Plant growth promotion of Miscanthus × giganteus by endophytic bacteria and fungi on non-polluted and polluted soils

  • Christoph Stephan Schmidt
  • Libor Mrnka
  • Tomáš Frantík
  • Petra Lovecká
  • Miroslav Vosátka
Original Paper


Putative endophytes of Miscanthus × giganteus were isolated, and screened in the laboratory, greenhouse and field for their plant growth promoting properties in this host. Pantoea ananatis and Pseudomonas savastanoi were the predominant bacteria in leaves whereas other pseudomonads prevailed in roots. Almost all fungal endophytes belonged to the Pezizomycotina and most were isolated from roots; Fusarium oxysporum was most abundant, followed by the genera Periconia, Exophiala, Microdochium and Leptodontidium. All endophytic groups produced phytohormones and some bacteria also produced siderophores, solubilised P and exhibited ACC-deaminase activity in vitro. In subsequent pot experiments with pre-selected endophytes, several isolates including pseudomonads, Variovorax paradoxus, Verticillium leptobactrum, Halenospora sp. and Exophiala sp. enhanced Miscanthus growth in gamma-sterilised soil. These promising Miscanthus-derived isolates were tested either as single or mixed inocula along with a mixed bacterial inoculum originating from poplar. No significant effects of inocula were detected in a pot experiment in non-sterilised soil. On two marginal field sites the mixture of bacterial endophytes from poplar had a consistently negative effect on survival and growth of Miscanthus. Contrarily, mixtures consisting of bacteria or fungi originating from Miscanthus promoted growth of their host, especially on the heavy metals-polluted site. The combination of bacteria and fungi was inferior to the mixtures consisting of bacteria or fungi alone. Our observations indicate extensive potential of mixed bacterial and fungal endophytic inocula to promote establishment and yield of Miscanthus grown on marginal and polluted land and emphasise the necessity to test particular microbial-plant host combinations.

Graphical Abstract

Morphotypes of fungi isolates from Miscanthus × giganteus


Antioxidative activity Heavy metals Miscanthus Plant growth promotion Variovorax Halenospora Verticillium leptobactrum 



We thank MSc. Dušan Kunc and RNDr. Helena Koblihová for skilful technical assistance.


This research was funded by the Technological Agency of the Czech Republic, Contract No. TA03011184, and by the Czech Academy of Sciences (long-term research development project RVO 67985939).

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.

Research involving human participants and/or animals

No animals or data from human participants were involved in the study.

Supplementary material

11274_2018_2426_MOESM1_ESM.xls (616 kb)
Supplementary material 1 (XLS 616 KB)
11274_2018_2426_MOESM2_ESM.xls (332 kb)
Supplementary material 2 (XLS 332 KB)
11274_2018_2426_MOESM3_ESM.pdf (127 kb)
Supplementary material 3 (PDF 126 KB)


  1. Alexander DB, Zuberer DA (1991) Use of chrome azurol S reagents to evaluate siderophore production by rhizosphere bacteria. Biol Fertil Soils 12:39–45CrossRefGoogle Scholar
  2. Babu AG, Shea PJ, Sudhakar D, Jung I-B, Oh B-T (2015) Potential use of Pseudomonas koreensis AGB-1 in association with Miscanthus sinensis to remediate heavy metal(loid)-contaminated mining site soil. J Environ Manage 151:160–166. CrossRefGoogle Scholar
  3. Bakker PAHM, Pieterse CMJ, van Loon LC (2007) Induced systemic resistance by fluorescent Pseudomonas sp. Phytopathology 97:239–243. CrossRefGoogle Scholar
  4. Bashan Y, Kamnev AA, de-Bashan LE (2013) Tricalcium phosphate is inappropriate as a universal selection factor for isolating and testing phosphate-solubilizing bacteria that enhance plant growth: a proposal for an alternative procedure. Biol Fertil Soils 49(4):65–479. CrossRefGoogle Scholar
  5. Batzer JC, Weber RWS, Mayfield DA, Gleason ML (2016) Diversity of the sooty blotch and flyspeck complex on apple in Germany. Mycol Prog 15:2. CrossRefGoogle Scholar
  6. Berbee ML (2001) The phylogeny of plant and animal pathogens in the Ascomycota. Physiol Mol Plant P 59:165–187CrossRefGoogle Scholar
  7. Bills G, Platas G, Pelaez F, Masurekar P (1999) Reclassification of a pneumocandin-producing anamorph, Glarea lozoyensis gen. et sp. nov., previously identifed as Zalerion arboricola. Mycol Res 103:179–192CrossRefGoogle Scholar
  8. Burd GI, Dixon DG, Glick BR (2000) Plant growth-promoting bacteria that decrease heavy metal toxicity in plants. Can J Microbiol 46:237–245CrossRefGoogle Scholar
  9. Cherkaoui A, Hibbs J, Emonet S, Tangomo M, Girard M, Francois P, Jacques Schrenzel J (2016) Comparison of two matrix-assisted laser desorption ionization–time of flight mass spectrometry methods with conventional phenotypic identification for routine identification of bacteria to the species level. J Clinic Microbiol 48:1169–1175CrossRefGoogle Scholar
  10. Cole J, Chai B, Farris RJ, Wang Q, Kulam SA, McGarrell MD, Garrity GM, Tiedje JM (2005) The Ribosomal Database Project (RDP-II): sequences and tools for high-throughput rRNA analysis. Nucleic Acids Res 33:294–296CrossRefGoogle Scholar
  11. Cope-Selby N, Cookson A, Squance M, Donnison I, Flavell R, Farrar K (2017) Endophytic bacteria in Miscanthus seed: implications for germination, vertical inheritance of endophytes, plant evolution and breeding. GCB Bioenergy 9:57–77. CrossRefGoogle Scholar
  12. Covarelli L, Beccari G, Tosi L (2012) Miscanthus rhizome rot: a potential threat for the establishment and the development of biomass cultivations. Biomass Bioenergy 46:263–269. CrossRefGoogle Scholar
  13. Davis MP, David MB, Voigt TB, Mitchell CA (2015) Effect of nitrogen addition on Miscanthus × giganteus yield, nitrogen losses, and soil organic matter across five sites. GCB Bioenergy 7:1222–1231. CrossRefGoogle Scholar
  14. de Freitas JR, Germida JJ (1991) Pseudomonas cepacia and Pseudomonas putida as winter wheat inoculants for biocontrol of Rhizoctonia solani. Can J Microbiol 37:780–784CrossRefGoogle Scholar
  15. de Abreu LM, Almeida AR, Salgado M, Pfenning LH (2010) Fungal endophytes associated with the mistletoe Phoradendron perrottettii and its host tree Tapirira guianensis. Fungal Progress 4:559–566. Google Scholar
  16. Dimkpa CO, Merten D, Svatoš A, Büchel G, Kothe E (2009) Metal-induced oxidative stress impacting plant growth in contaminated soil is alleviated by microbial siderophores. Soil Biol Biochem 41:154–162CrossRefGoogle Scholar
  17. Duffy BK, Defago G (1997) Zinc improves biocontrol of Fusarium crown and root rot of tomato by Pseudomonas fluorescens and represses the production of pathogen metabolites inhibitory to bacterial antibiotic biosynthesis. Phytopathology 87:1250–1257CrossRefGoogle Scholar
  18. Eskes AB, Mendes MDL, Robbs CF (1991) Laboratory and field studies on parasitism of Hemileia vastatrix with Verticillium lecani and V. leptobactrum. Café Cacao Thé 35:275–282Google Scholar
  19. Etesami H, Alikhani HA, Hosseini HM (2015) Indole-3-acetic acid (IAA) production trait, a useful screening to select endophytic and rhizosphere competent bacteria for rice growth promoting agents. MethodsX 2:72–78. CrossRefGoogle Scholar
  20. FAO (2006) World reference base for soil resources. A framework for international classification, correlation and communication. Food and Agriculture Organisation of the United Nations, RomeGoogle Scholar
  21. Farrar K, Bryant D, Cope-Selby N (2014) Understanding and engineering beneficial plant –microbe interactions: plant growth promotion in energy crops. Plant Biotechnol J 12:1193–1206. CrossRefGoogle Scholar
  22. Garrido-Sanz D, Meier-Kolthoff JP, Göker M, Martín M, Rivilla R, Redondo-Nieto M (2016) Genomic and genetic diversity within the Pseudomonas fluorescens complex. PLoS ONE 11(2):e0150183. CrossRefGoogle Scholar
  23. Hajšlova J, Fenclova M, Zachariašova M (2013) Methodology for the rapid screening of isolates of endophytic microorganisms and identification of strains with phytohormonal activity [in Czech]. ISBN 978-80-7080-869-6Google Scholar
  24. Heaton EA, Dohleman FG, Long SP (2008) Meeting US biofuel goals with less land: the potential of Miscanthus. Glob Change Biol 14:2000–2014. CrossRefGoogle Scholar
  25. Hoffman MT, Gunatilaka MK, Wijeratne K, Gunatilaka L, Arnold AE (2013) Endohyphal bacterium enhances production of indole-3-acetic acid by a foliar fungal endophyte. PLoS ONE 8:e73132. doi. CrossRefGoogle Scholar
  26. Indrasumunar A, Dart PJ, Menzies NW (2011) Symbiotic effectiveness of Bradyrhizobium japonicum in acid soils can be predicted from their sensitivity to acid soil stress factors in acidic agar media. Soil Biol Biochem 43:2046–2052. Google Scholar
  27. ISO 10390 (2005) Soil quality: determination of pH. International Organization for Standardization, ISOGoogle Scholar
  28. Jiang F, Chen L, Belimov AA, Shaposhnikov AI, Gong F, Meng X, Hartung W, Jeschke DW, Davies WJ, Dodd IC (2012) Multiple impacts of the plant growth-promoting rhizobacterium Variovorax paradoxus 5C-2 on nutrient and ABA relations of Pisum sativum. J Exp Bot 63:6421–6430. CrossRefGoogle Scholar
  29. Kempf H-J, Wolf G (1989) Erwinia herbicola as a biocontrol agent of Fusarium culmorum and Puccinia recondita f. sp. tritici on Wheat. Phytopathology 79:990–994CrossRefGoogle Scholar
  30. Khan Z, Doty SL (2009) Characterisation of bacterial endophytes in sweet potato plants. Plant Soil 322:197–207. CrossRefGoogle Scholar
  31. Khan AL, Hamayun M, Waqas M, Kang S-M, Kim Y-H, Kim D-H, Lee I-J (2012a) Exophiala sp. LHL08 association gives heat stress tolerance by avoiding oxidative damage to cucumber plants. Biol Fertil Soils 48:519–529. CrossRefGoogle Scholar
  32. Khan AL, Hamayun M, Kang S-M, Kim Y-H, Jung H-Y, Lee J-H, Lee I-J (2012b) Endophytic fungal association via gibberellins and indole acetic acid can improve plant growth under abiotic stress: an example of Paecilomyces formosus LHL10. BMC Microbiol 12:32. CrossRefGoogle Scholar
  33. Kim HJ, Lee JH, Kang BR, Rong X, Gardener BBM, Ji HJ, Park CS, Kim YC (2012) Draft genome sequence of Pantoea ananatis B1-9, a nonpathogenic plant growth-promoting bacterium. J Bacteriol 194:729. CrossRefGoogle Scholar
  34. Knoth JL, Kim S-H, Tell GJ, Dothy SL (2013) Effects of cross host species inoculation of nitrogen fixing endophytes on growth and leaf physiology of maize. GCB Bioenergy 5:408–418. CrossRefGoogle Scholar
  35. Koubek J, Uhlík O, Jecná K, Junková P, Vrkoslavová J, Lipov J, Kurzawova V, Macek T, Macková M (2012) Whole-cell MALDI-TOF: rapid screening method in environmental microbiology. Int Biodeter Biodegr 69:82–86. CrossRefGoogle Scholar
  36. Kuffner M, De Maria S, Puschenreiter M, Fallmann K, Wieshammer G, Gorfer M, Strauss J, Rivelli AR, Sessitsch A (2010) Culturable bacteria from Zn- and Cd-accumulating Salix caprea with differential effects on plant growth and heavy metal availability. J Appl Microbiol 108:1471–1484. CrossRefGoogle Scholar
  37. Lane DJ (1991) 16S/23S rRNA sequencing. Nucleic acid techniques. In: Stackebrandt E, Goodfellow M (eds) Bacterial systematics. Wiley, New York, pp 115–175Google Scholar
  38. Li J, Kremer RJ (2006) Growth response of weed and crop seedlings to deleterious rhizobacteria. Biol Control 39:58–65CrossRefGoogle Scholar
  39. Li Z, Chang S, Lin L, Li Y, An Q (2011a) A colorimetric assay of 1-aminocyclopropane-1-carboxylate (ACC) based on ninhydrin reaction for rapid screening of bacteria containing ACC deaminase. Lett Appl Microbiol 53:178–185. CrossRefGoogle Scholar
  40. Li L, Liu MJ, Zhang XT, Zhang HB, Sha T, Zhao ZW (2011b) Improved tolerance of maize (Zea mays L.) to heavy metals by colonization of a dark septate endophyte (DSE) Exophiala pisciphila. Sci Total Environ 409:1069–1074. CrossRefGoogle Scholar
  41. Li D, Voigt TB, Kent AD (2016) Plant and soil effects on bacterial communities associated with Miscanthus × giganteus rhizosphere and rhizomes. GCB Bioenergy 8:183–193. CrossRefGoogle Scholar
  42. Likar M, Regvar M (2013) Isolates of dark septate endophytes reduce metal uptake and improve physiology of Salix caprea L. Plant Soil 370:593–604. CrossRefGoogle Scholar
  43. Linde-Laursen I (1993) Cytogenetic analysis of MiscanthusGiganteus’, an interspecific hybrid. Hereditas 119:297–300CrossRefGoogle Scholar
  44. Ma Y, Prasad MNV, Rajkumar M, Freitas H (2011) Plant growth promoting rhizobacteria and endophytes accelerate phytoremediation of metalliferous soils. Biotechnol Adv 29:248–258. CrossRefGoogle Scholar
  45. Mandyam KG, Jumpponen A (2015) Mutualism-parasitism paradigm synthesized from results of root endophyte models. Front Microbiol 5:776. CrossRefGoogle Scholar
  46. Mejri D, Gamalero E, Tombolini R, Musso C, Massa N, Berta G, Souissi T (2010) Biological control of great brome (Bromus diandrus) in durum wheat (Triticum durum): specificity, physiological traits and impact on plant growth and root architecture of the fluorescent pseudomonad strain X33d. Biocontrol 55:561–572. CrossRefGoogle Scholar
  47. Moll J, Hoppe B, Konig S, Wubet T, Buscot F, Kruger D (2016) Spatial distribution of fungal communities in an arable soil. PLoS ONE 11:e0148130. doi. CrossRefGoogle Scholar
  48. Moore PD, Chapman SB (1986) Methods in plant ecology, 2nd edn. Blackwell, OxfordGoogle Scholar
  49. Nair A, Juwarkar AA, Singh SK (2007) Production and characterization of siderophores and its application in arsenic removal from contaminated soil. Water Air Soil Poll 180:199–212. CrossRefGoogle Scholar
  50. Nirenberg HI, O’Donnell K (1998) New Fusarium species and combinations within the Gibberella fujikuroi species complex. Mycologia 90:434–458CrossRefGoogle Scholar
  51. Nsanganwimana F, Pourrot B, Mensch M, Douay F (2014) Suitability of Miscanthus species for managing inorganic and organic contaminated land and restoring ecosystem services: a review. J Environ Manage 143:123–124. CrossRefGoogle Scholar
  52. Olsen RS, Sommers LE (1982) Phosphorus. In: Page AL et al (eds) Methods in soil analysis, Part 2, chemical and microbiological properties, agronomy monograph 9.2. Agronomy series 9, ASAS publications. American Society of Agronomy, Soil Science Society of America. Madison, pp 403–430Google Scholar
  53. Penrose DM, Glick BR (2003) Methods for isolating and characterizing ACC deaminase-containing plant growth-promoting rhizobacteria. Physiol Plant 118:10–15CrossRefGoogle Scholar
  54. Pereira SIA, Castro PML (2014) Diversity and characterization of culturable bacterial endophytes from Zea mays and their potential as plant growth-promoting agents in metal-degraded soils. Environ Sci Pollut R 21:14110–14123. CrossRefGoogle Scholar
  55. Pii Y, Mimmo T, Tomasi N, Terzano R, Cesco S, Crecchio C (2015) Microbial interactions in the rhizosphere: beneficial influences of plant growth-promoting rhizobacteria on nutrient acquisition process. A review. Biol Fertil Soils 51:403–415. CrossRefGoogle Scholar
  56. Rajkumar M, Ae N, Freitas H (2009) Endophytic bacteria and their potential to enhance heavy metal phytoextraction. Chemosphere 77:153–160. CrossRefGoogle Scholar
  57. Reddy CA, Saravanan RS (2013) Polymicrobial multi-functional approach for enhancement of crop productivity. In: Gadd GM, Sariaslani S (eds) Advances in applied microbiology. Oxford Academic, Oxford, pp 53–113Google Scholar
  58. Regaieg H, Ciancio A, Raouani NH, Rosso L (2011) Detection and biocontrol potential of Verticillium leptobactrum parasitizing Meloidogyne spp. World J Microb Biot 27:1615–1623. CrossRefGoogle Scholar
  59. Schmidt CS, Lovecká P, Mrnka L, Vychodilová A, Strejček M, Fenclová M, Demnerová K (2017) Distinct communities of poplar endophytes on an unpolluted and a risk elements-polluted site and their plant growth promoting potential in vitro. Microb Ecol, Google Scholar
  60. Schulz B, Boyle C (2005) The endophytic continuum. Mycol Res 109:661–686CrossRefGoogle Scholar
  61. Sharp RG, Chen L, Davis WJ (2011) Inoculation of growing media with the rhizobacterium Variovorax paradoxus 5C-2 reduces unwanted stress responses in hardy ornamental species. Sci Hortic 129:804–811CrossRefGoogle Scholar
  62. Shrestha P, Szaro TM, Bruns TD, Taylor JW (2011) Systematic search for cultivatable fungi that best deconstruct cell walls of Miscanthus and sugarcane in the field. Appl Environ Microb 77:5490–5504. CrossRefGoogle Scholar
  63. Stenstrom E, Ndobe NE, Jonsson M, Stenlid J, Menkis A (2013) Root-associated fungi of healthy-looking Pinus sylvestris and Picea abies seedlings in Swedish forest nurseries. Scand J Forest Res 29:12–21CrossRefGoogle Scholar
  64. Sun X, Ding Q, Hyde KD, Guo LD (2012) Community structure and preference of endophytic fungi of three woody plants in a mixed forest. Fungal Ecol 5:624–632CrossRefGoogle Scholar
  65. Sundara-Rao WVB, Sinha MK (1963) Phosphate dissolving microorganisms in the soil and rhizosphere. Indian J Agric Sci 33:272–278Google Scholar
  66. Tóth B, Csösz M, Dijksterhuis J, Frisvad JC, Varga J (2007) Pithomyces chartarum as pathogen on wheat. J Plant Pathol 89:405–408Google Scholar
  67. Unterseher M, Schnittler M (2009) Dilution-to-extinction cultivation of leaf-inhabiting endophytic fungi in beech (Fagus sylvatica L.)—different cultivation techniques influence fungal biodiversity assessment. Mycol Res 113:645–654. CrossRefGoogle Scholar
  68. Villaño D, Fernández-Pachón MS, Moyá ML, Troncoso AM, García-Parrilla MC (2007) Radical scavenging ability of polyphenolic compounds towards DPPH free radical 1. Talanta 71:230–235. CrossRefGoogle Scholar
  69. Wald J, Hroudová M, Jansa J, Vrchotová B, Macek T, Uhlík O (2015) Pseudomonads rule degradation of polyaromatic hydrocarbons in aerated sediment. Front Microbiol 6:1268. doi. CrossRefGoogle Scholar
  70. Wanat N, Austruy A, Joussein E, Soubrand M, Hitmi A, Gauthier-Moussard C, Lenain J-F, Vernay P, Munch JC, Pichon M (2013) Potentials of Miscanthus × giganteus grown on highly contaminated technosols. J Geochem Explor 126–127:78–84. CrossRefGoogle Scholar
  71. Weller DM (2007) Pseudomonas biocontrol agents of soilborne pathogens: looking back over 30 years. Phytopathology 97:250–256. CrossRefGoogle Scholar
  72. Weyens N, van der Lelie D, Taghavi S, Newman L, Vangronsveld J (2009) Exploiting plant–microbe partnerships to improve biomass production and remediation. Trends Biotechnol 27:591–598. CrossRefGoogle Scholar
  73. White TJ, Bruns TD, Lee S, Taylor J (1990) Analysis of phylogenetic relationship by amplification and direct sequencing of ribosomal RNA genes. In: Innis MA, Gelfand DH, Sninsky JJ, White TJ (eds) PCR protocols: a guide to methods and applications. Academic Press Inc., New York, pp 315–322Google Scholar
  74. Zadok JC, Chang TT, Konzak A (1974) A decimal code for the growth stages of cereals. Weed Res 14:415–421CrossRefGoogle Scholar
  75. Zdor RE, Alexander CM, Kremer RJ (2007) Weed suppression by soil bacteria is affected by formulation and soil properties. Commun Soil Sci Plan 36:1289–1299. CrossRefGoogle Scholar
  76. Zinniel DK, Lambrecht P, Harris NB, Feng Z, Kuczmarski D, Higley P, Ishimaru CA, Arunakumari A, Barletta RG, Vidaver AK (2002) Isolation and characterization of endophytic colonizing bacteria from agronomic crops and prairie plants. Appl Environ Microbiol 68:2198–2208CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media B.V., part of Springer Nature 2018

Authors and Affiliations

  1. 1.Institute of Botany, Czech Academy of Sciences, Department of Mycorrhizal SymbiosesPrůhoniceCzech Republic
  2. 2.University of Chemistry and Technology PraguePraha 6Czech Republic

Personalised recommendations