Sensitive and reliable detection of grapevine fanleaf virus in a single Xiphinema index nematode vector

Grapevine fanleaf virus (GFLV) is specifically transmitted from plant to plant by the ectoparasitic nematode Xiphinema index. A sensitive and reliable procedure was developed to readily detect GFLV in a single viruliferous X. index, regardless of the nematode origin, i.e. greenhouse rearings or vineyard soils. The assay is based on bead milling to disrupt nematodes extracted from soil samples, solid-phase extraction of total nematode RNAs, and amplification of a 555bp fragment of the coat protein (CP) gene by reverse transcription-polymerase chain reaction with two primers designed from conserved sequences. This procedure is sensitive since the CP gene fragment is amplified from an artificial sample consisting of one viruliferous nematode mixed with 3000 aviruliferous individuals. In addition, StyI RFLP analysis of the CP amplicon enables the GFLV isolate carried by a single viruliferous X. index to be characterized. This GFLV detection assay opens new avenues for epidemiological studies and for molecular investigations on the mechanism of X. index-mediated GFLV transmission.