Skip to main content
SpringerLink
Log in

Two seasons’ study on nifH gene expression and nitrogen fixation by diazotrophic endophytes in sugarcane (Saccharum spp. hybrids): expression of nifH genes similar to those of rhizobia

  • Regular Article
  • Published:
Plant and Soil Aims and scope Submit manuscript

Abstract

To explore the presence and expression of the nifH gene of diazotrophic endophytes in sugarcane (Saccharum spp. hybrids) cv. NiF8, we conducted pot experiments for two seasons in a glasshouse from 10 June to 17 September (high temperature and long days) in 2005 and 1 September to 9 December (low temperature and short days) in 2006. The expression of nifH genes in the stems and roots of sugarcane plants at 50 (or 59) and 100 days after transplanting was investigated by reverse transcription (RT)–PCR and by examining the nifH nucleotide sequence diversity. N2 fixation was assessed by the 15N-dilution method. The nifH RNA sequences in the stems and roots of the first experiment were similar to those of Bradyrhizobium sp. and Azorhizobium caulinodans. In the second experiment, nifH expression from these bacteria was not detected in the stems. nifH gene expression in the stems was in accordance with 18–24% N derived from air in the high-temperature season and negligible in the low-temperature season. Both the proliferation of N2 fixers and expression of the nifH gene indicated that bacteria similar to rhizobia may contribute to N2 fixation in sugarcane under our experimental conditions.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Fig. 1

Similar content being viewed by others

Abbreviations

BNF:

Biological nitrogen fixation

DAT:

Days after transplanting

%Ndfa:

Percent nitrogen derived from air

RT-PCR:

Reverse transcription-polymerase chain reaction

References

  • Altschul SF, Madden TL, Schäffer 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. doi:10.1093/nar/25.17.3389

    Article  CAS  PubMed  Google Scholar 

  • Amann RI, Ludwig W, Schleifer K-H (1995) Phylogenetic identification and in situ detection of individual microbial cells without cultivation. Microbiol Rev 59:143–169

    CAS  PubMed  Google Scholar 

  • Ando S, Goto M, Meunchang S, Thongra-ar P, Fujiwara T, Hayashi H, Yoneyama T (2005) Detection of nifH sequences in sugarcane (Saccharum officinarum L.) and pineapple (Ananas comosus [L.] Merr.). Soil Sci Plant Nutr 51:303-308. doi:10.1111/j.1747-0765.2005.tb00034.x

  • Asis CA Jr, Kubota M, Ohta H, Arima Y, Ohwaki Y, Yoneyama T, Tsuchiya K, Hayashi N, Nakanishi Y, Akao S (2002) Estimation of the nitrogen fixation by sugarcane cultivar NiF-8 using 15N dilution and natural 15N abundance techniques. Soil Sci Plant Nutr 48:283–285

    Google Scholar 

  • Baldani JI, Reis VM, Baldani VLD, Döbereiner J (2002) A brief story of nitrogen fixation in sugarcane—reasons for success in Brazil. Funct Plant Biol 29:417–423. doi:10.1071/PP01083

    Article  Google Scholar 

  • Biggs IM, Stewart GR, Wilson JR, Critchley C (2002) 15N natural abundance studies in Australian commercial sugarcane. Plant Soil 238:21–30. doi:10.1023/A:1014280420779

    Article  CAS  Google Scholar 

  • Boddey RM, Polidoro JC, Resende AS, Alves BJR, Urquiaga S (2001) Use of the 15N natural abundance technique for the quantification of the contribution of N2 fixation to sugar cane and other grasses. Aust J Plant Physiol 28:889–895. doi:10.1071/PP01058

    Google Scholar 

  • Boddey RM, Urquiaga S, Alves BJR, Reis V (2003) Endophytic nitrogen fixation in sugarcane: present knowledge and future applications. Plant Soil 252:139–149. doi:10.1023/A:1024152126541

    Article  CAS  Google Scholar 

  • Brown MM, Friez MJ, Lovell CR (2003) Expression of nifH genes by diazotrophic bacteria in the rhizosphere of short form Spartina alterniflora. FEMS Microbiol Ecol 43:411–417. doi:10.1111/j.1574-6941.2003.tb01081.x

    Article  CAS  PubMed  Google Scholar 

  • Caballero-Mellado J, Martínez-Aguilar L, Paredes-Valdez G, Estrada-de los Santos P (2004) Burkholderia unamae sp. nov., an N2-fixing rhizospheric and endophytic species. Int J Syst Evol Microbiol 54:1165–1172. doi:10.1099/ijs.0.02951-0

    Article  CAS  PubMed  Google Scholar 

  • Cantera JJL, Kawasaki H, Seki T (2004) The nitrogen-fixing gene (nifH) of Rhodopseudomonas palustris: a case of lateral gene transfer? Microbiology 150:2237–2246. doi:10.1099/mic.0.26940-0

    Article  CAS  PubMed  Google Scholar 

  • Cavalcante VA, Döbereiner J (1988) A new acid-tolerant nitrogen-fixing bacterium associated with sugarcane. Plant Soil 108:23–31. doi:10.1007/BF02370096

    Article  Google Scholar 

  • 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

    Article  CAS  PubMed  Google Scholar 

  • Chen W-M, de Faria SM, Straliotto R, Pitard RM, Simões-Araùjo JL, Chou J-H, Chou Y-J, Barrios E, Prescott AR, Elliott GN, Sprent JI, Young JPW, James EK (2005) Proof that Burkholderia strains form effective symbioses with legumes: a study of novel Mimosa-nodulating strains from South America. Appl Environ Microbiol 71:7461–7471. doi:10.1128/AEM.71.11.7461-7471.2005

    Article  CAS  PubMed  Google Scholar 

  • Döbereiner J (1997) Biological nitrogen fixation in the tropics: social and economic contributions. Soil Biol Biochem 29:771–774. doi:10.1016/S0038-0717(96)00226-X

    Article  Google Scholar 

  • Döbereiner J, Day JM, Dart PJ (1972) Nitrogenase activity in the rhizosphere of sugar cane and some other tropical grasses. Plant Soil 37:191–196. doi:10.1007/BF01578494

    Article  Google Scholar 

  • Dreyfus BL, Elmerich C, Dommergues YR (1983) Free-living Rhizobium strain able to grow on N2 as the sole nitrogen source. Appl Environ Micribiol 45:711–713

    CAS  Google Scholar 

  • Dreyfus B, Garcia JL, Gillis M (1988) Characterization of Azorhizobium caulinodans gen. nov., sp. nov., a stem-nodulating nitrogen-fixing bacterium isolated from Sesbania rostrata. Int J Syst Bactriol 38:89–98. doi:10.1099/00207713-38-1-89

    Article  CAS  Google Scholar 

  • Felsenstein J (2008) Phylip version 3.68. Department of Genome Sciences, University of Washington, Seattle, WA

  • Giller KE (2001) Nitrogen fixation in tropical cropping systems, 2nd edn. London, UK

    Book  Google Scholar 

  • Giraud E, Moulin L, Vallenet D, Barbe V, Cytryn E, Avarre J-C, Jaubert M, Simon D, Cartieaux F, Prin Y, Bena G, Hannibal L, Fardoux J, Kojadinovic M, Vuillet L, Lajus A, Cruveiller S, Rouy Z, Mangenot S, Segurens B, Dossat C, Franck WL, Chang W-S, Saunders E, Bruce D, Richardson P, Normand P, Dreyfus B, Pignol D, Stacey G, Emerich D, Verméglio A, Médigue C, Sadowsky M (2007) Legumes symbioses: absence of Nod genes in photosynthetic bradyrhizobia. Science 316:1307–1312. doi:10.1126/science.1139548

    Article  PubMed  Google Scholar 

  • Hoefsloot G, Termorshuizen AJ, Watt DA, Cramer MD (2005) Biological nitrogen fixation is not a major contributor to the nitrogen demand of a commercially grown south African sugarcane cultivar. Plant Soil 277:85–96. doi:10.1007/s11104-005-2581-0

    Article  CAS  Google Scholar 

  • Hürek T, Handley LL, Reinhold-Hurek B, Piché Y (2002) Azoarcus grass endophytes contribute fixed nitrogen to the plant in an unculturable state. Mol Plant Microbe Interact 15:233–242. doi:10.1094/MPMI.2002.15.3.233

    Article  PubMed  Google Scholar 

  • James EK (2000) Nitrogen fixation in endophytic and associative symbiosis. Field Crops Res 65:197–209. doi:10.1016/S0378-4290(99)00087-8

    Article  Google Scholar 

  • Jourand P, Giraud E, Giraud E, Béna G, Sy A, Willems A, Gillis M, Dreyfus B, de Lajudie P (2004) Methylobacterium nodulans sp. nov., for a group of aerobic, facultatively methylotrophic, legume root-nodule-forming and nitrogen-fixing bacteria. Int J Syst Evol Microbial 54:2269–2273. doi:10.1099/ijs.0.02902-0

    Article  CAS  Google Scholar 

  • Kimura M (1980) A simple method for estimating evolutionary rates of base substitutions through comparative studies of nucleotide sequences. J Mol Evol 16:111–120

    Article  CAS  PubMed  Google Scholar 

  • Ladha JK, So RB (1994) Numerical taxonomy of photosynthetic rhizobia nodulating Aeschynomene species. Int J Syst Bacteriol 44:62–73. doi:10.1099/00207713-44-1-62

    Article  Google Scholar 

  • Lima E, Boddey RM, Döbereiner J (1987) Quantification of biological nitrogen fixation associated with sugar cane using a 15N aided nitrogen balance. Soil Biol Biochem 19:165–170. doi:10.1016/0038-0717(87)90077-0

    Article  CAS  Google Scholar 

  • Loiret FG, Ortega E, Kleiner D, Ortega-Rodés P, Rodés R, Dong Z (2004) A putative new endophytic nitrogen-fixing bacterium Pantoea sp. from sugarcane. J Appl Microbiol 97:504–511. doi:0.1111/j.1365-2672.2004.02329.x

    Article  CAS  PubMed  Google Scholar 

  • Martínez-Aguilar L, Díaz R, Peña-Cabriales JJ, Santos PE, Dunn MF, Caballero-Mellado J (2008) Multichromosomal genome structure and confirmation of diazotrophy in novel plant-associated Burkholderia species. Appl Environ Microbiol 74:4574–4579. doi:10.1128/AEM.00201-08

    Article  PubMed  Google Scholar 

  • Noda S, Ohkuma M, Usami R, Horikoshi K, Kudo T (1999) Culture-independent characterization of a gene responsible for nitrogen fixation in the symbiotic microbial community in the gut of the termite Neotermes koshunensis. Appl Environ Microbiol 65:4935–4942

    CAS  PubMed  Google Scholar 

  • Olivares FL, James EK, Baldani JI, Döbereiner J (1997) Infection of mottled stripe disease-susceptible and resistant sugar cane varieties by the endophytic diazotroph Herbaspirillum. New Phytol 135:723–737. doi:10.1046/j.1469-8137.1997.00684.x

    Article  Google Scholar 

  • Oliveira ALM, Urquiaga S, Döbereiner J, Baldani JI (2002) The effect of inoculating endophytic N2-fixing bacteria on micropropagated sugarcane plants. Plant Soil 242:205–215. doi:10.1023/A:1016249704336

    Article  CAS  Google Scholar 

  • Perin L, Martínez-Aguilar L, Paredes-Valdez G, Baldani JI, Estrada-de los Santos P, Reis VM, Caballero-Mellado J (2006) Burkholderia silvatlantica sp. nov., a diazotrophic bacterium associated with sugar cane and maize. Int J Syst Evol Microbiol 56:1931–1937. doi:10.1099/ijs.0.64362-0

    Article  CAS  PubMed  Google Scholar 

  • Quan Z-X, Bae H-S, Baek J-H, Chen W-F, Im W-T, Lee S-T (2005) Rhizobium daejeonense sp. nov. isolated from a cyanide treatment bioreactor. Int J Syst Evol Microbiol 55:2543–2549. doi:10.1099/ijs.0.63667-0

    Article  CAS  PubMed  Google Scholar 

  • Rasolomampianina R, Bailly X, Fetiarison R, Rabevohitra R, Béna G, Ramaroson L, Raherimandimby M, Moulin L, de Lajudie P, Dreyfus B, Avarre J-C (2005) Nitrogen-fixing nodules from rose wood legume trees (Dalbergia spp.) endemic to Madagascar host seven different genera belonging to α- and β-proteobacteria. Mol Ecol 14:4135–4146. doi:10.1111/j.1365-294X.2005.02730.x

    Article  CAS  PubMed  Google Scholar 

  • Reinhold-Hurek B, Hurek T (1998) Life in grasses: diazotrophic endophytes. Trends Microbiol 6:139–144. doi:10.1016/S0966-842X(98)01229-3

    Article  CAS  PubMed  Google Scholar 

  • Reis VM, Estrada Santos P, Tenorio-Salgado S, Vogel J, Stoffels M, Guyon S, Mavingui P, Baldani VLD, Schmid M, Baldani JI, Balandreau J, Hartmann A, Caballero-Mellado J (2004) Burkholderia tropica sp. nov., a novel nitrogen-fixing, plant-associated bacterium. Int J Syst Evol Microbiol 54:2155–2162. doi:10.1099/ijs.0.02879-0

    Article  CAS  PubMed  Google Scholar 

  • Reiter B, Bürgmann H, Burg K, Sessitsch A (2003) Endophytic nifH gene diversity in African sweet potato. Can J Microbiol 49:549–555. doi:10.1139/w03-070

    Article  CAS  PubMed  Google Scholar 

  • Roesch LFW, Camargo FAO, Bento FM, Triplett EW (2008) Biodiversity of diazotrophic bacteria within the soil, root and stem of field-grown maize. Plant Soil 302:91–104. doi:10.1007/s11104-007-9458-3

    Article  CAS  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 

  • Sy A, Giraud E, Jourand P, Garcia N, Willems A, de Lajudie P, Prin Y, Neyra M, Gillis M, Boivin-Masson C, Dreyfus B (2001) Methylotrophic Methylobacterium bacteria nodulate and fix nitrogen in symbiosis with legumes. J Bacteriol 183:214–220. doi:10.1128/JB.183.1.214-220.2001

    Article  CAS  PubMed  Google Scholar 

  • Terakado-Tonooka J, Ohwaki Y, Yamakawa H, Tanaka F, Yoneyama T, Fujihara S (2008) Expressed nifH genes of endophytic bacteria detected in field-grown sweet potatoes (Ipomoea batatas L.). Microbes Environ 23:89–93. doi:10.1264/jsme2.23.89

    Article  Google Scholar 

  • Thompson JD, Higgins DG, Gibson TJ (1994) CLUSTAL W: Improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res 22:4673–4680. doi:10.1093/nar/22.22.4673

    Article  CAS  PubMed  Google Scholar 

  • Urquiaga S, Cruz KHS, Boddey RM (1992) Contribution of nitrogen fixation to sugarcane: nitrogen-15 and nitrogen-balance estimates. Soil Sci Soc Am J 56:105–114. doi:10.2136/sssaj1992.03615995005600010017x

    Article  Google Scholar 

  • Velázquez E, Rojas M, Lorite MJ, Rivas R, Zurdo-Piñeiro JL, Heydrich M, Bedmar EJ (2008) Genetic diversity of endophytic bacteria which could be found in the apoplastic sap of the medullary parenchyma of the stem of healthy sugarcane plants. J Basic Microbiol 48:118–124. doi:10.1002/jobm.200700161

    Article  PubMed  Google Scholar 

  • Yoneyama T (2002) N2 fixation by three types of plant-microbe interaction: carbon as the major limiting. In: Proceeding of Plant-Microbe Interaction. Chonnam National University, Korea, pp 33–44

  • Yoneyama T, Muraoka T, Kim TH, Dacanay EV, Nakanishi Y (1997) The natural 15N abundance of sugarcane and neighbouring plants in Brazil, the Philippines and Miyako (Japan). Plant Soil 189:239–244. doi:10.1023/A:1004288008199

    Article  CAS  Google Scholar 

Download references

Acknowledgments

We would like to thank Drs. Yasuhiro Nakanishi, Mitsunori Sato, Yoshifumi Terajima, and Makoto Matsuoka for supplying the sugarcane stems and soils from sugarcane fields. This study was partially supported by a research project on Development of New Biorational Techniques for Sustainable Agriculture of the Agriculture, Forestry and Fisheries Research Council, Japan, and by a research fund (B-1538047) from the Ministry of Education, Culture, Sports, Sciences and Technology, Japan.

Author information

Author notes

  1. Tadakatsu Yoneyama

    Present address: , Tsukuho 3-3-12, Tsukuba, Ibaraki, 300-3257, Japan

Authors and Affiliations

  1. Department of Applied Biological Chemistry, The University of Tokyo, Yayoi 1-1-1, Bunkyo-ku, Tokyo, 113-8657, Japan

    Nisarat Thaweenut, Yusuke Hachisuka, Shuichi Yanagisawa & Tadakatsu Yoneyama

  2. National Institute of Livestock and Grassland Science, National Agriculture and Food Research Organization, Senbonmatsu 768, Nasushiobara, Tochigi, 329-2793, Japan

    Nisarat Thaweenut & Shotaro Ando

Authors
  1. Nisarat Thaweenut
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Yusuke Hachisuka
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Shotaro Ando
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Shuichi Yanagisawa
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Tadakatsu Yoneyama
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Tadakatsu Yoneyama.

Additional information

Responsible Editor: Euan K. James.

Nisarat Thaweenut and Yusuke Hachisuka contributed equally to this work.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Thaweenut, N., Hachisuka, Y., Ando, S. et al. Two seasons’ study on nifH gene expression and nitrogen fixation by diazotrophic endophytes in sugarcane (Saccharum spp. hybrids): expression of nifH genes similar to those of rhizobia. Plant Soil 338, 435–449 (2011). https://doi.org/10.1007/s11104-010-0557-1

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s11104-010-0557-1

Keywords

Search

Navigation