Nodulation is an essential process for biological nitrogen (N 2 ) fixation in legumes, but its regulation remains poorly understood. Here, a b-expansin gene, GmEXPB2, was found to be critical for soybean (Glycine max) nodulation. GmEXPB2 was preferentially expressed at the early stage of nodule development. b-Glucuronidase staining further showed that GmEXPB2 was mainly localized to the nodule vascular trace and nodule vascular bundles, as well as nodule cortical and parenchyma cells, suggesting that GmEXPB2 might be involved in cell wall modification and extension during nodule formation and development. Overexpression of GmEXPB2 dramatically modified soybean root architecture, increasing the size and number of cortical cells in the root meristematic and elongation zones and expanding root hair density and size of the root hair zone. Confocal microscopy with green fluorescent protein-labeled rhizobium USDA110 cells showed that the infection events were significantly enhanced in the GmEXPB2-overexpressing lines. Moreover, nodule primordium development was earlier in overexpressing lines compared with wild-type plants. Thereby, overexpression of GmEXPB2 in either transgenic soybean hairy roots or whole plants resulted in increased nodule number, nodule mass, and nitrogenase activity and thus elevated plant N and phosphorus content as well as biomass. In contrast, suppression of GmEXPB2 in soybean transgenic composite plants led to smaller infected cells and thus reduced number of big nodules, nodule mass, and nitrogenase activity, thereby inhibiting soybean growth. Taken together, we conclude that GmEXPB2 critically affects soybean nodulation through modifying root architecture and promoting nodule formation and development and subsequently impacts biological N 2 fixation and growth of soybean.
Fifteen bacterial strains isolated from molasses grass (Melinis minutiflora Beauv.) were identified as nitrogen-fixers by using the acetylene-reduction assay and PCR amplification of nifH gene fragments. These strains were classified as a unique group by insertion sequence-PCR fingerprinting, SDS-PAGE protein patterns, DNA-DNA hybridization, 16S rRNA gene sequencing and morphological characterization. Phylogenetic analysis of the 16S rRNA gene indicated that these diazotrophic strains belonged to the genus Azospirillum and were closely related to Azospirillum lipoferum (with 97?5 % similarity). In all the analyses, including in addition phenotypic characterization using Biolog MicroPlates and comparison of cellular fatty acids, this novel group was found to be different from the most closely related species, Azospirillum lipoferum. Based on these data, a novel species, Azospirillum melinis sp. nov., is proposed for these endophytic diazotrophs of M. minutiflora, with TMCY 0552 T (=CCBAU 5106001 T =LMG 23364 T =CGMCC 1.5340 T ) as the type strain. INTRODUCTIONAssociation of nitrogen-fixing bacteria and herbaceous plants is a common phenomenon in nature. From this association, wild grasses can obtain nitrogen fixed by the bacteria and grow in nitrogen-deficient soils. Diverse endophytic diazotrophs have been isolated from maize, rice, sorghum, sugar cane, cameroon grass and other gramineous plants (Baldani et al., 1986 Olivares et al., 1996;Reis et al., 2004). Some of these plants could associate with a wide range of bacteria, such as in the case of Kallar grass [Leptochloa fusca (L.) Kunth], a pioneer plant grown on salt-affected, often flooded, low-fertility soils in the Punjab of Pakistan, which has been found to be associated with five nitrogen-fixing endophytic bacterial species (ReinholdHurek et al., 1993;Tan & Reinhold-Hurek, 2003).To date, diverse nitrogen-fixing bacteria, including Azospirillum lipoferum, Azospirillum brasilense, Azospirillum halopraeferens, Azoarcus indigens, Azoarcus communis, Azovibrio restrictus, Azospira oryzae and Burkholderia tropica, have been isolated from the roots of numerous wild and cultivated grasses grown in tropical, subtropical and temperate regions all over the world (Kirchhof et al., 1997;Reinhold et al., 1986Reinhold et al., , 1987Reinhold & Hurek, 1988;Reinhold-Hurek et al., 1993;Reis et al., 2004;Tarrand et al., 1978). Among these bacteria, Azospirillum species have been isolated from roots of numerous wild and cultivated grasses, cereals, food crops and soils in various regions. Based on their microaerophilic and nitrogen-fixing characteristics, semi-solid nitrogen-free medium (Döbereiner, 1980) was the key to the successful isolation of these bacteria. At present, eight species have been described within this genus, including the two original species, Azospirillum lipoferum and Azospirillum brasilense (Tarrand et al., 1978), and the later-described species Azospirillum amazonense (Magalhães et al., 1983), Azospirillum halopraeferens (Reinhold et al., 1987) supplie...
During a study of endophytic nitrogen-fixing bacteria present in the wild rice species Oryza alta, eight novel isolates were obtained from surface-sterilized roots and classified in the genus Rhizobium on the basis of almost-complete 16S rRNA gene sequence analysis. These strains can nodulate Phaseolus vulgaris and Glycine max. The highly similar protein patterns, DNA fingerprint patterns of insertion sequence-based PCR (IS-PCR) and DNA-DNA hybridizations showed that these novel isolates were members of the same species. The closest phylogenetic relatives of the representative strain Alt 505T of the novel group were Rhizobium etli CFN 42 T and Rhizobium indigoferae CCBAU 71714 T , with 96.2 and 96.0 % 16S rRNA gene sequence similarity, respectively. Low DNA-DNA relatedness with the type strains of R. etli, R. indigoferae, Rhizobium hainanense, Rhizobium mongolense and Rhizobium galegae and differences in IS-PCR fingerprinting patterns, SDS-PAGE of proteins, antibiotic resistance, phenotypic tests and comparison of cellular fatty acids with Rhizobium species indicated that the novel group of isolates were distinct from previously described species. Based on these results, we propose to place them in a novel species, as Rhizobium oryzae sp. nov. The type strain is Alt 505 T (5LMG 24253 T 5CGMCC 1.7048 T ).
Twenty-nine rhizobial isolates from root nodules of Astragalus and Lespedeza spp. growing in the Loess Plateau of China were characterized by numerical taxonomy, RFLP and sequencing of PCR-amplified 16S rRNA genes, measurement of DNA G+C content, DNA-DNA relatedness and cross-nodulation with selected legume species. Based on the results of numerical taxonomy, the isolates formed two clusters (1 and 2) with some single isolates at a similarity level of 82 %. Cluster 1 contained six isolates from Astragalus and Lespedeza spp. Cluster 2 consisted of nine isolates from Astragalus spp. DNA relatedness was greater than 80 % among isolates within cluster 2. Phylogenetic analysis based on 16S rRNA gene sequences showed that CCBAU 7190B T , representing cluster 2, was closely related to Rhizobium galegae and Rhizobium huautlense. DNA-DNA relatedness between CCBAU 7190BT and reference strains of R. galegae, R. huautlense and other related species ranged from 0 to 48?6 %. The cluster 2 isolates could also be differentiated phenotypically from related species. Based on these data, a novel species, Rhizobium loessense sp. nov., is proposed for cluster 2, with the type strain CCBAU 7190B
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