Development and Implementation of Management Strategies for Grapevine Leafroll Disease and Mealybug Vectors

This project continues to inform and support the adoption of management strategies to minimize the incidence of leafroll disease spread in California vineyards. During the 2014-15 project year, we completed several objectives: (1) we finalized our analyses of the factors impacting leafroll disease spread, (2) developed methods for hyperspectral airborne imaging to identify diseased vines, and (3) supported the efforts of a group of growers working on regional leafroll disease management strategies. Our analyses of factors affecting disease spread suggest that the most important determinant of new disease is “block”—that is, there is great variability in the incidence of new disease explained by factors that are specific to the block and vintage (management practices, locationparticularly in relation to other diseased blocks, variety, vine age, vine health, etc.) Disease pressure, defined as the number of diseased vines present in the vineyard in the prior year, also impacts the incidence of new disease. And, in vineyards with medium to high disease incidence, mealybug populations affect disease spread. This suggests that in vineyards with low levels of disease, management practices that support vine health and decrease the number of diseased vines are critical to minimize disease spread.

In vineyards with greater incidence of disease, management practices may also include strategies to minimize mealybug populations. These observations are consistent with similar studies in other grape growing regions around the world, and have clear implications for management: in order to determine which management strategies should be adopted, growers must evaluate disease pressure and GMB populations. In Napa County, where neighboring vineyards share the burden of disease management, growers may jointly launch regional responses to GLD. We have developed a template for these regional efforts, with a pioneering group of 20 grape growers farming 1900 contiguous acres. The grower group (LAMBA) is committed to implementation of coordinated, regional GLD management strategies that include monitoring of GLD and GMB, as well as lowering disease and vector pressure. The group shares information and develops goals and activities at regular meetings and focuses on the implementation of GLD management strategies at a regional level.

We have also developed the use of hyperspectral imaging to map the incidence of GLD in commercial vineyards and nurseries. Imaging spectroscopy provides a potentially valuable alternative to lab testing and field scouting in that it is efficient, non-destructive and relatively inexpensive. From an aircraft thousands of acres may be imaged in a single three-hour flight. Our preliminary analysis of hyperspectral images of Cabernet Sauvignon vineyards have shown 75 to 90{aed9a53339cdfc54d53cc0c4af03c96668ab007d9c364a7466e3349a91bf0a23} correlation between leafroll-diseased vines identified in ground surveys with those identified through aerial imaging. Further analyses planned for 2015 will refine these figures and help develop the use of this technology for this and other grape varieties.

Development and Implementation of Management Strategies for Grapevine Leafroll Disease and Mealybug Vectors

This project continues to develop management strategies for grape growers with grapevine leafroll disease. We recently completed a mapping project spanning 4 grape growing seasons (2010-2013) to determine the relationship between grape mealybug (GMB) populations and the incidence of grapevine leafroll disease (GLD) in Napa County vineyards. We surveyed 10  vineyards and conducted a preliminary analysis to demonstrate the link between the vector, GMB, and disease incidence. In vineyards with medium to high levels of GLD, rates of disease spread are influenced mainly by (1) the initial number of vines in a vineyard with GLD (disease pressure) and (2) GMB populations in the previous growing season. In vineyards with low levels of GLD, rates of disease spread are influenced by the initial disease incidence as well as the number of GMB found in the current season. These observations are consistent with similar studies in other grape growing regions around the world, and have clear implications for management: in order to successfully manage GLD incidence and spread, growers must evaluate disease pressure and GMB populations. Our preliminary analysis suggests that managing the vector alone may not be sufficient to decrease rates of disease spread, particularly in vineyards with medium to high GLD incidence.

Studies of other pests and diseases have demonstrated that a regional approach to disease management has more potential for success than individual efforts. This is especially true in Napa County, where a single or few growers rarely control large swaths of contiguous vineyard acreage. In Napa County, where neighboring vineyards share the burden of disease management, growers may jointly launch regional responses to GLD. We are developing a template for these regional efforts, with a pioneering group of 20 grape growers farming 1900 contiguous acres. The grower group is committed to implementation of coordinated, regional GLD management strategies that include monitoring of GLD and GMB, as well as lowering disease and vector pressure. Most importantly, the group shares information, communications and develops goals and activities at regular meetings (4-6 meetings per year) focusing on the implementation of GLD management strategies at a regional level.

This project is also developing the use of hyperspectral imaging to map the incidence of GLD in commercial vineyards and nurseries. Imaging spectroscopy provides a potentially valuable alternative to lab testing and field scouting in that it is efficient, non-destructive and relatively inexpensive. From an aircraft thousands of acres may be imaged in a single three-hour flight. Additionally, remote sensing provides continuous measurements to construct a map of the target vineyard. Our studies in Cabernet Sauvignon vineyards have shown a 94.7{aed9a53339cdfc54d53cc0c4af03c96668ab007d9c364a7466e3349a91bf0a23} correlation between leafroll-diseased vines identified in ground surveys with those identified through aerial imaging, suggesting a high level of confidence in the accuracy of our measurements for this variety, and potential to develop the use of this technology for other grape varieties. Hyperspectral airborne imaging could revolutionize the the collection of disease incidence data by increasing ease, accuracy and efficiency of measurements.

Management of Grapevine Leafroll Disease – What Level of Mealybug Control is Needed?

Grapevine leafroll-associated viruses (GLRaV) are a complex of viruses that cause leaf chlorosis and leaf margins to “roll” downward.  GLRaVs can reduce yields, delay fruit maturity, and impede fruit pigmentation. Our work focused on control measures for the insect vectors of GLRaV. In 2013 we monitored changes in vine mealybug (VMB) populations in three areas that were formerly part of an areawide mating disruption program and showed that populations have increased in two out of the three of the areas since the removal of pheromone dispensers. Trap catches surrounding historical “hotspots” were also elevated, suggesting that mealybug populations in these areas are spreading. We continued a six-year field study mapping the establishment and spread of GLRaV in a 20 acre vineyard planted in 2008 from certified virus-free scion and bordered by blocks with GLRaV-infected vines and mealybugs. PCR tests revealed that 17 out of 25 vines that were visually identified as symptomatic for GLRaV in 2011 and 2012 were in fact infected by red blotch, which was present in the vineyard by at least 2011. Red blotch-infected vines were randomly distributed inside the mapped plot.

We conducted a study of aerial grape and vine mealybug dispersal at two 10-acre vineyard sites in the Napa Valley, using sticky traps that were deployed in a grid pattern throughout each site. We have not observed mealybugs on the sticky traps that have been processed thus far, suggesting that aerial dispersal by mealybugs is uncommon in Napa vineyards. Work examining the temperature development rates of grape, obscure and Gills mealybugs is ongoing; we began this work with grape mealybug but were unable to maintain sufficient numbers on vines, and will attempt the work again in 2014 with added improvements.

We conducted several transmission experiments to: 1) compare GLRaV-3 transmission efficiency of mealybugs of different ages; 2) detect GLRaV-3 in various grapevine tissues after first inoculation; and 3) determine latency period between first inoculation and successful transmission of GLRaV-3 by vine mealybug (VMB) crawlers. The results indicate that VMBs of all ages are capable of transmitting GLRaV-3 in grapevines, but that first instar VMBs (crawlers) are the most efficient stage. The detection experiments showed that GLRaV-3 can be detected in potted grapevines 3-4 weeks after inoculation and crawlers can acquire and transmit GLRaV-3 from newly inoculated vines 2 weeks after first inoculation. The findings indicate that the infected grapevines could serve as a virus source long before the appearance of GLD symptoms. The stage-specific transmission studies showed that VMB (crawlers) are the most efficient stage and based on biology of VMB crawlers are also the most dispersible stage. Therefore, the GLD management efforts should focus primarily on effective control of VMB crawlers. However, previous studies have shown that application of even some of the highly effective insecticides after VMB infestation may not prevent GLRAV-3 inoculation by VMB crawlers in newly infested blocks. In this situation, application of fast acting products in a proactive manner would be the best approach to manage GLD in vineyards.

Mealybug Transmission of Grapevine Leafroll-Associated Virus 3

The overarching goal of this research is to obtain information about transmission of Grapevine leafroll-associated virus 3 (GLRaV-3), the primary virus species associated with spread of the economically damaging Grapevine Leafroll Disease (GLD) in Napa Valley. Such information is necessary to inform control strategies, it is clear that knowledge-based management of vector-borne diseases requires a robust understanding of how the pathogen spreads in vineyards. Mealybugs are the vectors associated with spread of GLD, but nothing is known about differences in transmission efficiency among mealybug species inhabiting vines in California. Furthermore, genetically distinct variants of GLRaV-3 exist but nothing is known about differences among these variants in terms of their ability to spread, or what the relevance of that variation is to GLD epidemiology. Lastly, all previous GLRaV-3 transmission studies were done under greenhouse conditions, and it is not known how well the results of such studies predict transmission in vineyards. This research addresses these significant gaps in knowledge.

We have completed proposed single and simultaneous mixed GLRaV-3 variant inoculations in greenhouse trials, using grape and vine mealybugs. Though two GLRaV-3 variants from singly infected source plants did not differ in transmission efficiency, the transmission efficiency of one variant was substantially lower when acquisition occurred from a co-infected source plant, indicating inhibition of transmission by the other variant. This may mean that one variant can be transmitted more efficiently than another and increase its incidence in the landscape (e.g. Napa Valley). It is not known whether some GLRaV-3 variants are more pathogenic than others.

We also set up an experiment in Napa Valley in summer 2011, inoculating 60 mature grapevines with GLRaV-3 using grape mealybugs as vectors. Each vine was inoculated using 10 first instar mealybugs, and then treated with insecticide two days later. Three months after inoculation, 20 of 60 plants tested positive for GLRaV-3 from our inoculations. No symptoms appeared in 2011. During the following growing season, GLD symptoms first began to appear in our experimental vines in June. By July, symptoms appeared in 29 of 60 experimental vines, and no other vines became symptomatic in 2012. Berry quality was affected in symptomatic vines compared to asymptomatic vines in the experiment.

Transmission in a parallel greenhouse experiment was higher than in the vineyard. In 2012 we set up a second field inoculation experiment in Napa to compare transmission of two different GLRaV-3 variants, by grape and vine mealybugs, in both Chardonnay and Pinot Noir. Inoculations were completed in July. Results from the 2012 inoculations are pending. This is the first time it has been shown that GLD symptoms due to mealybug inoculation of GLRaV-3 into established mature vines (~15 years old) in commercial vineyards are expressed in the following growing season. Results also showed that the entire vines were symptomatic in 2012, instead of just the inoculation site. Lastly, transmission success in the field was about 6{aed9a53339cdfc54d53cc0c4af03c96668ab007d9c364a7466e3349a91bf0a23} per individual mealybug.

Mealybug vectors and Grapevine Mealybug Vectors and Grapevine Leafroll Dsease: Temporal and Spatial Studies

Grapevine leafroll disease, caused by a complex known as grapevine leafroll-associated viruses, is a worldwide threat to vineyard health and sustainability. Mealybugs are the main agents (vectors) responsible for virus movement between vines in the field. This project continues to study the relationship between mealybug populations and the incidence of leafroll disease in vineyards. The ultimate goal is to develop and deploy best management practices for leafroll disease and vineyard mealybugs as vectors of leafroll viruses. The project not only focuses on development of these practices, but also works with grape growers to use these tools in a disease management program. For example, we have successfully developed the use of male GMB traps as a monitoring and decision-making tool and provided significant formal and informal educational efforts on the use of these traps. Traps are more sensitive detection tools that can supplement labor-intensive ground surveys. We provided training sessions on trap deployment and male mealybug identification to ensure that the traps are being used correctly, at the appropriate time of year, and that the trap data are useful to growers. Grape growers are increasingly adopting the use of GMB traps as a management tool. We have also worked with growers to explore and implement other effective and timely management practices, such as insecticide sprays and vine removal.

Our preliminary analysis has shown that prior infection rates of leafroll disease and mealybug populations in the current year will affect the number of vines that develop leafroll disease symptoms in the following year. This has implications for vine removal programs: in a given year, there may be a percentage of vines that contain virus particles but are not showing disease symptoms. This and other complementary projects worldwide are developing best management practices for leafroll disease management that include monitoring and management of vector populations, identification and removal of diseased vines or vineyards, planting of material free of known grapevine pathogens, and regional approaches to management. In future studies we propose to continue to develop new practices, fine-tune current practices to determine when, where and how they can be used most effectively, develop a regional approach to management, and strengthen our educational efforts.

Mealybug Pests and an Emerging Viral Disease: Vector Ecology and Their Role in Grape Leafroll Associated Virus Epidemiology

Grapevine leafroll-associated viruses (GLRaV) are a complex of viruses that cause leaf chlorosis and leaf margins to “roll” downward.  GLRaVs can reduce yields, delay fruit maturity, and impede fruit pigmentation.  Our work concerns GLRaV field epidemiology with respect to its insect vectors. In field studies, we continued evaluating seasonal densities of grape mealybugs and leafroll virus in five vineyards with varying levels of leafroll infections and found 43{aed9a53339cdfc54d53cc0c4af03c96668ab007d9c364a7466e3349a91bf0a23} of grape mealybugs collected on leafroll-infected vines tested positive for GLRaV-3. The proportion of leafroll-infective mealybugs was related to the number of leafroll-positive vines in the vineyards surveyed. Vineyards were mapped for leafroll virus symptoms in fall 2012 but the presence of Red Blotch Virus at two of the sites made accurate mapping difficult.

We continued a five-year field trial testing the impacts of “zero tolerance” for mealybugs on GLRaV infection establishment and spread. A 20 acre vineyard planted in 2008 from certified virus-free scion, and bordered by blocks with GLRaV-infected vines and mealybugs, received pesticide treatments in 2009, 2010, 2011 and 2012. No mealybugs were found in harvest counts; however, pheromone traps showed the presence of male grape mealybugs. All vines were inspected annually for GLRaV symptoms. In 2009 and 2010, there was one new infected vine, with strong leafroll symptoms and positive PCR test, each year in 2009 and 2010, six new vines in 2011, and 2 in 2012. GLRaV-3 variants in the spray trial block (-3a, -3c, and -3d) were different than the GLRaV-3 variants in adjacent virus-infected blocks to the north and west (-3b). The leafroll-infected vines were randomly distributed inside the mapped plot, with similar numbers in insecticide and untreated rows. Our hypothesis is that virus-carrying grape mealybug crawlers were “blown” into the plot, and infecting previously-healthy vines during feeding.

We conducted mealybug behavior studies in the greenhouse to determine if mealybugs prefer vines without GLRaV; this would result in movement of vectors away from infected vines to healthy vines. In non-choice tests, mealybugs survived equally well on infected and uninfected vines, while in choice tests, equal numbers of mealybugs were chose infected and healthy vines, indicating no preference for one over another.

In side-by-side comparisons, the five major GLRaV vector species found in California, EFLS, OMB, LTMB, GMB, VMB and GiMB all transmitted GLRaV-3 but at different rates. We showed that GLRaV-3 can be detected 3-4 weeks after first inoculation in any part of the inoculated vines using crude extraction of RNA and RT-PCR. The latency period studies (collected thus far) further signify the fact that VMB crawlers can acquire leafroll virus (GLRaV­3) from newly inoculated vines as soon as two weeks after first inoculation and then successfully transmit it to the healthy grapevines.  Ongoing studies are investigating the effectiveness of different insecticides to not simply kill mealybugs but reduce transmission rates.

Mealybug Vectors and Grapevine Leafroll Disease: Temporal and Spatial Studies to Understand the Relationship Between Vector Populations and Disease Incidence

Grapevine leafroll disease is a threat to vineyard health and sustainability worldwide. Mealybugs are the main vector of consequence. This project continues to provide critical information on the relationship between mealybug populations and incidence of leafroll disease in commercial vineyards. In 2011, we completed our 2nd year of data collection in fourteen, 10 acre study areas in Napa County. Near harvest time in each of the previous two years, we have collected data on (1) mealybug populations in grape clusters and (2) symptoms of leafroll disease incidence. Over the course of the study period, this work will accurately map chronological changes in vector density and location with reference to leafroll disease incidence. This study is the first of its kind; ultimately, differences in leafroll disease incidence among sampled vineyards can be used to model leafroll disease epidemiology, as related to vector density and location, virus species, and vineyard cultural practices. A similar study with a collaborator in Oregon is modeled on this work. These data, in combination with information on virus transmission and mealybug control strategies, will facilitate the development of management strategies for grapevine leafroll disease. We are also developing the use of sticky traps as sampling tools. Baited with pheromone lures, these traps attract and capture male grape mealybugs, and are used to follow the flight patterns of the male. Consistent with previous field observations on sessile grape mealybug populations, we have verified that there are two male grape mealybug flights per year in Napa County. Peak flights occur in June and September/October, respectively. Sticky traps have the potential to be more sensitive than field surveys for detecting small populations of grape mealybug. As historical trap catch data are collected, they may also be used to track changes in mealybug populations over time. Additionally, trap catch data may be used to predict events in the life cycle of the grape mealybug. The ability to accurately predict these events is essential for appropriate timing of insecticide applications. In 2012 and 2013, we propose to work with Dr. Daane to develop a predictive model for grape mealybug development, similar to work that was completed for the invasive vine mealybug. Once laboratory studies of grape mealybug development are complete, we will use the trap catch data generated in this study to verify the development model under field conditions. Once developed, the model has the potential to allow growers to predict events in the life cycle of the grape mealybug, eventually leading to optimal timing of insecticide applications on a field-scale.

Mealybug Pests and an Emerging Viral Disease: Temporal and Spatial Studies of Leafroll Disease and Mealybug Vectors

Grapevine leafroll disease is a threat to vineyard health and sustainability worldwide. Mealybugs are the main vector of consequence. This project is providing critical information on the relationship between mealybug populations and incidence of leafroll disease in Napa County vineyards. In the first of a three-year project, we identified thirteen vineyard study sites in Napa County. We collected data on mealybug damage to the fruit and disease incidence at each site, just prior to harvest. We will continue to do this each year during the next two growing seasons. We will then analyze the data to correlate vector incidence to disease. We are also developing the use of pheromone-baited sticky traps as sampling tools. Growers have traditionally relied on field sampling to provide information on mealybug life cycle and density. Sticky traps have the potential to be more sensitive than field surveys for detecting small populations of grape mealybug. We have continuously deployed pheromone-baited sticky traps at the thirteen vineyard study sites in Napa County. We detected grape mealybug males at all field sites. We gathered preliminary information on male flight patterns from June to December, 2010. One peak in the male flight was detected near harvest, corresponding to published information on the grape mealybug life cycle.

Sustainable Controls for Vine Mealybug

The 2006-2010 vine mealybug studies were funded, for one or more seasons, by the American Vineyard Foundation, Central Valley Table Grape Pest and Disease District, California Table Grape Commission, California Raisin Marketing Board, Viticulture Consortium West, and CDFA?s Biological Control Program. Sustainable control research focused on development of a mating disruption program and improving biological controls. The potential for mating disruption was initially studied from 2004-2006, with field experiments located in the Central Valley, the Northern Interior Winegrape region, and the Central and Northern Coastal Winegrape regions. Results showed mating disruption, in combination with an in-season insecticide, often reduced numbers of pheromone trap catches (adult males) and crop damage. Development of a commercial program was studied in 2007 and 2008, after which we successfully wrote a Section 18 to allow the commercial sale of mating disruption. In the 2009 and 2010 seasons, we investigated the use of mating disruption with reduced insecticide use. Results suggest mating disruption can be a part of sustainable mealybug control. Keys to its success include pre-conditioning the vineyard to lower mealybug density (e.g., insecticides), multi-year applications of dispensers, early dispenser deployment (e.g., typically May), in-season insecticide applications as needed, and management practices that promote natural enemies. Understanding and improving biological controls of vine mealybug began in 2003, with surveys of resident natural enemies. In 2005, we released commercially available parasitoids from Europe (Leptomastidea abnormis and Leptomastix dactylopii). In 2005, we also began foreign exploration and importation of parasitoids, eventually collected material in the Mediterranean (Spain, Portugal, France, northern Italy, and Sicily), Middle East (Israel, Egypt and Iran), and South Africa. In 2006 and 2007 studies focuses on production and release of A. pseudococci (from northern Italy) and Coccidoxenoides perminutus (from South Africa); ~75,000 A. pseudococci and ~500,000 C. perminutus were released in California vineyards. Recoveries of these parasitoids suggest all four species have established in California vineyards, with A. pseudococci the most important species. In 2009 and 2010, studies investigated A. pseudococci geographic strains, with molecular work showing distinct separation of populations (e.g., eastern Spain compared with Israel). Observations in the insectary and field cage studies suggest that A. pseudococci material from Spain may be best able to suppress vine mealybug populations in California. This parasitoid material has recently been released to commercial insectaries for greater statewide distribution. In 2009 and 2010, we investigated the combined use of mating disruption and biological controls. Studies in commercial vineyards showed little impact of parasitoids, in part, due to low mealybug densities and the use of non-selective insecticide materials. Field studies showed no increase in parasitism on vines with a mating disruption dispenser.

Mealybug Pests and an Emerging Viral Disease: Vector Ecology and Their Role in Grape Leafroll Associated Virus Epidemiology

Grapevine leafroll-associated viruses (GLRaV) are a complex of viruses that cause leaf chlorosis and leaf margins to ‘roll’ downward. GLRaVs can reduce berry yields up to 40{aed9a53339cdfc54d53cc0c4af03c96668ab007d9c364a7466e3349a91bf0a23}, delay fruit maturity, and impede fruit pigmentation. During the past decade, there has been an unexplained increase in disease incidence and damage. GLRaV can be spread from vine to vine by several species of mealybugs and soft scales. Our work concerns GLRaV field epidemiology with respect to its insect vectors. In field studies, we evaluated grape mealybug acquisition and transmission of GLRaV-3 from trunks, spurs, canes, and leaves. Less than 2.7{aed9a53339cdfc54d53cc0c4af03c96668ab007d9c364a7466e3349a91bf0a23} of mealybug crawlers, acquired GLRaV-3 from vines in the field and transmitted leafroll virus to vines in the greenhouse, compared to 0.7{aed9a53339cdfc54d53cc0c4af03c96668ab007d9c364a7466e3349a91bf0a23} in 2009. This is a lower acquisition rate than previously reported from greenhouse and laboratory studies. This acquisition percentage is likely influenced by the seasonal variation of GLRaV titer in the plant, and the feeding and survival of mealybugs on field-grown grapevines. Future studies will determine seasonal changes in the acquisition and transmission rate to provide better guidelines for insecticide applications for vector control. We continued a five-year field trial testing the impacts of ?zero tolerance? for mealybugs on GLRaV infection establishment and spread. A newly-established 20 acre vineyard, bordered by older blocks that contain both GLRaV-infected vines and mealybugs, received selective pesticide treatments in 2009 and 2010. No mealybugs were found in visual inspections of control and treatment plots in June and August. However, pheromone traps showed the presence of male grape mealybugs in both treatments, indicating the possibility of an ephemeral mealybug population moving into the vineyard block, or a resident population that was too small to find using a visual search. In the first year of the trial, all vines were inspected for GLRaV symptoms and 1 vine tested positive for GLRaV-3, while in the second year, 2 vines tested positive for GLRaV-3. The trial will continue for three more years and the results will show whether blocks can be established free of GLRaV though the use of annual insecticide treatments to eliminate mealybug vectors. We investigated grape phylloxera as a possible vector of GLRaV. Previous studies in New Zealand excluded this insect as a vector and we consider this to be the standard guideline. Nevertheless, we are conducting trials to alleviate grower concerns and eliminate the possibility that phylloxera play a role in GLRaV transmission in California. In 2009, 5{aed9a53339cdfc54d53cc0c4af03c96668ab007d9c364a7466e3349a91bf0a23} of grape phylloxera tested positive for GLRaV-3 and none tested positive for GLRaV-2, after six months on plants with GLRaV-2 and -3. In 2010, additional plants with GLRaV-1,-2-3, and -5 were infested with phylloxera in the greenhouse, and none of the 125 insects tested positive for GLRaV after one generation on the leafroll-positive plants. We note that this does not show that phylloxera can transmit GLRaV, and pathogen acquisition is only the first step in transmission by a potential vector. We stress that at this point in time we the standard guideline remains in place and we do not consider phylloxera to be an important GLRaV vector.