Pierce's Disease of Grapes Caused by Xylella Fastidiosa: A Survey for the Pathogen, Identification of Reservoir Hosts, and Identification of Insect Vectors in Kentucky Vineyards

A new j;!rapedisease for Kentucky. In September, 2001, Pierce's disease of grapes was discovered along the Ohio River in a western Kentucky vineyard. Confirmation of the causal agent, Xylella fastidiosa; was made using rapid cycling real-time polymerase chain reaction (PCR) technology, by using a serological (ELISA) test, and by electron microscopic observation of the characteristic undulating cell wall of the causal agent in grape leafpetioles. Pierce's disease of grapes is endemic to the southeastern United States and has recently become a major threat to California grapes because of the introduction of a new insect vector. Pierce's disease had never been found in the Ohio River Valley as far north as Kentucky. Indeed, analyisis of minimum winter temperature data published by other researchers suggest that most of Kentucky would be outside the geographic range for this disease. The Kentucky and eastern U.S. grape and wine industry needs research-based information on the biology and management of Pierce's disease. Pathogen detection is complicated. Current assays such aswe have done in our laboratory and through commercial laboratories only test for X fastidiosa. Bacterial leaf scorch, another devastating disease caused by X fastidiosa, is widespread in landscape trees in Kentucky. It is likely that the newly-found grape disease is caused by the Pierce's disease strain of X fastidiosa, but we really don't know for sure. The grape industry will need answers as to whether or not the pathogen we see in grapes or in vegetation near vineyards is actually Pierce's disease or another landscape strain. We have preliminary evidence that X fastidiosa is present in certain symptomless weeds in the vineyard and in symptomless woody vegetation growing nearby as well. This is not surprising given that we also find that in landscapes having trees with bacterial leaf scorch, the pathogen can be found in symptomless grasses, lawn weeds, and landscape shrubs and vines. A brief summary of pathogen detection using molecular methods. PCR-based identification provides a means of accurate detection of extremely low levels of a target organism, and real-time PCR technologies such as use of TaqMan primers (Heid et al. 1996; Nasarabadi et al. 1999), allow definitive identification of species or strain-specific DNA sequences in biological samples. For the research proposed here, it will be necessary to identify and survey species and individual strains of X fastidiosa in plants and insects. Species identification can be based on comparisons of gene sequences such as the ribosomal RNA operons (rDNA), whereas strain identification utilizes the more variable non-coding regions such as rDNA transcribed spacers. Although extensive databases have many such sequences, DNA sequences from particular target strains are needed. The most facile way to obtain such sequences is by a PCR~based approach (Schardl et al. 1997). Based on the sequences obtained, PCR primer sets both for species detection and strain identification can be synthesized, tested, and used in surveys of plants and insect vectors. A useful way to accomplish the PCR step for sequence determination, as well as to optimize the PCR diagnosis procedure, is with a real-time PCR instrument. Thus, research providing needed information for practical disease control is made feasible with molecular technology.