Rhizobium hidalgonense and Rhizobium redzepovicii as faba bean (Vicia faba L.) microsymbionts in Mexican soils.

As a legume crop widely cultured in the world, faba bean (Vicia faba L.) forms root nodules with diverse Rhizobium species in different regions. However, the symbionts associated with this plant in Mexico have not been studied. To investigate the diversity and species/symbiovar affiliations of rhizobia associated with faba bean in Mexico, rhizobia were isolated from this plant grown in two Mexican sites in the present study. Based upon the analysis of recA gene phylogeny, two genotypes were distinguished among a total of 35 isolates, and they were identified as Rhizobium hidalgonense and Rhizobium redzepovicii, respectively, by the whole genomic sequence analysis. Both the species harbored identical nod gene cluster and the same phylogenetic positions of nodC and nifH. So, all of them were identified into the symbiovar viciae. As a minor group, R. hidalgonense was only isolated from slightly acid soil and R. redzepovicii was the dominant group in both the acid and neutral soils. In addition, several genes related to resistance to metals (zinc, copper etc.) and metalloids (arsenic) were detected in genomes of the reference isolates, which might offer them some adaptation benefits. As conclusion, the community composition of faba bean rhizobia in Mexico was different from those reported in other regions. Furthermore, our study identified sv. viciae as the second symbiovar in the species R. redzepovicii. These results added novel evidence about the co-evolution, diversification and biogeographic patterns of rhizobia in association with their host legumes in distinct geographic regions.

Diversity of rhizobial and non-rhizobial bacteria nodulating wild ancestors of grain legume crop plants.

Chickpeas, lentils, and peas are the oldest grain legume species that spread to other regions after their first domestication in Fertile Crescent, and they could reveal the rhizobial evolution in relation to the microsymbionts of wild species in this region. This study investigated the phenotypic and genotypic diversity of the nodule-forming rhizobial bacteria recovered from Pisum sativum subsp., Cicer pinnatifidum, and Lens culinaris subsp. orientalis exhibiting natural distribution in the Gaziantep province of Turkey. PCA analyses of rhizobial isolates, which were tested to be highly resistant to stress conditions, showed that especially pH and salt concentrations had an important effect on these bacteria. Phylogenetic analysis based on 16S rRNA determined that these wild species were nodulated by at least 7 groups including Rhizobium and non-Rhizobium. The largest group comprised of Rhizobium leguminosarum and Rhizobium sp. while R. pusense, which was previously determined as non-symbiotic species, was found to nodulate C. pinnatifidum and L. culinaris subsp. orientalis. In recent studies, Klebsiella sp., which is stated to be able to nodulate different species, strong evidences have been obtained in present study exhibiting that Klebsiella sp. can nodulate C. pinnatifidum and Pseudomonas sp. was able to nodulate C. pinnatifidum and P. sativum subsp. Additionally, L. culinaris subsp. orientalis unlike other plant species, was nodulated by Burkholderia sp. and Serratia sp. associated isolates. Some isolates could not be characterized at the species level since the 16S rRNA sequence similarity rate was low and the fact that they were in a separate group supported with high bootstrap values in the phylogenetic tree may indicate that these isolates could be new species. The REP-PCR fingerprinting provided results supporting the existence of new species nodulating wild ancestors.

Rhizobium dioscoreae sp. nov., a plant growth-promoting bacterium isolated from yam (Dioscorea species).

This study investigated endophytic nitrogen-fixing bacteria isolated from two species of yam (water yam, Dioscorea alata L.; lesser yam, Dioscorea esculenta L.) grown in nutrient-poor alkaline soil conditions on Miyako Island, Okinawa, Japan. Two bacterial strains of the genus Rhizobium, S-93T and S-62, were isolated. The phylogenetic tree, based on the almost-complete 16S rRNA gene sequences (1476 bp for each strain), placed them in a distinct clade, with Rhizobium miluonense CCBAU 41251T, Rhizobium hainanense I66T, Rhizobium multihospitium HAMBI 2975T, Rhizobium freirei PRF 81T and Rhizobium tropici CIAT 899T being their closest species. Their bacterial fatty acid profile, with major components of C19 : 0 cyclo ω8c and summed feature 8, as well as other phenotypic characteristics and DNA G+C content (59.65 mol%) indicated that the novel strains belong to the genus Rhizobium. Pairwise average nucleotide identity analyses separated the novel strains from their most closely related species with similarity values of 90.5, 88.9, 88.5, 84.5 and 84.4 % for R. multihospitium HAMBI 2975T, R. tropici CIAT 899T, R. hainanense CCBAU 57015T, R. miluonense HAMBI 2971T and R. freirei PRF 81T, respectively; digital DNA-DNA hybridization values were in the range of 26-42 %. Considering the phenotypic characteristics as well as the genomic data, it is suggested that strains S-93T and S-62 represent a new species, for which the name Rhizobium dioscoreae is proposed. The type strain is S-93T (=NRIC 0988T=NBRC 114257T=DSM 110498T).

Rhizobium rhizophilum sp. nov., an indole acetic acid-producing bacterium isolated from rape (Brassica napus L.) rhizosphere soil.

A novel Gram-stain-negative, aerobic, rod-shaped and indole acetic acid-producing strain, designated 7209-2T, was isolated from rhizosphere of rape (Brassica napus L.) grown in the Yakeshi City, Inner Mongolia, PR China. The 16S rRNA gene sequence analysis indicated that strain 7209-2T belongs to the genus Rhizobium and is closely related to Rhizobium rosettiformans W3T, Rhizobium ipomoeae shin9-1T and Rhizobium wuzhouense W44T with sequence similarities of 98.2, 98.1 and 97.9 %, respectively. Phylogenetic analysis based on concatenated housekeeping recA and atpD gene sequences showed that strain 7209-2T formed a group together with R. wuzhouense W44T and R. rosettiformans W3T, with sequences similarities of 92.6 and 91.1 %, respectively. The genome size of strain 7209-2T was 5.25 Mb, comprising 5027 predicted genes with a DNA G+C content of 61.2 mol%. The average nucleotide identity and digital DNA-DNA hybridization comparisons among 7209-2T and reference strains for the most closely related species showed values below the accepted threshold for species discrimination. The major fatty acids of strain 7209-2T were summed feature 8 (C18 : 1 ω7c and/or C18 : 1 ω6c) and summed feature 2 (C12 : 0 aldehyde and/or unknown 10.953) . The major polar lipids were found to consist of phosphatidylethanolamine, diphosphatidylglycerol, phosphatidylglycerol, phosphatidylcholine and an unidentified aminophospholipid. The predominant ubiquinone was identified as quinone 10. Based on all the above results, strain 7209-2T represents a novel species of the genus Rhizobium, for which the name Rhizobium rhizophilum sp. nov. is proposed with 7209-2T (=CGMCC 1.15691T=DSM 103161T) as the type strain.

Molecular Diversity Analysis of Plant Growth Promoting Rhizobium Isolated from Groundnut and Evaluation of Their Field Efficacy.

Rhizobium are nitrogen-fixing bacteria which possess the nif gene that codes for the nitrogenase enzyme involved in the reduction of atmospheric dinitrogen (N2) to ammonia. Thirty rhizobial strains were identified from ten groundnut plant root nodules collected from semi-arid regions of Rajasthan, India. The isolates were initially identified on the basis of morphological, biochemical, and molecular characteristics. These rhizobium strains were further screened for plant growth promoting activities. Twenty-eight strains were able to produce indole acetic acid, nine strains could solubilize phosphate, and twenty-nine strains exhibited positive results for siderophore and ammonia production. All the bacterial strains were able to efficiently nodulate the groundnut under pot conditions and based on multiple PGP activities six strains were selected for field evaluation. Field experiments confirmed the effectiveness of these selected rhizobium strains resulted in significantly higher nodule number, nodule dry weight, grain yield, and yield components of inoculated plants. Inoculation of the rhizobium strain GN223 followed by GN221 resulted in high yield and field efficiency. Isolation of effective microbial strains is the prerequisite to increase the yield which is evident from the field data of the present study. Hence, these strains might serve as proficient inoculants.

Rhizobium deserti sp. Nov Isolated from Biological Soil Crusts Collected at Mu Us Sandy Land, China.

A novel gram-negative, aerobic, non-spore-forming, rod-shaped, and non-nitrogen-fixing bacterium, named SPY-1T, was isolated from biological soil crusts collected at Mu Us Sandy Land, China. Based on 16S rRNA sequence similarity, strain SPY-1T was most closely related to Neorhizobium alkalisoli CCTCC AB 2014138T (98.7%), Neorhizobium huautlense CGMCC 1.2538T (98.6%), Neorhizobium galegae DSM 11542T (98.4%), Rhizobium wenxiniae 166T (97.9%), and Rhizobium smilacinae CCTCC AB 2013016T (97.5%). Phylogenetic analysis based on 16S rRNA sequencing and multilocus sequence analysis of partial sequences of atpD-glnII-glnA-recA-ropD-thrC housekeeping genes both indicated that strain SPY-1T was a member of the genus Rhizobium. The draft genome of strain SPY-1T was 4.75 Mb in size, and the G + C content was 60.0%. The average nucleotide identity (ANI) values to N. alkalisoli CCTCC AB 2014138T and R. smilacinae CCTCC AB 2013016T were both 84.0%. The digital DNA-DNA hybridization (dDDH) values to N. alkalisoli CCTCC AB 2014138T and R. smilacinae CCTCC AB 2013016T were 20.9% and 20.2%, respectively. The major cellular fatty acids were summed feature 8 (C18:1ω7c and/or C18:1ω6c) and C16:0. Based on the data from chemotaxonomic, phylogenetic, and phenotypic evidence, strain SPY-1T represents a novel species in the genus Rhizobium, for which the name Rhizobium deserti sp. nov. is proposed. The type strain is SPY-1T (= ACCC 61627T = JCM 33732T).

The promiscuity of Phaseolus vulgaris L. (common bean) for nodulation with rhizobia: a review.

Phaseolus vulgaris L. (common bean) is a legume indigenous to American countries currently cultivated in all continents, which is nodulated by different rhizobial species and symbiovars. Most of species able to nodulate this legume worldwide belong to the genus Rhizobium, followed by those belonging to the genera Ensifer (formerly Sinorhizobium) and Pararhizobium (formerly Rhizobium) and minority by species of the genus Bradyrhizobium. All these genera belong to the phylum alpha-Proteobacteria, but the nodulation of P. vulgaris has also been reported for some species belonging to Paraburkholderia and Cupriavidus from the beta-Proteobacteria. Several species nodulating P. vulgaris were originally isolated from nodules of this legume in American countries and are linked to the symbiovars phaseoli and tropici, which are currently present in other continents probably because they were spread in their soils together with the P. vulgaris seeds. In addition, this legume can be nodulated by species and symbiovars originally isolated from nodules of other legumes due its high promiscuity, a concept currently related with the ability of a legume to be nodulated by several symbiovars rather than by several species. In this article we review the species and symbiovars able to nodulate P. vulgaris in different countries and continents and the challenges on the study of the P. vulgaris endosymbionts diversity in those countries where they have not been studied yet, that will allow to select highly effective rhizobial strains in order to guarantee the success of P. vulgaris inoculation.

Insights into the Phylogeny, Nodule Function, and Biogeographic Distribution of Microsymbionts Nodulating the Orphan Kersting's Groundnut [Macrotyloma geocarpum (Harms) Marechal & Baudet] in African Soils.

Kersting's groundnut [Macrotyloma geocarpum (Harms) Marechal & Baudet] is a neglected indigenous African legume adapted to growth in N-deficient soils due to its ability to fix atmospheric N2 via symbiosis with rhizobia. Despite its nutritional and medicinal uses, to date there is little information on the phylogeny and functional traits of its microsymbionts, aspects that are much needed for its conservation and improvement. This study explored the morphogenetic diversity, phylogenetic relationships, and N2-fixing efficiency of Kersting's groundnut rhizobial isolates from contrasting environments in Ghana, South Africa, and Mozambique. BOX-PCR fingerprinting revealed high diversity among the rhizobial populations, which was influenced by geographic origin. Of the 164 isolates evaluated, 130 BOX-PCR types were identified at a 70% similarity coefficient, indicating that they were not clones. Soil pH and mineral concentrations were found to influence the distribution of bradyrhizobial populations in African soils. Phylogenetic analysis of 16S rRNA genes and multilocus sequence analysis of protein-coding genes (atpD, glnII, gyrB, and rpoB) and symbiotic genes (nifH and nodC) showed that Kersting's groundnut is primarily nodulated by members of the genus Bradyrhizobium, which are closely related to Bradyrhizobium vignae 7-2T, Bradyrhizobium kavangense 14-3T, Bradyrhizobium subterraneum 58-2-1T, Bradyrhizobium pachyrhizi PAC48T, the type strain of Bradyrhizobium elkanii, and novel groups of Bradyrhizobium species. The bradyrhizobial populations identified exhibited high N2 fixation and induced greater nodulation, leaf chlorophyll concentration, and photosynthetic rates in their homologous host than did the 5 mM KNO3-fed plants and/or the commercial Bradyrhizobium sp. strain CB756, suggesting that they could be good candidates for inoculant formulations upon field testing.IMPORTANCE Rhizobia play important roles in agroecosystems, where they contribute to improving overall soil health through their symbiotic relationship with legumes. This study explored the microsymbionts nodulating Kersting's groundnut, a neglected orphan legume. The results revealed the presence of different bradyrhizobial populations with high N2-fixing efficiencies as the dominant symbionts of this legume across diverse agroecologies in Africa. Our findings represent a useful contribution to the literature in terms of the community of microsymbionts nodulating a neglected cultivated legume and its potential for elevation as a major food crop. The presence of potentially novel bradyrhizobial symbionts of Kersting's groundnut found in this study offers an opportunity for future studies to properly describe, characterize, and delineate these isolates functionally and phylogenetically for use in inoculant production to enhance food/nutritional security.

Genomic insights into plant growth promoting rhizobia capable of enhancing soybean germination under drought stress.

BACKGROUND: The role of soil microorganisms in plant growth, nutrient utilization, drought tolerance as well as biocontrol activity cannot be over-emphasized, especially in this era when food crisis is a global challenge. This research was therefore designed to gain genomic insights into plant growth promoting (PGP) Rhizobium species capable of enhancing soybean (Glycine max L.) seeds germination under drought condition. RESULTS: Rhizobium sp. strain R1, Rhizobium tropici strain R2, Rhizobium cellulosilyticum strain R3, Rhizobium taibaishanense strain R4 and Ensifer meliloti strain R5 were found to possess the entire PGP traits tested. Specifically, these rhizobial strains were able to solubilize phosphate, produce exopolysaccharide (EPS), 1-aminocyclopropane-1-carboxylate (ACC), siderophore and indole-acetic-acid (IAA). These strains also survived and grew at a temperature of 45 °C and in an acidic condition with a pH 4. Consequently, all the Rhizobium strains enhanced the germination of soybean seeds (PAN 1532 R) under drought condition imposed by 4% poly-ethylene glycol (PEG); nevertheless, Rhizobium sp. strain R1 and R. cellulosilyticum strain R3 inoculations were able to improve seeds germination more than R2, R4 and R5 strains. Thus, genomic insights into Rhizobium sp. strain R1 and R. cellulosilyticum strain R3 revealed the presence of some genes with their respective proteins involved in symbiotic establishment, nitrogen fixation, drought tolerance and plant growth promotion. In particular, exoX, htrA, Nif, nodA, eptA, IAA and siderophore-producing genes were found in the two rhizobial strains. CONCLUSIONS: Therefore, the availability of the whole genome sequences of R1 and R3 strains may further be exploited to comprehend the interaction of drought tolerant rhizobia with soybean and other legumes and the PGP ability of these rhizobial strains can also be harnessed for biotechnological application in the field especially in semiarid and arid regions of the globe.

Rhizobium panacihumi sp. nov., an isolate from ginseng-cultivated soil, as a potential plant growth promoting bacterium.

A novel bacterial strain designated DCY116T was isolated from ginseng-cultivated soil in Gochang-gun, Republic of Korea. Strain DCY116T, belongs to the genus Rhizobium, and is closely related to Rhizobium yantingense H66T (98.3%), Neorhizobium huautlense S02T (98.2%), Rhizobium soli DS-42T (98.1%), Rhizobium smilacinae PTYR-5T (97.9%), and Neorhizobium alkalisoli CCBAU 01393T (97.9%) based on 16S rRNA gene sequence analysis. Analysis of the housekeeping genes atpD, recA, and glnII showed low levels of sequence similarity (96.8%) between strain DCY116T and other closely related species. Strain DCY116T was Gram-stain negative, motile by peritrichous flagella, rod-shaped, strictly aerobic, catalase- and oxidase-positive. Q-10 was the predominant ubiquinone. The major cellular fatty acids were identified as C16:0 and summed feature 8 (C18:1 ω7c and/or C18:1 ω6c). The major polar lipids were diphosphatidylglycerol, phosphatidylethanolamine, and an unknown lipid (L1-3). Genomic DNA G + C content of strain DCY116T was determined to be 57.2 mol%. DNA-DNA homology values between strain DCY116T and closely related species of the genus Rhizobium were lower than 40%. Strain DCY116T produced indole-3-acetic acid, siderophores, and was able to solubilize phosphate as a potential plant growth promoting bacterium. In conclusion, the results of this study support strain DCY116T as a novel species of the genus Rhizobium, for which the name Rhizobium panacihumi is proposed. The type strain is DCY116T (= KCTC 62017T = JCM 32251T).

Heavy metal accumulation in Lathyrus sativus growing in contaminated soils and identification of symbiotic resistant bacteria.

In this study, two populations of leguminous plants Lathyrus sativus were grown in four soils that were collected from sites differently contaminated by heavy metals. Evaluations included basic soil properties, concentrations of major nutrients and four metals (copper, zinc, lead and cadmium) in these soils. Investigation of Lathyrus sativus response to contamination showed that the increase of heavy metal concentration in soils affected biomass of plant, number of nodules and plant metal uptake. Heavy metal tolerance of 46 isolated bacteria from the root nodules was evaluated and demonstrated that the maximum concentration of Cd, Pb, Cu and Zn tolerated by strains were 0.8, 2.5, 0.2, and 0.5 mM, respectively. Twenty-two isolates were tested for their effects on plant biomass production and nodule formation and showed that only R. leguminosarum nodulated Lathyrus sativus, while some bacteria improved the shoot and root dry biomass. Sequences of their 16S rDNA gene fragments were also obtained and evaluated for tentative identification of the isolates which revealed different bacterial genera represented by Rhizobium sp, Rhizobium leguminosarum, Sinorhizobium meliloti, Pseudomonas sp, Pseudomonas fluorescens, Luteibacter sp, Variovorax sp, Bacillus simplex and Bacillus megaterium. The existence of Pb- and Cd-resistant genes (PbrA and CadA) in these bacteria was determined by PCR, and it showed high homology with PbrA and CadA genes from other bacteria. The tested resistant population was able to accumulate high concentrations of Pb and Cd in all plant parts and, therefore, can be classified as a strong metal accumulator with suitable potential for phytoremediation of Pb and Cd polluted sites. Heavy metal resistant and efficient bacteria isolated from root nodules were chosen with Lathyrus sativus to form symbiotic associations for eventual bioremediation program, which could be tested to remove pollutants from contaminated sites.

Rhizobium chutanense sp. nov., isolated from root nodules of Phaseolus vulgaris in China.

Two Gram-stain-negative, rod-shaped bacterial strains (C5T and C16), isolated from root nodules of Phaseolus vulgaris L. in Jiangxi Province, PR China, were characterized by using a polyphasic taxonomical approach. The phylogenetic analysis of the 16S rRNA gene and three concatenated housekeeping genes (recA-glnII-atpD) revealed that C5T and C16 were members of the genus Rhizobium, yet were distinct from known species. The case for strain C5T representing a novel species was supported by genomic results. Pairwise digital DNA-DNA hybridization and average nucleotide identity values were much lower than the proposed and generally accepted species boundaries. The genome-based phylogenetic tree reconstructed by using the up-to-date bacterial core gene set consisting of 92 genes showed that the strains formed a monophyletic branch, further supporting this result. The symbiotic genes of nodC and nifH were identified in both strains; each could nodulate Phaseolus vulgaris and Glycine max but not Leucaena leucocephala, Pisum sativum or Medicago sativa plants. Major cellular fatty acids of C5T were summed feature 8 (C18 : 1 ω7c/C18 : 1 ω6c; 58.8 %), C18 : 1 ω7c 11-methyl (14.2 %) and C18 : 0 (8.1 %). The DNA G+C content of C5T was 61.4 mol%. Based on these genomic, chemotaxonomic and phenotypic characteristics, we propose a novel species: Rhizobium chutanense sp. nov. The type strain is C5T (=CCTCC AB 2018143T=LMG 30777T).

Bradyrhizobium and Pseudomonas strains obtained from coal-mining areas nodulate and promote the growth of Calopogonium muconoides plants used in the reclamation of degraded areas.

AIMS: The objective of this work was to isolate and characterize indigenous rhizobia from coal-mining areas able to efficiently nodulate and fix nitrogen in association with Calopogonium mucunoides (calopo). METHODS AND RESULTS: Isolation, authentication and morphological, biochemical and molecular characterization of the autochthonous rhizobia were performed and their symbiotic efficiency (SE) evaluated. Efficient rhizobial isolates suitable for the inoculation of calopo in coal-mining regions were obtained. A total of 30 isolates were obtained after nodulation authentication, of which five presented high SE with plant-growth promoting traits such as indole-3-acetic acid production, phosphate solubilization and biofilm formation. These isolates were identified as belonging to Bradyrhizobium, Pseudomonas and Rhizobium. CONCLUSIONS: Bradyrhizobium sp. A2-10 and Pseudomonas sp. A6-05 were able to promote calopo plant growth using soil obtained from coal-mining degraded areas, thus indicating their potential as inoculants aiming at land reclamation. SIGNIFICANCE AND IMPACT OF THE STUDY: To our knowledge, this is the first report of Pseudomonas nodule formation in calopo. Furthermore, the results demonstrated that autochthonous rhizobia obtained from degraded soils presented high SE in calopo and possess a wide range of plant-growth promoting traits. Ultimately, they may all contribute to an increased leguminous plant growth under stress conditions. The selected rhizobia strains may be used as inoculants and present a valuable role in the development of strategies aiming to recover coal-mining degraded areas. Bacterial inoculants would greatly reduce the use of often harmful nitrogen fertilizers vastly employed in revegetation programmes of degraded areas.

Rhizobium album sp. nov., isolated from a propanil-contaminated soil.

A novel Gram-stain negative, facultatively anaerobic, non-spore-forming, motile and rod-shaped bacterium (NS-104T) was isolated from a propanil-contaminated soil in Nanjing, China. Growth occurred at pH 5.0-9.0 (optimum 6.0), 16-37 °C (optimum 30 °C) and in the presence of 0-2.0% (w/v) NaCl (optimum, without NaCl). Strain NS-104T showed high 16S rRNA gene sequence identity to Rhizobium azooxidifex DSM 100211T (96.7%). The phylogenetic analysis of the 16S rRNA gene as well as the housekeeping genes recA, atpD and glnA demonstrated that strain NS-104T belongs to the genus Rhizobium. Strain NS-104T did not form nodules on six different legumes, and the nodD, nodC and nifH genes were neither amplified by PCR nor found in the draft genome of strain NS-104T. The sole respiratory quinone was ubiquinone Q-10. The polar lipid profile included the major amounts phosphatidylmonomethylethanolamine, phosphatidylglycerol and moderate amounts of phosphatidylethanolamine, phosphatidylcholine, diphosphatidylglycerol and unidentified aminolipids. The major cellular fatty acids were C18:1ω7c (39.6%), C19:0 cyclo ω8c (29.8%) and C16:0 (11.5%). The G + C content of strain NS-104T was 61.9 mol%. Strain NS-104T therefore represents a new species, for which the name Rhizobium album sp. nov. is proposed, with the type strain NS-104T (= KCTC 62327T = CCTCC AB 2017250T).

Endophytic Bacteria Potentially Promote Plant Growth by Synthesizing Different Metabolites and their Phenotypic/Physiological Profiles in the Biolog GEN III MicroPlateTM Test.

Endophytic bacteria, as the most promising components of effective, biofertilizers biostimulating and biocontrol preparations, should be very intensively obtained from various plants and studied in terms of the conditions determining the potential ability to promote plant growth. For this reason, endophytic bacteria have been isolated from both stems and roots of up to six systematically distant species of vascular plants: one species belonging to the seedless vascular plants (Monilophyta), and five seed plants (Spermatophyta). The 23 isolated strains represented nine genera: Delftia, Stenotrophomonas, Rhizobium, Brevundimonas, Variovorax, Achromobacter, Novosphingobium, Comamonas and Collimonas, notably which were closely related-belonging to the phylum Proteobacteria. Stenotrophomonas sp. strains showed the greatest ability to synthesize indole-3-acetic acid (IAA)-like compounds, while Achromobacter sp. strains produced the highest levels of siderophores. The presence of the nifH gene and nitrogen binding activity was demonstrated for 95% of the strains tested. Stenotrophomonas maltophila (ES2 strain) showed the highest metabolic activity based on Biolog GEN III test. The ability to solubilize phosphate was determined only for three tested strains from genus: Delftia, Rhizobium and Novosphingobium. The presented work demonstrated that the metabolic and phenotypic properties of plant growth-promoting endophytes are correlated with the genus of bacteria and are not correlated with the host plant species or part of plant (stem, root).

Distribution and Phylogeny of Microsymbionts Associated with Cowpea (Vigna unguiculata) Nodulation in Three Agroecological Regions of Mozambique.

Cowpea derives most of its N nutrition from biological nitrogen fixation (BNF) via symbiotic bacteroids in root nodules. In Sub-Saharan Africa, the diversity and biogeographic distribution of bacterial microsymbionts nodulating cowpea and other indigenous legumes are not well understood, though needed for increased legume production. The aim of this study was to describe the distribution and phylogenies of rhizobia at different agroecological regions of Mozambique using PCR of the BOX element (BOX-PCR), restriction fragment length polymorphism of the internal transcribed spacer (ITS-RFLP), and sequence analysis of ribosomal, symbiotic, and housekeeping genes. A total of 122 microsymbionts isolated from two cowpea varieties (IT-1263 and IT-18) grouped into 17 clades within the BOX-PCR dendrogram. The PCR-ITS analysis yielded 17 ITS types for the bacterial isolates, while ITS-RFLP analysis placed all test isolates in six distinct clusters (I to VI). BLASTn sequence analysis of 16S rRNA and four housekeeping genes (glnII, gyrB, recA, and rpoB) showed their alignment with Rhizobium and Bradyrhizobium species. The results revealed a group of highly diverse and adapted cowpea-nodulating microsymbionts which included Bradyrhizobium pachyrhizi, Bradyrhizobium arachidis, Bradyrhizobium yuanmingense, and a novel Bradyrhizobium sp., as well as Rhizobium tropici, Rhizobium pusense, and Neorhizobium galegae in Mozambican soils. Discordances observed in single-gene phylogenies could be attributed to horizontal gene transfer and/or subsequent recombinations of the genes. Natural deletion of 60 bp of the gyrB region was observed in isolate TUTVU7; however, this deletion effect on DNA gyrase function still needs to be confirmed. The inconsistency of nifH with core gene phylogenies suggested differences in the evolutionary history of both chromosomal and symbiotic genes.IMPORTANCE A diverse group of both Bradyrhizobium and Rhizobium species responsible for cowpea nodulation in Mozambique was found in this study. Future studies could prove useful in evaluating these bacterial isolates for symbiotic efficiency and strain competitiveness in Mozambican soils.

Rhizobium wuzhouense sp. nov., isolated from roots of Oryza officinalis.

Three bacterial isolates, designated W44T, W15 and W11, were isolated from the root of Oryza officinalis grown in Wuzhou, China. These isolates were Gram-negative, aerobic, motile and rod-shaped; demonstrated cellulase and urea activities; and formed cream-coloured colonies. The 16S rRNA gene sequence analysis indicated that the similarities between strain W44T and strains W15 and W11 were 100 %; all of them belonged to the genus Rhizobium and had the highest sequence similarity to Rhizobium rosettiformans W3T (98.7 %), followed by Rhizobium ipomoeae shin9-1T (98.2 %). Sequencing of housekeeping genes (recA, atpD, rpoB and glnA) of the novel isolates revealed similarities to members of established Rhizobium species to be less than 94.3 %. The values of DNA-DNA hybridization between strain W44T and the reference strains (R. rosettiformans W3T and R. ipomoeae shin9-1T) were 41.3 and 29.2 %, respectively. The major cellular fatty acid of strain W44T was summed feature 8 (C18 : 1ω9t and/or C18 : 1ω9c and/or C18 : 1ω7c). The polar lipid profile of strain W44T consisted of phosphatidylglycerol, diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylcholine, two unidentified lipids and two unidentified aminophospholipids. The G+C content of strain W44T was 62.4 mol%. In nodulation tests, none of the three strains could induce nodule formation in Glycine max, Phaseolus vulgaris or Medicago sativa. The nodulation gene (nodA), nitrogenase reductase gene (nifH) and virulence gene (virC) were not detected by PCR in these strains. Based on the above results and phenotypic features, a novel species, Rhizobium wuzhouense sp. nov., is proposed, with strain W44T (=CCTCC AB 2017179T=GDMCC 1.1257T=KCTC 62194T) as the type strain.

A new Rhizobium species isolated from the water of a crater lake, description of Rhizobium aquaticum sp. nov.

A novel isolate, strain SA-276T, was isolated from the water of Lake St. Ana, a crater lake which is located in Romania. Phylogenetic analysis based on the 16S rRNA gene revealed that the new strain is a member of the family Rhizobiaceae, showing a high pairwise similarity value (97.65%) to Rhizobium tubonense CCBAU 85046T (= DSM 25379T), Rhizobium leguminosarum USDA 2370T (= LMG 14904T), Rhizobium anhuiense CCBAU 23252T and Rhizobium laguerreae FB206T. Cells of strain SA-276T were rod-shaped, motile, oxidase negative and weakly catalase positive. The predominant fatty acids were C18:1ω7c and cyclo C19:0ω8c, the major respiratory quinones were Q-10 and Q-9, and the main polar lipids were phosphatidylmonomethylethanolamine, phosphatidylglycerol and phosphatidylcholine. The G + C content of the genomic DNA of strain SA-276T was 60.8 mol%. The novel isolate can be distinguished from the closest related type strain R. tubonense DSM 25379T based on its broader substrate specificity and positive trypsin enzyme activity. On the basis of the phenotypic, chemotaxonomic and molecular data, strain SA-276T is considered to represent a new species, for which the name Rhizobium aquaticum sp. nov. is proposed. The type strain is SA-276T (= DSM 29780T = JCM 31760T).

Old meets new: most probable number validation of metagenomic and metatranscriptomic datasets in soil.

Metagenomics and metatranscriptomics provide insights into biological processes in complex substrates such as soil, but linking the presence and expression of genes with functions can be difficult. Here, we obtain traditional most probable number estimates (MPN) of Rhizobium abundance in soil as a form of sample validation. Our work shows that in the Highfield experiment at Rothamsted, which has three contrasting conditions (>50 years continual bare fallow, wheat and grassland), MPN based on host plant nodulation assays corroborate metagenomic and metatranscriptomic estimates for Rhizobium leguminosarum sv. trifolii abundance. This validation is important to legitimize soil metagenomics and metatranscriptomics for the study of complex relationships between gene function and phylogeny. SIGNIFICANCE AND IMPACT OF THE STUDY: This study has demonstrated for the first time a functional assay validation of metagenomic and metatranscriptomic datasets by utilizing the clover and Rhizobium leguminosarum sv. trifolii mutualism. The results show that the Most Probable Number results corroborate the results of the 'omics approaches and gives confidence to the study of other biological systems where such a cross-check is not available.

Mining alfalfa (Medicago sativa L.) nodules for salinity tolerant non-rhizobial bacteria to improve growth of alfalfa under salinity stress.

There are fewer reports on plant growth promoting (PGP) bacteria living in nodules as helper to tolerance to abiotic stress such as salinity and drought. The study was conducted to isolate rhizobial and non-rhizobial drought and salinity tolerant bacteria from the surface sterilized root nodules of alfalfa, grown in saline soils, and evaluate the effects of effective isolates on plant growth under salt stress. Based on drought and salinity tolerance of bacterial isolates and having multiple PGP traits, two non-rhizobial endophytic isolates and one rhizobial endophytic isolate were selected for further identification and characterization. Based on partial sequences of 16 S rRNA genes, non-rhizobial isolates and rhizobial isolate were closely related to Klebsiella sp., Kosakonia cowanii, and Sinorhizobium meliloti, respectively. None of the two non-rhizobial strains were able to form nodules on alfalfa roots under greenhouse and in vitro conditions. Co-inoculation of alfalfa plant with Klebsiella sp. A36, K. cowanii A37, and rhizobial strain S. meliloti ARh29 had a positive effect on plant growth indices under salinity stress. In addition, the single inoculation of non-rhizobial strains without rhizobial strain resulted in an increase in alfalfa growth indices compared to the plants non-inoculated and the ones inoculated with S. meliloti ARh29 alone under salinity stress, indicating that nodule non-rhizobial strains have PGP potentials and may be a promising way for improving effectiveness of Rhizobium bio-fertilizers in salt-affected soils.