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.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
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
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
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
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
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
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
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
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
Felsenstein J (1981) Evolutionary trees from DNA sequences: a maximum likelihood approach. J Mol Evol 17:368–376
Felsenstein J (1985) Confidence limits on phylogenies: an approach using the bootstrap. Evolution 39:783–791
Fitch W (1971) Toward defining the course of evolution: minimum change for a specific tree topology. Syst Zool 20:406–416
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
Heiner C, Hunkapiller L, Chen S, Glass J, Chen E (1998) Sequencing multimegabase-template DNA using BigDye terminator chemistry. Genome Res 8:557–561
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
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
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
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
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
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
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
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
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
Magalhães F, Baldani J, Souto S, Kuykendall J, Döbereiner J (1983) A new acid-tolerant Azospirillum species. An Acad Bras Cien 55:417–430
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
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
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
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
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
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
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
Okon Y, Vanderleyden J (1997) Root-associated Azospirillum species can stimulate plants. ASM News 63:366–370
Ostle A, Holt J (1982) Nile blue A as a fluorescent stain for poly-β-hydroxybutyrate. Appl Environ Microbiol 44:238–241
Paisley R (1996) MIS whole cell fatty acid analysis by gas chromatography training manual. MIDI, Newark
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
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
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
Saitou N, Nei M (1987) The neighbor-joining method: a new method for reconstructing phylogenetic trees. Mol Biol Evol 4:406–425
Sasser M (1990) Identification of bacteria by gas chromatography of cellular fatty acids. MIDI Technical Note 101. MIDI Inc, Newark
Saxena B, Modi M, Modi V (1986) Isolation and characterization of siderophores from Azospirillum lipoferum D-2. J Gen Microbiol 132:2219–2224
Scherer P, Kneifel H (1983) Distribution of polyamines in methanogenic bacteria. J Bacteriol 154:1315–1322
Schlegel H, Lafferty R, Krauss I (1970) The isolation of mutants not accumulating poly-β-hydroxybutyric acid. Arch Microbiol 71:283–294
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
Seshadri S, Muthukumarasamy R, Lakshinarasimhan C, Ignacimuthu S (2000) Solubilization of inorganic phosphates by Azospirillum halopraeferans. Curr Sci 79:565–567
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
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
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
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
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
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
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
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
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
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
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
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
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
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
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2015 Springer International Publishing Switzerland
About this chapter
Cite this chapter
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
Download citation
DOI: https://doi.org/10.1007/978-3-319-06542-7_2
Publisher Name: Springer, Cham
Print ISBN: 978-3-319-06541-0
Online ISBN: 978-3-319-06542-7
eBook Packages: Biomedical and Life SciencesBiomedical and Life Sciences (R0)