DEVELOPMENT OF A POLYMERASE CHAIN-REACTION TECHNIQUE FOR THE DETECTION OF GRAPEVINE FANLEAF VIRUS IN GRAPEVINE TISSUE

A polymerase chain reaction (PCR) method has been developed to detect grapevine fanleaf virus (GFLV) in GFLV-infected grape tissue. Four sam ple extraction methods for infected plant tissues were compared. Altho ugh PCR could readily detect RNA in samples of GFLV RNA and virion in GFLV-infected leaf samples of Gomphrena globosa with all four extracti on methods, only one method was useful for GFLV detection in grapevine tissue. Dilution of infected grape leaf samples by a 200-fold excess of healthy leaf tissue did not prevent GFLV detection by this method. Extracts from healthy grapevines prepared by methods 1, 2, and 3 preve nted detection of extracted GFLV genomic RNAs by PCR, demonstrating th at grape tissue extracts can inhibit either reverse-transcriptase reac tions or PCR. Using method 4, GFLV could be detected in all tested cul tivars of European grape, Vitis vinifera, and an American species, V. rupestris. Detection was possible in infected leaves, shoots, roots, a nd bark scrapings. PCR detection of as little as 128 fg of GFLV RNA wa s possible.