Evaluating Fungal Endophyte Isolation Techniques in Three Grape Species

Endophytes are either fungal or bacterial organisms living within plant tissues. Most plants which have been evaluated for the presence of endophytes have been shown to host endophytes.  The characterization and function of bacterial endophytes in wine grapes and their relation to certain plant pathogens have been examined. However, research investigating the role of fungal endophytes in grapes is limited. Our objective was to evaluate different methods for extracting fungal endophytes from grape leaf tissue leading to the development of efficient extraction protocol. We collected fresh leaf samples from three different grape species; Vitis cinerea, V. aestivalis, and Vitis X 'Chambourcin'.  Leaf tissue from each species was subjected to three surface sterilization techniques (dips in EtOH followed by 2.5, 5, and 10 minute dips in NaOCl.)  Following surface sterilization, leaf tissue was exposed to  three different antibiotics (streptomycin, neomycin, and ampicillin) in the growing media). There were six replications of each treatment, and the growing media was a 1% malt extract agar (MEA).  The Petri dishes containing the leaf tissue and growing media were placed in a 21°C environment and checked weekly for four weeks for the presence of fungal growth.  Data were recorded on the presence or absence of fungal growth, and the levels of contamination. Data from the experiment were subjected to analysis using SAS® statistical software.  Our findings provide new insight into techniques useful for fungal endophyte extraction in Vitis spp.