Development of a Simulation Environment for Pathogen and Pest Spread in Vineyards

The overall goal of this research has been to develop an innovative modeling platform that can accurately simulate pathogen and pest spread in vineyards. The simulation tool will serve to help producers evaluate disease and pest management decisions using “virtual” crops. This system will allow producers to evaluate “what if” scenarios and to examine how to isolate individual management decisions that influence disease, pest, and plant development. The system can also be used to examine how row orientation, training system, etc. interact with climate and geography at new vineyard locations.

Completed research to date has focused on model development and integration. The project has successfully produced a model framework that integrates previously developed models for climate, plant growth, spore dispersion, pathogen infection, and colony growth. The system is currently able to simulate plant growth and disease progression throughout a growing season. An initial “vineyard builder” tool has been developed to rapidly build up the geometry of a particular vineyard of interest within the simulation system. Work is also underway to develop improved sub-models for meteorology and turbulence. The meteorological model will predict the three-dimensional turbulent wind field, which drives the airborne dispersion model. This work has involved comparing model outputs to field measurements, and making necessary modifications to the model to improve agreement between the two. Other work is developing improved models for airborne particle deposition to plant surfaces.

The overall modeling platform consists of a suite of coupled sub-models that represent the most important physical processes of disease spread such as plant growth, local climate, airborne dispersal by the turbulent wind, pathogen infection and colony growth. These state-of-the-art sub-models are among the most detailed simulation tools that have ever been developed for agricultural crops. Since they require substantial computational resources not provided by the processors of a standard desktop or laptop computer, we have overcome this limitation by using standard computers with a gaming graphics card. We have used the graphics card to accelerate many of the sub-models, meaning that very large simulations can be performed in a matter of minutes.

Egg Parasitism of the Virginia Creeper (Erythroneura ziczac),A Newly Invasive Leafhopper Pest in California

Grape growers in Mendocino/Lake County are experiencing outbreaks of the Virginia creeper leafhopper (Erythroneura ziczac) [Hemiptera: Ciccadellidae]. Feeding by E. ziczac causes leaf stippling, loss of photosynthetic capacity and can ultimately reduce crop yield and quality. This leafhopper is also thought to transmit the newly discovered grapevine virus “RedBlotch Disease”. The primary egg parasitoids of the Virginia creeper leafhopper (VCLH) are Anagrus daanei and Anagrus tretiakovae [Hymenoptera: Mymaridae]. A related vineyard pest, the Western grape leafhopper (Erythroneura elegantula, WGLH) is also parasitized by A. daanei as well as Anagrus erythroneurae. VCLH and WGLH are commonly found together in many North Coast vineyards. In California, A. daanei is the parasitoid species of most importance for VCLH control, as A. tretiakovae has never been found in California.

Over the past year we focused on determining parasitism levels and parasitoid species present in vineyards infested with VCLH and WGLH. Mendocino County surveys found that VCLH parasitism was practically non-existent while parasitism of WGLH eggs occurred with relatively high frequency. We isolated and reared the Anagrus species attacking WGLH eggs in these vineyards and found 87{aed9a53339cdfc54d53cc0c4af03c96668ab007d9c364a7466e3349a91bf0a23} A. erythroneurae and 13{aed9a53339cdfc54d53cc0c4af03c96668ab007d9c364a7466e3349a91bf0a23} A. daanei. While A. daanei is known to attack both WGLH and VCLH eggs, they are only attacking WGLH in Mendocino County. We subsequently reared Anagrus specimens from parasitized VCLH eggs from a vineyard in Yolo County. These specimens were identified as A. daanei. This finding brings into question the A. daanei populations found in these two counties – why is A. daanei attacking VCLH in Yolo, but not in Mendocino County? We will address this with our work in 2014.

We sampled for Anagrus and leafhopper species in the natural and cultivated habitats surrounding North Coast vineyards. While A. erythroneurae could be found on many host plants, we found A. daanei was very restricted in host diversity and overall in low abundance, which could explain the lack of VCLH parasitism. While we did find small populations of VCLH and WGLH on a variety of non-crop plants during the growing season, both pests appeared to overwhelmingly prefer cultivated grapes during the growing season and in the winter reside in vineyard leaf litter. The most common non-crop host was wild grape and VCLH actually appears to be reproducing on it. Work in 2014 will further evaluate VCLH use of wild grapes as refugia and reproductive sites.

We conducted a spray trial to determine effectiveness of OMRI approved products for VCLH control. Three insecticides were tested: Pyganic®, Mycotrol® and Grandevo™. Applicationtiming was scheduled to target young leafhopper nymphs (mid-June). Pyganic® significantly reduced nymph populations compared to the control while Mycotrol® and Grandevo™ were not significantly different from the control after the first or the second application. Further trials are planned in 2014 to evaluate application timing and frequency for non-OMRI products.

Bio-Economic Analysis of Grape Leafroll Virus Epidemics in California

The work in this research project concerns three things. First, it is intended to improve understanding of what controls the spread of leafroll disease within and between vineyard blocks. Second, it aims to work out costs for finding and dealing with leafroll infections in California vineyards so that growers can make better-informed choices about disease management. Lastly, it is intended to look at some of the difficult issues concerning cooperation and shared costs and impacts in managing leafroll at a neighborhood level, and to act as a focus for outreach from UC Davis to support the grower community and UC Cooperative Extension in tackling leafroll disease.

Our analysis of leafroll disease progress data shows that the disease develops in a predictable way irrespective of grape variety. The disease is typically introduced to healthy vine blocks at random locations, consistent with dispersal of mealybug juveniles in wind gusts. Spread between infected and healthy blocks may cause these initial infections to edges of healthy blocks, but random infections, well away from the edges, are also possible. Random initial infections could also arise, in theory, from infected planting material, but cases where this happens would be expected to show up one to two years after block establishment or vine replacement and so should be identifiable by reference to block age when disease first appears. Once introduced to a block, disease intensifies around the initial infection in a way that is consistent with mostly plant-to-plant spread of mealybug crawlers.

The research on epidemic dynamics feeds into our second area of work. As part of the epidemiology studies we have characterized the degree of clumping of diseased vines around the initial infections. This statistical analysis of the pattern of diseased vines allows us to calculate the effect of clumping on sampling efficiency for detecting the disease. That is, we can work out how the tendency for diseased vines to occur in small focused patches initially affects the efficiency of time spent sampling for disease and also on the accuracy of estimates of the level of disease. In general, the level of patchiness we find for leafroll has significant impacts on both the efficiency of sampling and the certainty of estimates based on sampling. We provide some illustrative results from this analysis.Neighborhood groups for managing leafroll have now been established in the Napa region, partly in response to suggestions made in the early stages of this project. We have extended the work reported last year on attitudes among growers to include representatives of the grapevine nursery industry. The results show that individuals from nursery trade have a similar range of attitudes towards leafroll as growers. There was some evidence that different nursery companies may have a recognizable company-level collective attitude, but the sample size is small. Our modeling work of disease dynamics at the neighborhood scale has highlighted the importance of disease management within existing infected blocks. The contribution of new infections from infected planting material is relatively small when there is a high background level of disease from existing infections.

Grapevine Leafroll Disease: a Detailed, Broad-scope Study of Host and Pathogen Effects

Grapevine leafroll disease causes non-uniform maturation of fruit in Vitis vinifera, including poor color development in red grape varieties. The disease causes losses of as much as 20-40{aed9a53339cdfc54d53cc0c4af03c96668ab007d9c364a7466e3349a91bf0a23}, with delays of 3 weeks to a month in fruit maturation. To date 5 different viruses, namely Grapevine leafroll associated virus (GLRaV) types -1 through -4, and -7, have been conclusively shown to be associated with leafroll disease. In the case of GLRaV-4, several distinct leafroll disease-associated virus strains have been identified within the virus species. This project was planned as a detailed study of the effects of these viruses on variety Cabernet Franc grapevines. This grapevine produces a readily scored foliar response to leafroll virus infection. The analysis includes challenges with each agromonically significant GLRaV species, including types -1 and -2 (2 isolates each), -3 (3 isolates), -4, -5, -7 and -9 (one isolate each). Also, pairwise combinations of GLRaVs -1, -2, -3, -5 and -7 are being tested. The test vines are grafted onto a broad selection of different rootstock varieties. Nine different rootstocks are involved in the test, including AXR #1, Mgt 101-14, 110R, 3309C, 5BB, 420A, Freedom, St. George 15 and St. George 18. 15 replicates for each treatment are divided into three separate blocks each (5 replicate per treatment per block). The project has thus-far revealed a spectrum of differences in infection symptoms attributable to the different virus species, and to different combinations of these viruses and the grapevine varieties they infected. For example, it was observed that leaf symptoms produced by GLRaV-3 were more severe than those produced by GLRaV-4. In another example, it was found that GLRaV-2 induced more severe reactions on vines propagated specifically on rootstocks Freedom and 5BB. Those test vines exhibited red leaf symptoms, short internodes, and a near-lethal decline in vigor. Detailed analysis of these and other specific aspects of leafroll disease are on-going. Data collected from the experiment in 2011 revealed one particularly severe infective combination. Virus isolate LR132 (which contained both GLRaV-1 and Grapevine virus A) produced a severe infection in Cabernet Franc plants propagated on rootstocks 420A, Freedom, 3309C and 101-14. Many of these plants died a few months after inoculation. Whether the severity is due to a particular strain of GLRaV-1 found in the LR132 isolate, or to a synergy arising from the mixture of GVA with GLRaV-1 in the inoculums is under investigation.

Grapevine Canker Diseases in California

The screening for potential fungal and bacterial epiphytes and endophytes recovered from grapevine pruning wounds to use as a biocontrol agents continued. Fifty isolates of fungi and bacteria were isolated from grapevines of different ages and cultivars growing in California and were identified based on morphological characteristics and via PCR. The retrieved isolates were tested both in vitro and in planta as potential antagonists against Botryosphaeria sp. and Eutypa lata. The preliminary screening for antagonistic isolates was performed in vitro considering inhibition of mycelial growth, inhibition of spore germination, and production of volatile antifungal compounds. Among those tested, one bacterial isolate (bp1) and two Trichoderma viridae isolates (UCD1 and UCD2) showed a strong inhibitory effect in vitro against Botryosphaeria spp. and Eutypa lata. Macroscopic and microscopic observations suggested that the inhibition of pathogens growth by both the bacterial isolate and the Trichoderma viride could be due by the secretion of antifungal metabolites. A direct mycoparasitism was also observed shortly after the pathogens were put in contact with the fungal antagonist. Further in vitro experiments were conducted using French squared bottles to assess the ability of the biocontrol agents, as well as of new experimental products, to protect grape wood chips from pathogen colonization. Standard fungicides normally used in the control of grape canker diseases such as myclobutanil (Rally) and thiophanate methyl (Topsin M) were used as reference controls. Preliminary results showed that wood chips treated with biocontrol suspension (bp1, UCD1 and UCD2) exhibited the same level of protection as the fungicide mixture (Rally + Topsin) used as controls. Overall, fungal growth on wood chips was prevented with Bp1, UCD1 and UCD2 and experimental product mixture for a longer incubation time than wood chips treated with Rally and Topsin. These promising in vitro results were confirmed by in planta evaluation conducted in two field trials. Pathogens were not recovered from the pruning wounds treated with Bp1 bacterial suspension. Visual observation indicated no phytotoxicity on buds and leaves treated with Bp1. However, bp1 is a known human pathogen in compromised individuals so we are dropping the species from the testing. We are though still trying to see if the metabolites produced by this bacterium might be used for control. Both Trichoderma strains are currently under field investigation. Based on the obtained results we have been able to assess the optimal application time of biological agents in order to guarantee an adequate protection of the pruning wounds. In fact, the antagonist organisms need to fully colonize the pruning wound surface in order to function against canker pathogen infections. We are also evaluating dormant fungicide treatments using spray applications by tractor for canker disease prevention under ?natural? disease pressure. A fungicide trial (third year) is being conducted in a vineyard in Napa County as a five year study to asses this objective.

Grapevine Canker Diseases in California

Fungal and bacterial epiphytes and endophytes have been recovered from grapevine pruning wounds and potential biocontrol isolates were identified using both morphological and genetic characters. The retrieved isolates have been tested both in vitro and in planta as potential antagonists against Botryosphaeria sp. and Eutypa lata. Among the fungal isolates, Cladosporium sp., Penicillium sp., Trichoderma sp. and Aureobasidium sp., showed inhibitory in vitro activity against Botryosphaeria spp. Macroscopic observations suggested that the inhibition of pathogen growth could be due to secretion of antifungal metabolites. A direct mycoparasitism was also observed shortly after the pathogen was put in contact with the antagonist. A preliminary screening for antagonistic bacterial isolates was done considering radial growth inhibition zone, inhibition of mycelium growth, inhibition of spore germination and production of volatile antifungal compounds. Among all the bacterial isolates tested, one strongly inhibited pathogen mycelial growth. Furthermore, both fungal and bacterial endophytes/epiphytes as well as new experimental products were tested in vitro for their ability to protect grape wood chips from pathogen colonization. Standard fungicides normally used in the control of grape canker diseases were used as reference controls. Based on these tests we have identified new microorganisms and experimental products that function on the wood surface longer than the fungicide mixture used as a reference. These promising in vitro results have been confirmed by the first year field trial. Topsin+Rally, B-LOCK and Elite, among all treatments, offered the best level of control of the pruning wounds against Botryosphaeria sp. Among the bio-control organisms, the fungi Cladosporium sp. and Penicillium sp., exhibited levels of control similar to that of the fungicides used as references. The bacterial isolate was better than fungi and chemistry in preventing infection by either Eutypa or Botryosphaeria spp. Based on the obtained results we have been able to assess the optimal application time of biological agents in order to guarantee an adequate protection of the pruning wounds. In fact, the antagonist organisms need to fully colonize the pruning wound surface in order to function against canker pathogen infections. New experimental products for the control of the disease are also under field investigation and the preliminary results were excellent and their potential to be registered as new commercial products is good. We are also evaluating dormant fungicide treatments using spray applications by tractor for canker disease prevention under ?natural? disease pressure. A fungicide trial (second year) is being conducted in a vineyard in Napa County as a five year study to asses this tractor application objective. As far as the investigation on the secondary metabolites produced by L. theobromae is concerned, we have identified 17 specific metabolites which appear to be related to the maturity and developmental stage of the cells within the colony and thus might be associated with different metabolic processes during the life of the organism. These molecules bear very stable structures, typical for cyclic and aromatic compounds. Further elucidation needs accurate mass spectrometry to determine elemental compositions and to investigate the role of these metabolites in the infection process. Finally, we have shown that Phomopsis spp. contribute to grapevine canker formation. All isolates tested in fact, were pathogenic towards six different grapevine cultivars being capable of causing V-shape cankers in the inoculated canes.

Etiology and Detection of the Cause of Syrah Decline in Syrah Grapevine

Syrah decline disease is a well documented problem in California and France. It is characterized by swelling and cracking of the graft union, stem pitting and grooving, and premature leaf reddening. French scientist have been studying this problem since 1999 and have failed to find any correlation with genetic incompatibility, known pathogens (viruses, bacteria, fungi, viroids), or environmental conditions. The potential of the Syrah decline disease to be associated with a virus is suspicious and the symptoms observed support the hypothesis. In a search for viruses associated with decline symptoms of Syrah grapevines, we have undertaken an analysis of total plant RNA sequences using dsRNA as template and the Life Sciences 454 high-throughput sequencing. In phase 1 (year 1) of this project we selected FPS Syrah clone 6 which was showing sever pitting, grooving and wood necrosis on the woody cylinder including die back and declining. For control a healthy looking Syrah clone 8 was selected. The data revealed that the Syrah clone 6 supported a mixed infection that included seven different RNA genomes including 4 viruses and 3 viroids. In the second phase (year 2) of the project, 5 more syrah clones were selected. These clones included three which are reported to produce high incidence of Syrah decline syndrome (clons B0 and B1 obtained from France and clone 99) and 2 with moderate incidence (clones 525 and 877). Total of 76.6 megabases of sequence information, from 371,906 fragment reads (each approximately 200 bases long) were initially produced from these five source vines in which 354,441 were high quality reads. The assembly and sequence analysis of the quality reads showed that Grapevine rupestris stem pitting associated virus (GRSPaV) was present in all of these vines with the highest concentration (based on the number of reads) compared to any other viruses found in these vines. Grapevine rupestris vein feathering virus (GRVFV) and Grapevine redglobe virus (GRGV) were found in clones 99 and 877 and Grapevine syrah virus 1 (GSyV-1) was found only in clone 877 in a very low titer. In a different experiment detailed information was obtained in regards to the population of the strains of the viruses found in these plants. In this study, for example, the sequences of GRSPaV found in each Syrah clone were compared with the sequences of different strains of the virus found in the GenBank. All 7 Syrah clones used in our investigation were carrying all 9 different strains of the virus (reported up to date) except for clon 525 which lacked strains Hail and Char (both reported from Japan). However, large amount of GRSPaV sequences still available from each Syrah clone that did not match the sequences of any existing strain and they may belong to new strains of the virus.

Epidemiology of Botryosphaeria spp. and the control of trunk diseases

Botryosphaeria species have emerged as important diseases in vineyards throughout the world. Because there are no cures for these diseases, one possible control strategy is to surgically remove all of the infected tissue, retrain the vines, and then use a variety of cultural and/or chemical controls to manage the disease. In collaboration with Sutter Homes vineyard, we established two trials in Zinfandel vines, one in 2005 and the other 2006, in a vineyard that is challenged by Botryosphaeria obtusa (=Diplodia seriata). The first years of the trials were described previously Epstein et al. & Huffsmith et al. (2008); the vines have regrown vigorously. This year, vines surgically treated and retrained four years ago yielded an average of 54 pounds of fruit per vine. However, the vines have also become reinfected at a much faster rate than we anticipated, and we now postulate that a key to Botryosphaeria species control is to start with clean plants from the nursery. Our trial has demonstrated the following. Painting the pruning wounds with the durable paint Duration does not cause phytotoxicity and has significantly reduced disease, in some but not in all measures of incidence. Destructive sampling has indicated that 29{aed9a53339cdfc54d53cc0c4af03c96668ab007d9c364a7466e3349a91bf0a23} of the pruning cuts were not completely painted to the margins of the wound; the data are consistent with the notion that tiny crevices may be prime infection sites. In the unpainted vines, we estimate a minimum of approximately 25 infections and 8 infections per vine in the four and five-year-old trials. B. obtuse mycelium can grow and induce the plant to release the compounds that cause discoloration from infections that originated on other pruning wounds. That is, shoots in which there has never been a pruning cut can be discolored at the time of pruning. In both trials, in January 2010, over one-third to half of the new pruning cuts were apparently already infected. Currently we are culturing pathogens from the trial so that we can assess the reliability of our visual assessments, and can better evaluate the rapidity of pathogen dissemination in the vineyard. Our current hypothesis is that the surgically retrained vines were reinfected from conidia in pycnidia that remained on the rootstock after surgery; this is consistent with our previous spore collection data. In our final objective, we tested phosphite as a treatment for grapevine trunk diseases; although the literature only indicates that it can be effective against some of Oomycete pathogens, some growers are interested in its use. In a greenhouse trial, we inoculated five pathogens and a mock-inoculated control into 3309 rootstock, and then estimated the amount of fungal growth by measuring the amount of discoloration. Based on the amount of growth in the untreated tissue, we list the pathogens that we tested from most to least pathogenic: Phaoacremonium aleophilum; Phaeomoniella chlamydospora; Botryosphaeria obtusa, Botryosphaeria rhodina, and Eutypa lata. Phosphite (1{aed9a53339cdfc54d53cc0c4af03c96668ab007d9c364a7466e3349a91bf0a23} Nutri-Phite® P+K) was applied as a drench to soil three times at six week intervals. There was no evidence that phosphite reduced fungal growth in the rootstock.

Grapevine Canker Diseases in California

Since Eutypa lata (Diatrypaceae) was first identified in California in 1975, grapevine cankers and consequent dieback have been attributed mainly to Eutypa dieback. However, research conducted in our laboratory in the past few years has shown that dieback of grapevines in California is complex and more than one fungal genus contributes to the dieback observed in the field. Our studies have shown that grapevine cankers can be caused by at least 25 different fungi in the Diatrypaceae, Botryosphaeriaceae, and Valsaceae families. Furthermore, field studies revealed the family Botryosphaeriaceae to constitute the main fungi isolated from grapevine cankers statewide and species such as Lasiodiplodia theobromae, Neofusicoccum parvum, Neofusicoccum luteum, and Neofusicoccum australe were shown to be much more virulent than the well-known pathogen E. lata. In addition, we have recently shown the pathogenicity of 8 different Diatrypaceae species on grapevines in California. Moreover, less known fungi associated with grapevine canker diseases in California such as Eutypella (Diatrypaceae) and Phomopsis (Valsaceae) species were also frequently isolated from grapevine cankers in table-raisin cultivars in both the San Joaquin Valley and Southern California and their role in grapevine health in California is currently under investigation. Preliminary pathogenicity tests have suggested that these fungi constitute new pathogens in table grape areas capable of colonizing wood and producing cankers. Identification work suggested that these fungi may constitute new species and more work is being conducted to characterize these fungi. Spore trapping studies conducted for the family Botryosphaeriaceae during the past three years have allowed us to better understand the epidemiology of this new group of fungi in California. Spore trap studies have shown Botryosphaeriaceae spores to be mainly trapped following rainfall events and overhead sprinkler irrigation statewide. Botryosphaeriaceae spores were trapped frequently from the first fall rain through the last spring rains coinciding with September to April. Furthermore, these studies have allowed us to characterize low infection risk periods throughout the growing season and therefore, improving appropriate timing periods for pruning. Results from the spore trapping study conducted in Coachella Valley showed a high incidence of Eutypella species. In this case it was documented that spore release occurred during sprinkler irrigation and also by drip irrigation. Surveys for the perithecia of Botryosphaeriaceae in California have shown various grapevine cultivars with perithecia of B. dothidea suggesting that the sexual stage could also play an important role in the epidemiology of the disease. More work is being done to understand the role of native and ornamental trees adjacent to vineyards in the epidemiology and disease cycle of canker diseases. Finally, our laboratory has developed chemical, cultural and organically acceptable control methods to reduce infections caused by these fungi. Accordingly, we have identified boron as an organic molecule for the control of E. lata. Additionally, we have proven the efficacy of several fungicides in reducing infection caused by at least 10 different fungi in 4 different families when apply as pruning wound protectants or as dormant spray applications. Furthermore, we have shown both double pruning and late pruning as a very effective cultural practice to reduce infections caused by both Diatrypaceae and Botryosphaeriaceae fungi in California. More work is being done to improve and implement single and combined applications of different active materials to control canker diseases. Finally, we showed that dormant application of fungicides with a penetrating surfactant did not cause significant phytotoxicity hazard to grapevines.

Effect of leafroll disease on grapevine drought tolerance and cold hardiness

This project is part of an interdisciplinary study into the influence of one of the most devastating viral diseases of grapevines, namely grapevine leafroll disease (GLD), on vine performance, and the integration of the resulting information into decision management tools for growers. In 2009 we evaluated plant water status, leaf gas exchange, and cold hardiness of Merlot grapevines with and without GLD. The vines are grown in a commercial vineyard and drip-irrigated using regulated deficit irrigation. We found no differences in gas exchange and plant water status before veraison, but GLD was associated with lower photosynthesis during fruit ripening. This, in turn, led to partial closure of the stomata and decreased water loss by transpiration. This response, in combination with reduced shoot vigor, improved plant water status of GLD-affected vines late in the season. Thus far we have found no differences between healthy and infected vines in the cold hardiness of buds and cane bark (phloem) and wood (xylem), but we are only in the middle of the first winter season. Although they are, of course, still preliminary, the results from the first of three field seasons seem to confirm our expectation that GLD might improve drought tolerance of grapevines and that the virus may at least not have a detrimental effect on cold hardiness. We would like to caution growers not to draw any premature conclusions, before additional data confirm or otherwise these results.