Pseudomonas kirkiae sp. nov., a novel species isolated from oak in the United Kingdom, and phylogenetic considerations of the genera Pseudomonas, Azotobacter and Azomonas.

As the current episode of Acute Oak Decline (AOD) continues to affect native British oak in the United Kingdom, ongoing isolations from symptomatic and healthy oak have yielded a large Pseudomonas species population. These strains could be divided into taxa representing three potential novel species. Recently, two of these taxa were described as novel Pseudomonas species in the Pseudomonas fluorescens lineage. Here, we demonstrate using a polyphasic approach that the third taxon represents another novel Pseudomonas species. The 16S rRNA gene sequencing assigned the strains to the Pseudomonas aeruginosa lineage, while multilocus sequence analysis (based on partial gyrB, rpoB and rpoD sequences) placed the 13 strains in a single cluster on the border of the Pseudomonas stutzeri group. Whole genome intra-species comparisons (based on average nucleotide identity and in silico DNA-DNA hybridization) confirmed that the strains belong to a single taxon, while the inter-species comparisons with closest phylogenetic relatives yielded similarity values below the accepted species threshold. Therefore, we propose these strains as a novel species, namely Pseudomonas kirkiae sp. nov., with the type strain FRB 229T (P4CT=LMG 31089T=NCPPB 4674T). The phylogenetic analyses performed in this study highlighted the difficulties in assigning novel species to the genus Pseudomonas due to its polyphyletic nature and close relationship to the genus Azotobacter. We further propose that a thorough taxonomic re-evaluation of the genus Pseudomonas is essential and should be performed in the near future.

Description of Azotobacter chroococcum subsp. isscasi subsp. nov. isolated from paddy soil and establishment of Azotobacter chroococcum subsp. chroococcum subsp. nov.

Three aerobic, asymbiotic, N2-fixing bacterial strains, designated P205T, P204 and P207, were isolated from a paddy soil in Yanting County, China. Based on 16S rRNA gene sequences, the three strains were closely related to Azotobacter chroococcum IAM 12666T (=ATCC 9043T) (99.00-99.79 % similarities). Strain P205T formed an individual branch distinct from the other two newly isolated strains and other related type strains in phylogenetic analyses based on 16S rRNA gene and 92 core genes. The average nucleotide identity (ANI), average amino acid identity (AAI) and digital DNA-DNA hybridization (dDDH) values based on genome sequences of strain P205T and A. chroococcum ATCC 9043T, P204, P207 were near or slightly higher than the thresholds for species circumscription (95-96, 95-96 and 70 %, respectively), and the dDDH values were significantly lower than the threshold for delineating subspecies (79-80 %), which strongly supported that strain P205T belonged to A. chroococcum but was a novel subspecies distinct from the type strain of A. chroococcum. This finding was further corroborated by distinct phenotypic characteristics such as growth in Luria-Bertani (LB) medium, carbon source utilization and chemical sensitivity to vancomycin. Therefore, strain P205T represents a novel subspecies of Azotobacter chroococcum, for which the name Azotobacter chroococcum subsp. isscasi subsp. nov. is proposed with the type strain P205T (=KCTC 72233T=CGMCC 1.16846T=CCTCC AB 2019080T). The subspecies Azotobacter chroococcum subsp. chroococcum subsp. nov. is created automatically with the type strain ATCC 9043T (=DSM 2286T=JCM 20725T=JCM 21503T=LMG 8756T=NBRC 102613T=NCAIM B.01391T=NRRL B-14346T=VKM B-1616T).

Azotobacter bryophylli sp. nov., isolated from the succulent plant Bryophyllum pinnatum.

A Gram-stain-negative, aerobic, nitrogen-fixing bacterium, designated strain L461T, was isolated from leaves of Bryophyllum pinnatum growing at the South China Agricultural University. Phylogenetic analysis of the 16S rRNA gene sequence indicated it as a member of the genus Azotobacter closely related to Azotobacter beijerinckii JCM 20725T (97.82 % similarity) and Azotobacter chroococcum ATCC 9043T (97.34 %). Its major fatty acid components were C16 : 1 ω9c and C16 : 0. Its predominant isoprenoid quinone was Q-9. Its major polar lipids were phosphatidylethanolamine, phosphatidylglycerol, aminophospholipid, phospholipid and one unknown lipid. Its DNA G+C content was 64.9 mol% (Tm). DNA-DNA relatedness values between strain L461T and the reference strains of A. beijerinckii and A. chroococcum were 46.43 and 28.23 %, respectively. Biological and biochemical tests, protein patterns, genomic DNA fingerprinting, and comparison of cellular fatty acids distinguished strain L461T from the closely related Azotobacter species. Based on these data, the novel species Azotobacter bryophylli sp. nov. is proposed, with the type strain L461T (=KCTC 62195T=GDMCC 1.1250T).

PGP potential, abiotic stress tolerance and antifungal activity of Azotobacter strains isolated from paddy soils.

Azotobacter strains were isolated by serial dilution method and colonies were viscous, smooth, glistening, and brown to black colour on Jenson's N-free agar. Morphological and biochemical tests showed characteristic features of Azotobacter. Further, molecular analyses revealed the presence of different Azotobacter species viz., A. armeniacus, A. chroococcum, A. salinestris, A. tropicalis and A. vinelandii. The isolates were tested for their ability of nitrogen fixation, indole acetic acid (IAA), gibberllic acid production and phosphate solubilization. Four isolates (GVT-1, GVT-2 KOP-11 and SND-4) were efficient in fixation of highest amount of N2 (29.21 µg NmL(-1) day(-1)), produced IAA (25.50 µg mL(-1)), gibberllic acid (17.25 µg 25 mL(-1)) and formed larger P solubilizing zone (13.4 mm). Some of the Azotobacter strains were produced siderophores, hydrogen cyanide and were positive for ammonia production with respect to antifungal activity of Azotobacter was tested with dual culture method and A. tropicalis inhibited the growth of Fusarium, Aspergillus and Alternaria species. Azotobacter isolates were tested against salt (0-10%), temperature (4-55 degrees C), pH (5.0-10) and insecticide chloropyrifos (0-3%) tolerance study. Among them, A. chroococcum was found tolerant to a maximum of 6% NaCl with a temperature of 35-45 degrees C and to a pH up to 8. All the 4 strains showed effective growth against 3% chloropyrifos concentration. The studies revealed that the Azotobacter strains not only produced plant growth promoting substances but are also tolerant to abiotic stresses such as temperature, pH and insecticides.

Solubilization of tricalcium phosphate by P(3HB) accumulating Azotobacter chroococcum MAL-201.

Cells of Azotobacter chroococcum MAL-201 (MTCC 3853) are capable of accumulating the intracellular poly(3-hydroxybutyric acid) [P(3HB)], accounting for 65-71 % of its cell dry weight and also capable of synthesizing the enzyme alkaline phosphatase (APase), when grown in glucose and tricalcium phosphate containing nitrogen-free modified Stockdale medium. The concentration of insoluble phosphate in broth medium was optimized as 0.25 % (w/v) for growth and biosynthesis of APase. However, the suboptimal concentration of phosphate (0.1 %, w/v) appeared as the best suited for accumulation of P(3HB) by the strain. The significant differences were observed in biosynthesis of polymer and APase enzyme under variable phosphate concentrations. Glucose, 3.0 % (w/v) was recorded as the optimum concentration for all of the three parameters. The continuation of APase biosynthesis was observed during the period of significant decline in the cellular content of the polymer in the late phase of growth. In order to study the role of P(3HB), the rate of autodigestion of biopolymer and phosphate solubilization rate (k, mineralization constant) were determined in carbon-free medium under batch cultivation process and the parameters were found to be positively correlated. The maximum phosphate solubilization rate (k = 0.0154) by the strain MAL-201 timed at the 10th hour of incubation when the rate of polymer degradation concomitantly attained its peak corresponding to 87 mg/l/h and then declined gradually. Only a negligible amount of residual polymer remained undigested. These data strongly support the functional role of P(3HB) in response to multinutritional stress condition.

Pesticide tolerant Azotobacter isolates from paddy growing areas of northern Karnataka, India.

A total of 14 Azotobacter strains were isolated from different paddy cultivating soils with pH ranging from 6.5 to 9.5 by using serial dilution agar plate method. The strains were Gram negative, rod shaped, cyst forming and developed brown to black colored colonies, which were glistening, smooth, slimy on Ashby's agar plates. Biochemically they were positive for biochemical tests namely, indole production, citrate, catalase, carbohydrate fermentation and Voges-Proskauer test. Further, sequence analysis of PCR amplicons obtained from these cultures revealed the presence of five different Azotobacter species viz., Azotobacter vinelandii, Azotobacter salinestris, Azotobacter sp., Azotobacter nigricans subsp. nigricans and Azotobacter tropicalis. Phylogenetically these strains were grouped into two distinct clusters. These strains were tested for their ability to grow on a media containing four different pesticides such as pendimethalin, glyphosate, chloropyrifos and phorate, which are commonly used for the paddy. Out of 14 strains tested, 13 strains were able to grow on a media containing herbicides such as pendimethalin, glyphosate and insecticides like chloropyrifos and phorate. However, five Azotobacter strains were able to grow at higher concentration of 5% pesticides, without affecting their growth rate. Further, the effect of pesticides on the indole acetic acid (IAA) production by Azotobacter strains was also estimated. Azotobacter-16 strain was found to produce 34.4 µg ml(-l) of IAA in a media supplemented with 1,000 mg of tryptophan and 5% of pendimethalin. Present study reveals that species of Azotobacter are able to grow and survive in the presence of pesticides and no significant effects were observed on the metabolic activities of Azotobacter species.

Plant growth promoting characterization of indigenous Azotobacteria isolated from soils in Iran.

It has been well known that the bacteria of the genus Azotobacter, in addition to the beneficial N(2)-fixing activity, are able to improve plant growth by a number of direct and indirect mechanisms. To identify this potential in indigenous azotobacteria, the efficiency of 17 isolates of Azotobacter from the rhizosphere of wheat and barley plants cultivated in salt- and/or drought-affected soils in Iran were evaluated for their ability to dissolve inorganic and organic phosphates, siderophore secretion, indole acetic acid (IAA) production; and protease, chitinase, and ACC deaminase (ACCD) activities. First, they were biochemically characterized and one isolate (strain) was identified by 16S rDNA sequencing. Eight isolates were designated as Azotobacter vinelandii and the remaining isolates were identified as A. chroococcum. All isolates hydrolyzed the organic and inorganic phosphate compounds and effectively produced IAA. Fifteen isolates produced siderophore, but only one isolate showed protease activity which is being reported for the first time in relation to Azotobacter. None of the 17 isolates was capable of producing ACCD or chitinase. However, polymerase chain reaction amplification of the ACCD coding genes, by the use of the gene-specific primers, indicated that not all contain the ACCD gene. The standard screening methods with slight modifications, especially in the case of ACCD assay, were applied. The results showed that the use of specific screening methods, modified according to bacterial nutritional requirements, are the efficient methods for precise evaluation of the plant growth promoting rhizobacteria activity.

Chain transfer reaction catalyzed by various polyhydroxyalkanoate synthases with poly(ethylene glycol) as an exogenous chain transfer agent.

Polyhydroxyalkanoate (PHA) synthases catalyze chain transfer (CT) reaction after polymerization reaction of PHA by transferring PHA chain from PHA synthase to a CT agent, resulting in covalent bonding of CT agent to PHA chain at the carboxyl end. Previous studies have shown that poly(ethylene glycol) (PEG) is an effective exogenous CT agent. This study aimed to compare the effects of PEG on CT reaction during poly[(R)-3-hydroxybutyrate] [P(3HB)] synthesis by using six PHA synthases in Escherichia coli JM109. The synthesized P(3HB) polymers were characterized in terms of molecular weight and end-group structure. Supplementation of PEG to the culture medium reduced P(3HB) molecular weights by up to 96% due to PEG-induced CT reaction. The P(3HB) polymers were subjected to (1)H NMR analysis to confirm the formation of a covalent bond between PEG and P(3HB) chain at the carboxyl end. This study revealed the reactivity of PHA synthases to PEG with respect to CT reaction in E. coli.

NifH-based studies on azotobacterial diversity in cotton soils of India.

In order to promote the use of Azotobacter inoculants for cotton crop, a complete characterization of soil isolates of Azotobacter, isolated and screened on the basis of physiological properties, from four different cotton-wheat cropping regions of India was carried out, and their genetic diversity determined by RFLP (restriction fragment length polymorphism) analysis of the functional gene nifH. Genetic analysis of these isolates depicted a similarity coefficient of > or = 80% among them, suggesting that though the isolates were obtained from different cotton soils of India, still they have large commonality in the nifH gene and constituted a homogeneous nifH population.

Enhanced production and partial characterization of an extracellular polysaccharide from newly isolated Azotobacter sp. SSB81.

A strain was selected by its highest extracellular polysaccharide (EPS) production ability compare to other isolates from the same rhizospheric soil. The selected strain was identified by 16S rDNA sequencing and designated as SSB81. Phylogenetic analysis of the gene sequence showed its close relatedness with Azotobacter vinelandii and Azotobacter salinestris. Maximum EPS (2.52 g l(-1)) was recovered when the basal medium was supplemented with glucose (2.0%), riboflavin (1 mg l(-1)) and casamino acid (0.2%). The EPS showed a stable viscosity level at acidic pH (3.0-6.5) and the pyrolysis temperature was found to be at 116.73 degrees C with an enthalpy (DeltaH) of 1330.72 J g(-1). MALDI TOF mass spectrometric result suggests that polymer contained Hex(5)Pent(3) as oligomeric building subunit. SEM studies revealed that the polymer had a porous structure with small pore size distribution indicating the compactness of the polymer. This novel EPS may find possible application as a polymer for environmental bioremediation and biotechnological processes.

Diversity studies of Azotobacter spp. from cotton-wheat cropping systems of India.

Azotobacter are the favored bioinoculants being promoted for cotton crop in India. In order to develop bioinoculants, both metabolic fingerprinting and genetic fingerprinting have been used to study the diversity among Azotobacter spp. isolated from four different cotton-wheat cropping regions of India. On the basis of acetylene reduction, indole acetic acid production and ammonia excretion, from 76 free-living diazotrophs isolated from the rhizospheric soil of cotton, 20 efficient isolates were selected for further studies. Morphological characterization indicated a close resemblance of these isolates to Azotobacter spp. BIOLOG cataloguing divided them into two main groups, but amplified ribosomal DNA restriction analysis clustered the isolates from the four regions having different soil types into four separate sub-clusters. Metabolic fingerprinting was not able to detect subtle differences among the isolates as achieved with genetic fingerprinting. However, 16S rRNA is a highly conserved locus. Variations observed could be due to domestication of the isolates in different agro-ecological niches.

Probable synonymy of the nitrogen-fixing genus Azotobacter and the genus Pseudomonas.

The relationships of the genus Azotobacter, Azomonas macrocytogenes and the genus Pseudomonas were revealed by comparative analysis of partial 16S rRNA and atpD, carA and recA gene sequences and as concatenated nucleotide and peptide sequences. Sequence similarities of Azotobacter species and Azomonas macrocytogenes indicated that these may be considered to be synonyms at the molecular level. In addition, these species show an intimate relationship with species of Pseudomonas, especially P. aeruginosa (the type species of the genus). In terms of the current circumscription of the genus Pseudomonas, Azotobacter and Azomonas macrocytogenes should be considered for amalgamation with Pseudomonas. Azotobacter and Azomonas comprise nitrogen-fixing strains with large pleomorphic cells that form cysts, and peritrichous flagella insertion; characteristics not included in the current circumscription of Pseudomonas. The data are discussed in the light of whether lateral transfer of genes could be involved in the determination of significant morphological characteristics, thus leading to a problem that may be encountered more frequently: how to resolve classification of taxa based on conserved sequences with those based on their phenotype. More fundamentally, the results illuminate problems that will increasingly be encountered: by what criteria can taxa be delineated, what are the most appropriate methods for classification, and what are the proper assumptions of bacterial classification?

Evaluation of abundance of aerobic bacteria in the rhizosphere of transgenic and non-transgenic alfalfa lines.

Fourteen genetically modified lines of alfalfa (Medicago sativa) containing the gene Ov from Japanese quail, coding for a methionine-rich protein ovalbumin, were evaluated for nodulation ability and concentration of aerobic bacteria in the rhizosphere. The transgenic lines were derived from a highly regenerable genotype Rg9/I-14-22, selected from cv. Lucia. On selective media, a higher concentration of ammonifying bacteria, bacterial spores, denitrifying and nitrifying bacteria were observed in the rhizosphere of transgenic clonesand, on the other hand, lower concentration of cellulolytic bacteria and Azotobacter spp. compared with the rhizosphere of non-transgenic clone SE/22-GT2. A statistically significant difference in the concentration of all the bacterial types was found between samples taken from two types of substrates (i.e. sterile vs. nonsterile). Higher bacterial concentration (measured as colony forming units per g soil dry mass) were observed for all tested groups of culturable bacteria in the non-sterile substrate. The presence of Azotobacter spp. was found only in the rhizosphere of plants grown in non-sterile soil in which the highest number of fertile soil particles (97 %) was observed in transgenic clones SE/22-9-1-12 and SE/22-11-1-1S.1. Concentration of bacteria involved in the N cycle in the soil was increased in the rhizosphere of transgenic clones and decreased in the rhizosphere of non-transgenic plants compared with the average value. In spite of some differences in colony numbers in samples isolated from the root rhizosphere of transgenic and nontransgenic alfalfa plants, we could not detect any statistically significant difference between individual lines.