New isolation method for endophytes based on enzyme digestion
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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.
KeywordsEndophytes Fungi Strain cultivation Isolation method Enzyme digestion
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.
- 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
- 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
- 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
- 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