A catalogue of molecular, physiological and symbiotic properties of soybean-nodulating rhizobial strains from different soybean cropping areas of China.

We have analysed 198 fast-growing soybean-nodulating rhizobial strains from four different regions of China for the following characteristics: generation time; number of plasmids; lipopolysaccharide (LPS), nodulation factors (LCOs) and PCR profiles; acidification of growth medium; capacity to grow at acid, neutral, and alkaline pH; growth on LC medium; growth at 28 and 37 degrees C; melanin production capacity; Congo red absorption and symbiotic characteristics. These unbiased analyses of a total subset of strains isolated from specific soybean-cropping areas (an approach which could be called "strainomics") can be used to answer various biological questions. We illustrate this by a comparison of the molecular characteristics of five strains with interesting symbiotic properties. From this comparison we conclude, for instance, that differences in the efficiency of nitrogen fixation or competitiveness for nodulation of these strains are not apparently related to differences in Nod factor structure.

Survival of several Rhizobium/Bradyrhizobium strains on different inoculant formulations and inoculated seeds.

The effect of a variety factors on the survival of several rhizobia strains on inoculants and inoculated seeds has been evaluated. Since the rhizobia strains showed different cell-density-evolution patterns on peat-based inoculants and on inoculated seeds, several inoculant formulations with highly effective Rhizobium/Bradyrhizobium strains (for Lupinus, Hedysarum, Phaseolus and Glycine max.) were monitored under the following storage conditions: (a) the inoculants were kept refrigerated (at 4 degrees C), or (b) at room temperature (25 degrees C). The effect of water content (30-50%, w/w) in the inoculants as well as that of several seed-coating adhesives were also investigated. Alternative carriers including perlite and vermiculite were tested. For all of the strains, survival on sterile peat-based inoculants was higher than on the corresponding unsterile peat formulation; for the latter, refrigerated storage conditions are recommended to ensure high bacterial densities. The water content of the inoculants had a differential effect on strain survival depending on the sterility of the peat, such that a high water content was more detrimental when unsterilized peat was employed. The best adherent for rhizobia survival was a gum arabic/water solution. Perlite was as effective as peat in maintaining a high population of rhizobia, at least for 6 months of storage.

Soils of the Chinese Hubei province show a very high diversity of Sinorhizobium fredii strains.

Biodiversity studies of native soybean-nodulating rhizobia in soils from the Chinese Hubei province (Honghu county; pH 8, alluvial soil) have been carried out. Inoculation of an American (Williams) and an Asiatic (Peking) soybean cultivar with eleven soil samples led to the isolation of 167 rhizobia strains. The ratio (%) of slow-/fast-growing isolates was different depending on the trap plant used. All isolates were able to nodulate both cultivars, although the N2-fixation efficiency (measured as plant-top dry weight) was different among them. A total of thirty-three isolates were selected for further characterisation on the basis of physiological parameters, PCR-RFLP of symbiotic genes and Low Molecular Weight RNA, lipopolysaccharide, protein and plasmid profiles. Low Molecular Weight RNA profiling indicates that all the isolates belong to species Sinorhizobium fredii. The dendrogram obtained with the physiological parameters has been useful to classify the isolates at strain level, although plasmid profiling was the most discriminating technique to detect differences among the analysed soybean-rhizobia isolates, showing there is not two isolates identical each other. Plasmid profile analyses also revealed that some of the investigated strains contain low molecular weight plasmids (7-8-kb). They are, to our knowledge, the smallest ever found in rhizobia and they could be the starting point for the construction of the first group of vectors based on a native rhizobia replicon.

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.

Enhanced symbiotic performance by Rhizobium tropici glycogen synthase mutants.

We isolated a Tn5-induced Rhizobium tropici mutant that has enhanced capacity to oxidize N,N-dimethyl-p-phenylendiamine (DMPD) and therefore has enhanced respiration via cytochrome oxidase. The mutant had increased levels of the cytochromes c(1) and CycM and a small increase in the amount of cytochrome aa(3). In plant tests, the mutant increased the dry weight of Phaseolus vulgaris plants by 20 to 38% compared with the control strain, thus showing significantly enhanced symbiotic performance. The predicted product of the mutated gene is homologous to glycogen synthases from several bacteria, and the mutant lacked glycogen. The DNA sequence of the adjacent gene region revealed six genes predicted to encode products homologous to the following gene products from Escherichia coli: glycogen phosphorylase (glgP), glycogen branching enzyme (glgB), ADP glucose pyrophosphorylase (glgC), glycogen synthase (glgA), phosphoglucomutase (pgm), and glycogen debranching enzyme (glgX). All six genes are transcribed in the same direction, and analysis with lacZ gene fusions suggests that the first five genes are organized in one operon, although pgm appears to have an additional promoter; glgX is transcribed independently. Surprisingly, the glgA mutant had decreased levels of high-molecular-weight exopolysaccharide after growth on glucose, but levels were normal after growth on galactose. A deletion mutant was constructed in order to generate a nonpolar mutation in glgA. This mutant had a phenotype similar to that of the Tn5 mutant, indicating that the enhanced respiration and symbiotic nitrogen fixation and decreased exopolysaccharide were due to mutation of glgA and not to a polar effect on a downstream gene.

Co-inoculation with Bacillus sp. CECT 450 improves nodulation in Phaseolus vulgaris L.

The strain Bacillus sp. CECT 450 increased nodulation on bean (Phaseolus vulgaris L.) when co-inoculated with Rhizobium tropici CIAT 899. This positive effect occured under controlled conditions on perlite-vermiculite, sand, or in a mixture of soil and sand. This increase was also observed in a field assay. Nodulation kinetic studies suggested that the synergistic effect is pronounced during the latter stages of cultivation. In contrast, the same bacteria co-inoculated with Bradyrhizobium japonicum USDA 110 reduced nodulation on soybean (Glycine max (L.) Merr.). Inoculation with Bacillus sp. CECT 450 alone had no effect on bean plants, but reduced root growth in soybean. The survival of Bacillus sp. CECT 450 on inoculated seeds was high, even when inoculated seeds were maintained for several months at room temperature.

The soybean cultivar specificity gene nolX is present, expressed in a nodD-dependent manner, and of symbiotic significance in cultivar-nonspecific strains of Rhizobium (Sinorhizobium) fredii.

Rhizobium (now Sinorhizobium) fredii is a symbiotic nitrogen-fixing bacterium that can nodulate soybean in a cultivar-specific manner. This process is governed by a set of negatively acting nodulation genes termed nolXWBTUV. These genes prevent R. fredii strain USDA257 from infecting soybean cultivars such as McCall, but they do not block nodulation of cultivar Peking. R. fredii strain USDA191 contains DNA sequences that hybridize to nolXWBTUV, yet it forms normal nitrogen-fixing nodules on both McCall and Peking soybean. These sequences were isolated and their structure and function examined in comparison to nolXWBTUV of strain USDA257. Restriction maps of the two loci are identical, as is a 2-4 kb DNA sequence that corresponds to nolX and its promoter region. Expression of nolX by strain USDA191 is flavonoid-dependent in culture and readily detectable in nodules. The gene is not inducible in a mutant of strain USDA191 that lacks the regulatory nodD1 gene, and its expression is greatly attenuated in a nodD2 mutant. nolX is also present and flavonoid-inducible in HH103, a second R. fredii strain that nodulates McCall soybean normally. Inactivation of nolX in strain HH103, USDA191 or USDA257 leads to retardation of initial nodulation rates on soybean cultivars such as Peking and to acquisition of the capacity to form nitrogen-fixing nodules on two species of Erythrina. nolX is thus of symbiotic significance in all three strains, even though it regulates soybean cultivar specificity only in strain USDA257.