Progression of Xylella fastidiosa Infection in Grapevines Under Field Conditions.

The pathogen Xylella fastidiosa subsp. fastidiosa has circulated through California's vineyards since its introduction from Central America in the 1800s. This pathogen is responsible for a bacterial disease called Pierce's disease (PD) of grapevine. With no known cure, PD has had devastating effects on some vineyards. Important factors that impact disease severity and persistence include: the presence of insect vectors, grapevine cultivar, management, ecology, and winter temperatures. Removal of infected vines is critical for reducing pathogen spread but relies on accurate and rapid pathogen detection. In this study, we foster a greater understanding of disease symptom emergence by way of a 3-year field inoculation project in Napa Valley. Although PD emergence and symptom progression have been studied in greenhouse and experimental plots, there is a large knowledge gap in quantifying disease progression under commercial conditions. After inoculating 80 mature Vitis vinifera vines in April 2017, we measured bacterial populations and six symptom types at four locations within each plant throughout the subsequent three growing seasons. The main foci of the project were understanding X. fastidiosa movement through the plants, infection, overwinter curing, and symptom development. We observed greater winter recovery than expected, and shriveled grape clusters proved to be a more reliable early indication of infection than other more commonly used symptoms. Although there were differences among wine grape cultivars, this work suggests that disease progression in the field may not fit the paradigm of predominant leaf scorch and low recovery rates as neatly as has been previously believed.

Phenotyping grapevine red blotch virus and grapevine leafroll-associated viruses before and after symptom expression through machine-learning analysis of hyperspectral images.

Introduction: Grapevine leafroll-associated viruses (GLRaVs) and grapevine red blotch virus (GRBV) cause substantial economic losses and concern to North America's grape and wine industries. Fast and accurate identification of these two groups of viruses is key to informing disease management strategies and limiting their spread by insect vectors in the vineyard. Hyperspectral imaging offers new opportunities for virus disease scouting. Methods: Here we used two machine learning methods, i.e., Random Forest (RF) and 3D-Convolutional Neural Network (CNN), to identify and distinguish leaves from red blotch-infected vines, leafroll-infected vines, and vines co-infected with both viruses using spatiospectral information in the visible domain (510-710nm). We captured hyperspectral images of about 500 leaves from 250 vines at two sampling times during the growing season (a pre-symptomatic stage at veraison and a symptomatic stage at mid-ripening). Concurrently, viral infections were determined in leaf petioles by polymerase chain reaction (PCR) based assays using virus-specific primers and by visual assessment of disease symptoms. Results: When binarily classifying infected vs. non-infected leaves, the CNN model reaches an overall maximum accuracy of 87% versus 82.8% for the RF model. Using the symptomatic dataset lowers the rate of false negatives. Based on a multiclass categorization of leaves, the CNN and RF models had a maximum accuracy of 77.7% and 76.9% (averaged across both healthy and infected leaf categories). Both CNN and RF outperformed visual assessment of symptoms by experts when using RGB segmented images. Interpretation of the RF data showed that the most important wavelengths were in the green, orange, and red subregions. Discussion: While differentiation between plants co-infected with GLRaVs and GRBV proved to be relatively challenging, both models showed promising accuracies across infection categories.

Identification of Putative SDHI Target Site Mutations in the SDHB, SDHC, and SDHD Subunits of the Grape Powdery Mildew Pathogen Erysiphe necator.

Succinate dehydrogenase inhibitors (SDHIs) are fungicides used in control of numerous fungal plant pathogens, including Erysiphe necator, the causal agent of grapevine powdery mildew (GPM). Here, the sdhb, sdhc, and sdhd genes of E. necator were screened for mutations that may be associated with SDHI resistance. GPM samples were collected from 2017 to 2020 from the U.S. states of California, Oregon, Washington, and Michigan, and the Canadian province of British Columbia. Forty-five polymorphisms were identified in the three sdh genes, 17 of which caused missense mutations. Of these, the SDHC-p.I244V substitution was shown in this study to reduce sensitivity of E. necator to boscalid and fluopyram, whereas the SDHC-p.G25R substitution did not affect SDHI sensitivity. Of the other 15 missense mutations, the SDHC-p.H242R substitution was shown in previous studies to reduce sensitivity of E. necator toward boscalid, whereas the equivalents of the SDHB-p.H242L, SDHC-p.A83V, and SDHD-p.I71F substitutions were shown to reduce sensitivity to SDHIs in other fungi. Generally, only a single amino acid substitution was present in the SDHB, SDHC, or SDHD subunit of E. necator isolates, but missense mutations putatively associated with SDHI resistance were widely distributed in the sampled areas and increased in frequency over time. Finally, isolates that had decreased sensitivity to boscalid or fluopyram were identified but with no or only the SDHC-p.G25R amino acid substitution present in SDHB, SDHC, and SDHD subunits. This suggests that target site mutations probably are not the only mechanism conferring resistance to SDHIs in E. necator.

Pheromone Deployment Strategies for Mating Disruption of a Vineyard Mealybug.

The mealybug, Planococcus ficus (Signoret), is a primary vineyard pest in California and other grape-growing regions throughout the World. Mating disruption programs are commercially available to manage Pl. ficus, but widespread adoption has been limited, in part, by high costs compared with insecticide programs. To improve mating disruption economic effectiveness, different deployment technologies (passive, aerosol, and microencapsulated formulations) were individually examined. Adult male Pl. ficus captures in pheromone traps and categorical ratings of vine infestation or crop damage suggest that all deployment strategies lowered mealybug densities or damage. Using passive dispensers, deployment rates of 310 and 465 per ha lowered Pl. ficus crop damage similar to 615 per ha, a rate commonly used in commercial operations; reduced rates would lower product and deployment costs. Meso dispensers, containing more a.i., deployed at 35 per ha did not have a treatment impact, but a microencapsulated formulation and aerosol canisters lowered male flight captures and/or crop damage. Male mealybug flight activity was greatest from 0500-1100 hr, which coincided with temperatures >16° and <32°C. These restricted times and temperatures suggest programable dispensers might allow pheromone deployment to coincide only with flight patterns. A large field trial using passive dispensers found greater treatment separation after 3 yr of pheromone deployment. Discrepancies in results among vineyards may be related to Pl. ficus density, but combined results from all trials suggest that different deployment technologies can be used to impact Pl. ficus densities and damage, even at reduced rates, especially with continued use over multiple seasons.

Impact of Rootstock and Season on Red Blotch Disease Expression in Cabernet Sauvignon (V. vinifera).

Grapevine red blotch virus (GRBV), the causative agent of grapevine red blotch disease, is widespread across the United States and causes a delay in ripening events in grapes. This study evaluates the effects of GRBV on Cabernet Sauvignon grape berry composition, grafted on two different rootstocks (110R and 420A) in two seasons (2016 and 2017). Total soluble solids, acidity, and anthocyanin concentrations were monitored through ripening and at harvest. Phenolic and volatile compounds were also analyzed at harvest to determine genotypic and environmental influences on disease outcome. Sugar accumulation through ripening was lower in diseased fruit (RB (+)) than healthy fruit across rootstock and season. GRBV impact was larger in 2016 than 2017, indicating a seasonal effect on disease expression. In general, anthocyanin levels and volatile compound accumulation was lower in RB (+) fruit than healthy fruit. Total phenolic composition and tannin content was higher in RB (+) fruit than healthy fruit in only 110R rootstock. Overall, GRBV impacted Cabernet Sauvignon grape composition crafted on rootstock 110R more than those crafted on rootstock 420A.

Assessment of Nymphal Ecology and Adult Xylella fastidiosa Transmission Biology of Aphrophora nr. permutata (Hemiptera: Aphrophoridae) in California Vineyards.

Although Aphrophora nr. permutata (Hemiptera: Aphrophoridae) is a reported vector of the plant pathogen Xylella fastidiosa (Wells) (Xanthomonadales: Xanthomonadaceae), its ecology and role in Pierce's disease dynamics in coastal California vineyards are poorly understood. From 2016 to 2020, we surveyed the abundance of A. nr. permutata nymphs among potential host plants along the vineyard floor, the vineyard edges, and adjacent vegetation in vineyards in Napa and Sonoma county. In 2019 and 2020, vineyards adjacent to woodland habitat hosted larger A. nr. permutata populations than those next to riparian habitat, while in 2017 and 2018, the nymphal populations were similar among riparian and woodland sites. Among 2020 plant cover taxa, nymph abundance was positively associated with Helminthotheca echioides, Vicia sativa, and Daucus carota cover and negatively associated with Taraxacum officinale cover. In 2018 and 2019, we also tracked early-season occurrence and development of A. nr. permutata nymphs among potential host plants. Analyses showed a significant effect of site, year, and plant taxa on the first detection of nymphs and a significant effect of site and year on the estimated development time between first and fifth instars. In 2019, we conducted grapevine to grapevine X. fastidiosa transmission experiments with individuals and groups of five A. nr. permutata adults. In the transmission experiment, 5% (3 of 60) individual A. nr. permutata and 7.7% (1 of 13) of groups successfully transmitted X. fastidiosa. This study provides preliminary evidence of potential host plant associations with A. nr. permutata abundance and phenology that should be explored further with field and greenhouse-based approaches.

Exploring Grower-sourced Data to Understand Spatiotemporal Trends in the Occurrence of a Vector, Pseudococcus maritimus (Hemiptera: Pseudococcidae) and Improve Grapevine Leafroll Disease Management.

Grapevine leafroll disease is a significant concern in the wine grape industry, as it spreads rapidly and contributes to economically significant reductions in yield and grape quality. Our objective was to utilize 5 yr of grower-sourced data from Napa (California, USA) to improve local and regional disease management efforts. Specifically, we applied a spatially integrated multivariate clustering technique to improve understanding of spatiotemporal trends in Pseudococcus maritimus (Ehrhorn) male populations-the primary vector in the region. We also implemented generalized linear mixed models to evaluate the effects of two key practices, insecticide sprays and roguing, on disease incidence. Results show P. maritimus has a biannual flight pattern in the study area, with the first flight peaking in early May and the second between early August and early September. Clusters of P. maritimus flight data fall largely within the vineyard footprints of individual growers, but also showed clear neighborhood effects. We found that when disease incidence within a block is <1%, consistent monitoring and removal of diseased vines is required to contain within-block spread. As within-block disease incidence grows to 1-20%, both insecticide applications and roguing are effective practices to reduce spread. At incidence levels >20%, roguing is a critical practice. Our results emphasize the importance of individual management efforts, but also the value of programs that engage the wider neighboring community and highlight the power of community data collection to guide decision-making.

Quantifying Planococcus ficus (Hemiptera: Pseudococcidae) Invasion in Northern California Vineyards to Inform Management Strategy.

The spread and impact of invasive species in exotic ranges can be mitigated by increased understanding of pest invasion dynamics. Here, we used geospatial analyses and habitat suitability modeling to characterize the invasion of an important vineyard pest, vine mealybug (Planococcus ficus Signoret, Hemiptera: Pseudococcidae), using nearly 15,000 trapping records from throughout Napa County, California, between 2012 and 2017. Spatial autocorrelation among P. ficus detections was strongest at distances of ~250 m and detectable at regional scales (up to 40 km), estimates of the rate and directionality of spread were highly idiosyncratic, and P. ficus detection hotspots were spatiotemporally dynamic. Generalized linear model, boosted regression tree, and random forest modeling methods performed well in predicting habitat suitability for P. ficus. The most important predictors of P. ficus occurrence were a positive effect of precipitation in the driest month, and negative effects of elevation and distance to nearest winery. Our results indicate that 250-m quarantine and treatment zones around P. ficus detections are likely sufficient to encompass most local establishment and spread, and that implementing localized regulatory procedures may limit inadvertent P. ficus spread via anthropogenic pathways. Finally, surveys of P. ficus presence at >300 vineyard sites validated that habitat suitability estimates were significantly and positively associated with P. ficus frequency of occurrence. Our findings indicate that habitat suitability predictions may offer a robust tool for identifying areas in the study region at risk to future P. ficus invasion and prioritizing locations for early detection and preventative management efforts.

Development of a Mating Disruption Program for a Mealybug, Planococcus ficus, in Vineyards.

The vine mealybug (VMB), Planococcus ficus (Hemiptera: Pseudococcidae), is a key insect pest of vineyards, and improvements in sustainable control of this pest are needed to meet increasing consumer demand for organically farmed products. One promising option is mating disruption. In a series of experiments conducted from 2004 to 2007, we tested the effects of mating disruption on trap captures of Pl. ficus males in pheromone-baited traps, on Pl. ficus numbers and age structure on vines, and on damage to grape clusters. From 2004 to 2005, the effects of dispenser load (mg active ingredient per dispenser) were also assessed, and dispensers were compared to a flowable formulation. Across all trials, mating disruption consistently reduced pheromone trap captures and often reduced mealybug numbers on vines and/or crop damage, regardless of the pheromone dose that was applied. Reductions in Pl. ficus densities in mating disruption plots were not accompanied by clear effects on mealybug population age structure; however, production of non-viable ovisacs by unmated females may have obscured differences in proportional representation of ovisacs. Pheromone trap captures were never lowered to zero (often called trap shut down), possibly because trials were conducted in vineyards with unusually high Pl. ficus densities. Trap-capture patterns in both treated and control plots commonly began low in April-May, increased in mid-July or August, and often decreased in September-October when post-harvest insecticides were applied. During the four-year trial, the release rate from plastic sachet dispensers was improved by industry cooperators as pheromone was released too quickly (2004) or not completely released during the season (2005-2006). The flowable formulation performed slightly better than dispensers at the same application dose. Results over all years suggest season-long coverage or late-season coverage may be as or more important than dose per hectare. Development of a dispenser with optimized season-long pheromone emission or targeted seasonal periods should be a future goal.

Impact of Grapevine Red Blotch Disease on Cabernet Sauvignon and Merlot Wine Composition and Sensory Attributes.

Grapevine red blotch disease (GRBD) is a recently identified viral disease that affects grapevines. GRBD has been shown to impact grapevine physiology and grape composition by altering specific ripening events. However, no studies have been reported on the impact of GRBD on wine composition and its sensory attributes. This study evaluated the impact of GRBD on wine primary and secondary metabolites, in addition to its sensory properties, when making wines from Cabernet Sauvignon and Merlot grapes during two seasons. Wines made with GRBD-impacted fruit were lower in ethanol content when compared to wines made with grapes from healthy grapevines. This was attributed to the lower total soluble sugar (TSS) levels of diseased grapes due to delayed ripening at harvest. GRBD impacted wine phenolic composition by decreasing anthocyanin concentrations and increasing flavonol concentrations in some instances. Additionally, proanthocyanidin concentrations were also consistently higher in GRBD wines compared to wines made from healthy fruit. Descriptive analysis demonstrated that GRBD can impact wine style by altering aroma, flavor, and mouthfeel attributes. However, the extent of GRBD impact on wine composition and sensory properties were site and season dependent.

Impact of Grapevine Red Blotch Disease on Grape Composition of Vitis vinifera Cabernet Sauvignon, Merlot, and Chardonnay.

Grapevine red blotch disease (GRBD) is a recently recognized viral disease that affects grapevines ( Vitis vinifera L.). Currently little is known about its impact on grape composition. This study focused on the impact of GRBD on grape primary and secondary metabolites (mainly phenolic compounds) of three Vitis vinifera L. cultivars during two seasons. Grapes from symptomatic red blotch diseased vines (RB (+)) mostly had lower concentration of total soluble solids (TSS) and higher titratable acidity (TA) levels when compared to grapes from healthy vines (RB (-)) at harvest. GRBD impacted grape phenolic composition by mostly decreasing anthocyanin and increasing flavonol and proanthocyanidin (PA) contents in berry skins. No major impacts were observed on seed phenolics. RB (+) grapes contained more amino and carboxylic acids, while RB (-) grapes contained more oligosaccharides, polyols, and some specific monosaccharides at harvest. The impact of GRBD on grape composition was variable and dependent on the cultivar, site, and season.

Grapevine Red Blotch Virus May Reduce Carbon Translocation Leading to Impaired Grape Berry Ripening.

Grapevine red blotch virus (GRBV) is suspected to alter berry ripening and chemistry. This study performed a physiological characterization of GRBV infected grapevines with attention to the factors leading to chemical changes during ripening of Cabernet Sauvignon in two rootstocks, 110R and 420A. RB(+) grapevines had transiently lower net photosynthesis; however, berry total soluble solids (TSS) accumulation was consistently reduced in the two years of study. Accumulation of anthocyanins and loss of titratable acidity and proanthocyanins were also delayed in RB(+) plants. However, the comparison of samples with the same TSS led to lower pH and anthocyanins content. The reduction in carbon import into berries under mild and transient reductions in carbon fixation suggested an impairment of translocation mechanisms with RB(+), leading into a desynchronization of ripening-related processes.

Characterization of grapevine leafroll-associated virus 3 genetic variants and application towards RT-qPCR assay design.

Grapevine leafroll-associated virus 3 (GLRaV-3) is the most widely prevalent and economically important of the complex of RNA viruses associated with grapevine leafroll disease (GLD). Phylogenetic studies have grouped GLRaV-3 isolates into nine different monophyletic groups and four supergroups, making GLRaV-3 a genetically highly diverse virus species. In addition, new divergent variants have been discovered recently around the world. Accurate identification of the virus is an essential component in the management and control of GLRaV-3; however, the diversity of GLRaV-3, coupled with the limited sequence information, have complicated the development of a reliable detection assay. In this study, GLRaV-3 sequence data available in GenBank and those generated at Foundation Plant Services, University of California-Davis, was used to develop a new RT-qPCR assay with the capacity to detect all known GLRaV-3 variants. The new assay, referred to as FPST, was challenged against samples that included plants infected with different GLRaV-3 variants and originating from 46 countries. The FPST assay detected all known GLRaV-3 variants, including the highly divergent variants, by amplifying a small highly conserved region in the 3' untranslated terminal region (UTR) of the virus genome. The reliability of the new RT-qPCR assay was confirmed by an enzyme linked immunosorbent assay (ELISA) that can detect all known GLRaV-3 variants characterized to date. Additionally, three new GLRaV-3 divergent variants, represented by four isolates, were identified using a hierarchical testing process involving the FPST assay, GLRaV-3 variant-specific assays and high-throughput sequencing analysis. These variants were distantly related to groups I, II, III, V, VI, VII and IX, but much similar to GLRaV-3 variants with no assigned group; thus, they may represent new clades. Finally, based on the phylogenetic analysis, a new GLRaV-3 subclade is proposed and named as group X.

Incidence of Grapevine Leafroll Disease: Effects of Grape Mealybug (Pseudococcus maritimus) Abundance and Pathogen Supply.

Studies of spatiotemporal dynamics are central to efforts to characterize the epidemiology of infectious disease, such as mechanism of pathogen spread and pathogen or vector sources in the landscape, and are critical to the development of effective disease management programs. To that end, we conducted a multi-year study of 20 vineyard blocks in coastal northern California to relate the dynamics of a mealybug vector, Pseudococcus maritimus (Ehrhorn) (Hemiptera: Pseudococcidae), to incidence of grapevine leafroll disease (GLD). In each vineyard block, a subset of vines were scored visually for relative mealybug abundance, disease was quantified by visual assessment, and virus presence was verified using standard laboratory molecular assays. GLD incidence was analyzed with a classification and regression tree, and with a hierarchical model that also captured variability among blocks and heterogeneity within blocks. Both analyses found strong interannual variability in incidence, with the hierarchical model also capturing substantial between- and within-block heterogeneity, but with significant contributions of vector abundance and pathogen supply (prior disease incidence) to the frequency of newly diseased vines. These results strengthen further the conclusion that mealybug vectors are causally related to pathogen spread in this system and are therefore an important target for management. Moreover, they are consistent with relatively efficient secondary spread of the pathogen, suggesting an important role for the removal of diseased vines as a tool to mitigate further damage.

Relative Prevalence of Grapevine Leafroll-Associated Virus Species in Wine Grape-Growing Regions of California.

Some diseases manifest as one characteristic set of symptoms to the host, but can be caused by multiple pathogens. Control treatments based on plant symptoms can make it difficult to effectively manage such diseases, as the biology of the underlying pathogens can vary. Grapevine leafroll disease affects grapes worldwide, and is associated with several viral species in the family Closteroviridae. Whereas some of the viruses associated with this disease are transmitted by insect vectors, others are only graft-transmissible. In three regions of California, we surveyed vineyards containing diseased vines and screened symptomatic plants for all known viral species associated with grapevine leafroll disease. Relative incidence of each virus species differed among the three regions regions, particularly in relation to species with known vectors compared with those only known to be graft-transmitted. In one region, the pathogen population was dominated by species not known to have an insect vector. In contrast, populations in the other surveyed regions were dominated by virus species that are vector-transmissible. Our survey did not detect viruses associated with grapevine leafroll disease at some sites with characteristic disease symptoms. This could be explained either by undescribed genetic diversity among these viruses that prevented detection with available molecular tools at the time the survey was performed, or a misidentification of visual symptoms that may have had other underlying causes. Based on the differences in relative prevalence of each virus species among regions and among vineyards within regions, we expect that region and site-specific management strategies are needed for effective disease control.

Seasonal biology of Ferrisia gilli (Hemiptera: Pseudococcidae) in California Sierra foothill vineyards.

The mealybug Ferrisia gilli Gullan is an emerging pest of wine grapes grown in California's Sierra foothills. A relatively new species, it had previously been recorded as a pest of pistachio, almond, and ornamentals. It was first reported on grape in El Dorado County in 2003 and has since established and spread. Nondestructive monitoring of grape vine sections was conducted in untreated vineyard plots and compared with destructive sampling conducted in grower-treated plots in 2008 and 2009 to determine F. gilli life stage seasonal presence, number of generations, location on the vine during the season, and damage potential to fruit clusters. Two generations were observed to be completed during the season. F. gilli overwintered under the bark at the base of the trunk, trunk, and cordon as second and third instars. Adults were found at the base of emerging shoots (spring) or on and under bark of old and new spurs. Live crawlers were born in June (first generation) and late August to September (second generation), and migrated to leaves to feed before moving to protected locations under bark or into fruit clusters. Lower mealybug densities and fruit damage were recorded on vines with than without insecticide treatment(s). Parasitized mealybugs were collected in low numbers and an Acerophagus sp. was the dominant parasitoid.

Ecology and management of grapevine leafroll disease.

Grapevine leafroll disease (GLD) is caused by a complex of vector-borne virus species in the family Closteroviridae. GLD is present in all grape-growing regions of the world, primarily affecting wine grape varieties. The disease has emerged in the last two decades as one of the major factors affecting grape fruit quality, leading to research efforts aimed at reducing its economic impact. Most research has focused on the pathogens themselves, such as improved detection protocols, with limited work directed toward disease ecology and the development of management practices. Here we discuss the ecology and management of GLD, focusing primarily on Grapevine leafroll-associated virus 3, the most important virus species within the complex. We contextualize research done on this system within an ecological framework that forms the backbone of the discussion regarding current and potential GLD management strategies. To reach this goal, we introduce various aspects of GLD biology and ecology, followed by disease management case studies from four different countries and continents (South Africa, New Zealand, California-USA, and France). We review ongoing regional efforts that serve as models for improved strategies to control this economically important and worldwide disease, highlighting scientific gaps that must be filled for the development of knowledge-based sustainable GLD management practices.

Occurrence of grapevine leafroll-associated virus complex in Napa Valley.

Grapevine leafroll disease (GLD) is caused by a complex of several virus species (grapevine leafroll-associated viruses, GLRaV) in the family Closteroviridae. Because of its increasing importance, it is critical to determine which species of GLRaV is predominant in each region where this disease is occurring. A structured sampling design, utilizing a combination of RT-PCR based testing and sequencing methods, was used to survey GLRaVs in Napa Valley (California, USA) vineyards (n = 36). Of the 216 samples tested for GLRaV-1, -2, -3, -4, -5, and -9, 62% (n = 134) were GLRaV positive. Of the positives, 81% (n = 109) were single infections with GLRaV-3, followed by GLRaV-2 (4%, n = 5), while the remaining samples (15%, n = 20) were mixed infections of GLRaV-3 with GLRaV-1, 2, 4, or 9. Additionally, 468 samples were tested for genetic variants of GLRaV-3, and of the 65% (n = 306) of samples positive for GLRaV-3, 22% were infected with multiple GLRaV-3 variants. Phylogenetic analysis utilizing sequence data from the single infection GLRaV-3 samples produced seven well-supported GLRaV-3 variants, of which three represented 71% of all GLRaV-3 positive samples in Napa Valley. Furthermore, two novel variants, which grouped with a divergent isolate from New Zealand (NZ-1), were identified, and these variants comprised 6% of all positive GLRaV-3 samples. Spatial analyses showed that GLRaV-3a, 3b, and 3c were not homogeneously distributed across Napa Valley. Overall, 86% of all blocks (n = 31) were positive for GLRaVs and 90% of positive blocks (n = 28) had two or more GLRaV-3 variants, suggesting complex disease dynamics that might include multiple insect-mediated introduction events.

Development of a multiplex PCR for identification of vineyard mealybugs.

A simple molecular tool was developed and tested to identify seven mealybug species found in North American vineyards: Pseudococcus maritimus Ehrhorn, Pseudococcus viburni (Signoret), Pseudococcus longispinus (Targioni-Tozzeti), Pseudococcus calceolariae (Maskell), Planococcus ficus (Signoret), Planococcus citri (Risso), and Ferrisia gilli Gullan. The developed multiplex PCR is based on the mitochondrial cytochrome c oxidase subunit one gene. In tests, this single-step multiplex PCR correctly identified 95 of 95 mealybug samples, representing all seven species and collected from diverse geographic regions. To test the sensitivity, single specimen samples with different Pl. ficus developmental stages (egg to adult female and adult male) were processed PCR and the resulting output provided consistent positive identification. To test the utility of this protocol for adult males caught in sex baited pheromone traps, Pl. ficus adult males were placed in pheromone traps, aged at a constant temperature of 26±2°C, and processed with the multiplex each day thereafter for 8 d. Results showed consistent positive identification for up to 6 d (range, 6-8 d). Results are discussed with respect to the usefulness of this molecular tool for the identification of mealybugs in pest management programs and biosecurity of invasive mealybugs.

Testing baits to control Argentine ants (Hymenoptera: Formicidae) in vineyards.

Liquid baits were evaluated for control of the Argentine ant, Linepithema humile (Mayr) (Hymenoptera: Formicidae), and associated mealybug and soft scale pests in California vineyards. In 2003, liquid baits with small doses ofimidacloprid, boric acid, or thiamethoxam dissolved in 25% sucrose water resulted in lower ant and mealybug densities and fruit damage, compared with an untreated control. Similar treatments in a soft scale-infested vineyard showed only a reduction of ant density and fruit infestation in only the boric acid and thiamethoxam treatments. In 2004, commercial and noncommercial formulations of liquid baits reduced ant densities in three separate trials, but they had inconsistent effects on mealybug densities and fruit infestation; granular protein bait had no effect. Using large plots and commercial application methodologies, liquid bait deployed in June resulted in lower ant density and fruit infestation, but it had no effect on mealybug density. Across all trials, liquid bait treatments resulted in lower ant density (12 of 14 trials) and fruit damage (11 of 14 sites), presenting the first report of liquid baits applied using commercial methodologies that resulted in a reduction of ants and their associated hemipteran crop damage. For commercialization of liquid baits, we showed that any of the tested insecticides can suppress Argentine ants when properly delivered in the crop system. For imidacloprid, bait dispensers must be protected from sunlight to reduce photodegradation. Results suggest that incomplete ant suppression can suppress mealybug densities. However, after ant populations are suppressed, there may be a longer period before hemipteran populations are effectively suppressed. Therefore, liquid baits should be considered part of a multiseason program rather than a direct, in-season control of hemipteran pest populations.