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Molecular Tools for Identification and Characterization of Plant Growth Promoting Rhizobacteria with Emphasis in Azospirillum spp.

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Handbook for Azospirillum

Abstract

Azospirillum is considered an important genus among plant growth promoting rhizobacteria (PGPR). After the recent reclassification of Azospirillum irakense to Niveispirillum irakense and Azospirillum amazonense to Nitrospirillum amazonense based on their polyphasic taxonomic characteristics, at present this genus encompasses 15 valid species. In this chapter, the identification and characterization of the genus Azospirillum through genotypic, phenotypic or chemotaxonomic approaches were reviewed. Under the given set of PCR condition, the genus specific primers Azo494-F/Azo756-R were sufficient to differentiate Azospirillum and other closely related genera such as Rhodocista and Skermanella. Along with PCR—denaturing gradient gel electrophoresis (PCR-DGGE) or real-time quantitative PCR (qPCR), the specific primers were useful to detect and identify Azospirillum in a short time no matter pure cultures or environmental samples were used. The minimum detection limit in real-time quantitative PCR analysis is 102 CFU g−1 in the seeded soil sample. Cells of the genus Azospirillum are Gram-stained negative, spiral or rod-shaped and non-spore-forming diazotrophic. Poly-β-hydroxybutyrate granules were observed after few days of incubation. The major fatty acids were C16:0, C16:0 3-OH, C18:1 2-OH, C14:0 3-OH/C16:1 iso I, C16:1 ω7c/C16:1 ω6c and C18:1 ω7c/C18:1 ω6c; the predominant polar lipids included phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylglycerol (PG), diphosphatidylglycerol (DPG), phosphatidyldimethylethanolamine (PDE) and unidentified aminolipid (AL) and phospholipids (PL); the common major respiratory quinone was ubiquinone Q-10 and predominant polyamines were sym-homospermidine and putrescine. These features are also useful to provide bases in the description of members belonging to the genus Azospirillum.

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References

  • Aziz A, Martin-Tanguy J, Larher F (1997) Plasticity of polyamine metabolism associated with high osmotic stress in rape leaf discs and with ethylene treatment. Plant Growth Regul 21:153–163

    Article  CAS  Google Scholar 

  • Bashan Y, Holguin G, de-Bashan L (2004) Azospirillum–plant relationships: physiological, molecular, agricultural, and environmental advances (1997–2003). Can J Microbiol 50:521–577

    Article  CAS  PubMed  Google Scholar 

  • Ben Dekhil S, Cahill M, Stackebrandt E, Sly LI (1997) Transfer of Conglomeromonas largomobilis subsp. largomobilis to the genus Azospirillum as Azospirillum largomobile comb. nov., and elevation of Conglomeromonas largomobilis subsp. parooensis to the new type species of Conglomeromonas, Conglomeromonas parooensis sp. nov. Syst Appl Microbiol 20:72–77

    Article  Google Scholar 

  • Döbereiner J, Day JM (1976) Associative symbioses in tropical grasses: characterization of microorganisms and dinitrogen-fixing sites. In: Newton WE, Nyman CJ (eds) Proceedings of the first international symposium on N2 fixation, Washington State University Press, Pullman, pp 518–538

    Google Scholar 

  • Eckert B, Weber OB, Kirchhof G, Halbritter A, Stoffels M, Hartmann A (2001) Azospirillum doebereinerae sp. nov., a nitrogen-fixing bacterium associated with the C(4)-grass Miscanthus. Int J Syst Evol Microbiol 51:17–26

    CAS  PubMed  Google Scholar 

  • Edwards U, Rogall T, Blocker H, Emde M, Bottger EC (1989) Isolation and direct complete nucleotide determination of entire genes. Characterization of a gene coding for 16S ribosomal RNA. Nucleic Acids Res 17:7843–7853

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • Falk EC, Döbereiner J, Johnson JL, Krieg NR (1985) Deoxyribonucleic acid homology of Azospirillum amazonense Magalhães et al. 1984 and emendation of the description of the genus Azospirillum. Int J Syst Bacteriol 35:117–118

    Article  CAS  Google Scholar 

  • Falk EC, Johnson JL, Baldani VLD, Döbereiner J, Krieg NR (1986) Deoxyribonucleic and ribonucleic acid homology studies of the genera Azospirillum and Conglomeromonas. Int J Syst Bacteriol 36:80–85

    Article  CAS  Google Scholar 

  • Felsenstein J (1981) Evolutionary trees from DNA sequences: a maximum likelihood approach. J Mol Evol 17:368–376

    Article  CAS  PubMed  Google Scholar 

  • Felsenstein J (1985) Confidence limits on phylogenies: an approach using the bootstrap. Evolution 39:783–791

    Article  Google Scholar 

  • Fitch W (1971) Toward defining the course of evolution: minimum change for a specific tree topology. Syst Zool 20:406–416

    Article  Google Scholar 

  • Hartmann A, Baldani J (2003) The genus Azospirillum. In: Dworkin M, Falkow S, Rosenberg E, Schleifer K-H, Stackebrandt E (eds) The prokaryotes. Wiley, New York, pp 114–140

    Google Scholar 

  • Heiner C, Hunkapiller L, Chen S, Glass J, Chen E (1998) Sequencing multimegabase-template DNA using BigDye terminator chemistry. Genome Res 8:557–561

    PubMed Central  CAS  PubMed  Google Scholar 

  • Khammas K, Ageron E, Grimont P, Kaiser P (1989) Azospirillum irakense sp. nov., a new nitrogen-fixing bacterium associated with rice roots and rhizosphere soil. Res Microbiol 140:679–693

    CAS  PubMed  Google Scholar 

  • Kirchhof G, Reis V, Baldan J, Eckert B, Döbereiner J, Hartmann A (1997) Occurrence, physiological and molecular analysis of endophytic diazotrophic bacteria in gramineous energy plants. Plant Soil 194:45–55

    Article  CAS  Google Scholar 

  • Ladha J, So R, Watanabe I (1987) Composition of Azospirillum species associated with wetland rice plants grown in different soils. Plant Soil 102:127–129

    Article  Google Scholar 

  • Lavrinenko K, Chernousova E, Gridneva E, Dubinina G, Akimov V, Kuever J, Lysenko A, Grabovich M (2010) Azospirillum thiophilum sp. nov., a novel diazotrophic bacterium isolated from a sulfide spring. Int J Syst Evol Microbiol 60:2832–2837

    Article  CAS  PubMed  Google Scholar 

  • Lin S-Y, Young C-C, Hupfer H, Siering C, Arun AB, Chen W-M, Lai W-A, Shen F-T, Rekha PD, Yassin AF (2009) Azospirillum picis sp. nov., isolated from discarded tar. Int J Syst Evol Microbiol 59:761–765

    Article  CAS  PubMed  Google Scholar 

  • Lin S-Y, Shen F-T, Young C-C (2011) Rapid detection and identification of the free-living nitrogen fixing genus Azospirillum by 16S rRNA-gene-targeted genus-specific primers. Antonie Van Leeuwenhoek 99:837–844

    Article  CAS  PubMed  Google Scholar 

  • Lin S-Y, Shen F-T, Young L-S, Zhu Z-L, Chen W-M, Young C-C (2012) Azospirillum formosense sp. nov., a novel diazotrophic bacterium isolated from agricultural soil. Int J Syst Evol Microbiol 62:1185–1190

    Article  CAS  PubMed  Google Scholar 

  • Lin S-Y, Liu Y-C, Hameed A, Hsu Y-H, Lai W-A, Shen F-T, Young C-C (2013) Azospirillum fermentarium sp. nov., a nitrogen-fixing species isolated from a fermenter. Int J Syst Evol Microbiol 63:3762–3768

    Article  CAS  PubMed  Google Scholar 

  • Lin S-Y, Hameed A, Shen F-T, Liu Y-C, Hsu Y-H, Shahina M, Lai W-A, Young C-C (2014) Description of Niveispirillum fermenti gen. nov., sp. nov., isolated from a fermentor in Taiwan, transfer of Azospirillum irakense (1989) as Niveispirillum irakense comb. nov., and reclassification of Azospirillum amazonense (1983) as Nitrospirillum amazonense gen. nov. Antonie Van Leeuwenhoek 105:1149–1162

    Article  CAS  PubMed  Google Scholar 

  • Magalhães F, Baldani J, Souto S, Kuykendall J, Döbereiner J (1983) A new acid-tolerant Azospirillum species. An Acad Bras Cien 55:417–430

    Google Scholar 

  • Mehnaz S, Weselowski B, Lazarovits G (2007a) Azospirillum canadense sp. nov., a nitrogen-fixing bacterium isolated from corn rhizosphere. Int J Syst Evol Microbiol 57:620–624

    Article  CAS  PubMed  Google Scholar 

  • Mehnaz S, Weselowski B, Lazarovits G (2007b) Azospirillum zeae sp. nov., a diazotrophic bacterium isolated from rhizosphere soil of Zea mays. Int J Syst Evol Microbiol 57:2805–2809

    Article  CAS  PubMed  Google Scholar 

  • Mesbah M, Premachandran U, Whitman W (1989) Precise measurement of the G + C content of deoxyribonucleic acid by high-performance liquid chromatography. Int J Syst Bacteriol 39:159–167

    Article  CAS  Google Scholar 

  • Miller L (1982) Single derivatization method for routine analysis of bacterial whole-cell fatty acid methyl esters, including hydroxyl acids. J Clin Microbiol 16:584–586

    PubMed Central  CAS  PubMed  Google Scholar 

  • Minnikin D, O’Donnell A, Goodfellow M, Alderson G, Athalye M, Schaal K, Parlett J (1984) An integrated procedure for the extraction of bacterial isoprenoid quinones and polar lipids. J Microbiol Methods 2:233–241

    Article  CAS  Google Scholar 

  • Murray R, Doetsch R, Robinow C (1994) Methods for general and molecular bacteriology. In: Gerhardt P, Murray RGE, Wood WA, Krieg NR (eds) Determination and cytological light microscopy. American Society for Microbiology, Washington, DC, pp 31–32

    Google Scholar 

  • Okon Y, Itzigsohn R (1992) Poly-β-hydroxybutyrate metabolism in Azospirillum brasilense and the ecological role of PHB in the rhizosphere. FEMS Microbiol Lett 103:131–139

    CAS  Google Scholar 

  • Okon Y, Vanderleyden J (1997) Root-associated Azospirillum species can stimulate plants. ASM News 63:366–370

    Google Scholar 

  • Ostle A, Holt J (1982) Nile blue A as a fluorescent stain for poly-β-hydroxybutyrate. Appl Environ Microbiol 44:238–241

    PubMed Central  CAS  PubMed  Google Scholar 

  • Paisley R (1996) MIS whole cell fatty acid analysis by gas chromatography training manual. MIDI, Newark

    Google Scholar 

  • Peng G, Wang H, Zhang G, Hou W, Liu Y, Wang ET, Tan Z (2006) Azospirillum melinis sp. nov., a group of diazotrophs isolated from tropical molasses grass. Int J Syst Evol Microbiol 56:1263–1271

    Article  CAS  PubMed  Google Scholar 

  • Poly F, Monrozier L, Bally R (2001) Improvement in the RFLP procedure for studying the diversity of nifH genes in communities of nitrogen fixers in soil. Res Microbiol 152:95–103

    Article  CAS  PubMed  Google Scholar 

  • Reinhold B, Hurek T, Fendrik I, Pot B, Gillis M, Kersters K, Thielemans S, Ley J (1987) Azospirillum halopraeferens sp. nov., a nitrogen-fixing organism associated with roots of Kallar grass (Leptochloa fusca (L.) Kunth). Int J Syst Bacteriol 37:43–51

    Article  Google Scholar 

  • Saitou N, Nei M (1987) The neighbor-joining method: a new method for reconstructing phylogenetic trees. Mol Biol Evol 4:406–425

    CAS  PubMed  Google Scholar 

  • Sasser M (1990) Identification of bacteria by gas chromatography of cellular fatty acids. MIDI Technical Note 101. MIDI Inc, Newark

    Google Scholar 

  • Saxena B, Modi M, Modi V (1986) Isolation and characterization of siderophores from Azospirillum lipoferum D-2. J Gen Microbiol 132:2219–2224

    CAS  Google Scholar 

  • Scherer P, Kneifel H (1983) Distribution of polyamines in methanogenic bacteria. J Bacteriol 154:1315–1322

    PubMed Central  CAS  PubMed  Google Scholar 

  • Schlegel H, Lafferty R, Krauss I (1970) The isolation of mutants not accumulating poly-β-hydroxybutyric acid. Arch Microbiol 71:283–294

    CAS  Google Scholar 

  • Seldin L, Dubnau D (1985) Deoxyribonucleic acid homology among Bacillus polymyxa, Bacillus macerans, Bacillus azotofixans, and other nitrogen-fixing Bacillus strains. Int Syst Bacteriol 35:151–154

    Article  CAS  Google Scholar 

  • Seshadri S, Muthukumarasamy R, Lakshinarasimhan C, Ignacimuthu S (2000) Solubilization of inorganic phosphates by Azospirillum halopraeferans. Curr Sci 79:565–567

    CAS  Google Scholar 

  • Shen F-T, Young C-C (2005) Rapid detection and identification of the metabolically diverse genus Gordonia by 16S rRNA-gene-targeted genus-specific primers. FEMS Microbiol Lett 250:221–227

    Article  CAS  PubMed  Google Scholar 

  • Stahl D, Flesher B, Mansfield H, Montgomery L (1988) Use of phylogenetically based hybridization probes for studies of ruminal microbial ecology. Appl Environ Microbiol 54:1079–1084

    PubMed Central  CAS  PubMed  Google Scholar 

  • Steenhoudt O, Vanderleyden J (2000) Azospirillum, a free-living nitrogen-fixing bacterium closely associated with grasses: genetic, biochemical and ecological aspects. FEMS Microbiol Rev 24:487–506

    Article  CAS  PubMed  Google Scholar 

  • Stoffels M, Castellanos T, Hartmann A (2001) Design and application of new 16S rRNA-targeted oligonucleotide probes for the Azospirillum-Skermanella-Rhodocista-cluster. Syst Appl Microbiol 24:83–97

    Article  CAS  PubMed  Google Scholar 

  • Tamura K, Stecher G, Peterson D, Filipski A, Kumar S (2013) MEGA6: molecular evolutionary genetics analysis version 6.0. Mol Biol Evol 30:2725–2729

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • Tarrand J, Krieg N, Döbereiner J (1978) A taxonomic study of the Spirillum lipoferum group, with descriptions of a new genus, Azospirillum gen. nov., and two species, Azospirillum lipoferum (Beijerinck) comb. nov. and Azospirillum brasilense sp. nov. Can J Microbiol 24:967–980

    Article  CAS  PubMed  Google Scholar 

  • Thompson J, Gibson TJ, Plewniak F, Jeanmougin F, Higgins D (1997) The CLUSTAL_X windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucleic Acids Res 25:4876–4882

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • Thuler D, Flosh E, Handro W, Barbosa M (2003) Plant growth regulators and amino acids released by Azospirillum sp. in chemically defined medium. Lett Appl Microbiol 37:174–178

    Article  CAS  PubMed  Google Scholar 

  • Tien TM, Gaskins M, Hubbell D (1979) Plant growth substances produced by Azospirillum brasilense and their effect on the growth of pearl millet (Pennisetum americanum L.). Appl Environ Microbiol 37:1016–1024

    PubMed Central  CAS  PubMed  Google Scholar 

  • Woese C, Kandler O, Wheelis M (1990) Towards a natural system of organisms: proposal for the domains Archaea, Bacteria, and Eucarya. Proc Natl Acad Sci 87:4576–4579

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • Xie C, Yokota A (2005) Azospirillum oryzae sp. nov., a nitrogen-fixing bacterium isolated from the roots of the rice plant Oryza sativa. Int J Syst Evol Microbiol 55:1435–1438

    Article  CAS  PubMed  Google Scholar 

  • Young C-C, Hupfer H, Siering C, Ho M-J, Arun AB, Lai W-A, Rekha PD, Shen F-T, Hung M-H, Chen W-M, Yassin AF (2008) Azospirillum rugosum sp. nov., isolated from oil-contaminated soil. Int J Syst Evol Microbiol 58:959–963

    Article  CAS  PubMed  Google Scholar 

  • Zehr J, McReynolds L (1989) Use of degenerate oligonucleotides for amplification of the nifH gene from the marine cyanobacterium Trichodesmium thiebautii. Appl Environ Microbiol 55:2522–2526

    PubMed Central  CAS  PubMed  Google Scholar 

  • Zhou S, Han L, Wang Y, Yang G, Zhuang L, Hu P (2013) Azospirillum humicireducens sp. nov., a nitrogen-fixing bacterium isolated from a microbial fuel cell. Int J Syst Evol Microbiol 63:2618–2624

    Article  CAS  PubMed  Google Scholar 

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Young, CC., Lin, SY., Shen, FT., Lai, WA. (2015). Molecular Tools for Identification and Characterization of Plant Growth Promoting Rhizobacteria with Emphasis in Azospirillum spp.. In: Cassán, F., Okon, Y., Creus, C. (eds) Handbook for Azospirillum. Springer, Cham. https://doi.org/10.1007/978-3-319-06542-7_2

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