Nitrogen Fixing Endophytes in Forest Trees

  • Rómulo Oses
  • A. Carolin Frank
  • Sofía ValenzuelaEmail author
  • Jaime Rodríguez
Part of the Forestry Sciences book series (FOSC, volume 86)


Nitrogen (N) is the most growth-limiting nutrient in most terrestrial and aquatic ecosystems, with new nitrogen is brought in primarily through biological nitrogen fixation (BNF) performed by bacteria and archaea. In addition to the well-studied nodulating symbioses between bacteria and legumes or actinorhizal plants, many plants, from grasses to trees, appear to meet some of their N demand by hosting N2-fixing endophytes above- or belowground. Most studies on endophytic N2 fixation come from grasses, but knowledge about endophytic N2 fixation in forest trees, including both conifers and woody angiosperms, is emerging. Studies of how the diazotroph Paenibacillus polymyxa strain P2b-2R, interacts with its host lodgepole pine as well as other plants, suggest that diazotrophs can colonize their host intracellulary; that conifers can derive a significant part of N from the atmosphere; and that the association can take months to establish and may depend on N soil content. P. polymyxa strain P2b-2R has also been shown to colonize, promote growth and fix N2 in crops, demonstrating that endophytic diazotrophs can be generalists. Culture independent studies suggest that conifers growing in N limited high altitude environments consistently host foliar endophytes related to the diazotroph Gluconacetobacter in their needles, and that nitrogenase is active within pine foliage, suggesting that endophytes may represent an N2-fixing strategy for long-living conifers to meet their N demand in N limited subalpine ecosystems. Diazotrophs have also been isolated from poplar and willow growing in N limited riparian ecosystems. These strains are also generalists and can promote growth and provide N to both poplar clones and crops. Direct evidence of N2 fixation and incorporation by native endophytes has been demonstrated in poplar using labelling with the stable nitrogen isotope 15N2. Enrichment of 15N was highly variable among samples, potentially as a result of differences in the endophyte community structure and abundance. We provide suggestions for research efforts that can take advantage of this new knowledge with the goal of reducing the use of chemical fertilizer in agriculture and forestry.


  1. Ahemad M, Kibret M (2014) Mechanisms and applications of plant growth promoting rhizobacteria: current perspective. J King Saud Univ-Sci 26(1):1–20CrossRefGoogle Scholar
  2. Anand R, Chanway CP (2013a) Detection of GFP-labeled Paenibacillus polymyxa in auto-fluorescing pine seedling tissues. Biol Fertil Soils 49:111–118CrossRefGoogle Scholar
  3. Anand R, Chanway CP (2013b) N2-fixation and growth promotion in cedar colonized by an endophytic strain of Paenibacillus polymyxa. Biol Fertil Soils 49:235–239CrossRefGoogle Scholar
  4. Anand R, Grayston S, Chanway CP (2013) N2-fixation and seedling growth promotion of lodgepole pine by endophytic Paenibacillus polymyxa. Microb Ecol 66:369–374CrossRefPubMedGoogle Scholar
  5. Bacon CW, White JF (2000) Microbial endophytes. Marcel Dekker Inc., New YorkGoogle Scholar
  6. Bal AS, Anand R, Berge O, Chanway CP (2012) Isolation and identification of diazotrophic bacteria from internal tissues of Pinus contorta and Thuja plicata. Can J For Res 42:807–813CrossRefGoogle Scholar
  7. Bal AS, Chanway CP (2012a) Evidence of nitrogen fixation in lodgepole pine inoculated with diazotrophic Paenibacillus polymyxa. Botany 90:891–896CrossRefGoogle Scholar
  8. Bal A, Chanway CP (2012b) 15N foliar dilution of western red cedar in response to seed inoculation with diazotrophic Paenibacillus polymyxa. Biol Fertil Soils 48:967–971CrossRefGoogle Scholar
  9. Baldotto LEB, Olivares FL, Bressan-Smith R (2011) Structural interaction between GFP-labeled diazotrophic endophytic bacterium Herbaspirillum seropedicae RAM10 and pineapple plantlets’ Vitória’. Braz J Microbiol 42(1):114–125CrossRefGoogle Scholar
  10. Bezdicek DF, Kennedy AC (1998) In: Lynch JM, Hobbie JE (eds) Microorganisms in action. Blackwell, OxfordGoogle Scholar
  11. Bhattacharjee RB, Singh A, Mukhopadhyay SN (2008) Use of nitrogen-fixing bacteria as biofertilizer for non-legumes: prospects and challenges. Appl Microbiol Biotechnol 80:199–209CrossRefPubMedGoogle Scholar
  12. Binkley D, Son Y, Valentine D (2000) Do forest receive occult inputs of nitrogen? Ecosystems 3:321–331CrossRefGoogle Scholar
  13. Boddey RM, Urquiaga S, Reis VM, Döbereiner J (1991) Biological nitrogen fixation associated with sugarcane. Plant Soil 37:111–117CrossRefGoogle Scholar
  14. Bormann B, Keller C, Wang D, Bormann H (2002) Lessons from the sandbox: is unexplained nitrogen real? Ecosystems 5:727–733CrossRefGoogle Scholar
  15. Boyd ES, Peters JW (2013) New insights into the evolutionary history of biological nitrogen fixation. Front Microbiol 4:201PubMedPubMedCentralGoogle Scholar
  16. Carrell AA, Frank AC (2014) Pinus flexilis and Picea engelmannii share a simple and consistent needle endophyte microbiota with a potential role in nitrogen fixation. Front Microbiol 5:333CrossRefPubMedPubMedCentralGoogle Scholar
  17. Carrell AA, Frank AC (2015) Bacterial endophyte communities in the foliage of coast redwood and giant sequoia. Front Microbiol 6:1008CrossRefPubMedPubMedCentralGoogle Scholar
  18. Carrell AA, Carper DL, Frank AC (2016). Subalpine conifers in different geographical locations host highly similar foliar bacterial endophyte communities. FEMS Microbiol Ecol 92(8), fiw124CrossRefPubMedGoogle Scholar
  19. Chanway CP, Anand R, Yang H (2014). Nitrogen Fixation Outside and Inside Plant Tissues. In: Ohyama T (ed) Advances in biology and ecology of nitrogen fixation InTech, ISBN: 978-953-51-1216-7Google Scholar
  20. Chanway CP, Holl FB (1991) Biomass increase and associative nitrogen fixation of mycorrhizal Pinus contorta seedlings inoculated with a plant growth promoting Bacillus strain. Can J Bot 69:507–511CrossRefGoogle Scholar
  21. Chapman WK, Paul L (2012) Evidence that northern pioneering pines with tuberculate mycorrhizae are unaffected by varying soil nitrogen levels. Microbiol Ecol 64:964–972CrossRefGoogle Scholar
  22. Compant S, Reiter B, Sessitsch A, Nowak J, Clement C, Aitsa E (2005) Endophytic colonization of Vitis vinifera L. by plant growth-promoting bacterium Burkholderia sp. strain PsJN. Appl Environ Microbiol 71:1685–1693CrossRefPubMedPubMedCentralGoogle Scholar
  23. Compant S, Clément C, Sessitsch A (2010) Plant growth-promoting bacteria in the rhizo-and endosphere of plants: their role, colonization, mechanisms involved and prospects for utilization. Soil Biol Biochem 42(5):669–678CrossRefGoogle Scholar
  24. Da Siva Fonseca E, Peixoto RS, Rosado AS, de Carvalho Balieiro F, Tiedje JM, da Costa Rachid CTC (2017) The Microbiome of eucalyptus roots under different management conditions and its potential for biological nitrogen fixation. Microbiol Ecol: 1–9Google Scholar
  25. Dalton DA, Kramer S (2006) Nitrogen-fixing bacteria in non-legumes. Springer, Dordrecht, pp 105–130Google Scholar
  26. Dos Santos P, Fang Z, Mason S, Setubal J, Dixon R (2012) Distribution of nitrogen fixation and nitrogenase-like sequences amongst microbial genomes. BMC Genom 13:162CrossRefGoogle Scholar
  27. Doty SL, Oakley B, Xin G, Kang JW, Singleton G, Khan Z, Vajzovic A, Staley JT (2009) Diazotrophic endophytes of native black cottonwood and willow. Symbiosis 47(1):23–33CrossRefGoogle Scholar
  28. Doty SL, Sher AW, Fleck ND, Khorasani M, Bumgarner RE, Khan Z, DeLuca TH (2016) Variable nitrogen fixation in wild Populus. PLoS ONE 11(5):e0155979CrossRefPubMedPubMedCentralGoogle Scholar
  29. Elbeltagy A, Nishioka K, Sato T, Suzuki H, Ye B, 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 67:5285–5293CrossRefPubMedPubMedCentralGoogle Scholar
  30. Eskin N, Vessey K, Tian L (2014). Research progress and perspectives of nitrogen fixing bacterium, Gluconacetobacter diazotrophicus, in monocot plants. Int J Agron ID 208383Google Scholar
  31. Galloway JN, Townsend AR, Erisman JW, Bekunda M, Cai Z, Freney JR, Martinelli LA, Seitzinger SP, Sutton MA (2008) Transformation of the nitrogen cycle: recent trends, questions, and potential solutions. Science 320:889–892CrossRefPubMedGoogle Scholar
  32. Gruber N, Galloway JN (2008) An Earth-system perspective of the global nitrogen cycle. Nature 451:293CrossRefPubMedGoogle Scholar
  33. Haber F (1922) Naturwissenschaften 10:1041CrossRefGoogle Scholar
  34. Hardoim PR, van Overbeek LS, Elsas JD (2008) Properties of bacterial endophytes and their proposed role in plant growth. Trends Microbiol 16:463–471CrossRefPubMedGoogle Scholar
  35. Hardoim PR, van Overbeek LS, Berg G, Pirttilä AM, Compant S, Campisano A, Sessitsch A (2015) The hidden world within plants: ecological and evolutionary considerations for defining functioning of microbial endophytes. Microbiol Mol Biol Rev 79(3):293–320CrossRefPubMedPubMedCentralGoogle Scholar
  36. Hurek TB, Reinhold-Hurek B, Montagu MB, Kellenberger E (1994) Root colonization and systematic spreading of Azoarcus sp strain BH72 in grasses. J Bacteriol 176:1913–1923CrossRefPubMedPubMedCentralGoogle Scholar
  37. Izumi H (2011) Diversity of endophytic bacteria in forest trees. In: Endophytes of forest trees. Springer, Dordrecht, pp 95–105CrossRefGoogle Scholar
  38. James K, Olivares FL (1997) Infection and colonization of sugar cane and other Graminaceous plants by endophytic diazotrophs. Crit Rev Plant Sci 17:77–119CrossRefGoogle Scholar
  39. Khan Z, Guelich G, Phan H, Redman R, Doty S (2012) Bacterial and yeast endophytes from poplar and willow promote growth in crop plants and grasses. ISRN Agron 11Google Scholar
  40. Khan Z, Rho H, Firrincieli A, Hung SH, Luna V, Masciarelli O, Kim SH, Doty SL (2016) Growth enhancement and drought tolerance of hybrid poplar upon inoculation with endophyte consortia. Curr Opin Plant Biol 6:38–47CrossRefGoogle Scholar
  41. Knoth JL, Kim SH, Ettl GJ, Doty SL (2013) Effects of cross host species inoculation of nitrogen-fixing endophytes on growth and leaf physiology of maize. Glob Change Biol Bioenergy 5(4):408–418CrossRefGoogle Scholar
  42. Knoth JL, Kim SH, Ettl GJ, Doty SL (2014) Biological nitrogen fixation and biomass accumulation within poplar clones as a result of inoculations with diazotrophic endophyte consortia. New Phytol 201(2):599–609CrossRefPubMedGoogle Scholar
  43. Koskimäki JJ, Pirttilä AM, Ihantola E-L, Halonen O, Frank AC (2015) The intracellular scots pine shoot symbiont Methylobacterium extorquens DSM13060 aggregates around the host nucleus and encodes eukaryote-like proteins. MBio. 6(2):e00039–15. Scholar
  44. Moyes AB, Kueppers LM, Pett-Ridge J, Carper DL, Vandehey N, O’Neil J, Frank AC (2016) Evidence for foliar endophytic nitrogen fixation in a widely distributed subalpine conifer. New Phytol 210(2):657–668CrossRefPubMedGoogle Scholar
  45. Myrold DD, Bottomley PJ (2007) Biological N inputs. Soil microbiology, ecology and biochemistry. Elsevier, Burlington, pp 365–388Google Scholar
  46. Mus F, Crook MB, Garcia K, Costas AG, Geddes BA, Kouri ED, Udvardi MK et al (2016) Symbiotic nitrogen fixation and the challenges to its extension to nonlegumes. Appl Environ Microbiol 82(13):3698–3710CrossRefPubMedPubMedCentralGoogle Scholar
  47. Nair DN, Padmavathy S (2014) Impact of endophytic microorganisms on plants, environment and humans. Sci World J 250693Google Scholar
  48. Oliveira ALM, Urquiaga S, Dobereiner J, Baldani JI (2002) The effect of inoculating endophytic N2-fixing bacteria on micropropagated sugarcane plants. Plant Soil 242:205–215CrossRefGoogle Scholar
  49. Pirttilä AM, Laukkanen H, Pospiech H, Myllylä R, Hohtola A (2000) Detection of intracellular bacteria in the buds of Scotch Pine (Pinus sylvestris L.) by In Situ Hybridization. Appl Environ Microbiol 66(7):3073–3077CrossRefPubMedPubMedCentralGoogle Scholar
  50. Padda KP, Puri A, Chanway CP (2016a) Plant growth promotion and nitrogen fixation in canola by an endophytic strain of Paenibacillus polymyxa and its GFP-tagged derivative in a long-term study. Botany 94:1209–1217CrossRefGoogle Scholar
  51. Padda KP, Puri A, Chanway CP (2016b) Effect of GFP tagging of Paenibacillus polymyxa P2b-2R on its ability to promote growth of canola and tomato seedlings. Biol Fertil Soils 52:377–387CrossRefGoogle Scholar
  52. Puri A, Padda KP, Chanway CP (2016) Seedling growth promotion and nitrogen fixation by a bacterial endophyte Paenibacillus polymyxa P2b-2R and its GFP derivative in corn in a long-term trial. Symbiosis 69:123–129CrossRefGoogle Scholar
  53. Remigi P, Zhu J, Young JPW, Masson-Boivin C (2016) Symbiosis within symbiosis: evolving nitrogen-fixing legume symbionts. Trends Microbiol 24(1):63–75CrossRefPubMedGoogle Scholar
  54. Rubio LM, Ludden PW (2008) Biosynthesis of the iron-molybdenum cofactor of nitrogenase. Annu Rev Microbiol 62:93–111CrossRefPubMedGoogle Scholar
  55. Santi C, Bogusz D, Franche C (2013) Biological nitrogen fixation in non-legume plants. Ann Bot 111(5):743–767CrossRefPubMedPubMedCentralGoogle Scholar
  56. Schmid M, Hartmann A (2007) Molecular phylogeny and ecology of root associated diazotrophic α-and β-proteobacteria. In: Associative and endophytic nitrogen-fixing bacteria and cyanobacterial associations. Springer, Dordrecht, pp. 21–40Google Scholar
  57. Sevilla M, Burris RH, Gunapala N, Kennedy C (2001) Comparison of benefit to sugarcane plant growth and 15N2 incorporation following inoculation of sterile plants with wild-type and nif¯ mutant strains. Mol Plant-Microbe Interact 14(3):358–366Google Scholar
  58. Shishido M, Chanway CP (1999) Spruce growth response specificity after treatment with plant growth-promoting pseudomonads. Can J Bot 77:22–31Google Scholar
  59. Smil V (2004) Enriching the earth: Fritz Haber, Carl Bosch, and the transformation of world food production. MIT Press, CambridgeGoogle Scholar
  60. Tanabe Y, Nishibayashi Y (2013) Developing more sustainable processes for ammonia synthesis. Coord Chem Rev 257:2551–2564CrossRefGoogle Scholar
  61. Tang Q, Puri A, Padda KP, Chanway CP (2017) Biological nitrogen fixation and plant growth promotion of lodgepole pine by an endophytic diazotroph Paenibacillus polymyxa and its GFP-tagged derivative. Botany 95(6):611–619CrossRefGoogle Scholar
  62. Thamdrup B (2012) New pathways and processes in the global nitrogen cycle. Annu Rev Ecol Evol Syst 43:407–428CrossRefGoogle Scholar
  63. Thomas P, Sekhar AC (2014) Live cell imaging reveals extensive intracellular cytoplasmic colonization of banana by normally non-cultivable endophytic bacteria. AoB Plants 6: plu002Google Scholar
  64. Urquiaga S, Cruz KHS, Boddey RM (1992) Contribution of nitrogen fixation to sugar cane: nitrogen-15 and nitrogen-balance estimates. Soil Sci Soc Am J 56:105–114CrossRefGoogle Scholar
  65. Van der Lelie D, Taghavi S, Monchy S, Schwender J, Miller L, Ferrieri R, Vangronsveld J (2009) Poplar and its bacterial endophytes: coexistence and harmony. Crit Rev Plant Sci 28(5):346–358CrossRefGoogle Scholar
  66. Van Nguyen T, Pawlowski K (2017) Frankia and Actinorhizal Plants: Symbiotic Nitrogen Fixation. In: Rhizotrophs: plant growth promotion to bioremediation. Springer, Singapore, pp 237–261Google Scholar
  67. Vitousek PM, Menge DN, Reed SC, Cleveland CC (2013) Biological nitrogen fixation: rates, patterns and ecological controls in terrestrial ecosystems. Philos Trans R Soc Lond B Biol Sci: Biol Sci 368(1621):20130119CrossRefGoogle Scholar
  68. Werner GD, Cornwell WK, Sprent JI, Kattge J, Kiers ET (2014) A single evolutionary innovation drives the deep evolution of symbiotic N2-fixation in angiosperms. Nat Commun 5:4087CrossRefPubMedPubMedCentralGoogle Scholar
  69. Wilson D (1993) Fungal endophytes: out of sight but should not be out of mind. Oikos 68(2):379–384CrossRefGoogle Scholar
  70. Wurzburger N (2016) Old-growth temperate forests harbor hidden nitrogen-fixing bacteria. New Phytol 210(2):374–376CrossRefPubMedGoogle Scholar
  71. Yang H, Puri A, Padda KP, Chanway CP (2016) Effects of Paenibacillus polymyxa inoculation and different soil nitrogen treatments on lodgepole pine seedling growth. Can J For Res 46(6):816–821CrossRefGoogle Scholar
  72. Yang H, Puri A, Padda KP, Chanway CP (2017) Substrate utilization by endophytic bacteria Paenibacillus polymyxa P2b-2R that may facilitate bacterial entrance and survival inside diverse plant hosts. Facets 2(1):120–130CrossRefGoogle Scholar

Copyright information

© Springer International Publishing AG, part of Springer Nature 2018

Authors and Affiliations

  • Rómulo Oses
    • 1
  • A. Carolin Frank
    • 2
  • Sofía Valenzuela
    • 1
    Email author
  • Jaime Rodríguez
    • 1
  1. 1.Biotechnology Center and Forest Science FacultyUniversidad de ConcepciónConcepciónChile
  2. 2.School of Natural Sciences, Life and Environmental Sciences and Sierra Nevada Research InstituteUniversity of California, MercedMercedUSA

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