Azospirillum Endophyticum sp. nov., an Endophyte of Paris Polyphylla Smith var. Yunnanensis.

A Gram-negative, facultative anaerobic bacterial strain, designated YIM B02556T, 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 B02556T belonged to the genus Azospirillum and the 16S rRNA gene sequence similarity values of strain YIM B02556T 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 B02556T 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 B02556T can tolerate 2% NaCl concentration. Strain YIM B02556T contained Q-10 as the major ubiquinone. The major fatty acids were C18:1 ω7c and summed feature three (C16:1 ω7c and/or C16:1 ω6c). The major polar lipids were diphosphatidylglycerol, phosphatidylglycerol, and phosphatidylethanolamine. Based on polyphasic analysis, strain YIM B02556T 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 B02556T (=JCM 34631T=CGMCC 1.18654T).

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 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)).

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)).

Azospirillum picis sp. nov., isolated from discarded tar.

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)).

Associative diazotrophic bacteria in grass roots and soils from heavy metal contaminated sites

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.

Objetivou-se avaliar a densidade de populações de bactérias diazotróficas associativas em amostras de solos e de raízes de gramíneas oriundas de sítios contaminados com metais pesados, e caracterizar isolados destas populações através da análise fenotípica (tolerância aos metais pesados zinco e cádmio e à NaCl in vitro, perfis protéicos), e genotípica (seqüenciamento de 16S rDNA), comparados às estirpes tipo das mesmas espécies. As densidades foram avaliadas nos meios NFb, Fam e LGI, comumente utilizados para culturas de enriquecimento de populações de bactérias diazotróficas associativas. As densidades encontradas em amostras de solo e raiz de sítios contaminados foram semelhantes àquelas relatadas na literatura para solos agrícolas. Isolados de Azospirillum spp. de solos contaminados e estirpes tipo oriundas de solos não contaminados variaram substancialmente com relação à tolerância a Zn+2 e Cd+2, sendo que Cd+2 mais tóxico que Zn+2. Dentre os isolados mais tolerantes (UFLA 1S, 1R, S181, S34, e S22), alguns(1R, S34 e S22) foram mais tolerantes a metais pesados que rizóbios isolados de solos de áreas tropicais e temperadas. A maioria dos isolados mais tolerantes a metais pesados também foi tolerante ao estresse salino, o que foi indicado por seu crescimento em meio sólido suplementado com 30 g L-1 de NaCl in vitro. Cinco isolados apresentaram alta dissimilaridade em perfis protéicos e o seqüenciamento do gene 16S rDNA em dois deles revelou que apresentam novas seqüências de Azospirillum.

Azospirillum rugosum sp. nov., isolated from oil-contaminated soil.

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).

Azospirillum zeae sp. nov., a diazotrophic bacterium isolated from rhizosphere soil of Zea mays.

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.

Azospirillum canadense sp. nov., a nitrogen-fixing bacterium isolated from corn rhizosphere.

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)).

Isolation and characterization of a diazotrophic, oxalate-oxidizing bacterium from sour grass (Oxalis pes-caprae L.).

A new type of nitrogen-fixing, oxalate-oxidizing Azospirillum sp. was isolated from the roots of Oxalis pes-caprae. Polyphasic taxonomy was performed, including auxanography using API galleries, physiological tests and 16S rRNA sequence comparison. Optimum growth occurred at 30 degrees C, pH 7.5. Growth was observed at 37 and 42 degrees C with oxalate and in the presence of 3-4% NaCl and 2% potassium oxalate. In liquid culture, the doubling time (t(d)) with oxalate was 9 h. Its closest phylogenetic neighbors, as deduced by 16S rDNA-based analysis, were Azospirillum brasilense, Azospirillum doebereinerae and Azospirillum lipoferum, with 99.5, 98.4 and 96.7% sequence similarity, respectively. The strain differed from A. brasilense by its ability to use N-acetylglucosamine, D-glucose and D-mannitol. It may be a variant strain of A. brasilense. Oxalotrophic, N2-fixing species of the genus Azospirillum may be important contributors to soil formation, soil fertility, and retention and/or cycling of elements necessary for plant growth.

Partial characterization of nif genes from the bacterium Azospirillum amazonense

Azospirillum amazonense revealed genomic organization patterns of the nitrogen fixation genes similar to those of the distantly related species A. brasilense. Our work suggests that A. brasilense nifHDK, nifENX, fixABC operons and nifA and glnB genes may be structurally homologous to the counterpart genes of A. amazonense. This is the first analysis revealing homology between A. brasilense nif genes and the A. amazonense genome. Sequence analysis of PCR amplification products revealed similarities between the amino acid sequences of the highly conserved nifD and glnB genes of A. amazonense and related genes of A. brasilense and other bacteria. However, the A. amazonense non-coding regions (the upstream activator sequence region and the region between the nifH and nifD genes) differed from related regions of A. brasilense even in nitrogenase structural genes which are highly conserved among diazotrophic bacteria. The feasibility of the 16S ribosomal RNA gene-based PCR system for specific detection of A. amazonense was shown. Our results indicate that the PCR primers for 16S rDNA defined in this article are highly specific to A. amazonense and can distinguish this species from A. brasilense

Azospirillum doebereinerae sp. nov., a nitrogen-fixing bacterium associated with the C4-grass Miscanthus.

A new group of nitrogen-fixing Azospirillum sp. bacteria was isolated from the roots of the C4-gramineous plant Miscanthus. Polyphasic taxonomy was performed, including auxanography using API galleries, physiological tests and 16S rRNA sequence comparison. The ability of the isolates to fix dinitrogen was evaluated by amplification of the nifD gene, immunodetection of the dinitrogenase reductase and acetylene-reduction assay. On the basis of these results, the nitrogen-fixing isolates represent a new species within the genus Azospirillum. Its closest phylogenetic neighbours, as deduced by 16S rDNA-based analysis, are Azospirillum lipoferum, Azospirillum largimobile and Azospirillum brasilense with 96.6, 96.6 and 95.9% sequence similarity, respectively. Two 16S rRNA-targeting oligonucleotide probes were developed which differentiate the new species from the other Azospirillum species by whole-cell fluorescence hybridization. Strains of the new species are curved rods or S-shaped, 1.0-1.5 microm in width and 2,0-3.0 microm in length, Gram-negative and motile with a single polar flagellum. Optimum growth occurs at 30 degrees C and at pH values between 6.0 and 7.0. No growth takes place at 37 degrees C. They have a respiratory type of metabolism, grow well on arabinose, D-fructose, gluconate, glucose, glycerol, malate, mannitol and sorbitol. They differ from A. largimobile and A. lipoferum by their inability to use N-acetylglucosamine and D-ribose, from A. lipoferum by their ability to grow without biotin supplementation and from A. brasilense by their growth with D-mannitol and D-sorbitol as sole carbon sources. Nitrogen fixation occurs in microaerobic nitrogen-limited conditions. For this species, the name Azospirillum doebereinerae sp. nov. is suggested, with strain GSF71T as the type strain (= DSM 13131T; reference strain Ma4 = DSM 13400). Its G+C content is 70.7 mol%.

Azospirillum, a free-living nitrogen-fixing bacterium closely associated with grasses: genetic, biochemical and ecological aspects.

Azospirillum represents the best characterized genus of plant growth-promoting rhizobacteria. Other free-living diazotrophs repeatedly detected in association with plant roots, include Acetobacter diazotrophicus, Herbaspirillum seropedicae, Azoarcus spp. and Azotobacter. Four aspects of the Azospirillum-plant root interaction are highlighted: natural habitat, plant root interaction, nitrogen fixation and biosynthesis of plant growth hormones. Each of these aspects is dealt with in a comparative way. Azospirilla are predominantly surface-colonizing bacteria, whereas A. diazotrophicus, H. seropedicae and Azoarcus sp. are endophytic diazotrophs. The attachment of Azospirillum cells to plant roots occurs in two steps. The polar flagellum, of which the flagellin was shown to be a glycoprotein, mediates the adsorption step. An as yet unidentified surface polysaccharide is believed to be essential in the subsequent anchoring phase. In Azoarcus sp. the attachment process is mediated by type IV pili. Nitrogen fixation structural genes (nif) are highly conserved among all nitrogen-fixing bacteria, and in all diazotrophic species of the class of proteobacteria examined, the transcriptional activator NifA is required for expression of other nif genes in response to two major environmental signals (oxygen and fixed N). However, the mechanisms involved in this control can vary in different organisms. In Azospirillum brasilense and H. seropedicae (alpha- and beta-subgroup, respectively), NifA is inactive in conditions of excess nitrogen. Activation of NifA upon removal of fixed N seems to involve, either directly or indirectly, the signal transduction protein P(II). The presence of four conserved cysteine residues in the NifA protein might be an indication that NifA is directly sensitive to oxygen. In Azotobacter vinelandii (gamma-subgroup) nifA is cotranscribed with a second gene nifL. The nifL gene product inactivates NifA in response to high oxygen tension and cellular nitrogen-status. NifL was found to be a redox-sensitive flavoprotein. The relief of NifL inhibition on NifA activity, in response to N-limitation, is suggested to involve a P(II)-like protein. Moreover, nitrogenase activity is regulated according to the intracellular nitrogen and O(2) level. In A. brasilense and Azospirillum lipoferum posttranslational control of nitrogenase, in response to ammonium and anaerobiosis, involves ADP-ribosylation of the nitrogenase iron protein, mediated by the enzymes DraT and DraG. At least three pathways for indole-3-acetic acid (IAA) biosynthesis in A. brasilense exist: two Trp-dependent (the indole-3-pyruvic acid and presumably the indole-3-acetamide pathway) and one Trp-independent pathway. The occurrence of an IAA biosynthetic pathway not using Trp (tryptophan) as precursor is highly unusual in bacteria. Nevertheless, the indole-3-pyruvate decarboxylase encoding ipdC gene is crucial in the overall IAA biosynthesis in Azospirillum. A number of genes essential for Trp production have been isolated in A. brasilense, including trpE(G) which codes for anthranilate synthase, the key enzyme in Trp biosynthesis. The relevance of each of these four aspects for plant growth promotion by Azospirillum is discussed.