New isolation method for endophytes based on enzyme digestion.

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. [ABSTRACT FROM AUTHOR]