A survey of soyabean rhizobia was carried out with six Asian and one modern soyabean genotypes as trap hosts. Soyabean seedlings were inoculated with soils from 22 Brazilian field sites, including undisturbed areas or areas traditionally cropped with this legume. A total of 30 fast growing strains, able to establish an effective symbiosis with both types of genotypes, were isolated from 12 of the 22 soils, representing 17% of the population in cropped areas and 24% in undisturbed soils. The bacterial mean generation time varied from 85 to 225 min and after 4 days of growth in YM medium the final pH ranged from 3.7 to 6.9. Although isolated from acid soils, only 37% of the strains were able to grow in TY or YM media at pH 4.0, while 60% were alkaline tolerant (pH 9.5). Most strains produced abundant extracellular polysaccharides (73%), were tolerant to 0.5 M NaCl (60%) and a temperature of 40°C (77%), grew in LB medium (67%) and synthesized melanin (53%). The strains differed in the use of C compounds supplied as sole C sources. The majority of the strains showed an intrinsic resistance to the antibiotics (μg ml −1 ) chloramphenicol (10), erythromycin (50), gentamicin (20), kanamicin (30), rifampicin (20) and tetracycline (10) and to the heavy metals cobalt chloride (0.5 mM) and potassium chromate (0.25 mM). A cluster analysis with 81 morphological and physiological parameters placed the strains from undisturbed soils in the central part of the dendrogram, even when isolated from distant areas, indicating that they had a common background. The relatedness decreased with the use of the soils for agriculture. Most of the strains isolated from cropped soils under conventional tillage were characterised by a high tolerance to stressful conditions and the ability to grow with several C sources, contrary to most of the strains isolated from soils under no-tillage management system. The strains differed from S. fredii in several characteristics and the protein and lipopolysaccharide profiles showed that each strain was unique. Therefore, although soyabean is an exotic plant in Brazil, several indigenous rhizobial strains may also establish an effective symbiosis with this legume.

Preliminary characterization of fast growing rhizobial strains isolated from soyabean nodules in Brazil

Soybean, Glycine max (L.) Merrill, is one of the most important food crops in the world. High soybean yields require large amounts of N fertilizers, which are expensive and can cause environmental problems. The industrial fixation of nitrogen accounts for about 50% of fossil fuel usage in agriculture. In contrast, biological fixation of N2 is a low-cost source of N for soybean cropping through the symbiotic association between the plant and soil bacteria belonging to the genera Bradyrhizobium and Sinorhizobium, which are collectively called “soybean rhizobia“. In general, symbiotic nitrogen fixation in crop legumes not only reduces fertilizer costs but also improves soil fertility through crop rotation and intercropping. Biological nitrogen fixation is due to symbioses between leguminous plants and species of Rhizobium bacteria. Replacing this natural N source by synthetic N fertilizers would cost around 10 billion dollars annually. Moreover, legume seed and foliage have a higher protein content than that of non-legumes, and this makes them desirable protein crops. There is a wide knowledge of the industrial elaboration and use of commercial soybean inoculants based on bradyrhizobia strains. At present, the technology to prepare different types of inoculants, either solid or liquid, is sufficiently developed to meet market requirements, although further research and investments are still required to improve the symbiotic efficacy of rhizobial inoculants. Inoculation of soybeans under field conditions has been successful in the USA, Brazil and Argentina, which are the world leaders in soybean cultivation in terms of acreage and grain yields. There are, however, limitations to a wider use of rhizobial inoculants: the size of indigenous soil rhizobial populations can prevent the successful use of inoculants in some particular areas. For example, many Chinese soils contain more than 105 soybean rhizobia per gram of soil, which imposes a serious barrier for nodule occupancy by the soybean rhizobia used as an inoculant. The use of inoculants based on soil bacteria other than rhizobia has also increased in the last decades. An example is the genus Azospirillum, which can be used for its capacity to increase plant growth and seed yields through different mechanisms, such as the production of plant hormones and the increase in phosphate uptake by roots. In addition, co-inoculation with Azospirillum and rhizobia enhances nodulation and nitrogen fixation. Although less developed, it is expected that inoculants based on mycorrhizal fungi will also play a relevant role in sustainable agriculture and forestry. In spite of any possible limitations, the use of inoculants appears compulsory in a frame of sustainable agriculture, which seeks to increase crop yields and nutrient-use efficiency while reducing the environmental costs associated with agriculture intensification. This review also summarizes some of the most relevant genetic aspects of soybean rhizobia in relation to their symbiosis with soybeans. They can be listed as follows: (1) legume roots exude flavonoids, which are able to activate the transcription of nodulation (nod, nol, noe) genes; (2) expression of nodulation genes results in the production and secretion of lipo-chitin oligosaccharide signal molecules, called LCOs or “Nod factors”, which activate nodule organogenesis in the legume root; (3) LCOs induce numerous responses of the legume roots, such as hair curling and the formation of nodule primordia in the inner or outer cortex; (4) the function of many soybean rhizobia nod genes is known and the chemical structure of the LCOs produced has been determined; (5) in addition to LCOs, different soybean rhizobia surface polysaccharides are required for the formation of nitrogenfixing nodules; (6) surface polysaccharides might act as signal molecules or could prevent plant defense reactions. Cyclic glucans, capsular polysaccharides and lipopolysaccharides appear to play relevant roles in the soybean nodulation process since rhizobial mutants affected in any of these surface polysaccharides are symbiotically impaired. Present knowledge of the molecular bases determining cultivar-strain specificity and nodule occupancy by soybean rhizobia competitors is clearly insufficient. This lack of information is a serious barrier for developing strategies aimed at improving nodulation and symbiotic nitrogen fixation of commercial inoculants. In spite of these difficulties, recent studies have shown that the signaling pathway involved in triggering nodule organogenesis is independent of that operating in bacterial entry through infection thread formation. Theses facts might offer new insights for improving symbiotic nitrogen fixation and also for the feasibility of transferring nodule organogenesis, a first step in expanding this symbiotic interaction into other agriculturally important species.

/pdf/soybean-interactions-with-soil-microbes-agronomical-and-2b2hmq68hh.pdf

Soybean interactions with soil microbes, agronomical and molecular aspects

The Sinorhizobium fredii HH103 rkp-1 region, which is involved in capsular polysaccharides (KPS) production, was isolated and sequenced. The organization of the S. fredii genes identified, rkpUAGHIJ and kpsF3, was identical to that described for S. meliloti 1021 but different from that of S. meliloti AK631. The long rkpA gene (7.5 kb) of S. fredii HH103 and S. meliloti 1021 appears as a fusion of six clustered AK631 genes, rkpABCDEF. S. fredii HH103-Rif(r) mutants affected in rkpH or rkpG were constructed. An exoA mutant unable to produce exopolysaccharide (EPS) and a double mutant exoA rkpH also were obtained. Glycine max (soybean) and Cajanus cajan (pigeon pea) plants inoculated with the rkpH, rkpG, and rkpH exoA derivatives of S. fredii HH103 showed reduced nodulation and severe symptoms of nitrogen starvation. The symbiotic capacity of the exoA mutant was not significantly altered. All these results indicate that KPS, but not EPS, is of crucial importance for the symbiotic capacity of S. fredii HH103-Rif(r). S. meliloti strains that produce only EPS or KPS are still effective with alfalfa. In S. fredii HH103, however, EPS and KPS are not equivalent, because mutants in rkp genes are symbiotically impaired regardless of whether or not EPS is produced.

https://apsjournals.apsnet.org/doi/pdf/10.1094/MPMI-19-0043

Sinorhizobium fredii HH103 mutants affected in capsular polysaccharide (KPS) are impaired for nodulation with soybean and Cajanus cajan.

We mutagenized Sinorhizobium fredii HH103-1 with Tn5-B20 and screened about 2,000 colonies for increased beta-galactosidase activity in the presence of the flavonoid naringenin. One mutant, designated SVQ287, produces lipochitooligosaccharide Nod factors (LCOs) that differ from those of the parental strain. The nonreducing N-acetylglucosamine residues of all of the LCOs of mutant SVQ287 lack fucose and 2-O-methylfucose substituents. In addition, SVQ287 synthesizes an LCO with an unusually long, C20:1 fatty acyl side chain. The transposon insertion of mutant SVQ287 lies within a 1.1-kb HindIII fragment. This and an adjacent 2.4-kb HindIII fragment were sequenced. The sequence contains the 3' end of noeK, nodZ, and noeL (the gene interrupted by Tn5-B20), and the 5' end of nolK, all in the same orientation. Although each of these genes has a similarly oriented counterpart on the symbiosis plasmid of the broad-host-range Rhizobium sp. strain NGR234, there are significant differences in the noeK/nodZ intergenic region. Based on amino acid sequence homology, noeL encodes GDP-D-mannose dehydratase, an enzyme involved in the synthesis of GDP-L-fucose, and nolK encodes a NAD-dependent nucleotide sugar epimerase/dehydrogenase. We show that expression of the noeL gene is under the control of NodD1 in S. fredii and is most probably mediated by the nod box that precedes nodZ. Transposon insertion into neoL has two impacts on symbiosis with Williams soybean: nodulation rate is reduced slightly and competitiveness for nodulation is decreased significantly. Mutant SVQ287 retains its ability to form nitrogen-fixing nodules on other legumes, but final nodule number is attenuated on Cajanus cajan.

/pdf/mutation-in-gdp-fucose-synthesis-genes-of-sinorhizobium-137szuuuar.pdf

Mutation in GDP-fucose synthesis genes of Sinorhizobium fredii alters Nod factors and significantly decreases competitiveness to nodulate soybeans.

The pleiotropic phenotype of an auxotrophic purL mutant (SVQ295) of Sinorhizobium fredii HH103 has been investigated. SVQ295 forms colonies that are translucent, produce more slime and absorb less Congo red than those of wild-type strain HH103. SVQ295 did not grow in minimal medium unless the culture was supplemented with thiamin and adenine or with thiamin and AICA-riboside (5-aminoimidazole-4-carboxamide 1-β-d-ribofuranoside), an intermediate of purine biosynthesis. Bacterial cultures supplemented with AICA-riboside or adenine reached the same culture density, although the doubling time of SVQ295 cultures containing AICA-riboside was clearly longer. S. fredii SVQ295 induced pseudonodules on Glycine max and failed to nodulate six different legumes. On Glycyrrhiza uralensis, however, nodules showing nitrogenase activity and containing infected plant cells were formed. SVQ295 showed auto-agglutination when grown in liquid TY medium and its lipopolysaccharide (LPS) electrophoretic profile differed from that of its parental strain HH103-1. In addition, four monoclonal antibodies that recognize the LPS of S. fredii HH103 failed to recognize the LPS produced by SVQ295. In contrast, 1H-NMR spectra of K-antigen capsular polysaccharides (KPS) produced by SVQ295 and the wild-type strain HH103 were similar. Co-inoculation of soybean plants with SVQ295 and SVQ116 (a nodA mutant derivative of HH103) produced nitrogen-fixing nodules that were only occupied by SVQ116.

/pdf/a-purl-mutant-of-sinorhizobium-fredii-hh103-is-symbiotically-qt9daml3k0.pdf

Manuel Chamber

Papers

A Comparative Study of the Physiological Characteristics, Plasmid Content and Symbiotic Properties of Different Rhizobium fredii Strains