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%, 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% of mealybug crawlers, acquired GLRaV-3 from vines in the field and transmitted leafroll virus to vines in the greenhouse, compared to 0.7% 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% 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.

Reproductive Biology of Vine and Grape Mealybugs and its Possible Effects on Detection, Sampling, and Control of Mealybugs

The goal of this project is to elucidate the reproductive biology of mealybugs infesting vineyards in the western United States. We were particularly interested in reproductive parameters that might have implications for the use of pheromones for monitoring or control of these mealybugs. Despite their pest status, remarkably little information is available about reproduction in mealybugs, such as whether males and females mate only once or multiple times and the frequency of mating events. During the 2010 funding cycle, the following progress was made: ? We determined that vine and grape mealybug females must be mated in order to reproduce. There is no parthenogenetic reproduction. ? We showed that both sexes of vine and grape mealybugs can mate multiple times, both on the same day and over a period of several days. ? After emergence from the cocoon, males of vine and grape mealybugs can live up to 4.5 and 7 days, respectively, whereas females live much longer (up to several months). ? Exposure to the sex pheromone produced by female mealybugs did not accelerate the development or emergence of male vine mealybugs, whereas grape mealybug males became active more quickly when exposed to pheromone. However, there was no effect of pheromone exposure on male longevity for either species. ? Although female vine and grape mealybugs will mate multiple times, they only need to mate once in order to maximize their production of offspring. Overall, these results have important implications for using pheromone for monitoring or control of these mealybug species. For example, because male vine and grape mealybugs can mate multiple times over a period of days, a relatively small number of males can inseminate most of the available females. Thus, for effective pheromone-based mating disruption, the pheromone coverage must be good, so that all males are prevented from finding mates for their entire flight period/generation. Thus, a method such as attract-and-kill using pheromone baits treated with a toxicant, that removes males from the population as soon as they contact the lure, might be as or more effective than mating disruption. In terms of using pheromone-based methods for detection and monitoring of populations, when crops are treated are not treated with pesticides, pheromone-baited traps should provide a very sensitive, reliable, and species-specific method of detection of mealybug infestations, and possibly an estimate of population sizes. However, in areas with frequent insecticide use, trap catches may be artificially low due to the greater sensitivity of male mealybugs to insecticides than females. In such cases, manual sampling or visual inspections of plants for infestations can be used as a backup.

Sustainable Controls for Vine Mealybug

As part of an ongoing foreign exploration and importation initiative for vine mealybug, Planococcus ficus, parasitoids collected in the Mediterranean (Spain, northern Italy, and Sicily) and South Africa were screened for their potential to lower mealybug populations in California. Studies in 2009 focused on geographic strains of Anagyrus pseudococci. Molecular studies showed clear separation of geographic populations in northern Italy, eastern Spain, Israel and California more similar than material collected in Argentina, Sicily. Studies conducted in large field cages suggest that A. pseudococci material from Spain may be better suited to control vine mealybug populations in California, although these results are preliminary. Contacts have been made with entomologists in Iran, Spain and Argentina for the importation of novel vine mealybug parasitoids in 2010. Contacts have also been made with entomologists in Chile and New Zealand for the importation of novel obscure mealybug parasitoids in 2010. In 2009, releases of A. pseudococci and Coccidoxenoides perminutus were made in vineyards in the in San Joaquin Valley (SJV), the Northern Interior Winegrape Region (Sacramento and San Joaquin Counties), and the Central Coast (San Luis Obispo County) regions. There were approximately 10,000 A. pseudococci Sicily, 2,000 A. pseudococci Spain, and 10,000 C. perminutus released in both open-air and caged vine trials. To date, we have recovered A. pseudococci at all sites. We have recovered C. perminutus from all regions, but not from all sites. The releases were made in combination with sustainable control tools (mating disruption and, when needed, Argentine ant controls) and a planned reduction in pesticide use, particularly organophosphates. In each region, a paired-plot design was used with sustainable and conventional treatments paired in each vineyard block, and 5-6 vineyards as replicates in each region. There were few differences between treatments as, in most cases, insecticide use did not vary between sustainable and conventional treatments. Trials investigating the rate of dispensers used in mating disruption programs showed lower trap catches when dispensers were released at 188 and 250 per acre than at 50 and 125 per acre (250 per acre is label rate). This trial will continue for two years to assess application rate for amting disruption, and the impact on parasitism levels.

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%, 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 apical, middle, and basal leaves. We found <7% of surviving mealybug crawlers, inoculated on vines in July and August trials, were able to acquire and transmit GLRaV-3. This is a lower acquisition rate than previously reported from greenhouse and laboratory studies, as would be expected. The seasonal variation of GLRaV titer in the plant, mealybug feeding performance and survival in the field, and trial conditions are possible factors, Future studies will determine seasonal changes in the acquisition and transmission rate in order to provide better guidelines for vector control decisions with respect to insecticide timing. A field trial was conducted in a newly established 20 acre vineyard, which is bordered by older blocks that contain both GLRaV-infected vines and mealybugs. In this five year trial, we are testing a ‘zero tolerance’ for mealybugs, established using selective pesticides applications, as a GLRaV control for new plantings. A June and August inspection of vines found no mealybugs in either control or treatment plots. However, data from 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 this first year trial, all vines were inspected for GLRaV symptoms and 1 vine tested positive for GLRaV. The trial will continue for four 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 began studies of grape phylloxera as a possible vector of GLRaV. Previous studies in New Zealand excluded this insect as a probably vector and we consider this to be the standard guideline. Nevertheless, we are conducting trials to alleviate grower concerns of phylloxera as a possible vector. In laboratory trials, grape phylloxera were placed on plants with either GLRaV-2 or GLRaV-3. After six months, no phylloxera tested positive for GLRaV-2, while 5% tested positive for GLRaV-3. 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.

Reproductive biology of obscure, longtailed, and vine mealybugs and its possible effects on detection, sampling, and control of mealybugs

Our goal is to elucidate parameters of the reproductive biology of mealybugs infesting vineyards, particularly those parameters that have implications for the use of pheromones for monitoring or control of mealybugs. Despite their pest status, little information is available about reproduction in mealybugs, such as whether males and females mate only once or multiple times and the frequency of mating events. Our initial target species were the obscure, longtailed, and grape mealybugs, but we have expanded the study to include vine mealybugs, based on comments received from the review panels last year. During the 2009 funding cycle, we made excellent progress in gaining a better understanding of reproduction in these insects, as follows: ? We showed that both sexes of obscure, longtailed, and vine mealybugs can mate multiple times on the same day. Male obscure mealybugs can also mate again on subsequent days, whereas male longtailed mealybugs that multiply mated on day 1 died by the following morning. Studies to determine whether male vine mealybugs mate on more than one day are in progress. For all three species, females could remate 1 or more days after the first mating. ? After emergence from the cocoon, males of longtailed and obscure mealybugs can live up to 5 days, whereas females live much longer, for several weeks to many months. ? Contrary to expectations, exposure to pheromone did not accelerate the development or emergence of male longtailed and obscure mealybugs, nor did it affect male longevity. ? Although females can and will mate multiple times, they only need to mate once in order to produce a full complement of offspring. ? We were not able to run the corresponding experiments with grape mealybug because of difficulties in establishing a stable colony of this species. Overall, these results have important implications for using pheromone for monitoring or control of mealybugs. For example, because males can mate multiple times over a period of days, a relatively small number of males can inseminate most of the available females. Thus, although males are more susceptible to insecticides than females, even if a relatively small percentage of males survived an insecticide treatment, it may still be sufficient to mate the female population. If pheromone-based mating disruption were attempted, the pheromone coverage must be very good, because males would have to be disrupted for their entire lifetimes of 4-5 days, and because males can mate multiple times. Thus, a method such as attract-and-kill, that takes males out of the population, might be a more effective strategy. In terms of using pheromone-based methods for detection and monitoring of populations, in the absence of pesticide treatments, pheromone-baited traps should provide a sensitive, reliable, and species-specific method of detection of mealybug infestations, and possibly an estimate of population sizes. However, in areas with frequent insecticide use, trap catches may be artificially low due to the greater sensitivity of male mealybugs to insecticides than females. In such cases, manual sampling for mealybugs can be used as a backup.

Spider Mite Control in Vineyards (Year 2).

Efficacy of the conventional acaricides abamectin (Agri-mek), etoxazole (Zeal), fenpyroximate (Fujimite), spirodiclofen (Envidor), acequinocyl (Kanemite), bifenazate (Acramite), hexythiazon (Onager), propargite, and horticultural mineral oil were evaluated for their effect in controlling a mixed population of Pacific spider mite and Willamette spider mite in an El Dorado Co. merlot vineyard during summer, 2007. All acaricides significantly reduced mite densities relative to the untreated control for all but one sampling date for 4 weeks following the application. During the course of the experiment, the Willamette spider mite was the dominant species present. We monitored a number of vineyards in the northern San Joaquin Valley and Napa Co. throughout the summer and were unsuccessful in our attempt to locate a treatable population of Pacific spider mite. We also evaluated efficacy of organic acaricides in a separate experiment in the El Dorado Co. vineyard. Treatments included Organic JMS Stylet Oil, GC-Mite, Ecotrol, Organocide, and M-pede. The mean motile spider mites in all of the organic treatments were significantly lower than the mean density of the untreated control plots (< 0.05) for 3 weeks following their application with the exception of M-pede on August 22. We completed a life table evaluation of the effects of newer acaricides on Pacific spider mite. At chemical concentrations equivalent to the high label rate of each product, only females exposed to Zeal, Envidor, Nexter and Agri-mek had any amount of survival as measured by longevity of females, and survival in all treatments was significantly (P<0.05) lower than observed in the control treatment. None of the females surviving except for those exposed to Zeal produced eggs, and all of those eggs were sterile. Reduced concentrations resulting in mortality of 20%of the Pacific mites treated with each acaricide clearly revealed mechanisms for the activity of these products.

Mealybug transmission of grapevine leafroll-associated viruses

Until we started working on this project, all that was known about vector transmission of Grapevine leafroll-associated viruses (GLRaV) was that mealybugs and scales were capable of transmitting this group of grape pathogens. We did not know how transmission occurred, if different mealybugs were capable of transmitting different viruses, if plant tissue affected transmission rates, etc. Our goal was to tackle some of these questions. We showed that transmission of GLRaV appears to lack vector specificity, in other words, different mealybugs may be capable of transmitting different GLRaV. We caution that this conclusion is based on limited data. We also showed, for the vine mealybug and GLRaV-3, that transmission occurs in a semi-persistent manner. That means insects can acquire and inoculate the pathogen within 1 hour of plant access, and that they loose infectivity over a few days. In addition, first instars seem to be better vectors than adults. Lastly, we analyzed the role of plant tissue on transmission rates. Under greenhouse conditions it seems that plant tissue does not affect transmission, but virus populations in the field vary dramatically and may affect disease spread rates.

Our work by no means fills all the gaps in knowledge emergently needed to develop management strategies for leafroll diseases, but it goes beyond the identification of vectors, adding information on i) what leafroll viruses may be mealybug-transmitted, ii) leafroll-mealybug specificity in relation to transmission, iii) conclusively demonstrating that transmission of at least one virus/vector combination in this system occurs in a semi-persistent manner, and iv) generating new methods and protocols, and using them, to study the ecology of leafroll diseases in relation to seasonality, virus strain, host variety, mealybug species, feeding preference. The last item will be an exciting and certainly fruitful research venue.

Sustainable Controls for Vine Mealybug: Biological Control – 2008

As part of an ongoing foreign exploration and importation initiative for vine mealybug, Planococcus ficus Signoret, parasitoids have been collected in the Mediterranean regions of Europe, northern Africa, and South Africa. Natural enemies were either sent first to the USDA-ARS laboratory in France, and then to the UC Berkeley Quarantine Facility (collections by Sforza) or sent directly to UC Berkeley Quarantine (collections by Daane). Our goals were to screen the imported natural enemies for their potential to lower mealybug populations in California, and for their possible non target impacts. In 2008, new material was collected in Spain, and a colony has been established in Quarantine. This material will undergo screening in 2009.

In 2008, releases of A. pseudococci and C. perminutus were made in vineyards the in San Joaquin Valley (SJV), the Northern Interior Winegrape Region (Sacramento and San Joaquin Counties), the North Coast (Sonoma and Napa Counties), and Central Coast (San Luis Obispo County) regions. There were nearly 54,000 female A. pseudococci produced and released, in both open-air and caged vine trials. A. pseudococci ?northern Italy? was produced in the UC Berkeley Insectary (12,200 females released) as well as the commercial ?Sterling Insectary? (36,100 females released). Field production of the A. pseudococci Sicily strain was directed from CDFA (5650 females released). From April to October, 2000-3000 female A. pseudococci per acre were released in the coastal plots, and ca. 700 females were released per acre in the interior plots. The more easily reared C. perminutus, of which 51,600 adults were released, were produced at the UC Berkeley Insectary. To date, we have recovered A. pseudococci at all sites, however, we have not yet completed the molecular work on samples to determine if the recovered material was from our release (northern Italy) or the A. pseudococci strain resident in California (Israel). We have recovered C. perminutus from all regions, but not from all sites.

Field cage studies show that combinations of the natural enemies tested (Anagyrus pseudococci, Coccidoxenoides perminutus and Cryptolaemus montrouzieri) most often work better than these species released as a monoculture. Field cage studies also suggest higher levels of parasitism and better levels of mealybug reduction when the mealybug sex pheromone was applied.

Molecular studies on vine mealybug have been completed and indicate that the material present in California and Mexico originated from Israel. Furthermore, there may be large enough differences between the European and Israel/California populations that biological difference may exist which will change the levels of damage and population sizes in the field, as well as control option. Current work is focusing on molecular difference in parasitoid populations.

Spider mites in California vineyards: temperature tolerance, effects of plant water status through leaf temperature, impact of novel pesticides and resistance management

Spider mites cause significant damage in California vineyards leading grape growers to treat more than 100,000ha annually with pesticides. The Pacific spider mite Tetranychus pacificus McGregor and the Willamette spider mite Eotetranychus willamettei (McGregor) (Acari: Tetranychidae) are the two most common spider mites in vineyards. The western predatory mite, Galendromus occidentalis (Nesbitt) (Acari: Phytoseiidae), is an important natural enemy of both spider mites.

Temperature is a critical factor influencing pest outbreaks. We evaluated the effects of temperature on life histories of the three mites and found that higher temperatures are more favorable for T. pacificus than for E. willamettei and G. occidentalis. The lower development threshold lay around 10oC for all three mites, while the upper development threshold was estimated to be 31, 37 and 40oC for E. willamettei, G. occidentalis and T. pacificus, respectively. T. pacificus completed its immature development significantly more rapidly than E. willamettei above 23oC, whereas G. occidentalis developed significantly faster than either spider mite from 11 to 36oC. The intrinsic rate of increase of the three mites followed a pattern similar to development time. The intrinsic rate of increase of G. occidentalis feeding on T. pacificus at 28oC was not significantly different than when feeding on E. willamettei.

Water stress is another factor influencing spider mite outbreaks. A field study showed that increases in ambient temperature and plant water stress increased grape leaf surface temperature. Densities of T. pacificus increased significantly with increasing frequency of leaf temperatures above 31oC while those of E. willamettei showed no relationship and those of predatory mites (Acari: Phytoseiidae) showed a negative relationship with high leaf temperatures. These results help to explain why outbreaks of T. pacificus occur in hot or water stressed vineyards, while E. willamettei develops higher populations in cool or well irrigated vineyards.

Applications of pesticides against other vineyard pests may affect biological control by G. occidentalis. The insecticides imidacloprid and buprofezin negatively affected the population growth of G. occidentalis but had no effect on T. pacificus. The fungicide wettable sulfur significantly decreased T. pacificus population growth but it did not affect G. occidentalis. In addition, the insecticides methoxyfenozide and the fungicides tebuconazole and trifloxystrobin had no effect on the population growth of G. occidentalis or T. pacificus.

Populations of T. pacificus from vineyards that reported miticide failures in recent years developed statistically significant 11-fold resistance to pyridaben, seven-fold resistance to bifenazate and four-fold resistance to propargite compared to a susceptible laboratory population. These results underline the importance of alternation of products from different mode of action groups to delay resistance development.

Seasonal biology of the Gill’s mealybug, Ferrisia gilli, in foothill vineyards

The seasonal biology of Gill’s mealybug, Ferrisia gilli, a new mealybug pest of grapes grown in the California Sierra foothills, was studied in five commercial vineyards located in El Dorado County during 2008. In each vineyard, untreated and treated vines were monitored bi-weekly by searching vine sections or the entire vine during timed searches and recording Gill’s mealybug life stage and numbers. Mealybug mummies were collected and held for parasitoid emergence and observations of predators in the field were recorded. Vineyard cooperators were kept apprised of the mealybug location and life stage and this information was used to time mealybug treatments, mostly consisting of applications of buprofezin and/or acetamiprid. Just prior to harvest, two hundred clusters were rated on a scale of 0 (no mealybugs present) to 3 (unacceptable damage) and the number of mealybugs found in fifty clusters was counted.

Specific accomplishments (2008):

  1. Tracking of Gill’s mealybug life stages during the season helped us determine it completed 2 full generations during the 2008 season.
  2. Early in the season Gill’s mealybug is best found on old and new spurs. This is important for monitoring and making insecticide treatment decisions in June. Observations showed that honeydew production does not generally take place until towards harvest, and is not a good clue to finding Gill’s early in the season.
  3. Treatments timed for the crawler/nymph stage out on leaves were targeted for late June, about a week earlier than previously targeted. Growers with neighboring blocks were well coordinated in their spray control program due to information and communication generated from this study.
  4. Cluster damage ratings showed that this pest appears similar to grape mealybug-if left untreated it can build to undesirable damage. We observed up to 42%of the clusters in untreated plots had Gill’smealybug, and 11%of these were rated either a “2” (more than ten mealybugs) or “3” (unacceptable damage).
  5. Insecticide treatments such as buprofezin (Applaud) and acetamiprid (Assail) appeared effective; however this combination is not desirable for a long term management plan due to effects of insecticide resistance and potential detriment to natural enemy populations.
  6. Parasitized Gill’s mealybugs were found at every site, however, numbers of mummies were very low in treated plots and only a few Acerophagus were reared out of collected mummies.
  7. Gill’s mealybug continues to be found at vineyards in the area. We now estimate at least 270 acres of grapes have the Gill’s mealybug in a portion of the vineyard.
  8. Our outreach strategy is to 1.) Communicate our monitoring information from this study to growers in order to properly time and increase the efficacy of their treatments and to 2.) Inform the greater extension, Pest Control Advisor, and research community in order to build awareness of Gill’s mealybug as an emerging pest in grapes. Several grower meetings, one popular press article (CAPCA Adviser magazine), one peer reviewed article (California Agriculture magazine), and a research presentation (Entomological Society of America) have been generated from this research thus far.