Abstract
Recent studies suggest a high diversity of diazotrophic bacteria in maize. However, none of these works have been based on a sufficient number of samples to provide reasonable quantitative estimates of diazotrophic bacterial diversity. Here we present the use of molecular tools and statistical inference to assess diazotrophic bacterial diversity within rhizosphere soils, roots and stems of field grown maize. DNA was isolated from the latter collected from six maize growing regions within the southern most state in Brazil, Rio Grande do Sul. Using conserved primers, nifH Cluster I gene fragments were amplified from each of the three zones, and the products cloned and sequenced. The majority of the sequences were classified within the Proteobacteria with the α-proteobacteria and β-proteobacteria being the most abundant in the rhizosphere soil and stem samples. The γ-proteobacteria were most abundant in rhizosphere soils, less so in roots, and least in the stem samples. According to three different diversity measures, the rhizosphere soil samples possessed greater diazotrophic bacterial diversity than the roots and stems of the maize plants. Only two genera, Azospirillum and Azotobacter, were found in virtually all samples at an abundance of over 1% of the total nifH sequences obtained. Other genera were largely restricted to soil (Methylocystis, Beijerinckia, Geobacter, Rhodovulum, Methylobacterium, Gluconacetobacter, Methylocella, and Delftia), roots (Dechloromonas), or stems (Methylosinus, Raoultella, and Rhizobium). Three genera, Herbaspirillum, Ideonella, and Klebsiella, appeared to dominate in the interior of the plant but were much rarer in soil.
Similar content being viewed by others
References
Altschul SF, Madden TL, Schaeffer AA, Zhang J, Zhang Z, Miller W, Lipman DJ (1997) Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res 25:3389–3402
Baldani VLD, Döbereiner J (1980) Host-plant specificity in the infection of cereals with Azospirillum spp. Soil Biol Biochem 12:433–439
Baldani VLD, Alvarez MAB, Baldani JI, Dobereiner J (1986a) Establishment of inoculated Azospirillum spp. in the rhizosphere and in roots of field-grown wheat and sorghum. Plant Soil 90:35–46
Baldani JI, Baldani VLD, Seldin L, Dobereiner J (1986b) Characterization of Herbaspirillum-seropedicae gen-nov, sp-nov, a root-associated nitrogen-fixing bacterium. Int J Syst Bacteriol 36:86–93
Baldani VLD, Baldani JI, Olivares FL, Döbereiner J (1992) Identification and ecology of Herbaspirillum seropedicae and closely related Pseudomonas rubrisubalbicans. Symbiosis 13:65–73
Bashan Y, Holguin G, de-Bashan LE (2004) Azospirillum-plant relationships: physiological, molecular, agricultural, and environmental advances (1997–2003). Can J Microbiol 50:521–577
Berge O, Heulin T, Balandreau J (1991) Diversity of diazotroph populations in the rhizosphere of maize (Zea mays L) growing on different French soils. Biol Fertil Soils 11:210–215
Bürgmann H, Widmer F, Von Sigler W, Zeyer J (2004) New molecular screening tools for analysis of free-living diazotrophs in soil. Appl Environ Microbiol 70:240–247
Chabot R, Antoun H, Kloepper JW, Beauchamp CJ (1996) Root colonization of maize and lettuce by bioluminescent Rhizobium leguminosarum biovar phaseoli. Appl Environ Microbiol 62:2767–2772
Chelius MK, Triplett EW (2000) Immunolocalization of dinitrogenase reductase produced by Klebsiella pneumoniae in association with Zea mays L. Appl Environ Microbiol 66:783–787
Chelius MK, Triplett EW (2001) The diversity of archaea and bacteria in association with the roots of Zea mays L. Microb Ecol 41:252–263
Chien YT, Zinder SH (1996) Cloning, functional organization, transcript studies, and phylogenetic analysis of the complete nitrogenase structural genes (nifHDK2) and associated genes in the archaeon Methanosarcina barkeri 227. J Bacteriol 178:143–148
Chaintreuil C, Giraud E, Prin Y, Lorquin J, Bâ A, Gillis M, de Lajudie P, Dreyfus B (2000) Photosynthetic bradyrhizobia are natural endophytes of the African wild rice Oryza breviligulata. Appl Environ Microbiol 66:5437–5447
Cocking EC (2003) Endophytic colonization of plant roots by nitrogen-fixing bacteria. Plant Soil 252:169–175
Dobbelaere S, Vanderleyden J, Okon Y (2003) Plant growth-promoting effects of diazotrophs in the rhizosphere. Crit Rev Plant Sci 22:107–149
Döbereiner J (1997) Biological nitrogen fixation in the tropics: social and economic contributions. Soil Biol Biochem 29:771–774
Drancourt M, Bollet C, Carta A, Rousselier P (2001) Phylogenetic analyses of Klebsiella species delineate Klebsiella and Raoultella gen. nov., with description of Raoultella ornithinolytica comb. nov., Raoultella terrigena comb. nov. and Raoultella planticola comb. nov. Int J Syst Evol Microbiol 51:925–932
Elbeltagy A, Nishioka K, Sato T, Suzuki H, Bin Y, Hamada T, Isawa T, Mitsui H, Minamisawa K (2001) Endophytic colonization and in planta nitrogen fixation by a Herbaspirillum sp. isolated from wild rice species. Appl Environ Microbiol 67:5285–5293
Excoffier L, Laval G, Schneider S (2005) Arlequin ver. 3.0: an integrated software package for population genetics data analysis. Evolutionary Bioinformatics Online 1:47–50
Felsenstein J (1989) PHYLIP-phylogeny inference package (version 3.2). Cladistics 5:164–166
Gans J, Wolinsky M, Dunbar J (2005) Computational improvements reveal great bacterial diversity and high metal toxicity in soil. Science 309:1387–1390
Gutierrez-Zamora ML, Martinez-Romero E (2001) Natural endophytic association between Rhizobium etli and maize (Zea mays L.). J Biotechnol 91:117–126
Gyaneshwar P, James EK, Reddy PM, Ladha JK (2002) Herbaspirillum colonization increases growth and nitrogen accumulation in aluminium-tolerant rice varieties. New Phytol 154:131–146
Hong SH, Bunge J, Jeon SO, Epstein SS (2006) Predicting microbial species richness. Proc Natl Acad Sci USA 103:117–122
Hughes JB, Hellmann JJ, Ricketts TH, Bohannan BJM (2001) Counting the uncountable: statistical approaches to estimating microbial diversity. Appl Environ Microbiol 67:4399–4406
James EK, Olivares FL (1998) Infection and colonization of sugarcane and other graminaceous plants by endophytic diazotrophs. Crit Rev Plant Sci 17:77–119
James EK, Olivares FL, Baldani JI, Döbereiner J (1997) Herbaspirillum, an endophytic diazotroph colonizing vascular tissue and leaves of Sorghum bicolor L. Moench. J Exp Bot 48:785–797
James EK, Gyaneshwar P, Mathan N, Barraquio WL, Reddy PM, Iannetta PPM, Olivares FL, Ladha JK (2002) Infection and colonization of rice seedlings by the plant growth-promoting bacterium Herbaspirillum seropedicae Z67. Mol Plant – Microb Interact 15:894–906
Kennedy IR, Choudhury ATMA, Kecskes ML (2004) Non-symbiotic bacterial diazotrophs in crop-farming systems: can their potential for plant growth promotion be better exploited? Soil Biol Biochem 36:1229–1244
Kumar S, Tamura K, Nei M (2004) MEGA3: integrated software for molecular evolutionary genetics analysis and sequence alignment. Brief Bioinform 5:150–163
Liu XM, Zhao HX, Chen SF (2006) Colonization of maize and rice plants by strain Bacillus megaterium C4. Curr Microbiol 52:186–190
Lodewyckx C, Vangronsveld J, Porteous F, Moore ERB, Taghavi S, Mezgeay M, van der Leite D (2002) Endophytic bacteria and their potential applications. Crit Rev Plant Sci 21:583–606
Lovell CR, Piceno YM, Quattro JM, Bagwell CE (2000) Molecular analysis of diazotroph diversity in the rhizosphere of the smooth cordgrass, Spartina alterniflora. Appl Environ Microbiol 66:3814–3822
Moore FP, Barac T, Borremans B, Oeyen L, Vangronsveld J, van der Lelie D, Campbell CD, Moore ER (2006) Endophytic bacterial diversity in poplar trees growing on a BTEX-contaminated site: the characterization of isolates with potential to enhance phytoremediation. Syst Appl Microbiol 29:539–556
Olivares FL, Baldani VLD, Reis VM, Baldani JI, Döbereiner J (1996) Occurrence of the endophytic diazotrophs Herbaspirillum spp. in roots, stems and leaves predominantly of Gramineae. Biol Fertil Soils 21:197–200
Oliveira ALM, Canuto EL, Urquiaga S, Reis VM, Baldani JI (2006) Yield of micropropagated sugarcane varieties in different soil types following inoculation with diazotrophic bacteria. Plant Soil 284:23–32
Pace NR, Stahl DA, Lane DJ, Olsen GJ (1997) The analysis of natural microbial-populations by ribosomal-RNA sequences. Adv Microbiol Ecol 9:1–55
Palus JA, Borneman J, Ludden PW, Triplett EW (1996) A diazotrophic bacterial endophyte isolated from stems of Zea mays L. and Zea luxurians Iltis and Doebley. Plant Soil 186:135–142
Poly F, Ranjard L, Nazaret S, Gourbiere F, Monrozier LJ (2001) Comparison of nifH gene pools in soils and soil microenvironments with contrasting properties. Appl Environ Microbiol 67:2255–2262
Reiter B, Bürgmann H, Burg K, Sessitsch A (2003) Endophytic nifH gene diversity in African sweet potato. Can J Microbiol 49:549–555
Roncato-Maccari LDB, Ramos HJO, Pedrosa FO, Alquini Y, Chubatsu LS, Yates MG, Rigo LU, Steffens MBR, Souza EM (2003) Endophytic Herbaspirillum seropedicae expresses nif genes in gramineous plants. FEMS Microbiol Ecol 45:39–47
Rösch C, Mergel A, Bothe H (2002) Biodiversity of denitrifying and dinitrogen-fixing bacteria in an acid forest soil. Appl Environ Microbiol 68:3818–3829
Rosenblueth M, Martinez-Romero E (2004) Rhizobium etli maize populations and their competitiveness for root colonization. Arch Microbiol 181:337–344
Rosenblueth M, Martinez-Romero E (2006) Bacterial endophytes and their interactions with hosts. Mol Plant – Microb Interact 19:827–837
Sanguin H, Remenant B, Dechesne A, Thioulouse J, Vogel TM, Nesme X, Moenne-Loccoz Y, Grundmann GL (2006) Potential of a 16S rRNA-based taxonomic microarray for analyzing the rhizosphere effects of maize on Agrobacterium spp and bacterial communities. Appl Environ Microbiol 72:4302–4312
Schloss PD, Handelsman J (2005) Introducing DOTUR, a computer program for defining operational taxonomic units and estimating species richness. Appl Environ Microbiol 71:1501–1506
Schloter M, Wiehe W, Assmus B, Steindl H, Becke H, Hoflich G, Hartmann A (1997) Root colonization of different plants by plant-growth-promoting Rhizobium leguminosarum bv trifolii R39 studied with monospecific polyclonal antisera. Appl Environ Microbiol 63:2038–2046
Schmalenberger A, Tebbe CC (2003) Bacterial diversity in maize rhizospheres: conclusions on the use of genetic profiles based on PCR-amplified partial small subunit rRNA genes in ecological studies. Mol Ecol 12:251–262
Seghers D, Wittebolle L, Top EM, Verstraete W, Siciliano SD (2004) Impact of agricultural practices on the Zea mays L. endophytic community. Appl Environ Microbiol 70:1475–1482
Soares RS, Roesch LFW, Zanatta G, Camargo FAO, Passaglia LMP (2006) Occurrence and distribution of nitrogen fixing bacterial community associated with oat (Avena sativa) assessed by molecular and microbiological techniques. Appl Soil Ecol 33:221–234
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
Steward GF, Jenkins BD, Ward BB, Zehr JP (2004) Development and testing of a DNA macroarray to assess nitrogenase (nifH) gene diversity. Appl Environ Microbiol 70:1455–1465
Sturz AV, Nowak J (2000) Endophytic communities of rhizobacteria and the strategies required to create yield enhancing associations with crops. Appl Soil Ecol 15:183–190
Sumner ME (1990) Crop responses to Azospirillum inoculation. Adv Soil Sci 12:53–123
Thompson JD, Gibson TJ, Plewniak F, Jeanmougin F, Higging DG (1997) The CLUSTAL-X windows interface: flexible strategies for multiple sequence alignment aided by quality tools. Nucleic Acid Res 25:4876–4882
Ueda T, Suga Y, Yahiro N, Matsuguchi (1995) Remarkable N2-fixing bacterial diversity detected in rice roots by molecular evolutionary analysis of nifH gene sequences. J Bacteriol 177:1414–1417
Welbaum GE, Sturz AV, Dong ZM, Nowak J (2004) Managing soil microorganisms to improve productivity of agro-ecosystems. Crit Rev Plant Sci 23:175–193
Widmer F, Shaffer BT, Porteous LA, Seidler RJ (1999) Analysis of nifH gene pool complexity in soil and litter at a Douglas fir forest site in the Oregon Cascade Mountain Range. Appl Environ Microbiol 65:374–380
Zehr JP, McReynolds LA (1989) Use of degenerate oligonucleotides for amplification of the NifH gene from the marine cyanobacterium Trichodesmium thiebautii. Appl Environ Microbiol 55:2522–2526
Zehr JP, Capone DG (1996) Problems and promises of assaying the genetic potential for nitrogen fixation in the marine environment. Microb Ecol 32:263–281
Zehr JP, Jenkins BD, Short SM, Steward GF (2003) Nitrogenase gene diversity and microbial community structure: a cross-system comparison. Environ Microbiol 5:539–554
Acknowledgments
L.F.W. Roesch was supported by the CAPES Foundation scholarship. This work was supported by the Florida Agricultural Experiment Station, NSF (MCB-0454030) and USDA-NRI (2005-35319-16300). We are grateful to Roberta R. Fulthorpe for the helpful discussions and the constructive suggestions.
Author information
Authors and Affiliations
Corresponding author
Additional information
Responsible Editor: Euan K. James
Rights and permissions
About this article
Cite this article
Roesch, L.F.W., Camargo, F.A.O., Bento, F.M. et al. Biodiversity of diazotrophic bacteria within the soil, root and stem of field-grown maize. Plant Soil 302, 91–104 (2008). https://doi.org/10.1007/s11104-007-9458-3
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s11104-007-9458-3