What Do We Know About the Publications Related with Azospirillum? A Metadata Analysis.

Azospirillum is one of the most successful plant growth-promoting bacteria (PGPB) genera and it is considered a study model for plant-bacteria interactions. Because of that, a wide broad of topics has been boarded and discussed in a significant number of publications in the last four decades. Using the Scopus® database, we conducted a bibliographic search in order to analyze the number and type of publications, the authors responsible of these contributions, and the origin of the researchers, as well as the keywords and journals selected by the authors, among other related characteristics, with the aim to understand some less addressed details about the work done with Azospirillum worldwide since its discovery in 1925. Despite that the largest numbers of publications about this bacterium were obtained between the 1970 and 1980s, there is still a linear increase tendency in the number of published works. Understanding the mechanisms involved in the ability of these bacteria to promote growth in a wide broad of plant species under both laboratory and field conditions has been a preferential target for these published articles. This tendency could be considered a cause or consequence of the current increase in the number of commercial products formulated with Azospirillum around the world and a catalyzer for the increase of published articles along time.

Genome-based reclassification of Azospirillum brasilense Sp245 as the type strain of Azospirillum baldaniorum sp. nov.

Azospirillum sp. strain Sp245T, originally identified as belonging to Azospirillum brasilense, is recognized as a plant-growth-promoting rhizobacterium due to its ability to fix atmospheric nitrogen and to produce plant-beneficial compounds. Azospirillum sp. Sp245T and other related strains were isolated from the root surfaces of different plants in Brazil. Cells are Gram-negative, curved or slightly curved rods, and motile with polar and lateral flagella. Their growth temperature varies between 20 to 38 °C and their carbon source utilization is similar to other Azospirillum species. A preliminary 16S rRNA sequence analysis showed that the new species is closely related to A. brasilense Sp7T and A. formosense CC-Nfb-7T. Housekeeping genes revealed that Azospirillum sp. Sp245T, BR 12001 and Vi22 form a separate cluster from strain A. formosense CC-Nfb-7T, and a group of strains closely related to A. brasilense Sp7T. Overall genome relatedness index (OGRI) analyses estimated based on average nucleotide identity (ANI) and digital DNA-DNA hybridization (dDDH) between Azospirillum sp. Sp245T and its close relatives to other Azospirillum species type strains, such as A. brasilense Sp7T and A. formosense CC-Nfb-7T , revealed values lower than the limit of species circumscription. Moreover, core-proteome phylogeny including 1079 common shared proteins showed the independent clusterization of A. brasilense Sp7T, A. formosense CC-Nfb-7T and Azospirillum sp. Sp245T, a finding that was corroborated by the genome clustering of OGRI values and housekeeping phylogenies. The DNA G+C content of the cluster of Sp245T was 68.4-68.6 %. Based on the phylogenetic, genomic, phenotypical and physiological analysis, we propose that strain Sp245T together with the strains Vi22 and BR12001 represent a novel species of the genus Azospirillum, for which the name Azospirillum baldaniorum sp. nov. is proposed. The type strain is Sp245T (=BR 11005T=IBPPM 219T) (GCF_007827915.1, GCF_000237365.1, and GCF_003119195.2).

Azospirillum thermophilum sp. nov., isolated from a hot spring.

A novel Gram-staining negative, aerobic, motile by flagellum, rod-shaped bacterium, designated CFH 70021T was isolated from a hot spring soil sample collected from Tengchong, Yunnan province, PR China. Growth of CFH 70021T occurred at 15-50 °C (optimum 50 °C), pH 5.0-7.0 (optimum pH 7.0) and with 0-3.0 % (w/v) NaCl (optimum 0 %, w/v). The genome of CFH 70021T consisted of four complete circular chromosomes and five plasmids, the genomic DNA G+C content was 69.3 mol%. Comparison of the 16S rRNA gene sequences indicated that CFH 70021T represented a member of the genus Azospirillum and showed close relationship with the type strains of Azospirillum agricola CC-HIH038T (97.8 %), Azospirillum rugosum IMMIB AFH-6T (97.6 %), Azospirillum doebereinerae GSF71T (97.6 %), Azospirillum thiophilum DSM 21654T (97.4 %) and Azospirillum picis IMMIB TAR-3T (97.2 %). The polar lipids of CFH 70021T contained diphosphatidylglycerol, phosphatidylmehtylethanolamine, phosphatidylglycerol, phosphatidylcholine, two aminolipids and an unidentified phospholipid. The predominant cellular fatty acids (>10 %) included C19:0cyclo ω8c (11.4 %), C16 : 0 (27.6 %) and summed feature 8 (C18:1ω7c/C18:1ω6c, 40.9 %). The major isoprenoid quinone was Q-10. On the basis of the low ANIb result (<78 %) and different phenotypic and chemotaxonomic characters, we conclude that strain CFH 70021T represents a novel member of the genus Azospirillum, for which the name Azospirillum thermophilum sp. nov. is proposed. The type strain is CFH 70021T (=KCTC 62259T= CCTCC AB2018121T).

Azospirillum palustre sp. nov., a methylotrophic nitrogen-fixing species isolated from raised bog.

Nitrogen-fixing bacterial strain, designated B2T, was isolated from methane-oxidation enrichment originating from a Sphagnum-dominated raised peatland in Tver region, Russia, and its phenotypic, chemotaxonomic and genomic characteristics were investigated. Cells of isolate were Gram-negative, aerobic, rod or spiral-shaped, with motility provided by a single polar flagellum in liquid media and peritrichous flagella on solid media. Strain was able to grow at 15-40 °C, pH 5.5-8.5 and tolerated NaCl to 2.0 % (w/v). Strain B2T gave positive amplification for dinitrogen reductase (nifH gene) and acetylene reduction activity was recorded up to 1250 nmol ethylene h-1 (mg protein)-1. Analysis of 16S rRNA showed that B2T represents a member of the genus Azospirillum and had the highest sequence similarity with A. humicireducens SgZ-5T (97.92 %). The predominant quinone system was ubiquinone Q-10 and the major fatty acids were C18 : 1ω7, C16 : 1ω7 and C16 : 0. The strain was facultative methylotrophic and used methanol and formate for the growth. Genome sequencing revealed a genome size of 8.0 Mbp and a G+C content of 67.8 mol%. The mxaFI genes encoding methanol dehydrogenase were absent, but a homologous xoxF gene was detected. The genes encoding enzymes involved in the biosynthesis of tetrahydromethanopterin (H4MPT) (formaldehyde oxidation) and NAD-linked formate dehydrogenase (fdsABG) were identified. Pairwise determined whole genome average nucleotide identity (gANI) values confirmed that strain B2T represents a novel species, for which we propose the name Azospirillum palustre sp. nov. with the type strain B2T (VKM B-3233T, КСТС 62613Т).

Azospirillum ramasamyi sp. nov., a novel diazotrophic bacterium isolated from fermented bovine products.

A Gram-stain-negative, rod-shaped, aerobic bacterium, designated M2T2B2T, was isolated from fermented bovine products in Suwon, Republic of Korea. The strain displayed growth at 15-45 °C (optimum, 28-30 °C), pH 6.0-10.0 (pH 7.0) and 0-2 % (w/v) NaCl (0 %). Colonies were light pink-coloured, round and convex. The cells were positive for oxidase and weakly positive for catalase. The major fatty acids in whole cells of strain M2T2B2T were summed feature 8 (C18 : 1ω7c and/or C18 : 1ω6c), followed by summed feature 3 (C16 : 1ω7c/C16 : 1ω6c), summed feature 2 (C12 : 0 aldehyde/unidentified 10.928/C14 : 0 3-OH/iso-C16 : 1 I), C16 : 0, C18 : 1 2-OH, C16 : 0 3-OH and C17 : 1ω6c. The polar lipids were phosphatidylethanolamine, diphosphatidylglycerol, phosphatidylcholine, phosphatidylglycerol, and three unidentified aminolipids. Ubiquinone 10 was the predominant ubiquinone. The DNA G+C content was 68.0 mol%. The strain could fix atmospheric nitrogen, which was evaluated by the acetylene reduction assay. Further, whole genome sequence analysis revealed the presence of a nif gene cluster. Strain M2T2B2T showed the highest 16S rRNA, rpoD and nifH gene sequence similarity to members of the genus Azospirillum, and showed 97.6 % 16S rRNA gene sequence similarity to Azospirillum oryzae COC8T. The phenotypic, phylogenetic and genomic analyses support the proposal of strain M2T2B2T as being a novel species of the genus Azospirillum, for which the name Azospirillumramasamyi sp. nov. is proposed. The type strain is M2T2B2T (=KACC 14063T=NBRC 106460T).

Azospirillum griseum sp. nov., isolated from lakewater.

A novel Gram-stain-negative, strictly aerobic, rod-shaped, gray-pigmented bacterial strain, designated L-25-5 w-1T, was isolated from the water at Baiyang Lake, PR China. Cells of strain L-25-5 w-1T were motile, with a single polar flagellum. L-25-5 w-1T was able to grow at 15-37 °C (optimum 37 °C) and pH 5-8 (optimum pH 6) in R2A medium. 16S rRNA gene sequence analysis and phylogenetic analysis of L-25-5 w-1T showed the highest relationship to Azospirillum doebereinerae GSF71T (97.9 %), Azospirillum thiophilum DSM 21654T (97.9 %) and Azospirillum agricola CC-HIH038T (97.0 %), with other species of the genus Azospirillum showing less than 97 % sequence similarity. The predominant polar lipids were phosphatidylethanolamine, phosphatidylcholine, phosphatidylglycerol, diphosphatidylglycerol and two unidentified aminolipids; the major cellular fatty acids were C16 : 0, C16 : 0 3-OH, C18 : 1 2-OH, iso-C18 : 0, summed feature 2 (C12 : 0 aldehyde and/or unknown 10.9525 and/or iso-C16 : 1I and/or C14 : 0 3-OH), summed feature 3 (C16 : 1ω7c and/or C16 : 1ω6c) and summed feature 8 (C18 : 1ω7c and/or C18 : 1ω6c); and the major respiratory quinone was ubiquinone 10. The draft genome size of L-25-5 w-1T was 5.8 Mbp, and the DNA G+C content was 66.6 %. The average nucleotide identity value and digital DNA-DNA hybridization relatedness value between L-25-5 w-1T and related type strains were 80.2 and 24.7 % with A. doebereinerae GSF71T and 78.8 and 23.6 % with A. thiophilum DSM 21654T, respectively. According to the phylogenetic, chemotaxonomic and genotypic properties, strain L-25-5 w-1T represents a novel species in the genus Azospirillum, for which the name Azospirillum griseum sp. nov. is proposed. The type strain is L-25-5 w-1T (=CGMCC 1.13672T=KCTC 62777T).

Azospirillum spp. from native forage grasses in Brazilian Pantanal floodplain: biodiversity and plant growth promotion potential.

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.

Development of sequence-characterized amplified region (SCAR) markers as a quality standard of inoculants based on Azospirillum.

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.

Azospirillum agricola sp. nov., a nitrogen-fixing species isolated from cultivated soil.

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 soli sp. nov., a nitrogen-fixing species isolated from agricultural soil.

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).

Azospirillum fermentarium sp. nov., a nitrogen-fixing species isolated from a fermenter.

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)).

Azospirillum humicireducens sp. nov., a nitrogen-fixing bacterium isolated from a microbial fuel cell.

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)).

Characterization of cellular, biochemical and genomic features of the diazotrophic plant growth-promoting bacterium Azospirillum sp. UENF-412522, a novel member of the Azospirillum genus.

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.

Genomic insights into the versatility of the plant growth-promoting bacterium Azospirillum amazonense.

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.

The genetic diversity of culturable nitrogen-fixing bacteria in the rhizosphere of wheat.

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.

A rapid and simple PCR method for identifying isolates of the genus Azospirillum within populations of rhizosphere bacteria.

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.

Azospirillum oleiclasticum sp. nov, a nitrogen-fixing and heavy oil degrading bacterium isolated from an oil production mixture of Yumen Oilfield.

Two nitrogen-fixing and heavy oil degrading strains, designated RWY-5-1-1T and ROY-1-1-2, were isolated from an oil production mixture from Yumen Oilfield in China. The 16S rRNA gene sequence showed they belong to Azospirillum and have less than 96.1 % pairwise similarity with each species in this genus. The average nucleotide identity and digital DNA-DNA hybridization values between them and other type strains of Azospirillum species were less than 75.69 % and 22.0 %, respectively, both below the species delineation threshold. Pan-genomic analysis showed that the novel isolate RWY-5-1-1T shared 2145 core gene families with other type strains in Azospirillum, and the number of strain-specific gene families was 1623, almost two times more than the number known from other species. Furthermore, genes related to nitrogenase, hydrocarbon degradation and biosurfactant production were found in the isolates' genomes. Also, this strain was capable of reducing acetylene to ethylene at a rate of 22nmol ethylene h-1 (108 cells) and degrading heavy oil at a rate of 36.2 %. The major fatty acids and polar lipids were summed feature 8 (C18:1ω7c/C18:1ω6c), and phosphatidylethanolamine, diphosphatidylglycerol, phosphatidylglycerol, and phosphatidylcholine. Furthermore, a combination of phenotypic, chemotaxonomic, phylogenetic and genotypic data clearly indicated that strains RWY-5-1-1T and ROY-1-1-2 represent a novel species, for which the name Azospirillum oleiclasticum sp. nov. is proposed. The type strain is RWY-5-1-1T (=CGMCC 1.13426T =KCTC 72259 T). Azospirillum novel strains with the ability of heavy oil degradation associated with the promotion of plant growth has never been reported to date.

Azospirillum thiophilum sp. nov., a diazotrophic bacterium isolated from a sulfide spring.

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 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)).

Isolation of functional single cells from environments using a micromanipulator: application to study denitrifying bacteria.

We developed a novel method to isolate functionally active single cells from environmental samples and named it the functional single-cell (FSC) isolation method. This method is based on a combination of substrate-responsive direct viable counts, live-cell staining with 5-carboxyfluorescein diacetate acetoxymethyl ester, and micromanipulation followed by cultivation in a medium. To evaluate this method, we applied it to study a denitrifying community in rice paddy soil. Similar denitrifier counts were obtained by the conventional most probable number analysis and our FSC isolation method. Using the FSC isolation method, 37 denitrifying bacteria were isolated, some of which harbored copper-containing nitrite reductase gene (nirK). The 16S rRNA gene analysis showed that members belonging to the genera Azospirillum and Ochrobactrum may be the major denitrifiers in the rice paddy soil. These results indicate that the FSC isolation method is a useful tool to obtain functionally active single cells from environmental samples.

Azospirillum palatum sp. nov., isolated from forest soil in Zhejiang province, China.

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)).