Grapevine Fleck Virus: Molecular Detection and Investigation of Strain Variability

Grapevine fleck is a graft-transmissible disease and does not show symptom on most grapevine scion and rootstock varieties (disease is latent). Disease symptom is very clearly shown on Vitis rupestris St. George. This rootstock is used as a biological indicator for disease detection. Although bioassays are likely to remain an important part of the procedure for detecting grapevine viruses, the use of relatively quick molecular techniques like polymerase chain reaction (PCR) could greatly streamline disease detection and elimination procedures. These tests require only a day or two in comparison to the 2 to 3 years for a field biological assay. The genomic composition of grapevine fleck virus (GFLKV) is quite different from many other plant viruses and tends to fold and form complex secondary structures. These secondary structures usually hinder the PCR process. However, in the past year we have been able to overcome this problem and develop a PCR method for the detection of this virus. We have also developed a simple extraction method for the preparation of samples for PCR and combined with a simple one-tube PCR protocol that allows for fast processing and testing of samples. This simplified sample preparation method and one tube PCR protocol is no more complex or labor intensive than sample preparation and testing by ELIS A. Our preliminary data show that variations exist among the genome of different isolates of GFLKV. In order to develop a reliable PCR method these variations must be studied and PCR primers designed accordingly. We are in the process of identifying some of these isolates and comparing their genomic sequences.