Nitrogen-fixing bacteria promote growth and bioactive components accumulation of Astragalus mongholicus by regulating plant metabolism and rhizosphere microbiota.

BACKGROUND: The excessive application of chemical fertilizers in the cultivation of Astragalus mongholicus Bunge results in a reduction in the quality of the medicinal plant and compromises the sustainable productivity of the soil. PGPB inoculant is a hot topic in ecological agriculture research. In the cultivation of Astragalus mongholicus, the screened nitrogen-fixing bacteria can promote plant growth, however, whether it can promote the accumulation of main bioactive components remains unknown. In this study, mixed inoculants containing 5 strains of growth promoting bacteria (Rhizobium T16 , Sinorhizobium T21 , Bacillus J1 , Bacillus G4 and Arthrobacter J2) were used in the field experiment. The metabolic substances in the root tissues of Astragalus mongholicus were identified during the harvest period by non-targeted metabolomics method, and the differential metabolites between groups were identified by statistical analysis. Meanwhile, high-throughput sequencing was performed to analyze the changes of rhizosphere soil and endophytic microbial community structure after mixed microbial treatment. RESULTS: The results of non-targeted metabolism indicated a significant increase in the levels of 26 metabolites after treatment including 13 flavonoids, 3 saponins and 10 other components. The contents of three plant hormones (abscisic acid, salicylic acid and spermidine) also increased after treatment, which presumed to play an important role in regulating plant growth and metabolism. Studies on endosphere and rhizosphere bacterial communities showed that Rhzobiaceae, Micromonosporaceae, and Hypomicrobiaceae in endophytic, and Oxalobactereae in rhizosphere were significantly increased after treatment. These findings suggest their potential importance in plant growth promotion and secondary metabolism regulation. CONCLUSIONS: This finding provides a basis for developing nitrogen-fixing bacteria fertilizer and improving the ecological planting efficiency of Astragalus mongholicus.

Horizontal gene transfer of the Mer operon is associated with large effects on the transcriptome and increased tolerance to mercury in nitrogen-fixing bacteria.

BACKGROUND: Mercury (Hg) is highly toxic and has the potential to cause severe health problems for humans and foraging animals when transported into edible plant parts. Soil rhizobia that form symbiosis with legumes may possess mechanisms to prevent heavy metal translocation from roots to shoots in plants by exporting metals from nodules or compartmentalizing metal ions inside nodules. Horizontal gene transfer has potential to confer immediate de novo adaptations to stress. We used comparative genomics of high quality de novo assemblies to identify structural differences in the genomes of nitrogen-fixing rhizobia that were isolated from a mercury (Hg) mine site that show high variation in their tolerance to Hg. RESULTS: Our analyses identified multiple structurally conserved merA homologs in the genomes of Sinorhizobium medicae and Rhizobium leguminosarum but only the strains that possessed a Mer operon exhibited 10-fold increased tolerance to Hg. RNAseq analysis revealed nearly all genes in the Mer operon were significantly up-regulated in response to Hg stress in free-living conditions and in nodules. In both free-living and nodule environments, we found the Hg-tolerant strains with a Mer operon exhibited the fewest number of differentially expressed genes (DEGs) in the genome, indicating a rapid and efficient detoxification of Hg from the cells that reduced general stress responses to the Hg-treatment. Expression changes in S. medicae while in bacteroids showed that both rhizobia strain and host-plant tolerance affected the number of DEGs. Aside from Mer operon genes, nif genes which are involved in nitrogenase activity in S. medicae showed significant up-regulation in the most Hg-tolerant strain while inside the most Hg-accumulating host-plant. Transfer of a plasmid containing the Mer operon from the most tolerant strain to low-tolerant strains resulted in an immediate increase in Hg tolerance, indicating that the Mer operon is able to confer hyper tolerance to Hg. CONCLUSIONS: Mer operons have not been previously reported in nitrogen-fixing rhizobia. This study demonstrates a pivotal role of the Mer operon in effective mercury detoxification and hypertolerance in nitrogen-fixing rhizobia. This finding has major implications not only for soil bioremediation, but also host plants growing in mercury contaminated soils.

Azotosporobacter soli gen. nov., sp. nov., a novel nitrogen-fixing bacterium isolated from paddy soil.

A nitrogen-fixing strain designated SG130T was isolated from paddy soil in Fujian Province, China. Strain SG130T was Gram-staining-negative, rod-shaped, and strictly anaerobic. Strain SG130T showed the highest 16S rRNA gene sequence similarities with the type strains Dendrosporobacter quercicolus DSM 1736T (91.7%), Anaeroarcus burkinensis DSM 6283T (91.0%) and Anaerospora hongkongensis HKU 15T (90.9%). Furthermore, the phylogenetic and phylogenomic analysis also suggested strain SG130T clustered with members of the family Sporomusaceae and was distinguished from other genera within this family. Growth of strain SG130T was observed at 25-45 °C (optimum 30 °C), pH 6.0-9.5 (optimum 7.0) and 0-1% (w/v) NaCl (optimum 0.1%). The quinones were Q-8 and Q-9. The polar lipids were phosphatidylserine (PS), phosphatidylethanolamine (PE), glycolipid (GL), phospholipid (PL) and an unidentified lipid (UL). The major fatty acids (> 10%) were iso-C13:0 3OH (26.6%), iso-C17:1 (15.6%) and iso-C15:1 F (11.4%). The genomic DNA G + C content was 50.7%. The average nucleotide identity (ANI) and digital DNA-DNA hybridization (dDDH) values between strain SG130T and the most closely related type strain D. quercicolus DSM 1736T (ANI 68.0% and dDDH 20.3%) were both below the cut-off level for species delineation. The average amino acid identity (AAI) between strain SG130T and the most closely related type strain D. quercicolus DSM 1736T was 63.2%, which was below the cut-off value for bacterial genus delineation (65%). Strain SG130T possessed core genes (nifHDK) involved in nitrogen fixation, and nitrogenase activity (106.38 µmol C2H4 g-1 protein h-1) was examined using the acetylene reduction assay. Based on the above results, strain SG130T is confirmed to represent a novel genus of the family Sporomusaceae, for which the name Azotosporobacter soli gen. nov., sp. nov. is proposed. The type strain is SG130T (= GDMCC 1.3312T = JCM 35641T).

Nitrogen-fixing bacterial communities differ between perennial agroecosystem crops.

Biocrusts, common in natural ecosystems, are specific assemblages of microorganisms at or on the soil surface with associated microorganisms extending into the top centimeter of soil. Agroecosystem biocrusts have similar rates of nitrogen (N) fixation as those in natural ecosystems, but it is unclear how agricultural management influences their composition and function. This study examined the total bacterial and diazotrophic communities of biocrusts in a citrus orchard and a vineyard that shared a similar climate and soil type but differed in management. To contrast climate and soil type, these biocrusts were also compared with those from an apple orchard. Unlike natural ecosystem biocrusts, these agroecosystem biocrusts were dominated by proteobacteria and had a lower abundance of cyanobacteria. All of the examined agroecosystem biocrust diazotroph communities were dominated by N-fixing cyanobacteria from the Nostocales order, similar to natural ecosystem cyanobacterial biocrusts. Lower irrigation and fertilizer in the vineyard compared with the citrus orchard could have contributed to biocrust microbial composition, whereas soil type and climate could have differentiated the apple orchard biocrust. Season did not influence the bacterial and diazotrophic community composition of any of these agroecosystem biocrusts. Overall, agricultural management and climatic and edaphic factors potentially influenced the community composition and function of these biocrusts.

Nodulation Experiment by Cross-Inoculation of Nitrogen-Fixing Bacteria Isolated from Root Nodules of Several Leguminous Plants.

Root-nodule nitrogen-fixing bacteria are known for being specific to particular legumes. This study isolated the endophytic root-nodule bacteria from the nodules of legumes and examined them to determine whether they could be used to promote the formation of nodules in other legumes. Forty-six isolates were collected from five leguminous plants and screened for housekeeping (16S rRNA), nitrogen fixation (nifH), and nodulation (nodC) genes. Based on the 16S rRNA gene sequencing and phylogenetic analysis, the bacterial isolates WC15, WC16, WC24, and GM5 were identified as Rhizobium, Sphingomonas, Methylobacterium, and Bradyrhizobium, respectively. The four isolates were found to have the nifH gene, and the study confirmed that one isolate (GM5) had both the nifH and nodC genes. The Salkowski method was used to measure the isolated bacteria for their capacity to produce phytohormone indole acetic acid (IAA). Additional experiments were performed to examine the effect of the isolated bacteria on root morphology and nodulation. Among the four tested isolates, both WC24 and GM5 induced nodulation in Glycine max. The gene expression studies revealed that GM5 had a higher expression of the nifH gene. The existence and expression of the nitrogen-fixing genes implied that the tested strain had the ability to fix the atmospheric nitrogen. These findings demonstrated that a nitrogen-fixing bacterium, Methylobacterium (WC24), isolated from a Trifolium repens, induced the formation of root nodules in non-host leguminous plants (Glycine max). This suggested the potential application of these rhizobia as biofertilizer. Further studies are required to verify the N2-fixing efficiency of the isolates.

An Integrated Affinity Chromatography-Based Approach to Unravel the sRNA Interactome in Nitrogen-Fixing Rhizobia.

The activity mechanism and function of bacterial base-pairing small non-coding RNA regulators (sRNAs) are largely shaped by their main interacting cellular partners, i.e., proteins and mRNAs. We describe here an MS2 affinity chromatography-based procedure adapted to unravel the sRNA interactome in nitrogen-fixing legume endosymbiotic bacteria. The method consists of tagging of the bait sRNA at its 5'-end with the MS2 aptamer followed by pulse overexpression and immobilization of the chimeric transcript from cell lysates by an MS2-MBP fusion protein conjugated to an amylose resin. The sRNA-binding proteins and target mRNAs are further profiled by mass spectrometry and RNAseq, respectively.

Rhizobium mayense sp. Nov., an efficient plant growth-promoting nitrogen-fixing bacteria isolated from rhizosphere soil.

The nitrogen-fixing bacterium has great prospects in replacing synthetic fertilizers with biofertilizers for plant growth. It would be a useful tool in eradicating chemical fertilizers from use. Five nitrogen-fixing bacteria were isolated from the Tea and Groundnut rhizosphere soil out of which RSKVG 02 proved to be the best. The optimized condition of RSKVG 02 was found to be pH 7 at 30 °C utilizing 1% glucose and 0.05% ammonium sulfate as the sole carbon and nitrogen source. Plant growth-promoting traits such as IAA and ammonia were estimated to be 82.97 ± 0.01254a µg/ml and 80.49 ± 0.23699a mg/ml respectively. Additionally, their phosphate and potassium solubilization efficiency were evaluated to be 46.69 ± 0.00125 b mg/ml and 50.29 ± 0.000266 mg/ml. Morphological, and biochemical methods characterized the isolated bacterial culture, and molecularly identified by 16 S rRNA sequencing as Rhizobium mayense. The isolate was further tested for its effects on the growth of Finger millet (Eleusine coracana) and Green gram (Vigna radiata) under pot conditions. The pot study experiments indicated that the bacterial isolates used as bio inoculants increased the total plant growth compared to the control and their dry weight showed similar results. The chlorophyll content of Green gram and Finger millet was estimated to be 19.54 ± 0.2784a mg/L and 15.3 ± 0.0035 mg/L which suggested that Rhizobium sp. Possesses high nitrogenase activity. The enzyme activity proved to use this bacterium as a biofertilizer property to enhance soil fertility, efficient farming, and an alternative chemical fertilizer. Therefore, Rhizobium mayense can be potentially used as an efficient biofertilizer for crop production and increase yield and soil fertility.

Isolation, identification and characterization of nitrogen fixing endophytic bacteria and their effects on cassava production.

BACKGROUND: Cassava (Manibot esculenta Crantz) is one of the most important among tuber crops. The amount of nitrogen fertilizer used for cassava production is relatively high (400 kg ha-1), but there are few studies on biological nitrogen fixation in this crop. Therefore, it is particularly important to study whether cassava and microorganisms have the associated nitrogen-fixing and other promoting effects of endophytic bacteria. METHODS: We screened 10 endophytic bacteria using the nitrogen-free culture method from the roots of seven cassava cultivars, and the nitrogenase activity of the A02 strain was the highest 95.81 nmol mL-1 h-1. The A02 strain was confirmed as Microbacteriaceae, Curtobacterium using 16S rRNA sequence alignment. The biological and morphological characteristics of strain A02 were further analyzed. RESULTS: The experimental results showed that the biomass of roots, stems, and leaves of cassava inoculated with A02 increased by 17.6%, 12.6%, and 10.3%, respectively, compared to that of the control (without A02 inoculation). These results were not only related to the secretion of auxin (IAA) and solubilization of phosphate but also in the promotion of biological nitrogen fixation of cassava leaves by strain A02. Moreover, the highest 95.81 nmol mL-1h-1 of nitrogenase activity was reported in strain A02, and thus more nitrogen fixation was observed in strain A02. In conclusion, A02 is a newly discovered endophytic nitrogen-fixing bacteria in cassava that can be further used in the research of biological bacterial fertilizers.

Description of two nitrogen-fixing bacteria, Geomonas fuzhouensis sp. nov. and Geomonas agri sp. nov., isolated from paddy soils.

Two strictly anaerobic nitrogen-fixing strains, designated RG17T and RG53T, were isolated from paddy soils in China. Strains RG17T and RG53T showed the highest 16S rRNA gene sequence similarities to the type strain Geomonas paludis (97.9-98.4%). Phylogenetic tree based on 16S rRNA gene sequences showed that two strains clustered with members of the genus Geomonas. Growth of strain RG17T was observed at 20-42 °C, pH 5.5-8.5 and 0-0.3% (w/v) NaCl while strain RG53T growth was observed at 20-42 °C, pH 5.5-9.5 and 0-0.7% (w/v) NaCl. Strains RG17T and RG53T contained MK-8 as main menaquinone and C15:1 ω6c, iso-C15:0, and Summed Feature 3 as the major fatty acids. The genomic DNA G + C content of strains RG17T and RG53T were 61.6 and 60.7%, respectively. The digital DNA-DNA hybridization (dDDH) and average nucleotide identity (ANI) values between the isolated strains and the closely related Geomonas species were lower than the cut-off value (dDDH 70% and ANI 95-96%) for prokaryotic species delineation. Both strains possessed nif genes nifHDK and nitrogenase activities. Based on the above results, the two strains represent two novel species of the genus Geomonas, for which the names Geomonas fuzhouensis sp. nov. and Geomonas agri sp. nov., are proposed. The type strains are RG17T (= GDMCC 1.2687T = KTCC 25332T) and RG53T (= GDMCC 1.2630T = KCTC 25331T), respectively.

Diversification of plasmids in a genus of pathogenic and nitrogen-fixing bacteria.

Members of the agrobacteria-rhizobia complex (ARC) have multiple and diverse plasmids. The extent to which these plasmids are shared and the consequences of their interactions are not well understood. We extracted over 4000 plasmid sequences from 1251 genome sequences and constructed a network to reveal interactions that have shaped the evolutionary histories of oncogenic virulence plasmids. One newly discovered type of oncogenic plasmid is a mosaic with three incomplete, but complementary and partially redundant virulence loci. Some types of oncogenic plasmids recombined with accessory plasmids or acquired large regions not known to be associated with pathogenicity. We also identified two classes of partial virulence plasmids. One class is potentially capable of transforming plants, but not inciting disease symptoms. Another class is inferred to be incomplete and non-functional but can be found as coresidents of the same strain and together are predicted to confer pathogenicity. The modularity and capacity for some plasmids to be transmitted broadly allow them to diversify, convergently evolve adaptive plasmids and shape the evolution of genomes across much of the ARC. This article is part of the theme issue 'The secret lives of microbial mobile genetic elements'.

The Flora Compositions of Nitrogen-Fixing Bacteria and the Differential Expression of nifH Gene in Pennisetum giganteum z.x.lin Roots.

The flora compositions of nitrogen-fixing bacteria in roots of Pennisetum giganteum z.x.lin at different growth stages and the expression and copy number of nitrogen-fixing gene nifH were studied by Illumina Miseq second-generation sequencing technology and qRT-PCR. The results showed that there were more than 40,000~50,000 effective sequences in 5 samples from the roots of P. giganteum, with Proteobacteria and Cyanobacteria as the dominant nitrogen-fixing bacteria based on the OTU species annotations for each sample and Bradyrhizobium as the core bacterial genera. The relative expression and quantitative change of nifH gene in roots of P. giganteum at different growth stages were consistent with the changes in the flora compositions of nitrogen-fixing microbia. Both revealed a changing trend with an initial increase and a sequential decrease, as well as changing order as jointing stage>maturation stage>tillering stage>seedling stage>dying stage. The relative expression and copy number of nifH gene were different in different growth stages, and the difference among groups basically reached a significant level (p < 0.05). The relative expression and copy number of nifH gene at the jointing stage were the highest, and the 2-△△CT value was 4.43 folds higher than that at the seedling stage, with a copy number of 1.32 × 107/g. While at the dying stage, it was the lowest, and the 2-△△CT value was 0.67 folds, with a copy number of 0.31 × 107/g.

Diversity of soil nitrogen-fixing bacteria in the rhizosphere and non-rhizophere soils of Ebinur Lake Wetland.

Ebinur Lake Wetland is an understudied desert wetland ecosystem, particularly regarding nitrogen cycling. This study aimed to ascertain the diversity and richness of nitrogen-fixing bacterial communities in the Ebinur Lake Wetland. The diversity of the nitrogen-fixing bacteria community of nifH genes from the rhizosphere and non-rhizosphere soils of four plants in different seasons were examined using Illumina HiSeq PE250 high-throughput sequencing technology. The correlation between soil environmental factors and diversity and richness of nitrogen-fixing bacteria was studied using the redundancy analysis (RDA). The results showed that the diversity of nitrogen-fixing bacteria in the rhizosphere soil of the constructive plants was higher than that in the non-rhizosphere soil; also, the diversity in July was higher than that in October and April. Geobacter, Pseudomonas and Bradyrhizobium were the dominant common bacteria in different samples of Ebinur Lake Wetland. The RDA showed that the total nitrogen, available potassium and available phosphoruswere significantly correlated with the diversity and richness of nitrogen-fixing bacteria. The diversity and community structure of nitrogen-fixing bacteria in soil samples also changed over time. The community structures of nitrogen-fixing bacteria in the rhizosphere and non-rhizosphere soils of the four plants were not the same during the same period. The correlation between soil environmental factors and the community structure and abundance of nitrogen-fixing bacteria can provide data basis and theoretical support for the degradation and restoration of Ebinur Lake Wetland.

Genomic characterization and computational phenotyping of nitrogen-fixing bacteria isolated from Colombian sugarcane fields.

Previous studies have shown the sugarcane microbiome harbors diverse plant growth promoting microorganisms, including nitrogen-fixing bacteria (diazotrophs), which can serve as biofertilizers. The genomes of 22 diazotrophs from Colombian sugarcane fields were sequenced to investigate potential biofertilizers. A genome-enabled computational phenotyping approach was developed to prioritize sugarcane associated diazotrophs according to their potential as biofertilizers. This method selects isolates that have potential for nitrogen fixation and other plant growth promoting (PGP) phenotypes while showing low risk for virulence and antibiotic resistance. Intact nitrogenase (nif) genes and operons were found in 18 of the isolates. Isolates also encode phosphate solubilization and siderophore production operons, and other PGP genes. The majority of sugarcane isolates showed uniformly low predicted virulence and antibiotic resistance compared to clinical isolates. Six strains with the highest overall genotype scores were experimentally evaluated for nitrogen fixation, phosphate solubilization, and the production of siderophores, gibberellic acid, and indole acetic acid. Results from the biochemical assays were consistent and validated computational phenotype predictions. A genotypic and phenotypic threshold was observed that separated strains by their potential for PGP versus predicted pathogenicity. Our results indicate that computational phenotyping is a promising tool for the assessment of bacteria detected in agricultural ecosystems.

Minimal gene set from Sinorhizobium (Ensifer) meliloti pSymA required for efficient symbiosis with Medicago.

Reduction of N2 gas to ammonia in legume root nodules is a key component of sustainable agricultural systems. Root nodules are the result of a symbiosis between leguminous plants and bacteria called rhizobia. Both symbiotic partners play active roles in establishing successful symbiosis and nitrogen fixation: while root nodule development is mostly controlled by the plant, the rhizobia induce nodule formation, invade, and perform N2 fixation once inside the plant cells. Many bacterial genes involved in the rhizobia-legume symbiosis are known, and there is much interest in engineering the symbiosis to include major nonlegume crops such as corn, wheat, and rice. We sought to identify and combine a minimal bacterial gene complement necessary and sufficient for symbiosis. We analyzed a model rhizobium, Sinorhizobium (Ensifer) meliloti, using a background strain in which the 1.35-Mb symbiotic megaplasmid pSymA was removed. Three regions representing 162 kb of pSymA were sufficient to recover a complete N2-fixing symbiosis with alfalfa, and a targeted assembly of this gene complement achieved high levels of symbiotic N2 fixation. The resulting gene set contained just 58 of 1,290 pSymA protein-coding genes. To generate a platform for future synthetic manipulation, the minimal symbiotic genes were reorganized into three discrete nod, nif, and fix modules. These constructs will facilitate directed studies toward expanding the symbiosis to other plant partners. They also enable forward-type approaches to identifying genetic components that may not be essential for symbiosis, but which modulate the rhizobium's competitiveness for nodulation and the effectiveness of particular rhizobia-plant symbioses.

Diversity and abundance of culturable nitrogen-fixing bacteria in the phyllosphere of maize.

AIMS: The present study aimed at gaining an insight into the abundance and genetic diversity of culturable N-fixing epiphyte bacteria on the phyllosphere of maize in arid and semi-arid regions of Iran. METHODS AND RESULTS: Leaf samples of the maize variety, 'single cross 704' (Zea mays L.) were collected from different locations in Iran. The community of culturable N-fixing epiphyte bacteria present was examined by 16S rRNA sequencing, BOXAIR-polymerase chain reaction (PCR) and restricted fragment length polymorphisms analysis of 16S rRNA gene (16S-RFLP). Approximately, 31·82% of the 242 isolates were identified as N-fixers by cultivation of bacteria in Rennie medium and detection of their nifH gene. The N-fixers were affiliated with four bacterial phyla: Firmicutes, Proteobacteria, Actinobacteria and Bacteroidetes. 16S rRNA sequencing detected 16 genera and 24 different species in the identified phyla. The most dominant genus was Bacillus and the species identified were B. pumilus, B. amyloliquefaciens, B. subtilis, B. paralicheniformis, B. licheniformis, B. niabensis and B. megaterium. In total, 22 RFLP groups were present among the isolates originally identified as N-fixing bacteria. BOXAIR-PCR showed that there was a low similarity level among the N-fixing bacteria isolates, and genetic differentiation of individual strains was relatively great. CONCLUSIONS: Our findings suggest that nitrogen-fixing epiphyte bacteria on the phyllosphere of maize may provide significant nitrogen input into arid and semi-arid ecosystem. SIGNIFICANCE AND IMPACT OF THE STUDY: This research implies that phyllosphere epiphyte diazotrophs have much to offer in sustainable agriculture and can be an alternative to chemical N-fertilizers for providing nitrogen to crops arid and semi-arid regions.

Effect of different short-term tillage management on nitrogen-fixing bacteria community in a double-cropping paddy field of southern China.

Soil nitrogen (N)-fixing bacteria community plays an important role in the N cycling process in soil, but there is still limited information about how the soil microbes that drive this process to respond to combined application of tillage and crop residue management under the double-cropping rice (Oryza sativa L.) paddy field in southern of China. Therefore, the effects of 6-years short-term tillage treatment on soil N-fixing bacteria community under the double-cropping rice paddy field in southern China were studied by using the polymerase chain reaction-denaturing gradient gel electrophoresis method. The field experiment included four tillage treatments: conventional tillage with crop residue incorporation (CT), rotary tillage with crop residue incorporation (RT), no-tillage with crop residue retention (NT), rotary tillage with crop residue removed as control (RTO). The results showed that the diversity index and richness index of cbbLR and nifH genes with CT, RT, and NT treatments were increased, compared with RTO treatment. Compared with RTO treatment, the abundance of cbbLR gene with CT, RT, and NT treatments were increased by 6.54, 4.73, and 2.78 times, respectively. Meanwhile, the abundance of nifH gene with CT, RT, and NT treatments were 5.32, 3.71, and 2.45 times higher than that of RTO treatment. The results also indicated that soil autotrophic Azotobacter and nitrogenase activity with CT and RT treatments were significantly higher (p < .05) than that of RTO treatment. There was an obvious difference in characteristic of soil N-fixing bacteria community between the application of crop residue and without crop residue input treatments. In summary, the results indicated that the abundance of N-fixing bacteria community in the double-cropping rice paddy field increased with conventional tillage and rotary tillage practice.

Bradyrhizobium campsiandrae sp. nov., a nitrogen-fixing bacterial strain isolated from a native leguminous tree from the Amazon adapted to flooded conditions.

The nitrogen-fixing bacterial strain UFLA 01-1174T was isolated from nodules of Campsiandra laurilifolia Benth. originating from the Amazon region, Brazil. Its taxonomic position was defined using a polyphasic approach. Analysis of the 16S rRNA gene placed the strain in the Bradyrhizobium genus, the closest species being B. guangdongense CCBAU 51649T and B. guangzhouense CCBAU 51670T, both with 99.8% similarity. Multilocus sequence analysis (MLSA) of recA, gyrB, glnII, rpoB, atpD, and dnaK indicated that UFLA 01-1174T is a new species, most closely related to B. stylosanthis BR 446T (94.4%) and B. manausense BR 3351T (93.7%). Average nucleotide identity (ANI) differentiated UFLA 01-1174T from the closest species with values lower than 90%. The G + C content in the DNA of UFLA 01-1174T is 63.6 mol%. Based on this data, we conclude that the strain represents a new species. The name proposed is Bradyrhizobium campsiandrae, with UFLA 01-1174T (= INPA 394BT = LMG 10099T) as type strain.

Paraburkholderia atlantica sp. nov. and Paraburkholderia franconis sp. nov., two new nitrogen-fixing nodulating species isolated from Atlantic forest soils in Brazil.

A polyphasic study was conducted with 11 strains trapped by Mimosa pudica and Phaseolus vulgaris grown in soils of the Brazilian Atlantic Forest. In the phylogenetic analysis of the 16S rRNA gene, one clade of strains (Psp1) showed higher similarity with Paraburkholderia piptadeniae STM7183T (99.6%), whereas the second (Psp6) was closely related to Paraburkholderia tuberum STM678T (99%). An MLSA (multilocus sequence analysis) with four (recA, gyrB, trpB and gltB) housekeeping genes placed both Psp1 and Psp6 strains in new clades, and BOX-PCR profiles indicated high intraspecific genetic diversity within each clade. Values of digital DNA-DNA hybridization (dDDH) and average nucleotide identity (ANI) of the whole genome sequences were of 56.9 and 94.4% between the Psp1 strain CNPSo 3157T and P. piptadeniae; and of 49.7% and 92.7% between the Psp6 strain CNPSo 3155T and P. tuberum, below the threshold for species delimitation. In the nodC analysis, Psp1 strains clustered together with P. piptadeniae, while Psp6 did not group with any symbiotic Paraburkholderia. Other phenotypic, genotypic and symbiotic properties were evaluated. The polyphasic analysis supports that the strains represent two novel species, for which the names Paraburkholderia franconis sp. nov. with type strain CNPSo 3157T (= ABIP 241, = LMG 31644) and Paraburkholderia atlantica sp. nov. with type strain CNPSo 3155T (= ABIP 236, = LMG 31643) are proposed.

Mining the roots of various species of the halophyte Suaeda for halotolerant nitrogen-fixing endophytic bacteria with the potential for promoting plant growth.

Saline area may tend to be a productive land; however, many of salt-affected soils have nitrogen limitation and depend on plant-associated diazotrophs as their source of 'new' nitrogen. Herein, a total of 316 salinity tolerant nitrogen-fixing endophytic bacteria were isolated from roots of the halophyte Suaeda sp. sampled from 22 different areas of Iran to prepare the collection of nitrogen-fixing bacterial endophytes and evaluate the plant growth-promoting effect of effective isolates on growth of the halophyte Suaeda maritima. All of the identified nitrogen-fixing endophytes were classified to Proteobacteria, Actinobacteria, Firmicutes, and Bacteroidetes phylum while we did not detect common nitrogen-fixing endophyte of glycophytes like Azospirillum. The genera Pseudomonas and Microbacterium were both encountered in high abundance in all samples, indicating that they might play an advanced role in the micro-ecosystem of the halophyte Suaeda. In addition, the results also showed that not only soil salinity can affect halophyte endophytic composition but also other factors such as geographical location, plant species, and other soil properties may be involved. Interestingly, only Zhihengliuella halotolerans and Brachybacterium sp. belonging to Actinobacteria could grow in semi-solid N-free (NFb) medium supplemented with 6% NaCl and highly enhanced growth of S. maritima in vitro. Overall, this study offers useful new resources for nitrogen-fixing endophytic bacteria which may be utilized to improve approaches for providing bio-fertilizer useful in saline-based agriculture.

Methods for Isolation and Characterization of Nitrogen-Fixing Legume-Nodulating Bacteria.

Symbiotic nitrogen fixation (SNF) is a characteristic feature of nodulating legumes. The wild legumes are comparatively less explored for their SNF ability; hence, it is essential to study nodulation and identify the microsymbiont diversity associated with them. This chapter aims to describe the methodology for nodule hunting; trapping, isolation, and characterization of root nodule bacteria (RNB) at phenotypic, genotypic, and symbiotic levels. The documentation of nodulating native legume species and the rhizobial diversity associated with them in various parts of world has gained attention as this symbiotic association provides fixed nitrogen, improves productivity of plants in an ecofriendly manner. Before field-based applications the symbiotic bacteria need to be assessed for their N fixing ability as well as characterized at molecular level. The phylogeny based on symbiosis-essential genes supplemented with the host-range studies helps in better understanding of the symbiotaxonomy of rhizobia. More efficient symbiotic couples need to be screened by cross-nodulation studies for their application in agricultural practices.