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Mycological Progress

, Volume 13, Issue 3, pp 849–856 | Cite as

New isolation method for endophytes based on enzyme digestion

  • René Prior
  • Katharina Görges
  • Andrey Yurkov
  • Dominik Begerow
Original Article

Abstract

Investigation of the diversity and ecology of endophytic fungi has gained popularity in recent decades. Thereby, culture-independent methods were very instrumental as they revealed greater species richness and challenged the efficiency of isolation methods. However, culture-based methods remain the only possibility to provide strains for future studies. To improve the efficiency of endophyte isolation, we compared seven different artificial media that included a variety of plant extracts. Furthermore, we developed a method based on enzymatic digestion of plant tissue to improve the isolation efficiency in terms of species richness. The effect of additional plant extract in media that contained yeast and malt extract was of minor significance, whereas isolations using enzymatic digestion of plant leaf tissue combined with a 1:10 dilution of nutrients revealed a much higher diversity of species. Using this protocol, we isolated three times as many species from the same leaves than with previously described methods. This is reflected by the large number of singletons obtained in culture-independent approaches to study the diversity of endophytic fungi.

Keywords

Endophytes Fungi Strain cultivation Isolation method Enzyme digestion 

Notes

Acknowledgments

We thank Danja Schünemann for providing principles and recipes of the enzymatic digestion method. We thank Dominik Schmidt, Moritz Mittelbach, Alistair McTaggart and Derek Peršoh for critically reading the manuscript and for their advice. We thank Ilse Weßel and Tanja Rollnik for technical support and the Deutsche Bundesstiftung Umwelt for financial support.

References

  1. Arnold AE, Herre EA (2003) Canopy cover and leaf age af- fect colonization by tropical fungal endophytes: ecological pattern and process in Theobroma cacao (Malvaceae). Mycologia 95(3):388–398PubMedCrossRefGoogle Scholar
  2. Arnold AE, Lutzoni F (2007) Diversity and host range of foliar fungal endophytes: are tropical leaves biodiversity hotspots? Ecology 88(3):541–549PubMedCrossRefGoogle Scholar
  3. Arnold AE, Maynard Z, Gilbert GS, Coley PD, Kursar TA (2000) Are tropical fungal endophytes hyperdiverse? Ecol Lett 3(4):267–274CrossRefGoogle Scholar
  4. Arnold AE, Mejía LC, Kyllo D, Rojas EI, Maynard Z, Robbins N, Herre EA (2003) Fungal endophytes limit pathogen damage in a tropical tree. PNAS 100(26):15649–15654PubMedCentralPubMedCrossRefGoogle Scholar
  5. Ban YH, Tang M, Chen H, Xu ZY, Zhang HH, Yang YR (2012) The response of dark septate endophytes (dse) to heavy metals in pure culture. Plos One 7(10):e47968PubMedCentralPubMedCrossRefGoogle Scholar
  6. Begerow D, Nilsson H, Unterseher M, Maier W (2010) Current state and perspectives of fungal DNA barcoding and rapid identification procedures. Appl Microbiol Biotechnol 87:99–108PubMedCrossRefGoogle Scholar
  7. Bernardi-Wenzel J, García A, Filho CJR, Prioli AJ, Pamphile JA (2010) Evaluation of foliar fungal endophyte diversity and colonization of medicinal plant Luehea divaricata (Martius et Zuccarini). Biol Res 43:375–384PubMedCrossRefGoogle Scholar
  8. Burmeister L, Hau B (2009) Control of the bean rust fungus Uromyces appendiculatus by means of Trichoderma harzianum: leaf disc assays on the antibiotic effect of spore suspensions and culture filtrates. Biol Control 54:575–585Google Scholar
  9. Chao A (1987) Estimating the population size for capture-recapture data with unequal catchability. Biometrics 43:783–791PubMedCrossRefGoogle Scholar
  10. Clay K, Schardl C (2002) Evolutionary origins and ecological consequences of endophyte symbiosis with grasses. Am Nat 160(Suppl 4):S99–S127PubMedCrossRefGoogle Scholar
  11. Fröhlich J, Hyde KD, Petrini O (2000) Endophytic fungi associated with palms. Mycol Res 104(10):1202–1212CrossRefGoogle Scholar
  12. Gadanho M, Sampaio JP (2002) Polyphasic taxonomy of the basidiomycetous yeast genus Rhodotorula : Rh. glutinis sensu stricto and Rh. dairenensis comb. nov. FEMS Yeast Res 2(1):47–58PubMedGoogle Scholar
  13. Gardes M, Bruns D (1993) ITS primers with enhanced specificity for basidiomycetes—application to the identification of mycorrhizae and rusts. Mol Ecol 2:113–118PubMedCrossRefGoogle Scholar
  14. Giovannoni S, Stingl U (2007) The importance of culturing bacterioplankton in the 'omics' age. Nat Rev Microbiol 5(10):820–826PubMedCrossRefGoogle Scholar
  15. Glushakova AM, Chernov IY (2004) Seasonal dynamics in a yeast population on leaves of the common wood sorrel Oxalis acetosella L. Microbiology 73(2):184–188CrossRefGoogle Scholar
  16. Guo LD, Huang GR, Wang Y (2008) Seasonal and tissue age influences on endophytic fungi of Pinus tabulaeformis (Pinaceae) in the Dongling Mountains, Beijing. J Integr Plant Biol 50(8):997–1003PubMedCrossRefGoogle Scholar
  17. Hamilton C, Bauerle T (2012) A new currency for mutualism ? Fungal endophytes alter antioxidant activity in hosts responding to drought. Fungal Divers 54(1):39–49CrossRefGoogle Scholar
  18. Helander M, Ahlholm J, Sieber TN, Hinneri S, Saikkonen K (2007) Fragmented environment affects birch leaf endophytes. New Phytol 175(3):547–553PubMedCrossRefGoogle Scholar
  19. Impullitti AE, Malvick DK (2013) Fungal endophyte diversity in soybean. J Appl Microbiol 114:1500–1506PubMedCrossRefGoogle Scholar
  20. Larran S, Perello A, Simo MR, Moreno V (2002) Isolation and analysis of endophytic microorganisms in wheat (Triticum aestivum L.) leaves. World J Microbiol Biotechnol 18(7):683–686CrossRefGoogle Scholar
  21. Moore D, Robson GD, Trinci APJ (2011) 21st Century guidebook to fungi. Cambridge University Press, CambridgeCrossRefGoogle Scholar
  22. Mülhardt C (2009) Der Experimentator - Molekularbiologie / Genomics. Spektrum Akademischer Verlag, Heidelberg, pp 27–29CrossRefGoogle Scholar
  23. Musetti R, Vecchione A, Stringher L, Borselli S, Zulini L, Marzani C, D’Ambrosio M, Sanità di Toppi L, Pertot I (2006) Inhibition of sporulation and ultrastructural alterations of grapevine downy mildew by the endophytic fungus Alternaria alternata. Am Phytopathol Soc 96(7):689–698CrossRefGoogle Scholar
  24. O’Donnell K (1993) Fusarium and its near relatives. In: Reynolds DR, Taylor JW (eds) The fungal holomorph: mitotic, meiotic and pleomorphic speciation in fungal systematics. CAB International, Wallingford, pp 225–233Google Scholar
  25. Osono T (2008) Endophytic and epiphytic phyllosphere fungi of Camellia japonica: seasonal and leaf age-dependent variations. Mycologia 100(3):387–391PubMedCrossRefGoogle Scholar
  26. Padhi S, Tayung K (2013) Antimicrobial activity and molecular characterization of an endophytic fungus, Quambalaria sp isolated from Ipomoea carnea. Ann Microbiol 63(2):793–800CrossRefGoogle Scholar
  27. Paulus B, Gadek P, Hyde K (2003) Estimation of microfungal diversity in tropical rainforest leaf litter using particle filtration: the effects of leaf storage and surface treatment. Mycol Res 107(6):748–756PubMedCrossRefGoogle Scholar
  28. Pérez L, Gundel C, Ghersa CM, Omacini M (2013) Family issues: fungal endophyte protects host grass from the closely related pathogen Claviceps purpurea. Fungal Ecol 6:379–386CrossRefGoogle Scholar
  29. Peršoh D (2013) Factors shaping community structure of endophytic fungi–evidence from the Pinus-Viscum-system. Fungal Divers 60(1):55–69CrossRefGoogle Scholar
  30. Rajala T, Velmala SM, Tuomivirta T, Haapanen M, Müller M, Pennanen T (2013) Endophyte communities vary in the needles of Norway spruce clones. Fungal Biol 117(3):182–190PubMedCrossRefGoogle Scholar
  31. Redman RS, Sheehan KB, Stout RG, Rodriguez RJ, Henson JM (2002) Thermotolerance generated by plant/fungal symbiosis. Science 298(5598):1581PubMedCrossRefGoogle Scholar
  32. Rodriguez RJ, White JF, Arnold AE, Redman RS (2009) Fungal endophytes: diversity and functional roles. New Phytol 182:314–330PubMedCrossRefGoogle Scholar
  33. Schulz B, Boyle C (2005) The endophytic continuum. Mycol Res 109(6):661–686PubMedCrossRefGoogle Scholar
  34. 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(5):645–654PubMedCrossRefGoogle Scholar
  35. Unterseher M, Peršoh D, Schnittler M (2013) Leaf-inhabiting endophytic fungi of European Beech (Fagus sylvatica L.) co-occur in leaf litter but are rare on decaying wood of the same host. Fungal Divers 60(1):43–54CrossRefGoogle Scholar
  36. Wang Y, Guo LD (2007) A comparative study of endophytic fungi in needles, bark, and xylem of Pinus tabulaeformis. Can J Bot 85(10):911–917CrossRefGoogle Scholar
  37. Watrud LS, Martin KD, Kelly KS, Jeffrey KC, Clarace G (2006) Comparison of taxonomic, colony morphotype and PCR-RFLP methods to characterize microfungal diversity. Mycologia 98(3):384–392PubMedCrossRefGoogle Scholar
  38. White TJ, Bruns T, Lee S, Taylor J (1990) Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. 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
  39. Xiao Y, Li H-X, Li C, Wang J-X, Li J, Wang M-H, Ye Y-H (2013) Antifungal screening of endophytic fungi from Ginkgo biloba for discovery of potent anti-phytopathogenic fungicides. FEMS Microbiol Lett 339(2):130–136PubMedCrossRefGoogle Scholar
  40. Yurkov AM, Kemler M, Begerow D (2011) Species accumulation curves and incidence-based species richness estimators to appraise the diversity of cultivable yeasts from beech forest soils. PLoS ONE 6(8):e23671. doi: 10.1371/journal.pone.0023671 PubMedCentralPubMedCrossRefGoogle Scholar

Copyright information

© German Mycological Society and Springer-Verlag Berlin Heidelberg 2014

Authors and Affiliations

  • René Prior
    • 1
  • Katharina Görges
    • 1
  • Andrey Yurkov
    • 2
  • Dominik Begerow
    • 1
  1. 1.Geobotany, Department of Evolution and Biodiversity of Plants, Faculty of Biology and BiotechnologyRuhr-Universität BochumBochumGermany
  2. 2.Leibniz Institute DSMZ-German Collection of Microorganisms and Cell CulturesBrunswickGermany

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