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It is known that members of the bacterial genus Azospirillum can promote the growth of a great variety of plants, an ability harnessed by the industry to create bioproducts aimed to enhance the yield of economically relevant crops. Its versatile metabolism allows this bacterium to adapt to numerous environments, from optimal to extreme or highly polluted. The fact of having been isolated from soil and rhizosphere samples collected worldwide and many other habitats proves its remarkable ubiquity. Azospirillum rhizospheric and endophytic lifestyles are governed by several mechanisms, leading to efficient niche colonization. These mechanisms include cell aggregation and biofilm formation, motility, chemotaxis, phytohormone and other signaling molecules production, and cell-to-cell communication, in turn, involved in regulating Azospirillum interactions with the surrounding microbial community. Despite being infrequently mentioned in metagenomics studies after its introduction as an inoculant, an increasing number of studies detected Azospirillum through molecular tools (mostly 16S rRNA sequencing) as part of diverse, even unexpected, microbiomes. This review focuses on Azospirillum traceability and the performance of the available methods, both classical and molecular. An overview of Azospirillum occurrence in diverse microbiomes and the less-known features explaining its notorious ability to colonize niches and prevail in multiple environments is provided., (© The Author(s) 2023. Published by Oxford University Press on behalf of Applied Microbiology International.)

A novel Gram-staining negative, aerobic, rod-shaped bacterium, designated strain YIM DDC1 T , was isolated from an estuary sediment sample of Dongda River flowing into Dianchi lake in Yunnan, southwest China. The strain displayed growth at 10-40 °C (optimum of 28 °C), pH 5.0-9.0 (optimum of 7.0-8.0) and in presence of 0-3% (w/v) NaCl (optimum of 0-1%). Strain YIM DDC1 T comprised diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine, phosphatidylcholine and two unidentified aminolipids as the predominant polar lipids; the ubiquinone 10 as the major respiratory quinone; and summed feature 8 (C 18:1 ω6c and/or C 18:1 ω7c), summed feature 3 (C 16:1 ω7c and/or C 16:1 ω6c) and C 18:1 2-OH as the major cellular fatty acids. Analysis of 16S rRNA showed that YIM DDC1 T represents a member of the genus Azospirillum, and was closely related to A. brasilense ATCC 29145  T (98.9%), A. baldaniorum Sp245 T (98.2%), A. argentinense Az39 T (98.2%) and A. formosense CC-Nfb-7  T  (98.2%). The draft genome size was 7.15 Mbp with a 68.4% G + C content. The average nucleotide identity (ANI) and digital DNA-DNA hybridization (dDDH) values between strain YIM DDC1 T and the aforementioned closely related strains exhibited similarity in the range of 93.8-93.5% and 53.7-52.7%, respectively. nif gene cluster (nifHDK) and denitrification genes ((napA, nirS, nirK, norBC and nosZ) detected in the genome indicated its potential nitrogen fixation and full-fledged denitrifying function. Based on combined genotypic and phenotypic data, strain YIM DDC1 T represents a novel species of the genus Azospirillum, for which the name Azospirillum aestuarii sp. nov. is proposed. The type strain is YIM DDC1 T (= KCTC 42887  T  = CGMCC 1.17325  T )., (© 2023. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

A Gram-negative, facultative anaerobic bacterial strain, designated YIM B02556 T , was isolated from the root of Paris polyphylla Smith var. yunnanensis collected from Yunnan Province, southwest China. By using a polyphasic approach, its taxonomic position was investigated. Phylogenetic analysis based on 16S rRNA gene sequences revealed that strain YIM B02556 T belonged to the genus Azospirillum and the 16S rRNA gene sequence similarity values of strain YIM B02556 T to the type strains of members of this genus ranged from 94.9 to 98.3%. Overall genome relatedness index (OGRI) analysis estimated based on average nucleotide identity (ANI) and digital DNA-DNA hybridization (dDDH) between YIM B02556 T and other Azospirillum species type strains were <90.8% and <37.8%, lower than the limit of species circumscription. Cells of the strain were characterized as oxidase- and catalase-positive, with motility provided by flagella. The growth conditions of the strain were found to occur at 20-40 °C (optimum, 35 °C), and pH 6.0-9.5 (optimum, pH 7.5). Strain YIM B02556 T can tolerate 2% NaCl concentration. Strain YIM B02556 T contained Q-10 as the major ubiquinone. The major fatty acids were C 18:1 ω7c and summed feature three (C 16:1 ω7c and/or C 16:1 ω6c). The major polar lipids were diphosphatidylglycerol, phosphatidylglycerol, and phosphatidylethanolamine. Based on polyphasic analysis, strain YIM B02556 T could be differentiated genotypically and phenotypically from recognized species of the genus Azospirillum. Therefore, the isolate represents a novel species, for which the name Azospirillum endophyticum is proposed. The type strain is YIM B02556 T (=JCM 34631 T =CGMCC 1.18654 T )., (© 2022. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

The use of inoculants carrying diazotrophic and other plant growth-promoting bacteria plays an essential role in the Brazilian agriculture, with a growing use of microorganism-based bioproducts. However, in the last few years, some farmers have multiplied microorganisms in the farm, known as "on farm" production, including inoculants of Bradyrhizobium spp. for soybean (Glycine max L. Merrill.) and Azospirillum brasilense for corn (Zea mays L.) or co-inoculation in soybean. The objective was to assess the microbiological quality of such inoculants concerning the target microorganisms and contaminants. In the laboratory, 18 samples taken in five states were serial diluted and spread on culture media for obtaining pure and morphologically distinct colonies of bacteria, totaling 85 isolates. Molecular analysis based on partial sequencing of the 16S rRNA gene revealed 25 genera of which 44% harbor species potentially pathogenic to humans; only one of the isolates was identified as Azospirillum brasilense, whereas no isolate was identified as Bradyrhizobium. Among 34 isolates belonging to genera harboring species potentially pathogenic to humans, 12 had no resistance to antibiotics, six presented intrinsic resistance, and 18 presented non-intrinsic resistance to at least one antibiotic. One of the samples analyzed with a shotgun-based metagenomics approach to check for the microbial diversity showed several genera of microorganisms, mainly Acetobacter (~ 32% of sequences) but not the target microorganism. The samples of inoculants produced on farm were highly contaminated with non-target microorganisms, some of them carrying multiple resistances to antibiotics., (© 2021. Sociedade Brasileira de Microbiologia.)

Given their remarkable beneficial effects on plant growth, several Azospirillum isolates currently integrate the formulations of various commercial inoculants. Our research group isolated a new strain, Azospirillum sp. UENF-412522, from passion fruit rhizoplane. This isolate uses carbon sources that are partially distinct from closely-related Azospirillum isolates. Scanning electron microscopy analysis and population counts demonstrate the ability of Azospirillum sp. UENF-412522 to colonize the surface of passion fruit roots. In vitro assays demonstrate the ability of Azospirillum sp. UENF-412522 to fix atmospheric nitrogen, to solubilize phosphate and to produce indole-acetic acid. Passion fruit plantlets inoculated with Azospirillum sp. UENF-41255 showed increased shoot and root fresh matter by 13,8% and 88,6% respectively, as well as root dry matter by 61,4%, further highlighting its biotechnological potential for agriculture. We sequenced the genome of Azospirillum sp. UENF-412522 to investigate the genetic basis of its plant-growth promotion properties. We identified the key nif genes for nitrogen fixation, the complete PQQ operon for phosphate solubilization, the acdS gene that alleviates ethylene effects on plant growth, and the napCAB operon, which produces nitrite under anoxic conditions. We also found several genes conferring resistance to common soil antibiotics, which are critical for Azospirillum sp. UENF-412522 survival in the rhizosphere. Finally, we also assessed the Azospirillum pangenome and highlighted key genes involved in plant growth promotion. A phylogenetic reconstruction of the genus was also conducted. Our results support Azospirillum sp. UENF-412522 as a good candidate for bioinoculant formulations focused on plant growth promotion in sustainable systems., (Copyright © 2021 Elsevier GmbH. All rights reserved.)

Denitrification ability is sporadically distributed among diverse bacteria, archaea, and fungi. In addition, disagreement has been found between denitrification gene phylogenies and the 16S rRNA gene phylogeny. These facts have suggested potential occurrences of horizontal gene transfer (HGT) for the denitrification genes. However, evidence of HGT has not been clearly presented thus far. In this study, we identified the sequences and the localization of the nitrite reductase genes in the genomes of 41 denitrifying Azospirillum sp. strains and searched for mobile genetic elements that contain denitrification genes. All Azospirillum sp. strains examined in this study possessed multiple replicons (4 to 11 replicons), with their sizes ranging from 7 to 1,031 kbp. Among those, the nitrite reductase gene nirK was located on large replicons (549 to 941 kbp). Genome sequencing showed that Azospirillum strains that had similar nirK sequences also shared similar nir-nor gene arrangements, especially between the TSH58, Sp7 T , and Sp245 strains. In addition to the high similarity between nir-nor gene clusters among the three Azospirillum strains, a composite transposon structure was identified in the genome of strain TSH58, which contains the nir-nor gene cluster and the novel IS 6 family insertion sequences (IS Az581 and IS Az582 ). The nirK gene within the composite transposon system was actively transcribed under denitrification-inducing conditions. Although not experimentally verified in this study, the composite transposon system containing the nir-nor gene cluster could be transferred to other cells if it is moved to a prophage region and the phage becomes activated and released outside the cells. Taken together, strain TSH58 most likely acquired its denitrification ability by HGT from closely related Azospirillum sp. denitrifiers. IMPORTANCE The evolutionary history of denitrification is complex. While the occurrence of horizontal gene transfer has been suggested for denitrification genes, most studies report circumstantial evidences, such as disagreement between denitrification gene phylogenies and the 16S rRNA gene phylogeny. Based on the comparative genome analyses of Azospirillum sp. denitrifiers, we identified denitrification genes, including nirK and norCBQD , located on a mobile genetic element in the genome of Azospirillum sp. strain TSH58. The nirK was actively transcribed under denitrification-inducing conditions. Since this gene was the sole nitrite reductase gene in strain TSH58, this strain most likely benefitted by acquiring denitrification genes via horizontal gene transfer. This finding will significantly advance our scientific knowledge regarding the ecology and evolution of denitrification., (Copyright © 2019 American Society for Microbiology.)

Background: The cyclic-di-GMP (c-di-GMP) second messenger exemplifies a signaling system that regulates many bacterial behaviors of key importance; among them, c-di-GMP controls the transition between motile and sessile life-styles in bacteria. Cellular c-di-GMP levels in bacteria are regulated by the opposite enzymatic activities of diguanylate cyclases and phosphodiesterases, which are proteins that have GGDEF and EAL domains, respectively. Azospirillum is a genus of plant-growth-promoting bacteria, and members of this genus have beneficial effects in many agronomically and ecologically essential plants. These bacteria also inhabit aquatic ecosystems, and have been isolated from humus-reducing habitats. Bioinformatic and structural approaches were used to identify genes predicted to encode GG[D/E]EF, EAL and GG[D/E]EF-EAL domain proteins from nine genome sequences., Results: The analyzed sequences revealed that the genomes of A. humicireducens SgZ-5 T , A. lipoferum 4B, Azospirillum sp. B510, A. thiophilum BV-S T , A. halopraeferens DSM3675, A. oryzae A2P, and A. brasilense Sp7, Sp245 and Az39 encode for 29 to 41 of these predicted proteins. Notably, only 15 proteins were conserved in all nine genomes: eight GGDEF, three EAL and four GGDEF-EAL hybrid domain proteins, all of which corresponded to core genes in the genomes. The predicted proteins exhibited variable lengths, architectures and sensor domains. In addition, the predicted cellular localizations showed that some of the proteins to contain transmembrane domains, suggesting that these proteins are anchored to the membrane. Therefore, as reported in other soil bacteria, the Azospirillum genomes encode a large number of proteins that are likely involved in c-di-GMP metabolism. In addition, the data obtained here strongly suggest host specificity and environment specific adaptation., Conclusions: Bacteria of the Azospirillum genus cope with diverse environmental conditions to survive in soil and aquatic habitats and, in certain cases, to colonize and benefit their host plant. Gaining information on the structures of proteins involved in c-di-GMP metabolism in Azospirillum appears to be an important step in determining the c-di-GMP signaling pathways, involved in the transition of a motile cell towards a biofilm life-style, as an example of microbial genome plasticity under diverse in situ environments.

Osmotic variations in the soil can affect bacterial growth diminishing the number of inoculated bacteria. In a scenario of water deficit having tolerant bacteria would be beneficial to achieve a better response of the plant to stress. Thus, selection of more resistant bacteria could be useful to design new inoculants to be used in arid zones. In this sense, a group of Azospirillum isolates deposited in INTA collection was characterized in order to select strains tolerant to osmotic stress. The results obtained demonstrated that Az19 strain has similar in vitro PGPR characteristics to Az39, the most used strain in Argentina for inoculants industries, with the advantage of a better tolerance to osmotic and salt stress. Inoculation of maize plants with this strain resulted in a better response against water deficit compared to Az39 strain, encouraging us to further study the behavior of this strain in greenhouse and field trials in view of developing new inoculants suitable for areas with water deficit., (Copyright © 2017 Elsevier GmbH. All rights reserved.)

A sustainable alternative to improve yield and the nutritive value of forage is the use of plant growth-promoting bacteria (PGPB) that release nutrients, synthesize plant hormones and protect against phytopathogens (among other mechanisms). Azospirillum genus is considered an important PGPB, due to the beneficial effects observed when inoculated in several plants. The aim of this study was to evaluate the diversity of new Azospirillum isolates and select bacteria according to the plant growth promotion ability in three forage species from the Brazilian Pantanal floodplain: Axonopus purpusii, Hymenachne amplexicaulis and Mesosetum chaseae. The identification of bacterial isolates was performed using specific primers for Azospirillum in PCR reactions and partial sequencing of the 16S rRNA and nifH genes. The isolates were evaluated in vitro considering biological nitrogen fixation (BNF) and indole-3-acetic acid (IAA) production. Based on the results of BNF and IAA, selected isolates and two reference strains were tested by inoculation. At 31 days after planting the plant height, shoot dry matter, shoot protein content and root volume were evaluated. All isolates were able to fix nitrogen and produce IAA, with values ranging from 25.86 to 51.26 mg N mL -1 and 107-1038 µmol L -1 , respectively. The inoculation of H. amplexicaulis and A. purpusii increased root volume and shoot dry matter. There were positive effects of Azospirillum inoculation on Mesosetum chaseae regarding plant height, shoot dry matter and root volume. Isolates MAY1, MAY3 and MAY12 were considered promising for subsequent inoculation studies in field conditions.

Since their discovery, plant-growth promoting rhizobacteria from the genus Azospirillum have been subjected to intensive research due to their biotechnological potential as crop inoculants. Phylogenetic analysis of Azospirillum spp. is carried out by 16S rRNA sequencing almost exclusively, but inconsistencies and low confidence often arise when working with close species. In this work, it was observed that these difficulties might be explained by a high number of rRNA operons with considerable inter-genic variability within Azospirillum genomes. To search for alternative genetic markers from a list of housekeeping genes, the correlation between pairwise gene and whole-genome similarities was examined. Due to its good performance, rpoD was selected for further analyses. Genus-specific primers for the PCR-amplification and sequencing of rpoD from Azospirillum spp. were designed and tested on 16 type strains of different species. The sequences obtained were used for inferring a phylogenetic tree of the genus, which was in turn used as a reference to successfully identify a collection of 31 azospirilla isolated from many different locations of Argentine. In addition, several strains that might represent novel species were detected. The results indicate that the sequencing of rpoD is a suitable alternative method for a confident molecular identification in Azospirillum spp., (Copyright © 2016 Elsevier GmbH. All rights reserved.)

Diazotrophic Alphaproteobacteria of the genus Azospirillum are usually organotrophs, although some strains of Azospirillum lipoferum are capable of hydrogen-dependent autotrophic growth. Azospirillum thiophilum strain was isolated from a mineral sulfide spring, a biotope highly unusual for azospirilla. Here, the metabolic pathways utilized by A. thiophilum were revealed based on comprehensive analysis of its genomic organization, together with physiological and biochemical approaches. The A. thiophilum genome contained all the genes encoding the enzymes of carbon metabolism via glycolysis, tricarboxylic acid cycle and glyoxylate cycle. Genes for a complete set of enzymes responsible for autotrophic growth, with an active Calvin-Benson-Bassham cycle, were also revealed, and activity of the key enzymes was determined. Microaerobic chemolithoautotrophic growth of A. thiophilum was detected in the presence of thiosulfate and molecular hydrogen, being in line with the discovery of the genes encoding the two enzymes involved in dissimilatory thiosulfate oxidation, the Sox-complex and thiosulfate dehydrogenase and Ni-Fe hydrogenases. Azospirillum thiophilum utilizes methanol and formate, producing CO 2 that can further be metabolized via the Calvin cycle. Finally, it is capable of anaerobic respiration, using tetrathionate as a terminal electron acceptor. Such metabolic versatility is of great importance for adaptation of A. thiophilum to constantly changing physicochemical environment., (© FEMS 2016. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

An attempt was made in this work to develop a strain-level molecular marker for unambiguous authentication of two Azospirillum inoculants, viz. A. lipoferum (strain Az204) and A. brasilense (strain Sp7). The sequence-characterized amplified region (SCAR) markers obtained from DNA fingerprints were designed for discrete detection of the strains. The SCAR primers could successfully amplify the target strain without cross-reaction with other Azospirillum strains, native isolates and other inoculants. The detection limit of SCAR primer for Az204 was 8.00 pg of DNA (approximately 10(5) cells per mL), and for Sp7, it was 0.49 pg of DNA (equal to 10(4) cells per mL). A simplified Sephadex G100-based crude DNA extraction protocol developed in this study was found suitable for SCAR marker-based strain authentication. Further, SCAR primers were assessed for simultaneous authentication as well as quantification of commercially prepared Azospirillum inoculants by quantitative real-time PCR (RT-PCR) and most-probable-number PCR (MPN-PCR). The RT-PCR assay can be able to quantify the commercial formulations as equal to culturable MPN method, while MPN-PCR failed for Az204. The SCAR marker-based strain authentication and presumptive quantification developed in the present work can contribute to improving the quality standard of commercial inoculants.

A polyphasic approach was used to characterize a novel nitrogen-fixing bacterial strain, designated CC-HIH038T, isolated from cultivated soil in Taiwan. Cells of strain CC-HIH038T were Gram-stain-negative, facultatively aerobic and spiral-shaped, with motility provided by a single polar flagellum. The 16S rRNA gene sequence analysis of strain CC-HIH038T showed highest sequence similarity to Azospirillum doebereinerae (98.0 %), Azospirillum thiophilum (97.5 %), Azospirillum rugosum (97.4 %) and Azospirillum zeae (97.2 %) and lower sequence similarity ( < 97.0 %) to all other species of the genus Azospirillum. According to DNA-DNA association, the relatedness values of strain CC-HIH038T with A. doebereinerae, A. thiophilum, A. rugosum and A. zeae were 51.8 %, 41.2 %, 56.5 % and 37.5 %, respectively. Strain CC-HIH038T was able to grow at 20-37 °C and pH 7.0-8.0. Strain CC-HIH038T gave positive amplification for dinitrogen reductase (nifH gene); the activity was recorded as 8.4 nmol ethylene h- 1. The predominant quinone system was ubiquinone Q-10 and the DNA G+C content was 68.8 mol%. The major fatty acids found in strain CC-HIH038T were C16 : 0, iso-C18 : 0, C16 : 0 3-OH, C14 : 0 3-OH/iso-C16 : 1 and C18 : 1ω7c/C18 : 1ω6c. Based on the distinct phylogenetic, phenotypic and chemotaxonomic traits together with results of comparative 16S rRNA gene sequence analysis, strain CC-HIH038T is considered to represent a novel species in the genus Azospirillum, for which the name Azospirillum agricola sp. nov. is proposed. The type strain is CC-HIH038T ( = BCRC 80909T = JCM 30827T).

Azospirillum thiophilum BV-S(T), isolated from a sulfide spring, is a novel nitrogen-fixing bacterium harboring sulfur-lithotrophy. In order to identify genetic characteristics with habitat- and metabolic features contrasting to those from terrestrial Azospirillum species, we present here the genome sequence of a novel species A. thiophilum BV-S(T), with a significance of first genome report in the aquatic Azospirillum species. The genome of strain BV-S(T) is comprised of 7.6Mb chromosome with a GC content of 68.2%. This information will contribute to expand understandings of sulfur-oxidizer microbes that preserve inherencies as a diazotroph, and further it will provide insights into genome plasticity of the genus Azospirillum for niche specific adaptations., (Copyright © 2015 Elsevier B.V. All rights reserved.)

An aerobic, Gram-stain-negative, rod or spiral-shaped diazotrophic bacterium (designated strain CC-LY788T), was isolated from agricultural soil in Taiwan. Strain CC-LY788T was able to grow at 25-40 °C, pH 6.0-8.0 and tolerated NaCl to 2.0% (w/v). Positive for nitrogen fixation with the activity recorded as 6.5 nmol ethylene h(-1). Strain CC-LY788T showed highest 16S rRNA gene sequence similarity to Azospirillum picis DSM 19922T (97.2%) and Azospirillum rugosum DSM 19657T (97.1%) and lower sequence similarities (<96.6%) to all other species of the genus Azospirillum. According to the DNA-DNA hybridization, the relatedness values of strain CC-LY788T with A. picis DSM 19922T and A. rugosum DSM 19657T were 51.1±5.5% and 46.8±2.1%, respectively. Strain CC-LY788T was positive for the rapid identification of the genus-specific primer set. The respiratory quinone system was ubiquinone (Q-10) and the DNAG+C content was 69.8 mol%. The major fatty acids found in strain CC-LY788T were C16 : 0, C18 : 1 2-OH, C14 : 0 3-OH/C16 : 1 iso I (summed feature 2), C16 : 1ω7c/C16 : 1ω6c (summed feature 3), C18 : 0 ante/C18 : 2ω6,9c (summed feature 5) and C18 : 1ω7c/C18 : 1ω6c (summed feature 8). Based on the phylogenetic, phenotypic and chemotaxonomic features, strain CC-LY788T represents a novel species of the genus Azospirillum, for which the name Azospirillum soli sp. nov. is proposed. The type strain is CC-LY788T (=BCRC 80569T=JCM 18820T).

Background: Two-component systems (TCS) play critical roles in sensing and responding to environmental cues. Azospirillum is a plant growth-promoting rhizobacterium living in the rhizosphere of many important crops. Despite numerous studies about its plant beneficial properties, little is known about how the bacterium senses and responds to its rhizospheric environment. The availability of complete genome sequenced from four Azospirillum strains (A. brasilense Sp245 and CBG 497, A. lipoferum 4B and Azospirillum sp. B510) offers the opportunity to conduct a comprehensive comparative analysis of the TCS gene family., Results: Azospirillum genomes harbour a very large number of genes encoding TCS, and are especially enriched in hybrid histidine kinases (HyHK) genes compared to other plant-associated bacteria of similar genome sizes. We gained further insight into HyHK structure and architecture, revealing an intriguing complexity of these systems. An unusual proportion of TCS genes were orphaned or in complex clusters, and a high proportion of predicted soluble HKs compared to other plant-associated bacteria are reported. Phylogenetic analyses of the transmitter and receiver domains of A. lipoferum 4B HyHK indicate that expansion of this family mainly arose through horizontal gene transfer but also through gene duplications all along the diversification of the Azospirillum genus. By performing a genome-wide comparison of TCS, we unraveled important 'genus-defining' and 'plant-specifying' TCS., Conclusions: This study shed light on Azospirillum TCS which may confer important regulatory flexibility. Collectively, these findings highlight that Azospirillum genomes have broad potential for adaptation to fluctuating environments.

A field study was conducted to compare the formationand bacterial communities of rhizosheaths of wheat grown under wheat-cotton and wheat-rice rotation and to study the effects of bacterial inoculation on plant growth. Inoculation of Azospirillum sp. WS-1 and Bacillus sp. T-34 to wheat plants increased root length, root and shoot dry weight and dry weight of rhizosheathsoil when compared to non-inoculated control plants, and under both crop rotations. Comparing both crop rotations, root length, root and shoot dry weight and dry weight of soil attached with roots were higher under wheat-cotton rotation. Organic acids (citric acid, malic acid, acetic acid and oxalic acid) were detected in rhizosheaths from both rotations, with malic acid being most abundant with 24.8±2 and 21.3±1.5 μg g(-1) dry soil in wheat-cotton and wheat-rice rotation, respectively. Two sugars (sucrose, glucose) were detected in wheat rhizosheath under both rotations, with highest concentrations of sucrose (4.08±0.5 μg g(-1) and 7.36±1.0 μg g(-1)) and glucose (3.12±0.5 μg g(-1) and 3.01± μg g(-1)) being detected in rhizosheaths of non-inoculated control plants under both rotations. Diversity of rhizosheath-associated bacteria was evaluated by cultivation, as well as by 454-pyrosequencing of PCR-tagged 16S rRNA gene amplicons. A total of 14 and 12 bacterial isolates predominantly belonging to the genera Arthrobacter, Azospirillum, Bacillus, Enterobacter and Pseudomonaswere obtained from the rhizosheath of wheat grown under wheat-cotton and wheat-rice rotation, respectively. Analysis of pyrosequencing data revealed Proteobacteria, Bacteriodetes and Verrucomicrobia as the most abundant phyla in wheat-rice rotation, whereas Actinobacteria, Firmicutes, Chloroflexi, Acidobacteria, Planctomycetes and Cyanobacteria were predominant in wheat-cotton rotation. From a total of 46,971 sequences, 10.9% showed ≥97% similarity with 16S rRNA genes of 32 genera previously shown to include isolates with plant growth promoting activity (nitrogen fixation, phosphate-solubilization, IAA production). Among these, the most predominant genera were Arthrobacter, Azoarcus, Azospirillum, Bacillus, Cyanobacterium, Paenibacillus, Pseudomonas and Rhizobium.

The reaction of soil microorganisms to the contamination of soil artificially polluted with polycyclic aromatic hydrocarbons (PAHs) was evaluated in pot experiments. The plant used in the tests was cock's foot (Dactylis glomerata). Three different soils artificially contaminated with PAHs were applied in the studies. Three selected PAHs (anthracene, phenanthrene, and pyrene) were used at the doses of 100, 500, and 1000 mg/kg d.m. of soil and diesel fuel at the doses of 100, 500, and 1000 mg/kg d.m. of soil. For evaluation of the synergistic effect of nitrogen fixing bacteria, the following strains were selected: associative Azospirillum spp. and Pseudomonas stutzerii. Additionally, in the bioremediation process, the inoculation of plants with a mixture of the bacterial strains in the amount of 1 ml suspension per 500 g of soil was used. Chamber pot-tests were carried out in controlled conditions during four weeks of plant growth period. The basic physical, microbiological and biochemical properties in contaminated soils were determined. The obtained results showed a statistically important increase in the physical properties of soils polluted with PAHs and diesel fuel compared with the control and also an important decrease in the content of PAHs and heavy metals in soils inoculated with Azospirillum spp. and P. stutzeri after cock's foot grass growth. The bioremediation processes were especially intensive in calcareous rendzina soil artificially polluted with PAHs.

The rhizosphere bacterium Azospirillum amazonense associates with plant roots to promote plant growth. Variation in replicon numbers and rearrangements is common among Azospirillum strains, and characterization of these naturally occurring differences can improve our understanding of genome evolution. We performed an in silico comparative genomic analysis to understand the genomic plasticity of A. amazonense. The number of A. amazonense-specific coding sequences was similar when compared with the six closely related bacteria regarding belonging or not to the Azospirillum genus. Our results suggest that the versatile gene repertoire found in A. amazonense genome could have been acquired from distantly related bacteria from horizontal transfer. Furthermore, the identification of coding sequence related to phytohormone production, such as flavin-monooxygenase and aldehyde oxidase, is likely to represent the tryptophan-dependent TAM pathway for auxin production in this bacterium. Moreover, the presence of the coding sequence for nitrilase indicates the presence of the alternative route that uses IAN as an intermediate for auxin synthesis, but it remains to be established whether the IAN pathway is the Trp-independent route. Future investigations are necessary to support the hypothesis that its genomic structure has evolved to meet the requirement for adaptation to the rhizosphere and interaction with host plants.

Beneficial microorganisms have been considered as an important tool for crop improvement. Native isolates of Azospirillum spp. were obtained from the rhizospheres of different rice fields. Phenotypic, biochemical and molecular characterizations of these isolates led to the identification of six efficient strain of Azospirillum. PCR amplification of the nif genes (nifH, nifD and nifK) and protein profile of Azospirillum strains revealed inter-generic and inter-specific diversity among the strains. In vitro nitrogen fixation performance and the plant growth promotion activities, viz. siderophore, HCN, salicylic acid, IAA, GA, zeatin, ABA, NH3, phosphorus metabolism, ACC deaminase and iron tolerance were found to vary among the Azospirillum strains. The effect of Azospirillum formulations on growth of rice var. Khandagiri under field condition was evaluated, which revealed that the native formulation of Azospirillum of CRRI field (As6) was most effective to elevate endogenous nutrient content, and improved growth and better yield are the result. The 16S rRNA sequence revealed novelty of native Azospirillum lipoferum (As6) (JQ796078) in the NCBI database.

An aerobic, Gram-stain-negative, spiral or rod-shaped, non-spore-forming, diazotrophic bacterium (strain CC-LY743(T)) was isolated from a fermentative tank in Taiwan. Strain CC-LY743(T) was able to grow at 20-37 °C and pH 6.0-8.0 and tolerated up to 3.0 % (w/v) NaCl. It was positive for nitrogen fixation, with activity of 10.6 nmol ethylene h(-1). 16S rRNA gene sequence analysis of strain CC-LY743(T) showed highest similarity to Azospirillum picis DSM 19922(T) (96.1 %), Azospirillum oryzae JCM 21588(T) (96.0 %) and Azospirillum rugosum DSM 19657(T) (96.0 %) and lower similarity (<96.0 %) to all other Azospirillum species. Highest nifH gene sequence similarities were obtained with Azospirillum brasilense BCRC 12270(T) (92.0 %), Azospirillum formosense BCRC 80273(T) (92.3 %) and A. rugosum DSM 19657(T) (91.8 %). It was positive in the rapid identification by a genus-specific primer set. The predominant quinone system was ubiquinone 10 (Q-10) and the DNA G+C content was 69.6±0.1 mol%. The major fatty acids found in strain CC-LY743(T) were n-C16 : 0, C19 : 0 cyclo ω8c, C14 : 0 3-OH/C16 : 1 iso I, C16 : 1ω7c/C16 : 1ω6c and C18 : 1ω7c/C18 : 1ω6c. Based on its phylogenetic, phenotypic and chemotaxonomic features, strain CC-LY743(T) is considered to represent a novel species within the genus Azospirillum for which the name Azospirillum fermentarium sp. nov. is proposed. The type strain is CC-LY743(T) ( = BCRC 80505(T) = JCM 18688(T) = LMG 27264(T)).

A Gram-negative, facultative anaerobic, motile, spiral, straight-to-slightly curved rod-shaped and nitrogen-fixing strain, designated SgZ-5(T), was isolated from a microbial fuel cell (MFC) and was characterized by means of a polyphasic approach. Growth occurred with 0-1 % (w/v) NaCl (optimum 1 %) and at pH 5.5-8.5 (optimum pH 7.2) and at 25-37 °C (optimum 30 °C) in nutrient broth (NB). The strain had the ability to grow under anaerobic conditions via the oxidation of various organic compounds coupled to the reduction of anthraquione-2,6-disulfonate (AQDS). Chemotaxonomic characteristics (main ubiquinone Q-10, major fatty acid C18 : 1ω7c/C18 : 1ω6c and DNA G+C content 67.7 mol%) were similar to those of members of the genus Azospirillum. According to the results of phylogenetic analyses, strain SgZ-5(T) belonged to the genus Azospirillum within the family Rhodospirillaceae of the class Alphaproteobacteria, and was related most closely to the type strains of Azospirillum lipoferum, Azospirillum thiophilum and Azospirillum oryzae (98.0, 97.6 and 97.1 % 16S rRNA gene sequence similarity, respectively). DNA-DNA pairing studies showed that the unidentified organism displayed reassociation values of 36.7 ± 3.7, 24.1 ± 2.2 and 22.3 ± 2.4 % to the type strains of A. lipoferum, A. thiophilum and A. oryzae, respectively. Similarities between nifH gene sequences of strain SgZ-5(T) and members of the genus Azospirillum ranged from 94.0 to 97.0 %. A combination of phenotypic, chemotaxonomic, phylogenetic and genotypic data clearly indicated that strain SgZ-5(T) represents a novel species, for which the name Azospirillum humicireducens sp. nov. is proposed. The type strain is SgZ-5(T) ( = CCTCC AB 2012021(T) = KACC 16605(T)).

The plant growth-promoting proteobacterium Azospirillum brasilense enhances growth of many economically important crops, such as wheat, maize, and rice. The sequencing and annotation of the 1.59-Mbp replicon of A. brasilense CBG497, a strain isolated from a maize rhizosphere grown on an alkaline soil in the northeast of Mexico, revealed a GC content of 68.7 % and the presence of 1,430 potential protein-encoding genes, 1,147 of them classified into clusters of orthologous groups categories, and 16 tRNA genes representing 11 tRNA species. The presence of sixty-two genes representatives of the minimal gene set and chromid core genes suggests its importance in bacterial survival. The phaAB → G operon, reported as involved in the bacterial adaptation to alkaline pH in the presence of K(+), was also found on this replicon and detected in several Azospirillum strains. Phylogenetic analysis suggests that it was laterally acquired. We were not able to show its inference on the adaptation to basic pH, giving a hint about the presence of an alternative system for adaptation to alkaline pH.

Dissolved N(2)O is occasionally detected in surface and ground water in rice paddy fields, whereas little or no N(2)O is emitted to the atmosphere above these fields. This indicates the occurrence of N(2)O reduction in rice paddy fields; however, identity of the N(2)O reducers is largely unknown. In this study, we employed both culture-dependent and culture-independent approaches to identify N(2)O reducers in rice paddy soil. In a soil microcosm, N(2)O and succinate were added as the electron acceptor and donor, respectively, for N(2)O reduction. For the stable isotope probing (SIP) experiment, (13)C-labeled succinate was used to identify succinate-assimilating microbes under N(2)O-reducing conditions. DNA was extracted 24  h after incubation, and heavy and light DNA fractions were separated by density gradient ultracentrifugation. Denaturing gradient gel electrophoresis and clone library analysis targeting the 16S rRNA and the N(2)O reductase gene were performed. For culture-dependent analysis, the microbes that elongated under N(2)O-reducing conditions in the presence of cell-division inhibitors were individually captured by a micromanipulator and transferred to a low-nutrient medium. The N(2)O-reducing ability of these strains was examined by gas chromatography/mass spectrometry. Results of the SIP analysis suggested that Burkholderiales and Rhodospirillales bacteria dominated the population under N(2)O-reducing conditions, in contrast to the control sample (soil incubated with only (13)C-succinate). Results of the single-cell isolation technique also indicated that the majority of the N(2)O-reducing strains belonged to the genera Herbaspirillum (Burkholderiales) and Azospirillum (Rhodospirillales). In addition, Herbaspirillum strains reduced N(2)O faster than Azospirillum strains. These results suggest that Herbaspirillum spp. may have an important role in N(2)O reduction in rice paddy soils.

Fossil records indicate that life appeared in marine environments ∼3.5 billion years ago (Gyr) and transitioned to terrestrial ecosystems nearly 2.5 Gyr. Sequence analysis suggests that "hydrobacteria" and "terrabacteria" might have diverged as early as 3 Gyr. Bacteria of the genus Azospirillum are associated with roots of terrestrial plants; however, virtually all their close relatives are aquatic. We obtained genome sequences of two Azospirillum species and analyzed their gene origins. While most Azospirillum house-keeping genes have orthologs in its close aquatic relatives, this lineage has obtained nearly half of its genome from terrestrial organisms. The majority of genes encoding functions critical for association with plants are among horizontally transferred genes. Our results show that transition of some aquatic bacteria to terrestrial habitats occurred much later than the suggested initial divergence of hydro- and terrabacterial clades. The birth of the genus Azospirillum approximately coincided with the emergence of vascular plants on land., Competing Interests: The authors have declared that no competing interests exist.

Aims: To develop a rapid and simple genus-specific polymerase chain reaction (PCR) method for detecting and identifying isolates of the genus Azospirillum which is well-recognized as plant growth-promoting rhizobacterium., Methods and Results: Nine pairs of PCR primers were designed based on the Azospirillum 16S rRNA, ipdC, nifA and nifH genes to assess their genus specificity by testing against 12 Azospirillum (from seven species) and 15 non-Azospirillum reference strains, as compared with the fAZO/rAZO pair reported by Baudoin et al. (J Appl Microbiol, 108, 2010, 25). Among the primer pairs assessed, the Az16S-A pair designed on the 16S rRNA gene sequence showed the highest genus specificity: it successfully yielded a single amplicon of the expected size in all the 12 Azospirillum strains and for a close relative, Rhodocista centenaria. The PCR with the Az16S-A primers generated a detectable amount of the amplicon from ≥10³ CFU ml⁻¹ of Azospirillum cell suspensions even in the presence of contaminants and accurately discriminated Azospirillum and non-Azospirillum species in both 35 Azospirillum-like and 70 unknown isolates from plant roots and rhizosphere soils., Conclusions: We developed a rapid and simple PCR method for detecting and identifying Azospirillum isolates within populations of rhizosphere bacteria., Significance and Impact of the Study: The method developed would serve as a useful tool for isolating a variety of indigenous Azospirillum bacteria from agricultural samples., (© 2011 The Authors. Journal of Applied Microbiology © 2011 The Society for Applied Microbiology.)

Background: The species Azospirillum amazonense belongs to a well-known genus of plant growth-promoting bacteria. This bacterium is found in association with several crops of economic importance; however, there is a lack of information on its physiology. In this work, we present a comprehensive analysis of the genomic features of this species., Results: Genes of A. amazonense related to nitrogen/carbon metabolism, energy production, phytohormone production, transport, quorum sensing, antibiotic resistance, chemotaxis/motility and bacteriophytochrome biosynthesis were identified. Noteworthy genes were the nitrogen fixation genes and the nitrilase gene, which could be directly implicated in plant growth promotion, and the carbon fixation genes, which had previously been poorly investigated in this genus. One important finding was that some A. amazonense genes, like the nitrogenase genes and RubisCO genes, were closer phylogenetically to Rhizobiales members than to species of its own order., Conclusion: The species A. amazonense presents a versatile repertoire of genes crucial for its plant-associated lifestyle.

Background: Azospirillum amazonense has potential to be used as agricultural inoculant since it promotes plant growth without causing pollution, unlike industrial fertilizers. Owing to this fact, the study of this species has gained interest. However, a detailed understanding of its genetics and physiology is limited by the absence of appropriate genetic tools for the study of this species., Results: Conjugation and electrotransformation methods were established utilizing vectors with broad host-replication origins (pVS1 and pBBR1). Two genes of interest--glnK and glnB, encoding PII regulatory proteins--were isolated. Furthermore, glnK-specific A. amazonense mutants were generated utilizing the pK19MOBSACB vector system. Finally, a promoter analysis protocol based on fluorescent protein expression was optimized to aid genetic regulation studies on this bacterium., Conclusion: In this work, genetic tools that can support the study of A. amazonense were described. These methods could provide a better understanding of the genetic mechanisms of this species that underlie its plant growth promotion.

The modern agricultural practice utilizing plant growth promoting rhizobacteria (PGPR) has brought great benefits in the promotion of crop growth. Among PGPR, Azospirillum is considered as an important genus which is not only closely-associated with plants but also shows potential in the degradation of organic contaminants. However, lack of media for selective isolation or techniques for specific detection or identification limit the exploration of these rhizobacteria. This motivated us to design a genus-specific oligonucleotide primer pair which could assist in rapid detection of species of the genus Azospirillum by means of PCR-specific amplification. The sensitivity and specificity of the newly designed primer pair Azo494-F/Azo756-R were tested against 12 Azospirillum type strains and other closely-related genera. The Azospirillum-specific 16S rRNA gene fragment (263 bp) was successfully amplified for all the reference Azospirillum species with the designed primer pair. No amplification was noted for closely-related species from other genera. The genus specificity was validated with 18 strains including environmental isolates. Interestingly, two strains assigned earlier as Azospirillum amazonense (DSM 2787(T)) and Azospirillum irakense (DSM 11586(T)) failed to produce an Azospirillum-specific fragment with this primer pair. Further phylogenetic analysis of these two isolates based on 16S rRNA gene sequences shows that these two strains might belong to other genera rather than Azospirillum. Preliminary screening of isolates and soil samples with the Azospirillum-specific primers was successful in terms of the rapid detection of Azospirillum isolates. By using real-time PCR analysis the minimum limit of Azospirillum detection was 10(2) CFU g(-1) in the seeded soil sample. The newly designed primers can be used to study the diversity of Azospirillum in ecosystems and aid in the exploration of novel species.

A total of 17 culturable nitrogen-fixing bacterial strains associated with the roots of wheat growing in different regions of Greece were isolated and characterized for plant-growth-promoting traits such as auxin production and phosphate solubilization. The phylogenetic position of the isolates was first assessed by the analysis of the PCR-amplified 16S rRNA gene. The comparative sequence analysis and phylogenetic analysis based on 16S rRNA gene sequences show that the isolates recovered in this study are grouped with Azospirillum brasilense, Azospirillum zeae, and Pseudomonas stutzeri. The diazotrophic nature of all isolates was confirmed by amplification of partial nifH gene sequences. The phylogenetic tree based on nifH gene sequences is consistent with 16S rRNA gene phylogeny. The isolates belonging to Azospirillum species were further characterized by examining the partial dnaK gene phylogenetic tree. Furthermore, it was demonstrated that the ipdC gene was present in all Azospirillum isolates, suggesting that auxin is mainly synthesized via the indole-3-pyruvate pathway. Although members of P. stutzeri and A. zeae are known diazotrophic bacteria, to the best of our knowledge, this is the first report of isolation and characterization of strains belonging to these bacterial genera associated with wheat.

During evolution, plants have become associated with guilds of plant-growth-promoting rhizobacteria (PGPR), which raises the possibility that individual PGPR populations may have developed mechanisms to cointeract with one another on plant roots. We hypothesize that this has resulted in signaling phenomena between different types of PGPR colonizing the same roots. Here, the objective was to determine whether the Pseudomonas secondary metabolite 2,4-diacetylphloroglucinol (DAPG) can act as a signal on Azospirillum PGPR and enhance the phytostimulation effects of the latter. On roots, the DAPG-producing Pseudomonas fluorescens F113 strain but not its phl-negative mutant enhanced the phytostimulatory effect of Azospirillum brasilense Sp245-Rif on wheat. Accordingly, DAPG enhanced Sp245-Rif traits involved in root colonization (cell motility, biofilm formation, and poly-β-hydroxybutyrate production) and phytostimulation (auxin production). A differential fluorescence induction promoter-trapping approach based on flow cytometry was then used to identify Sp245-Rif genes upregulated by DAPG. DAPG enhanced expression of a wide range of Sp245-Rif genes, including genes involved in phytostimulation. Four of them (i.e., ppdC, flgE, nirK, and nifX-nifB) tended to be upregulated on roots in the presence of P. fluorescens F113 compared with its phl-negative mutant. Our results indicate that DAPG can act as a signal by which some beneficial pseudomonads may stimulate plant-beneficial activities of Azospirillum PGPR.

Oligotrophic denitrifying bacteria, including those belonging to the genera Herbaspirillum, Azospirillum, and Bradyrhizobium, were obtained using a single-cell isolation technique. The taxonomic composition of the denitrifier population was similar to those assessed by previous culture-independent studies. The sequencing of nitrite reductase and N(2)O reductase genes of these strains revealed previously unknown links between 16S rRNA and the denitrification-functional gene phylogenies. In particular, we identified Bradyrhizobium strains that harbor nirS sequences previously detected only in culture-independent studies.

Presence of endophytic bacteria was reported in many crops including maize (Zea mays L.). Endophytes play a significant role in plant nutrient and pesticide uptake. Application of endophytic bacteria is a goal of sustainable agriculture. Occurrence of Azospirillum strains is often reported as tissue inhabiting bacteria of maize. The biological N2-fixation is one of most important processes assigned to this bacteria. The objective of this study was to examine the biodiversity of Azospirillum spp. isolated from the leaves of 6 cultivars of Zea mays L.. They were cultivated on two experimental fields at Smolice and Kobierzyce, (Poland). Strains of Azospirillum spp. were isolated on the solid RC medium. Forty four isolates grown as a small intensive red colonies were selected. To verify ability to N2-fixation isolates were analyzed based on nifH gene presence. Presence of nifH gene was tested using PCR method with PolF and PolR universal degenerate primers. The presence of nifH gene was found in 6 tested strains isolated from leaves of 3 cultivars (Cyrkon, Kosmo230, KB2704) from Smolice location, only. Our results suggest that selection of Azospirillum-like strains on RC solid medium based on appearance of colony is not correlated with theirs ability to nitrogen fixation or used degenerated primers (PolF, PolR) are not universal enough.

A novel nitrogen-fixing strain, designated BV-S(T), was isolated from a sulfur bacterial mat collected from a sulfide spring of the Stavropol Krai, North Caucasus, Russia. Strain BV-S(T) grew optimally at pH 7.5 and 37°C. According to the results of phylogenetic analysis, strain BV-S(T) belonged to the genus Azospirillum within the family Rhodospirillaceae of the class Alphaproteobacteria. Within the genus Azospirillum, strain BV-S(T) was most closely related to Azospirillum doebereinerae GSF71(T), A. picis IMMIB TAR-3(T) and A. lipoferum ATCC 29707(T) (97.7, 97.7 and 97.4 % 16S rRNA gene sequence similarity, respectively). DNA-DNA relatedness between strain BV-S(T) and A. doebereinerae DSM 13131(T), A. picis DSM 19922(T) and A. lipoferum ATCC 29707(T) was 38, 55 and 42 %, respectively. Similarities between nifH sequences of strain BV-S(T) and members of the genus Azospirillum ranged from 94.5 to 96.8 %. Chemotaxonomic characteristics (quinone Q-10, major fatty acid C(18 : 1)ω7c and G+C content 67&emsp14;mol%) were similar to those of members of the genus Azospirillum. In contrast to known Azospirillum species, strain BV-S(T) was capable of mixotrophic growth under microaerobic conditions with simultaneous utilization of organic substrates and thiosulfate as electron donors for energy conservation. Oxidation of sulfide was accompanied by deposits of sulfur globules within the cells. Based on these observations, strain BV-S(T) is considered as a representative of a novel species of the genus Azospirillum, for which the name Azospirillum thiophilum sp. nov. is proposed. The type strain is BV-S(T) (=DSM 21654(T) =VKM B-2513(T)).

Biodegradation of TCE was studied in a biofilter packed with wood charcoal and inoculated with diazotrophic bacterial community isolated from local soil. Steady state TCE removal efficiencies higher than 85% were observed up to inlet load of 2.866 g m(-3) h(-1). The maximum elimination capacity of 5.31 g m(-3) h(-1) was observed at an inlet load of more than 7.90 g m(-3) h(-1). The biofilter was sensitive to fluctuations in the process conditions but could easily recover its performance after 10 days shutdown. Almost constant and small pressure drop per unit length and very negligible compaction was observed during the whole experimental period. The molecular analyses such as RT-PCR and gene sequencing revealed the presence of functionally active Azospirillum species in the biofilm., (Copyright 2009 Elsevier Ltd. All rights reserved.)

Concise synthesis of a tetrasaccharide repeating unit of the LPS isolated from Azospirillum lipoferum SR65 has been accomplished through suitable protecting group manipulations and stereoselective glycosylation starting from commercially available L-rhamnose and D-glucose. The target oligosaccharide in the form of its p-methoxyphenyl glycoside is suitable for further glycoconjugate formation via selective cleavage of the OMP glycoside. Plant growth-promoting bacteria (PGPB) of genus Azospirillum plays important roles in the growth and development of plants. The interaction between the roots of the plants and the microbes is governed by the cell surface carbohydrate polymers (CPS, LPS, etc.). The present synthetic-based study elucidates aspects of plant-microbe interaction and future biofertiliser design., ((c) 2009 Elsevier Ltd. All rights reserved.)

We determined the nucleotide sequence of the entire genome of a diazotrophic endophyte, Azospirillum sp. B510. Strain B510 is an endophytic bacterium isolated from stems of rice plants (Oryza sativa cv. Nipponbare). The genome of B510 consisted of a single chromosome (3,311,395 bp) and six plasmids, designated as pAB510a (1,455,109 bp), pAB510b (723,779 bp), pAB510c (681,723 bp), pAB510d (628,837 bp), pAB510e (537,299 bp), and pAB510f (261,596 bp). The chromosome bears 2893 potential protein-encoding genes, two sets of rRNA gene clusters (rrns), and 45 tRNA genes representing 37 tRNA species. The genomes of the six plasmids contained a total of 3416 protein-encoding genes, seven sets of rrns, and 34 tRNAs representing 19 tRNA species. Eight genes for plasmid-specific tRNA species are located on either pAB510a or pAB510d. Two out of eight genomic islands are inserted in the plasmids, pAB510b and pAB510e, and one of the islands is inserted into trnfM-CAU in the rrn located on pAB510e. Genes other than the nif gene cluster that are involved in N(2) fixation and are homologues of Bradyrhizobium japonicum USDA110 include fixABCX, fixNOQP, fixHIS, fixG, and fixLJK. Three putative plant hormone-related genes encoding tryptophan 2-monooxytenase (iaaM) and indole-3-acetaldehyde hydrolase (iaaH), which are involved in IAA biosynthesis, and ACC deaminase (acdS), which reduces ethylene levels, were identified. Multiple gene-clusters for tripartite ATP-independent periplasmic-transport systems and a diverse set of malic enzymes were identified, suggesting that B510 utilizes C(4)-dicarboxylate during its symbiotic relationship with the host plant.

A polyphasic taxonomic study was performed on a pink-coloured unknown bacterium isolated from discarded road tar. Comparative analysis of the 16S rRNA gene sequence demonstrated that the isolate belongs phylogenetically to the genus Azospirillum with Azospirillum lipoferum, A. melinis and A. rugosum as its closest phylogenetic relatives (96.7, 96.6 and 96.6 % similarity to the respective type strains). The generic assignment was confirmed on the basis of chemotaxonomic data, which revealed a fatty acid profile characteristic for the genus Azospirillum, consisting of straight-chain saturated and unsaturated fatty acids, with C(18 : 1)omega7c as the major unsaturated non-hydroxylated fatty acid, and C(16 : 0) 3-OH as the major hydroxylated fatty acid, and a ubiquinone with ten isoprene units (Q-10) as the predominant respiratory quinone. On the basis of both the phenotypic and molecular genetic evidence, it is proposed that the unknown isolate should be classified within a novel species of the genus Azospirillum, for which the name Azospirillum picis sp. nov. is proposed. The type strain is IMMIB TAR-3(T) (=CCUG 55431(T) =DSM 19922(T)).

Bacteria with high N2-fixing activity were isolated from the root zone of spring wheat grown on leach chernozem and soddy podzolic soil in Ukrainian marshy woodlands. They were characterized by phenotypic signs and investigated with the help of molecular-genetic methods. On the basis of diagnostic signs the investigated strains were referred to Azospirillum brasilense from Azospirillum genus. Their 3'- and 5'-thermal 16S RNA hypervariable sites with length from 373 to 395 nucleotides were amplified and sequenced. The comparative analysis of results confirmed the 100% identity of 16S RNA sequences from investigated bacteria with the same sequences of A. brasilense from Gene Bank database. Thus the results of sequence analysis agree with results obtained during the investigation of phenotypic signs.

A Gram-negative bacterium designated ww 10(T) was isolated by plating dilutions from forest soil in Zhejiang province, China. Strain ww 10(T) was investigated by polyphasic taxonomic study including phenotypic and phylogenetic analysis. Cells of ww 10(T) were Gram-negative, strictly aerobic, motile with peritrichous flagella and rod-shaped. The strain grew optimally at 30-37 masculineC and pH 6.0-8.0. The major fatty acids were C(18:1)omega7C, cyclo-C(19:0) omega8C and C(16:0). The respiratory quinones contained a large amount of Q-10, a moderate component of Q-9 and a minor of Q-8. The G+C content of genomic DNA was about 67.3 mol%. Phylogenetic analysis revealed that strain ww 10(T) belongs to the phyletic cluster of genus Azospirillum and displayed 16S rRNA gene sequence similarity lower than 96.3% to the four closest described species of the genera Azospirillum and Roseomonas. Results of polyphasic taxonomic analysis showed that strain ww 10(T) represents a novel species in the genus Azospirillum, for which the name Azospirillum palatum sp. nov., is proposed. The type strain is ww 10(T) (=LMG 24444(T)=KCTC 13200(T)=CCTCC AB 207189(T)).

This work aimed to evaluate density of associative diazotrophic bacteria populations in soil and grass root samples from heavy metal contaminated sites, and to characterize isolates from these populations, both, phenotypically (Zinc, Cadmium and NaCl tolerance in vitro, and protein profiles) and genotypically (16S rDNA sequencing), as compared to type strains of known diazotrophic species. Densities were evaluated by using NFb, Fam and JNFb media, commonly used for enrichment cultures of diazotrophic bacteria. Bacterial densities found in soil and grass root samples from contaminated sites were similar to those reported for agricultural soils. Azospirillum spp. isolates from contaminated sites and type strains from non-contaminated sites varied substantially in their in vitro tolerance to Zn+2 and Cd+2, being Cd+2 more toxic than Zn+2. Among the most tolerant isolates (UFLA 1S, 1R, S181, S34 and S22), some (1R, S34 and S22) were more tolerant to heavy metals than rhizobia from tropical and temperate soils. The majority of the isolates tolerant to heavy metals were also tolerant to salt stress as indicated by their ability to grow in solid medium supplemented with 30 g L(-1) NaCl. Five isolates exhibited high dissimilarity in protein profiles, and the 16S rDNA sequence analysis of two of them revealed new sequences for Azospirillum.

The taxonomic status of a light-orange-coloured bacterial isolate from an oil-contaminated soil sample was characterized by using a polyphasic taxonomic approach. Comparative analysis of 16S rRNA gene sequences demonstrated that the isolate belonged phylogenetically to the genus Azospirillum, with Azospirillum canadense, Azospirillum brasilense and Azospirillum doebereinerae as its closest phylogenetic relatives (97.3, 97.0 and 97.0 % similarity, respectively). DNA-DNA pairing studies showed that the unidentified organism displayed 25.0, 17.0 and 19.0 % relatedness to the type strains of A. brasilense, A. canadense and A. doebereinerae, respectively. The generic assignment was confirmed by chemotaxonomic data, which revealed a fatty acid profile that was characteristic of the genus Azospirillum, consisting of straight-chain saturated and unsaturated fatty acids with C18 : 1 omega 7c as the major fatty acid, and ubiquinone with ten isoprene units (Q-10) as the predominant respiratory quinone. On the basis of both the phenotypic and molecular genetic evidence, it is proposed that the unknown isolate be classified as a representative of a novel species of the genus Azospirillum, for which the name Azospirillum rugosum sp. nov. is proposed. The type strain is IMMIB AFH-6T (=CCUG 53966T=DSM 19657T).

Two free-living nitrogen-fixing bacterial strains, N6 and N7(T), were isolated from corn rhizosphere. A polyphasic taxonomic approach, including morphological characterization, Biolog analysis, DNA-DNA hybridization, and 16S rRNA, cpn60 and nifH gene sequence analysis, was taken to analyse the two strains. 16S rRNA gene sequence analysis indicated that strains N6 and N7(T) both belonged to the genus Azospirillum and were closely related to Azospirillum oryzae (98.7 and 98.8 % similarity, respectively) and Azospirillum lipoferum (97.5 and 97.6 % similarity, respectively). DNA-DNA hybridization of strains N6 and N7(T) showed reassociation values of 48 and 37 %, respectively, with A. oryzae and 43 % with A. lipoferum. Sequences of the nifH and cpn60 genes of both strains showed 99 and approximately 95 % similarity, respectively, with those of A. oryzae. Chemotaxonomic characteristics (Q-10 as quinone system, 18 : 1omega7c as major fatty acid) and G+C content of the DNA (67.6 mol%) were also similar to those of members of the genus Azospirillum. Gene sequences and Biolog and fatty acid analysis showed that strains N6 and N7(T) differed from the closely related species A. lipoferum and A. oryzae. On the basis of these results, it is proposed that these nitrogen-fixing strains represent a novel species. The name Azospirillum zeae sp. nov. is suggested, with N7(T) (=NCCB 100147(T)=LMG 23989(T)) as the type strain.

Aims: To isolate micro-organisms capable of utilizing polyhexamethylene biguanide (PHMB) as a sole source of nitrogen, and to demonstrate biodegradation of the biocide., Methods and Results: Two consortia of bacteria were successfully enriched at the expense of PHMB, using sand from PHMB-treated swimming pools as inoculum. Both consortia were shown to contain bacteria belonging to the genera Sphingomonas, Azospirillum and Mesorhizobium. It was shown that the presence of both Sphingomonas and Azospirillum spp. was required for extensive growth of the consortia. In addition, the Sphingomonads were the only isolates capable of growth in axenic cultures dosed with PHMB. Using a stable isotope (15N)-labelled PHMB, metabolism of the biocide by both consortia was demonstrated. By comparing the level of 15N atom incorporation into bacterial DNA after growth on either 15N-PHMB or 15N-labelled NH4Cl, it was possible to estimate the percentage of PHMB biodegradation., Conclusions: The microbial metabolism of nitrogen from the biguanide moiety of PHMB has been demonstrated. It was revealed that Sphingomonas and Azospirillum spp. are the principal organisms responsible for growth at the expense of PHMB., Significance and Impact of the Study: This is the first study to demonstrate the microbial metabolism of PHMB.

A free-living diazotrophic strain, DS2(T), was isolated from corn rhizosphere. Polyphasic taxonomy was performed including morphological characterization, Biolog analysis, and 16S rRNA, cpn60 and nifH gene sequence analyses. 16S rRNA gene sequence analysis indicated that strain DS2(T) was closely related to the genus Azospirillum (96 % similarity). Chemotaxonomic characteristics (DNA G+C content 67.9 mol%; Q-10 quinone system; major fatty acid 18 : 1omega7c) were also similar to those of the genus Azospirillum. In all the analyses, including phenotypic characterization using Biolog analysis and comparison of cellular fatty acids, this isolate was found to be different from the closely related species Azospirillum lipoferum, Azospirillum oryzae and Azospirillum brasilense. On the basis of these results, a novel species is proposed for this nitrogen-fixing strain. The name Azospirillum canadense sp. nov. is suggested with the type strain DS2(T) (=NCCB 100108(T)=LMG 23617(T)).

Forty Azospirillum strains were tested for their ability to synthesize N-acyl-homoserine lactones (AHLs). AHL production was detected for four strains belonging to the lipoferum species and isolated from a rice rhizosphere. AHL molecules were structurally identified for two strains: Azospirillum lipoferum TVV3 produces 3O,C(8)-HSL (N-3-oxo-octanoyl-homoserine-lactone), C(8)-HSL (N-3-octanoyl-homoserine-lactone), 3O,C(10)-HSL (N-3-oxo-decanoyl-homoserine-lactone), 3OH,C(10)-HSL (N-3-hydroxy-decanoyl-homoserine-lactone) and C(10)-HSL (N-3-decanoyl-homoserine-lactone), whereas A. lipoferum B518 produced 3O,C(6)-HSL (N-3-oxo-hexanoyl-homoserine-lactone), C(6)-HSL (N-3-hexanoyl-homoserine-lactone), 3O,C(8)-HSL, 3OH,C(8)-HSL and C(8)-HSL. Genes involved in AHL production were characterized for A. lipoferum TVV3 by generating a genomic library and complementing an AHL-deficient strain with sensor capabilities. Those genes, designated alpI and alpR, were found to belong to the luxI and luxR families, respectively. When cloned in a suitable heterologous host, alpI and alpR could direct the synthesis of the five cognate AHLs present in A. lipoferum TVV3. These two adjacent genes were found to be located on a 85 kb plasmid. Southern hybridization experiments with probes alpI/R indicated that genes involved in AHL production in the three other AHL-producing strains were not closely related to alpI and alpR. This study demonstrates that AHL-based quorum-sensing is not widespread among the genus Azospirillum and could be found only in some A. lipoferum strains.

The plant growth-promoting rhizobacterium Azospirillum lipoferum 4B generates in vitro at high frequency a stable nonswimming phase variant designated 4V(I), which is distinguishable from the wild type by the differential absorption of dyes. The frequency of variants generated by a recA mutant of A. lipoferum 4B was increased up to 10-fold. The pleiotropic modifications characteristic of the phase variant are well documented, but the molecular processes involved are unknown. Here, the objective was to assess whether genomic rearrangements take place during phase variation of strain 4B. The random amplified polymorphic DNA (RAPD) profiles of strains 4B and 4V(I) differed. RAPD fragments observed only with the wild type were cloned, and three cosmids carrying the corresponding fragments were isolated. The three cosmids hybridized with a 750-kb plasmid and pulse-field gel electrophoresis analysis revealed that this replicon was missing in the 4V(I) genome. The same rearrangements took place during phase variation of 4BrecA. Large-scale genomic rearrangements during phase variation were demonstrated for two additional strains. In Azospirillum brasilense WN1, generation of stable variants was correlated with the disappearance of a replicon of 260 kb. For Azospirillum irakense KBC1, the variant was not stable and coincided with the formation of a new replicon, whereas the revertant recovered the parental genomic architecture. This study shows large-scale genomic rearrangements in Azospirillum strains and correlates them with phase variation.

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.

The taxonomic position of the free-living diazotrophic strain COC8(T) isolated from rice was investigated based on phylogenetic analyses. 16S rRNA gene sequence analyses indicated that strain COC8(T) was closely related to the genus Azospirillum (96% similarity). Chemotaxonomic characteristics (G+C content of the DNA 66.8 mol%, Q-10 quinone system, 18:1omega7c as the major fatty acid and 14:0 3-OH and 16:0 3-OH as the major hydroxy fatty acids) were also similar to those of the genus Azospirillum. Based on its physiological characteristics, strain COC8(T) can clearly be differentiated from recognized species of Azospirillum. The name Azospirillum oryzae sp. nov. is proposed to accommodate this bacterial strain; the type strain is COC8(T) (=IAM 15130(T)=CCTCC AB204051(T)).

Perchlorate contamination is a concern because of the increasing frequency of its detection in soils and groundwater and its presumed inhibitory effect on human thyroid hormone production. Although significant perchlorate contamination occurs in the vadose (unsaturated) zone, little is known about perchlorate biodegradation potential by indigenous microorganisms in these soils. We measured the effects of electron donor (acetate and hydrogen) and nitrate addition on perchlorate reduction rates and microbial community composition in microcosm incubations of vadose soil. Acetate and hydrogen addition enhanced perchlorate reduction, and a longer lag period was observed for hydrogen (41 days) than for acetate (14 days). Initially, nitrate suppressed perchlorate reduction, but once perchlorate started to be degraded, the process was stimulated by nitrate. Changes in the bacterial community composition were observed in microcosms enriched with perchlorate and either acetate or hydrogen. Denaturing gradient gel electrophoresis analysis and partial sequencing of 16S rRNA genes recovered from these microcosms indicated that formerly reported perchlorate-reducing bacteria were present in the soil and that microbial community compositions were different between acetate- and hydrogen-amended microcosms. These results indicate that there is potential for perchlorate bioremediation by native microbial communities in vadose soil.