P-179 - Development and use of a novel diagnostic assay of grapevine leafroll-associated virus 3 for epidemiological studies in diseased vineyards

Grapevine leafroll-associated virus 3 (GLRaV3, species Ampelovirus trivitis, genus Ampelovirus, family Closteroviridae) is the primary causal agent of grapevine leafroll disease, the most widespread and economically damaging viral disease of Vitis worldwide. Secondary spread of GLRaV3 in vineyards is mediated through mealybug and soft scale insect vectors, the most efficient of which is the vine mealybug, Plannococcus ficus (Hemiptera: Pseudococcidae). Leafroll disease epidemics usually start with randomly distributed diseased vines followed by spatiotemporal aggregations along rows due to resident mealybug vectors dispersing along trellis systems and vine canopies. Previous studies have identified four major genetic variants of GLRaV3 in Californian vineyards. Recent observations revealed random distribution patterns of newly infected vines with limited to no aggregations in some Californian vineyards, suggesting additional modes of GLRaV3 spread. To better understand this phenomenon, new diagnostic tools of GLRaV3 are needed for large scale epidemiological studies. Such tools should be more suitable than, but equally efficient to, high-throughput sequencing for the testing and characterization of GLRaV3 in large grapevine leaf sample sizes. Thus, a RT-PCR restriction fragment length polymorphism (RT-PCR RFLP) assay was developed for the identification of the major GLRaV3 genetic variants. This assay is essential to determine the variability of GLRaV3 isolates within and across diseased vineyards and accurately trace infection sources.