence of large numbers (5 × 1O7 cells/g soil) of added USDA 110.

Host Genetic Variation in the Early Nodulation and Dinitrogen Fixation of Soybean

Soybeans [Glycine max (L.) Merr.] grown on soils low in N can exhibit N deficiency during early plant development. Improved early N2 fixation may alleviate this problem. Our objectives were to (i) examine genetic variation in early nodulation and N2 fixation of soybean, and (ii) identify traits useful in breeding for early nodulation and N2 fixation in this crop. Eighty‐six soybean lines showed considerable genetic variation in efficiency and speed of nodulation with Bradyrhizobium japonicum strain USDA 110. Eighteen lines were then reevaluated for nodulation efficiency with the B. japonicum strains USDA 123, UMR 161, and NA 6407 for differences in nodule number 5 to 12 d after inoculation (DAI) and for differences in plant dry weight, nodule fresh weight, acetylene reduction activity (ARA), nodule enzyme activity, leghemoglobin concentration, and nodule soluble protein concentration (SOLP) 10 to 17 DAI. The 86 lines differed in percentage roots nodulated (PRN; 26–93%) and number of nodules above the root tip mark (NARTM; 0.3–3.0), and in uppermost nodule position (− 17.5 to 19.5 mm). For the 18 lines that were reevaluated, mean PRN and NARTM values were greater for USDA 123 than for USDA 110 (65.1 vs. 57.9%; 1.4 vs. 1.0, respectively). For these lines, correlation analyses showed that PRN correlated with nodule number at 5 (r = 0.69**) but not 12 DAI, while nodule number 12 DAI correlated positively with nodule fresh weight, ARA, enzyme activity, and SOLP 17 DAI. Plant dry weight, nodule fresh weight, and nodule SOLP assayed at 17 DAI are suggested as traits for use in a breeding program to improve early nodulation and N2 fixation in soybean.

Soybean Response to Nodulation by Wild‐Type and an Isogenic Bradyrhizobium elkanii Mutant Lacking Rhizobitoxine Production

Soybean [Glycine max (L.) Merr.] cultivars differ in their susceptibility to rhizobitoxine‐producing (RT+) bradyrhizobia, but the physiological basis for this variation is uncertain. We conducted greenhouse experiments to determine the response of six cultivars, that differ in their RT susceptibilities, to nodulation by four strains of Bradyrhizobium: USDA 61 and 94 (RT+ wild‐type B. elkanii strains), USDA 110 (RT wild‐type B. japonicum strain), and RX 18E (a Tn5‐induced mutant of USDA 61 lacking RT production in planta). At 45 d after planting, plants were rated for RT symptoms and analyzed for shoot N contents and shoot, root, and nodule biomass and RT concentrations. Foliar chlorosis was generally better correlated with shoot RT concentrations than with nodular concentrations. Strain USDA 94 generally produced more RT in planta than did USDA 61. When nodulated by USDA 61, cultivars rated as RT‐resistant often had lower nodular RT concentrations than did RT‐susceptible plants. Relative to RX 18E, nodulation by USDA 61 significantly reduced plant shoot weight for the RT‐sensitive cultivar Hutcheson and tended to reduce shoot weight for all remaining cultivars except the highly resistant SS FFR 565; decreased shoot N content by 50.5 and 17.4% in cultivar Hutcheson and Williams, respectively; and had no significant effect on nodule number and nodule weight. These results indicate that mechanisms of RT resistance may differ among the cultivar‐strain combinations examined and involve differences in RT production, RT export from the nodules, and RT detoxification during and following transport to the shoot.

Characterization of Fast-Growing Rhizobia from Nodulated Soybean [ Glycine max (L.) Merr.] in Vietnam

Summary We have examined six fast-growing Rhizobium strains isolated from nodulated soybean [Glycine max (L.) Merr.] plants growing in Vietnam, with special emphasis on two of them, SMX11 and SMH12. The abilities of strains SMX11 and SMH12 to utilize 11 carbon sources were indistinguishable from that of control Sinorhizobium fredii strains USDA257 and HH103, but their growth rates in standard media were considerably more rapid. The Vietnamese strains also were more salt-tolerant and antibiotic-resistant than the S. fredii reference strains, and their LPS banding patterns were less complex. Weak immunological cross-reactivity between the Vietnamese strains and S. fredii was evident. RFLP patterns of a series of nodulation genes and a repetitive sequence from S. fredii matched those of the Vietnamese strains, as did the sequence of a portion of a 16S rRNA gene. Analysis of cellular fatty acids was consistent with the assignment of strain SMH 12 and four other Vietnamese strains to S. fredii, but it aligned strain SMX11 more closely to other related groups of rhizobia. The Vietnamese strains have broad host ranges for nodulation of legumes, and their abilities to fix nitrogen in association with five soybean cultivars are equivalent to or surpass that of S. fredii.

PQQ-linked extracellular glucose oxidation and chemotaxis towards this cofactor in rhizobia

The presence of the pyrroloquinoline quinone (PQQ)-dependent glucose dehydrogenase in several strains of different Rhizobium species and of Bradyrhizobium japonicum was investigated. It was found that most strains were only able to synthesize the apo-enzyme, whereas in three strains, Rhizobium meliloti 102F34 and Bradyrhizobium japonicum strains USDA110 and USDA138 holo-enzyme activity could be observed. In washed suspensions, however, only Rhizobium meliloti 102F34 produced gluconate from glucose in the absence of added PQQ. All strains showed positive chemotaxis towards PQQ. This chemotaxis could already be observed with nanomolar concentrations of PQQ.

PQQ-linked extracellular glucose oxidation and chemotaxis towards this cofactor in rhizobia

The presence of the pyrroloquinoline quinone (PQQ)-dependent glucose dehydrogenase in several strains of different Rhizobium species and of Bradyrhizobium japonicum was investigated. It was found that most strains were only able to synthesize the apo-enzyme, whereas in three strains, Rhizobium meliloti 102F34 and Bradyrhizobium japonicum strains USDA110 and USDA138 holo-enzyme activity could be observed. In washed suspensions, however, only Rhizobium meliloti 102F34 produced gluconate from glucose in the absence of added PQQ. All strains showed positive chemotaxis towards PQQ. This chemotaxis could already be observed with nanomolar concentrations of PQQ.

Proteins from cells of Rhizobium fredii bind to DNA sequences precedingnolX, a flavonoid-inducible nod gene that is not associated with a nod box.

Rhizobium fredii strains USDA257 and USDA191 both contain a set of nodulation genes termed nolXWBTUV. In the USDA257 background, nolX prevents infection of soybean cultivars such as McCall, and in both backgrounds, it blocks nodulation of Erythrina spp. We report here that expression of nolX is differentially responsive to a panel of flavonoids, and that the most potent inducers are also the most active inducers of nodC, a conventional, nod box-associated gene. Cell-free protein extracts from uninduced and flavonoid-induced cells of strains USDA191 and USDA257 retard the electrophoretic mobility of DNA sequences that lie upstream of nolX. Binding is dependent both on nodD1 and nodD2, and it is abolished by the presence of a double-stranded, 23-bp oligonucleotide that lies within a 114-bp TaqI/SacII restriction fragment. This oligomer has significant sequence homology to A3, a putative negative regulatory element from R.leguminosarum bv. viciae. Deletion of the A3-homologous sequences elevates the basal and flavonoid-inducible expression of nolX by about 50%.

Cooperative Action of Lipo-Chitin Nodulation Signals on the Induction of the Early Nodulin, ENOD2, in Soybean Roots

Various lipo-chitin molecules were tested for their ability to induce the expression of the early nodulin, ENOD2, in Glycine soja roots. When inoculated separately onto G. soja roots, LCO-V (C18:1 delta 11,Mefuc), LCO-V (C18:1 delta 9,Mefuc), LCO-V (C16:0,Mefuc), and LCO-IV (C16:0) were unable to induce ENOD2 expression, even though these compounds had previously been shown to induce root hair curling, the formation of nodule-like primordia, and induction of the early nodulin, ENOD40. ENOD2 expression, however, was induced when any two of these molecules were inoculated in combination. Thus, the lipo-chitin nodulation signals appear to act cooperatively to induce ENOD2 expression. B. japonicum strains USDA110 and USDA135 and B. elkanii strain USDA61, all symbionts of soybean, were found to produce at least two distinct nod signals ([i.e., NodBj-V[C18:1,Mefuc] and NodBj-V[C16:0,Mefuc]). These two compounds were mixed in various ratios and tested for their ability to induce ENOD2 expression. The results indicate that the former compound must be present in equivalent or excess amount in order to obtain maximum ENOD2 expression. Additional nonspecific LCOs (e.g., LCO-IV[C16:2 delta 2,9; SO3]), incapable of inducing root hair curling or cortical cell division, were tested in combination with the four active LCOs listed above. It was found that any combination of one active LCO with a nonspecific LCO was sufficient to induce ENOD2 mRNA expression. The ENOD2 mRNA expression pattern detected by in situ hybridization closely resembled that found in bacterial-induced nodules with expression detected in cortical cells between primary and secondary meristems and around the vascular strands. These data demonstrate that the cooperative action of at least two LCO nodulation signals leads to a greater progression of nodule ontogeny as demonstrated by the expression of ENOD2, a marker gene for the differentiation of nodule parenchyma.

Nodule development induced by mutants of Bradyrhizobium japonicum defective in cyclic B-glucan synthesis.

The soybean response to inoculation with B. japonicum mutants defective in cyclic beta-(1-->3), beta-(1-->6) glucan synthesis was examined by electron microscopy and by monitoring the expression of early and late nodulin genes. Two mutants were examined. Strain AB-14 is an ndvB mutant and is unable to synthesize beta-glucans. Strain AB-1 is an ndvC mutant and produces cyclic glucans containing 95 to 100% beta-(1-->3) glycosidic linkages. Nodules formed by either mutant were defective in nitrogen fixation activity. Soybean plants inoculated with strain AB-14 formed nodules roughly at the same rate as the wild-type strain USDA110, but nodulation by strain AB-1 was significantly delayed. Microscopy of nodules formed by strain AB-14 showed an overall ultrastructure similar to nodules formed by the wild type. However, in some nodules bacteroids were limited to only one part of the infected cells and in others the few bacteroids present showed signs of degradation. Nodulelike structures were formed by strain AB-1 that showed some signs of cellular differentiation. For example, clear parenchyma and sclerenchyma tissue could be seen. However, no infection threads or bacteria were evident in these structures. The expression of early (e.g., ENOD2 and ENOD55) and late (e.g., NOD26 and leghemoglobin) nodulins was examined. Nodules formed by strain AB-14 expressed all of the nodulins tested but at a reduced level. Expression of late nodulins was delayed in strain AB-14-induced nodules. Nodules formed by strain AB-1 were more strongly affected in nodulin expression. Although leghemoglobin synthesis was not detected, infection-specific nodulin transcripts of GmN93 and ENOD55 were detected. However, expression of the early nodulins ENOD2 and ENOD55 was considerably delayed and only apparent when assayed 21 days postinoculation. A low level of expression of intermediate nodulin GmN70 and late nodulin NOD26 could also be detected by 21 days postinoculation. The microscopy data show that tissue differentiation occurs in these nodules even in the absence of active bacterial invasion. These results demonstrate the importance of cyclic beta-(1-->3), beta-(1-->6) glucan synthesis to symbiotic development in soybean.

Long term field study shows increased biomass production in tree legumes inoculated with Rhizobium

Nodulation potential, nitrogen fixation efficiency (nitrogenase activity) and biomass yield response of Leucaena leucocephala and Acacia nilotica to inoculation with 6 selected fast growing Rhizobium strains was explored in long-term (5 year) field trials. All the strains formed nodules and fixed nitrogen in L. leucocephala and A. nilotica. Seasonal effects on nitrogenase activity was observed and in winter (ambient temperature about 20 °C), nitrogenase activity could not be detected. However, with the onset of spring and a rise in temperature, fresh nodulation (renodulation) by all the inoculant rhizobial strains was observed in both the tree legumes. In L. leucocephala, maximum renodulation was exhibited by strain A1 while in A. nilotica, strain AB3 formed the maximum renodulation 24 months after transplantation. Dry matter yield of all the inoculated plants demonstrated a significant increase over that of the uninoculated plants at the end of five years after transplanting. In L. leucocephala, strain NGR8 gave the maximum response (45% more dry matter yield) in dry matter production while in A. nilotica, strain USDA 3325 showed a 25% increase in total dry matter yield five years after transplantation.

A Rhizobium strain that nodulates and fixes nitrogen in association with alfalfa and soybean plants.

A Rhizobium meliloti strain that forms effective nodules on soybean (Glycine max) and alfalfa (Medicago sativa) is described. The strain, 042B, was isolated from root nodules of alfalfa in Xinjiang Autonomous Region of China. Experiments showed that strain 042B was able to nodulate soybean as effectively as Bradyrhizobium japonicum USDA110, a widely used inoculant strain. Under hydroponic conditions, both strains performed similarly in many respects such as time required for the appearance of nodules, total nitrogenase activity, plant top dry weight and total plant nitrogen. In soil pot experiments, total nitrogen, seed weight and seed nitrogen of soybean plants inoculated with strains 042B or USDA110 were much higher than those of the uninoculated control, but there were no differences between plants with strain 042B relative to strain USDA110. However, when strain 042B was the inoculant, the number of nodules was greater, but the nodules were smaller than those of strain USDA110. Results from ELISA showed that nodule occupancy of strain 042B in soybean ranged from 82-90% and that of strain USDA110 ranged from 78-86%. Strain 042B can grow in the presence of 5% (w/v) NaCl, at 42 °C, and at pH 10·7. Strain 042B was shown to have two large plasmids (molecular sizes 200 kb and > 1000 kb, respectively). The DNA G + C mol% of strain 042B was 63·6. The DNA level of homology between strain 042B and type-strain R. meliloti USDA1002 was 76%, while DNA homologies of strain 042B were 10% and 12% with R. fredii USDA205 and B. japonicum USDA6, respectively.

Host-Controlled Restriction of Nodulation by Bradyrhizobium japonicum Strains in Serogroup 110

We previously reported the identification of a soybean plant introduction (PI) genotype, PI 417566, which restricts nodulation by Bradyrhizobium japonicum MN1-1c (USDA 430), strains in serogroup 129, and USDA 110 (P. B. Cregan, H. H. Keyser, and M. J. Sadowsky, Appl. Environ. Microbiol. 55:2532-2536, 1989, and Crop Sci. 29:307-312, 1989). In this study, we further characterized nodulation restriction by PI 417566. Twenty-four serogroup 110 isolates were tested for restricted nodulation on PI 417566. Of the 24 strains examined, 62.5% were restricted in nodulation by the PI genotype. The remainder of the serogroup 110 strains tested (37.5%), however, formed significant numbers of nodules on PI 417566, suggesting that host-controlled restriction of nodulation by members of serogroup 110 is strain dependent. Analysis of allelic variation at seven enzyme-encoding loci by multilocus enzyme electrophoresis indicated that the serogroup 110 isolates can be divided into two major groups. The majority of serogroup 110 isolates which nodulated PI 417566 belonged to the same multilocus enzyme electrophoresis group. B. japonicum USDA 110 and USDA 123 were used as coinoculants in competition-for-nodulation studies using PI 417566. Over 98% of the nodules formed on PI 417566 contained USDA 123, whereas less than 2% contained USDA 110. We also report the isolation of a Tn5 mutant of USDA 110 which has overcome nodulation restriction conditioned by PI 417566. This mutant, D4.2-5, contained a single Tn5 insertion and nodulated PI 417566 to an extent equal to that seen with the unrestricted strain USDA 123. The host range of D4.2-5 on soybean plants and other legumes was unchanged relative to that of USDA 110, except that the mutant nodulated Glycine max cv. Hill more efficiently. While strain USDA 110 has the ability to block nodulation by D4.2-5 on PI 417566, the nodulation-blocking phenomenon was not seen unless strain USDA 110 was inoculated at a 100-fold greater concentration than the mutant strain.