N2-fixation ability of Brazilian soybean cultivars with Sinorhizobium fredii and Sinorhizobium xinjiangensis

The main N₂-fixing symbiotic associations with soybean (Glycine max (L.) Merrill) plants are realized with bacteria belonging to the species Bradyrhizobium japonicum and B. elkanii. However, in 1982, fast-growing rhizobia were isolated from soybean root nodules collected in The People's Republic of China and these bacteria are today classified as Sinorhizobium fredii and S. xinjiangensis. The fast growing strains formed an effective symbiosis with primitive soybean cultivars such as Peking, but not with most North American cultivars, which are the progenitors of almost all Brazilian cultivars. The main purpose of this study was to evaluate the ability of 80 soybean cultivars from the Brazilian germplasm bank to produce effective nodules when inoculated with S. fredii or S. xinjiangensis strains. Sixty-six percent of the Brazilian genotypes formed effective nodules with both Sinorhizobium species. However, when 20 Fix⁺ genotypes were inoculated with a mixture of B. elkanii and S. fredii, at a ratio of 1:1, most or all nodules were occupied by B. elkanii. Consequently, there was no relationship between the growth rate in vitro and the ability to compete for nodule occupancy. Fast-growing strains have also been isolated from soybean nodules in Brazil, but the ecological importance of these symbiotic associations is still to be determined.     

Effect of pH and soybean cultivars on the quantitative analyses of soybean rhizobia populations

Quantitative analyses of fast- and slow-growing soybean rhizobia populations in soils of four different provinces of China (Hubei, Shan Dong, Henan, and Xinjiang) have been carried out using the most probable number technique (MPN). All soils contained fast- (FSR) and slow-growing (SSR) soybean rhizobia. Asiatic and American soybean cultivars grown at acid, neutral and alkaline pH were used as trapping hosts for FSR and SSR strains. The estimated total indigenous soybean-rhizobia populations of the Xinjiang and Shan Dong soil samples greatly varied with the different soybean cultivars used. The soybean cultivar and the pH at which plants were grown also showed clear effects on the FSR/SSR rations isolated from nodules. Results of competition experiments between FSR and SSR strains supported the importance of the soybean cultivar and the pH on the outcome of competition for nodulation between FSR and SSR strains. In general, nodule occupancy by FSRs significantly increased at alkaline pH. Bacterial isolates from soybean cultivar Jing Dou 19 inoculated with Xinjiang soil nodulate cultivars Heinong 33 and Williams very poorly. Plasmid and lipopolysaccharide (LPS) profiles and PCR-RAPD analyses showed that cultivar Jing Dou 19 had trapped a diversity of FSR strains. Most of the isolates from soybean cultivar Heinong 33 inoculated with Xinjiang soil were able to nodulate Heinong 33 and Williams showed very similar, or identical, plasmid, LPS and PCR-RAPD profiles. All the strains isolated from Xinjiang province, regardless of the soybean cultivar used for trapping, showed similar nodulation factor (LCO) profiles as judged by thin layer chromatographic analyses. These results indicate that the existence of soybean rhizobia sub-populations showing marked cultivar specificity, can affect the estimation of total soybean rhizobia populations indigenous to the soil, and can also affect the diversity of soybean rhizobial strains isolated from soybean nodules.     

Isolation of plant-growth-promoting Bacillus strains from soybean root nodules

Endophytic bacteria reside within plant tissues and have often been found to promote plant growth. Fourteen strains of putative endophytic bacteria, not including endosymbiotic Bradyrhizobium strains, were isolated from surface-sterilized soybean (Glycine max. (L.) Merr.) root nodules. These isolates were designated as non-Bradyrhizobium endophytic bacteria (NEB). Three isolates (NEB4, NEB5, and NEB17) were found to increase soybean weight when plants were co-inoculated with one of the isolates and Bradyrhizobium japonicum under nitrogen-free conditions, compared with plants inoculated with B. japonicum alone. In the absence of B. japonicum, these isolates neither nodulated soybean, nor did they affect soybean growth. All three isolates were Gram-positive spore-forming rods. While Biolog tests indicated that the three isolates belonged to the genus Bacillus, it was not possible to determine the species. Phylogenetic analysis of 16S rRNA gene hypervariant region sequences demonstrated that both NEB4 and NEB5 are Bacillus subtilis strains, and that NEB17 is a Bacillus thuringiensis strain.     

Response of introduced Bradyrhizobium strains infectinga promiscuous soybean cultivar

Two field experiments were established to assess the competitiveness of foreign bradyrhizobia in infecting the promiscuous soybean cultivar TGX 536-02D. Seeds were inoculated with antibiotic mutants of the bradyrhizobia strains before planting after land preparation. Soybean plants were harvested at pre-determined days after planting for estimating nodule number, nodule dry weight, nodule occupancy, shoot dry weight and seed yield. Results show that nodule number and dry weight significantly increased and showed great variability at 84 days after planting (DAP), probably due to differences in the ability of inoculant bradyrhizobia to form nodules with the soybean cultivar TGX 536-02D. Increased shoot dry weight, %N, total N and seed yield were a result of increased nodulation by the effective and competitive inoculant Bradyrhizobium strains. Strain USDA 110 occupied the highest percentage of nodule sites because it was more competitive than the other Bradyrhizobium strains. These results show that there was high potential for increasing growth and seed yield of the promiscuous soybean cultivar TGX 536-02D by inoculation with foreign Bradyrhizobium strains.     

Acetylene reduction by effective and ineffective clover and soybean root nodules

Summary Acetylene was reduced to ethylene by effective white clover nodules and by fully and partially effective intact nodules, nodule homogenates, and bacteroids of soybeans. Succinate and several amino acids markedly stimulated the reduction by effective soybean bacteroids, but the stimulation was slight with partially effective bacteroids. Acetylene metabolism by effective soybean bacteroids was also enhanced by excretions of in vitro-grown Rhizobium japonicum, excretions of bacteria derived from effective and ineffective nodules, and the soluble fraction from these nodules. Inhibitors of nitrogen fixation were not found in ineffective nodules. Ineffective soybean and white clover nodules and homogenates or isolated bacteria from ineffective soybean nodules did not reduce acetylene. Additions of succinate, amino acids, the soluble fraction of effective nodules, or excretions of effective bacteroids or of in vitro-grown cells of an effective R. japonicum strain did not promote nitrogen fixation by bacterial cells obtained from ineffective soybean nodules.     

Assessing host range,symbiotic effectiveness,and photosynthetic rates induced by native soybean rhizobia isolated from Mozambican and South African soils

Host range and cross-infectivity studies are important for identifying rhizobial strains with potential for use as inoculants. In this study, 10 native soybean rhizobia isolated from Mozambican and South African soils were evaluated for host range, symbiotic effectiveness and ability to induce high rates of photosynthesis leading to enhanced plant growth in cowpea (Vigna unguiculata L. Walp.), Bambara groundnut (Vigna subterranean L. Verdc.), Kersting’s groundnut (Macrotyloma geocarpum Harm) and soybean (Glycine max L. Merr). The test isolates had different growth rates and colony sizes. Molecular analysis based on enterobacterial repetitive intergenic consensus (ERIC)-PCR revealed high genetic diversity among the test isolates. The results further showed that isolate TUTLBC2B failed to elicit nodulation in all test plants, just as TUTNSN2A and TUTDAIAP3B were also unable to nodulate cowpea, Kersting’s bean and Bambara groundnut. Although the remaining strains formed ineffective nodules on cowpea and Kersting’s bean, they induced effective nodules on Bambara groundnut and the two soybean genotypes. Bacterial stimulation of nodule numbers, nodule dry weights and photosynthetic rates was generally greater with isolates TUTRSRH3A, TUTM19373A, TUTMCJ7B, TUTRLR3B and TUTRJN5A. As a result, these isolates elicited significantly increased accumulation of biomass in shoots and whole plants of Bambara groundnut and the two soybean genotypes. Whole-plant symbiotic nitrogen (N) of soybean and Bambara groundnut was highest for the commercial strains CB756 and WB74, as well as for TUTRLR3B, TUTMCJ7B and TUTRSRH3A, suggesting that the three native rhizobial isolates have potential for use as inoculants.     

Siderophore-bound iron in the peribacteriod space of soybean root nodules

Water-soluble, non-leghemoglobin iron (125 µmol kg^-1 wet weight nodule) is found in extracts of soybean root nodules. This iron is probably confined to the peribacteroid space of the symbiosome, where its estimated concentration is 0.5 – 2.5 mM. This iron is bound by siderophores (compounds binding ferric iron strongly) which are different for each of the three strains of Bradyrhizobium japonicum with which the plants were inoculated. One of these, that from nodules inoculated with strain CC 705, is tentatively identified as a member of the pseudobactin family of siderophores. Leghemoglobin is present in only very small amounts in the peribacteroid space of symbiosomes isolated from soybean root nodules, and may be absent from the peribacteroid space of the intact nodule.     

Isolation and characterization of rhizobitoxine mutants of Bradyrhizobium japonicum.

To explore the role of rhizobitoxine in Bradyrhizobium-legume symbiosis, 11 rhizobitoxine mutants of B. japonicum USDA61 were isolated on the basis of their inability to synthesize the toxin in culture. Each mutant is prototrophic and symbiotically effective on soybean, cowpea, siratro, and Glycine soja. The rhizobitoxine mutants differ in their chlorosis phenotypes and rhizobitoxine production in planta. As expected, one group of mutant fail to make toxin in planta, resulting in the absence of chlorosis. Another group of mutants causes severe chlorosis on all cultivars of soybean tested. Surprisingly, this group of mutants makes more rhizobitoxine in soybean nodules than the wild-type strain does. This phenotype is only observed on soybean and not on other hosts such as cowpea, siratro, or G. soja. The remaining mutants all produce rhizobitoxine in planta but vary in the amount of toxin they produce and the severity of chlorosis they induce in soybean plants. Biochemical analysis of mutants demonstrates that one mutant is unable to synthesize serinol, a molecule hypothesized to be an intermediate in rhizobitoxine biosynthesis. By using these mutants, it was found that rhizobitoxine plays no apparent role in the nodulation of rj1 soybeans. Recently, it was found that inhibition of ethylene biosynthesis allows Rhizobium meliloti to overcome nitrate inhibition of nodule formation on alfalfa. Because rhizobitoxine also inhibits ethylene biosynthesis, we tested the ability of mutants which accumulate high levels of toxin in planta to overcome nitrate inhibition of nodule formation on soybean plants and found that the nodule formation induced by the wild type and that induced by mutant strains were equally suppressed in the presence of nitrate.     

Strain-specific antisera to identify Thai Bradyrhizobium japonicum strains in preserved soybean nodules

Strain-specific antisera were produced against six Bradyrhizobium japonicum strains using two immunization procedures. These specific antisera were used for detection of bradyrhizobia in preserved soybean nodules. Antisera specific for two of these strains were either conjugated with a fluorescent dye or used with a fluorescent secondary antibody for identification of bradyrhizobia in soybean nodules that were preserved in four different storage conditions. Results show that soybean nodules dried in the oven, stored under room temperature, or at –20 °C are as suitable as fresh nodules for strain identification using fluorescent antisera.     

Mutants of Rhizobium japonicum defective in nodulation

Several mutants defective in nodulation were isolated from Rhizobium japonicum strains 3I1b110 and 61A 76. Mutants of class I do not form nodules after incubation with soybean [Glycine max (L.) Merrill] for 17 days, but will do so by 28 days. When host plants other than G. max are infected with several of these strains, there is no detectable difference in the time of nodulation or size of nodules as compared to the wild type. Two mutants of class I (i. e., SM1 and SM2) have been shown previously to be altered in the lipopolysaccharide portion of their cell wall. Mutants of class II are not slow to nodulate but form fewer nodules than the wild type on all the host plants tested. Mutants of class III are unable to form nodules. Some bacteriophage-resistant mutants, altered in cell surface structure, fall into this class. Two mutants of class III do not bind to soybean roots.     

Ineffective and non-nodulating mutant strains of Rhizobium japonicum.

Mutant strains of Rhizobium japonicum that were unable to allow the Corsoy cultivar of soybean to reduce acetylene or fix N2 were isolated. These strains grow as well as the wild type in a variety of media. Mutant strains SM1 and SM2 did not form nodules on the host plant; however, they reduced acetylene in the nonsymbiotic assay. Strains SM3 and SM4 produced nodules that did not have the characteristic pink pigment caused by leghemoglobin. The nodules formed by these strains also were small. One mutant strain, SM5, produced large pink nodules. The lesion in this strain seems to be in the gene that specifies nitrogenase component II.     

Induction of Symbiotically Defective Auxotrophic Mutants of Rhizobium fredii HH303 by Transposon Mutagenesis

Symbiotically defective auxotrophic mutants were isolated by transposon Tn5 mutagenesis of Rhizobium fredii HH303, a fast-growing microsymbiont of North American commercial soybean cultivars such as Glycine max cv. Williams. Three different Tn5-carrying suicide vectors, pBLK1-2, pSUP1011, and pGS9, were used for mutagenesis with transposition frequencies of 4 x 10, 3 x 10, and 1 x 10, respectively, while the frequency of background mutation resistant to 500 mug of kanamycin per ml was 1 x 10. From 2,600 Tn5-induced mutants, 14 auxotrophic mutants were isolated and classified in seven groups including adenosine (four), aspartate (two), cysteine or methionine (two), isoleucine and valine (two), nicotinic acid (one), pantothenic acid (one), and uracil (two). All the auxotrophs induced nodulation on soybean, but the symbiotic effectiveness of each mutant was different. Three auxotrophs (two cysteine or methionine and one pantothenic acid) formed effective nodules similar to those of the wild type. Three auxotrophs (one nicotinic acid and two aspartate) produced mature nodules like those of the wild type, but the nodules lacked the characteristic pink color inside and were unable to fix nitrogen. Four auxotrophs (two adenosine and two uracil) induced pseudonodules unable to fix nitrogen. The other four auxotrophs repeatedly induced both effective and ineffective nodules, but bacteroids isolated from the effective nodules were prototrophic revertants. The symbiotic phenotype and the degree of effectiveness of the auxotrophic mutants varied with the type of mutation.     

Diverse rhizobia that nodulate two species of Kummerowia in China

A total of 63 bacterial strains were isolated from root nodules of Kummerowia striata and K. stipulacea grown in different geographic regions of China. These bacteria could be divided into fast-growing (FG) rhizobia and slow-growing (SG) rhizobia according to their growth rate. Genetic diversity and taxonomic relationships among these rhizobia were revealed by PCR-based 16 S rDNA RFLP and sequencing, 16 S-IGS RFLP, SDS-PAGE of whole cell soluble proteins, BOX-PCR and symbiotic gene (nifH/nodC) analyses. The symbiotic FG strains were mainly isolated from temperate regions and they were identified as four genomic species in Rhizobium and Sinorhizobium meliloti based on the consensus of grouping results. The SG strains were classified as five genomic species within Bradyrhizobium and they were mainly isolated fron the subtropic and tropical regions. The phylogenetic analyses of nifH and nodC genes showed relationships similar to that of 16 S rDNA but the symbiotic genes of Bradyrhizobium strains isolated from Kummerowia were distinct from those isolated from Arachis and soybean. These results offered evidence for rhizobial biogeography and demonstrated that the Kummerowia-nodulating ability might have evolved independently in different regions in association with distinctive genomic species of rhizobia.     

Expression of nir, nor and nos denitrification genes from Bradyrhizobium japonicum in soybean root nodules

Expression of Bradyrhizobium japonicum wild-type strain USDA110 nirK , norC and nosZ denitrification genes in soybean root nodules was studied by in situ histochemical detection of β -galactosidase activity. Similarly, P_nirK- lacZ , P_norC- lacZ , and P_nosZ- lacZ fusions were also expressed in bacteroids isolated from root nodules. Levels of β -galactosidase activity were similar in both bacteroids and nodule sections from plants that were solely N₂-dependent or grown in the presence of 4 m M KNO₃. These findings suggest that oxygen, and not nitrate, is the main factor controlling expression of denitrification genes in soybean nodules. In plants not amended with nitrate, B. japonicum mutant strains GRK308, GRC131, and GRZ25, that were altered in the structural nirK , norC and nosZ genes, respectively, showed a wild-type phenotype with regard to nodule number and nodule dry weight as well as plant dry weight and nitrogen content. In the presence of 4 m M KNO₃, plants inoculated with either GRK308 or GRC131 showed less nodules, and lower plant dry weight and nitrogen content, relative to those of strains USDA110 and GRZ25. Taken together, the present results revealed that although not essential for nitrogen fixation, mutation of either the structural nirK or norC genes encoding respiratory nitrite reductase and nitric oxide reductase, respectively, confers B. japonicum reduced ability for nodulation in soybean plants grown with nitrate. Furthermore, because nodules formed by each the parental and mutant strains exhibited nitrogenase activity, it is possible that denitrification enzymes play a role in nodule formation rather than in nodule function.     

Changes in electrophoretic profiles of lipopolysaccharides from competitive strains of Bradyrhizobium spp. induced by soybean roots

The soybean Bradyrhizobium strain Semia 566 was introduced into soils of the Cerrados (Brazilian edaphic savannas) in the late 1960s. Then, nodule occupancy by this strain was not greater than 2%. Recently, this serogroup has been found in approximately 60% of nodules formed on soybeans cultivated in the Cerrados, replacing the strains 29W and Semia 587, the Brazilian commercial inoculant for soybean. Although some re-isolates of Semia 566, adapted to Cerrado soils, were more competitive than 29W under both field and aseptic conditions, they did not differ from the parental strain, based on their lipopolysaccharide (LPS) electrophoretic profile. The only exceptions were the isolates 4A-5 and CPAC-15 which presented an additional polysaccharidic band of low molecular weight or higher mobility. On the other hand, this same band may be induced and intensified in LPS extracted from competitive strains (29W, 220, 204, 370, 372, 516, 122 and CPAC-15) after bacterial contact with soybean roots for 6 or 12 h. In addition, a 29W Tn5 mutant with a phenotype of delayed nodulation showed a delayed induction of this polysaccharidic band. Conversely, the LPS of less competitive strains was not modified or showed a weak intensification of this band. As this band alteration was correlated with the concurrent elevation of dominance in nodules, it may be suggested that LPS plays a role in the competitive ability of rhizobia strains for nodulation.     

Nodulation of soybean byRhizobium japonicum mutants with altered capsule synthesis

Spontaneous mutants with altered capsule synthesis were isolated from a marked strain of the symbiont,Rhizobium japonicum. Differential centrifugation was used to enrich serially for mutants incapable of forming capsules. The desired mutants were detected by altered colony morphology and altered ability to bind host plant lectin. Three mutants failed to form detectable capsules at any growth phase when cultured in vitro or in association with the host (soybean,Glycine max (L.) Merr.) roots. These mutants were all capable of nodulating and attaching to soybean roots, indicating that the presence of a capsule physically surrounding the bacterium is not required for attachment or for infection and nodulation. Nodulation by several of the mutants was linearly proportional to the amount of acidic exopolysaccharide that they released into the culture medium during the exponential growth phase, indicating that such polysaccharide synthesis is important and perhaps required for nodulation. Two of the mutants appeared to synthesize normal lectin-binding capsules when cultured in association with host roots, but not when cultured in vitro. Nodulation by these mutants appeared to depend on how rapidly after inoculation they synthesized capsular polysaccharide.Abbreviations CPS capsular polysaccharide - EPS exopolysaccharide - FITC fluorescein isothiocyanate Contribution No. 719 of the C.F. Kettering Research Laboratory     

Genetic characterization of soybean rhizobia in Paraguay

The soybean is an exotic plant introduced in Paraguay in this century; commercial cropping expanded after the 1970s. Inoculation is practiced in just 15 to 20% of the cropping areas, but root nodulation occurs in most sites where soybeans grow. Little is known about rhizobial diversity in South America, and no study has been performed in Paraguay until this time. Therefore, in this study, the molecular characterization of 78 rhizobial isolates from soybean root nodules, collected under field conditions in 16 sites located in the two main producing states, Alto Paraná and Itapúa, was undertaken. A high level of genetic diversity was detected by an ERIC-REP-PCR analysis, with the majority of the isolates representing unique strains. Most of the 58 isolates characterized by slow growth and alkaline reactions in a medium containing mannitol as a carbon source were clustered with strains representative of the Bradyrhizobium japonicum and Bradyrhizobium elkanii species, and the 16S ribosomal DNA (rDNA) sequences of 5 of those isolates confirmed the species identities. However, slow growers were highly polymorphic in relation to the reference strains, including five carried in commercial inoculants in neighboring countries, thus indicating that the Paraguayan isolates might represent native bradyrhizobia. Twenty isolates highly polymorphic in the ERIC-REP-PCR profiles were characterized by fast growth and acid reactions in vitro, and two of them showed high 16S rDNA identities with Rhizobium genomic species Q. However, two other fast growers showed high 16S rDNA identity with Agrobacterium spp., and both of these strains established efficient symbioses with soybean plants.     

Gene centres,a source for genetic variants in symbiotic nitrogen fixation: Host-induced ineffectivity inPisum sativum ecotype fulvum

Summary Pisum sativum ecotype fulvum forms ineffective nodules with Rhizobium strains, isolated from effective nodules of the cultivated pea in Europe. Rhizobium strains isolated from nodules of fulvum peas in Israel are fully effective on this host plant, but in association with the cultivated pea they induce nodules of poor N₂-fixing activity. The distribution of these fulvum-specific Rhizobium strains is restricted to regions where the fulvum pea occurs naturally. Rhizobium strains from other geographical regions induce mainly ineffective, or partially effective nodules on fulvum plants.A wide genetic variation, with regard to symbiotic response to a standard set of Rhizobium strains, was demonstrated in the fulvum plants collected in Israel. Based on variation in N₂-fixation three groups of plants can be distinguished. These plants offer the possibility for the study of host-genetic control on symbiotic nitrogen fixation.     

Improvement of Rhizobium Inoculants

A practical approach was used to develop a Rhizobium (Bradyrhizobium) japonicum inoculant that increases soybean (Glycine max (L.) Merr.) yield in fields with indigenous Rhizobium populations, which typically outcompete strains present in existing commercial inoculants and therefore decrease the value of inoculant use. Field tests managed by several universities in the Mississippi delta region averaged a 169-kg/ha (P < 0.01) grain yield increase. The inoculant contains a mixture of mutants selected for increased nitrogen fixation ability. These mutants were derived from indigenous wild-type strains that are capable of high-level occupancy of nodules in soybean fields in the Mississippi delta region. To ensure microbiological purity, the inoculant is fermented directly in the point-of-use container with a vermiculite carrier (L. Graham-Weiss, M. L. Bennett, and A. S. Paau, Appl. Environ. Microbiol. 53:2138-2140, 1987). It should be possible to use this approach to produce more effective Rhizobium inoculants for any legume in any geographical area.     

Phosphatidylcholine levels in Bradyrhizobium japonicum membranes are critical for an efficient symbiosis with the soybean host plant

Phosphatidylcholine (PC), the major membrane phospholipid in eukaryotes, is found in only some bacteria including members of the family Rhizobiaceae. For this reason, it has long been speculated that rhizobial PC might be required for a successful interaction of rhizobia with their legume host plants in order to allow the formation of nitrogen-fixing root nodules. A major pathway for PC formation in prokaryotes involves a threefold methylation of the precursor phosphatidylethanolamine (PE). Here, we report on the isolation of a Bradyrhizobium japonicum gene (pmtA) encoding the phospholipid N-methyltransferase PmtA. Upon expression of the bradyrhizobial pmtA gene in Escherichia coli, predominantly monomethylphosphatidylethanolamine was formed from PE. PmtA-deficient B. japonicum mutants still produced low levels of PC by a second methylation pathway. The amount of PC formed in such mutants (6% of total phospholipids) was greatly decreased compared with the wild type (52% of total phospholipids). Root nodules of soybean plants infected with B. japonicum pmtA mutants showed a nitrogen fixation activity of only 18% of the wild-type level. The interior colour of the nodules was beige instead of red, suggesting decreased amounts of leghaemoglobin. Moreover, ultrastructure analysis of these nodules demonstrated a greatly reduced number of bacteroids within infected plant cells. These data suggest that the biosynthesis of wild-type amounts of PC are required to allow for an efficient symbiotic interaction of B. japonicum with its soybean host plant.