Effective disease management hinges on an accurate understanding of the ecological and epidemiological underpinnings of the pathosystem. New epidemics may prompt consideration of whether knowledge gaps or changes to the pathosystem warrant revision of management strategies. Pierce’s disease of grapevines (PD) is highly episodic in coastal Northern California vineyards, with modest incidence in most years punctuated by occasionally severe epidemics. To better understand what was driving a developing epidemic in the region, we revaluated what is known about the ecology and epidemiological role of the dominant vector, the blue-green sharpshooter Graphocephala atropunctata. We monitored vector spatiotemporal dynamics at 32 vineyards over three years, surveyed plant community composition in the adjacent habitat to understand its link to vector recruitment, and quantified patterns of natural infectivity for the pathogen Xylella fastidiosa. Overall, the results were consistent with past studies of G. atropunctata ecology. For example, the scale of dispersal from source habitat and seasonal patterns in activity were generally similar to those documented in prior studies. The results also confirmed the influence of adjacent plant community composition on G. atropunctata activity in vineyards, and the role of riparian habitat and select plant taxa as vector sources. Nonetheless, further consideration of the epidemiological significance of certain features of the pathosystem may be warranted, especially those related to seasonality in X. fastidiosa natural infectivity. A marked increase in infectious G. atropunctata late in the season likely reflects pathogen acquisition from infected grapevines, which may have implications for disease management strategies.

Graphocephala atropunctata monitoring:

 Monitoring for the dominant vector in the region, G. atropunctata, was conducted at 32 commercial vineyards in coastal Northern California, spread throughout the central viticultural regions in Napa and Sonoma counties. The vineyard sites were selected in part to capture variation in acreage, surrounding landscape context, vector abundance, and disease pressure. At each vineyard, we deployed between 6 and 15 yellow panel traps (10.2 x 17.8 cm double-sided yellow panel traps; Seabright Laboratories, Emeryville, CA), depending on vineyard size and shape, for a total of more than 375 unique traps. Traps were distributed in two to three transects per site running from the edge of the vineyard into the interior. At each site we also placed one to three traps on the periphery of the vineyard nearby the closest putative vector source habitat (“vegplot" traps). Each trap was checked twice per month over the growing season and once a month in the dormant season

Graphocephala atropunctata natural infectivity:

 To better understand patterns of natural infectivity in vector populations, we screened all G. atropunctata collected on traps for the presence of X. fastidiosa. After collecting sticky traps during the monitoring program described earlier, sharpshooters were individually removed from traps, cleaned, DNA was extracted and subjected to real-time PCR to determine the fraction positive for X. fastidiosa.

Insects were removed from sticky traps and agitated in a xylene-like solution, Citrisolv (Decon Laboratories Inc., King of Prussia, PA), for 30 seconds to remove adhesive residue. They were then subjected to three consecutive washes in 100% ethanol for a minute each to minimize residue carryover. Next, we used sterilized forceps to isolate individual sharpshooter heads. DNA was then extracted from insect heads using Qiagen’s DNeasy 96 Blood and Tissue Kits (Qiagen Inc., Santa Clarita, CA), eluted in 100uL buffer AE. Real-time PCR was run using Promega’s GoTaq® qPCR Master Mix reagents (Promega Corporation, Madison, WI) on an Applied Biosystems™ 7500 Real-Time PCR thermocycler under the following conditions: 95°C for 15min, then 40 cycles of 95°C for 10 secs, 60°C for 15 secs, and 72°C for 30 secs. The primers utilized were PD0059 at 0.30uM final concentration (PD0059_For GCTTCGGCCACTGACATATT, PD0059_Rev GCAAGGTTCACAAGCACGAT; Sicard et al. 2019). Positives samples were determined by the presence of a Cq value and a Tm between 82.24-84°C.