SUMMARY Spray-Induced Silencing of Grape Powdery Mildew Genes to Reduce Powdery Mildew Growth PI: Dr. Mary Wildermuth, University of California, Berkeley Senior researcher: Dr. Jyoti Taneja, University of California, Berkeley Powdery mildew is the dominant disease of grapevine. It infects all grape varietals and vines are typically treated 9-11 times over the growing season. Despite well-planned treatments, infection with powdery mildew can still occur and tolerance levels on grapes is very low. Many commonly used fungicides for powdery mildew control are becoming less effective as powdery mildews develop resistance and there is a demand for safer treatments. In this multi-year project, we are developing a novel biological powdery mildew control. The process or technology is called Spray Induced Gene Silencing (SIGS), where the RNAi molecules are designed to target powdery mildew genes that are essential for infection development and growth. Using endogenous RNAi machinery, the designed long double-stranded (ds) RNA is processed into multiple small interfering RNAs (siRNAs) that result in cleavage of the powdery mildew target gene transcript and its degradation resulting in reduced powdery mildew growth and reproduction. In previous funding years, we developed and optimized the method for SIGS assay using CYP51 as target gene, which is a target for FRAC group-3 fungicides. Using our optimized multi-pronged method for selecting conserved powdery mildew genes likely to be critical to powdery mildew growth and reproduction, we screened numerous target genes for their ability to control powdery mildew when silenced using SIGS, and have filed a PCT patent application with >200 of predicted and tested targets. We developed two pathosystems – the Arabidopsis-G. orontii powdery mildew system for ease of use and the grapevine-E. necator powdery mildew system for commercial relevance. Effective SIGS targets identified in the Arabidopsis powdery mildew system were also effective in the grapevine powdery mildew system. Furthermore, effective SIGS targets identified in growth chamber/greenhouse studies with Arabidopsis and grapevine powdery mildew systems were also effective in limiting powdery mildew in vineyard trials (performed at 2 sites in 2021 growing season). Our results suggest SIGS treatment could be used as a replacement for systemic fungicides in mid-growing season. Furthermore, the SIGS treatment had no impact on canopy or berry development, or berry chemistry. In 2022, we performed i) pilot studies to assess the systemic action of our applied dsRNA, ii) developed protocols to isolate high quality RNA from grape leaves and berries for quantification of the applied dsRNA and derived siRNAs, iii) tested two formulations/delivery methods in lab and one in a pilot field test with a new commercial collaborator to ascertain whether the formulation/delivery methods would be suitable for use and able to increase efficacy of the dsRNA treatment in reducing powdery mildew proliferation, and iv) tested two new dsRNAs against novel powdery mildew targets.
Three bacterial isolates selected from previous AVF grants (2019-2332 and 2021-2573) were utilized as biological control agents (BCAs) against fungal pathogens responsible for Grapevine Trunk Diseases (GTDs) in field conditions. The BCAs used in this study were obtained from woody tissues and the rhizosphere of grapevines grown in California; therefore, it is assumed that they have adaptations to their host and the environmental conditions of vineyards in California. The biocontrol agents were fermented for the production of secondary metabolites in liquid media and applied adopting four approaches: (i) sprayed onto pruning wounds in mature grapes; (ii) infiltrated in dormant propagation material in nurseries; (iii) injected in the trunk and cordon of mature grapevines; and (iv) poured as a soil drench treatment in the vineyard. Results of first year trials are herein presented and discussed. Among the three bacterial isolates, a positive effect was observed in treatments with Bacillus velezensis and Pseudomonas chlororaphis. These bacteria are known biocontrol agents in other plant hosts and results suggest a better performance against slow-growing GTD pathogens such as Eutypa lata and Phaeoacremonium minimum. These trials will be repeated in 2023 in order to determine the consistency of the effect of the beneficial bacteria and the influence of the season.
In the 2021 field season, eleven field sites from nine different vineyards (eight commercial vineyards and one research vineyard) in the Willamette Valley were sampled for Botrytis from May to September. Grape inflorescence, clusters, vineyard floor debris (grapevine rachis), and nearby wild blackberries were collected, incubated, and then visually accessed for Botrytis. Botrytis incidence on clusters over the field season ranged from <1% to 11%. Botrytis isolates generated were screened for fungicide resistance to Benomyl (FRAC 1), Iprodione (FRAC 2), Myclobutanil, Tebuconazole, Difenoconazole (FRAC 3), Fluopyram, Boscalid (FRAC 7), Cyprodinil (FRAC 9), Trifloxystrobin, Azoxystrobin (FRAC 11), Fenhexamid (FRAC 17) and Polyoxin-D (FRAC 19). In 2020 isolate collection, some level of tolerance was seen to all fungicide classes examined, with tolerance to more than one fungicide class observed in 35% of the 144 isolates examined to date. In 2021 isolate collection, all but FRAC 9 fungicide classes tested had some level of tolerance, with tolerance to more than one chemistry seen in 18% of tested 48 isolates examined to date. Monitoring for sources of inoculum was done by sampling dead grape rachis and cane tissue from vineyard floor and wild blackberries adjacent to the vineyard. Incidence of Botrytis on vineyard floor debris in all but one site was over 75% in late April and all sites sampled decreased over time to under 25% by September. For field sites with wild blackberries (3 vineyard sites), Botrytis on Blackberry flower parts and berries was found at low levels throughout the season. The results from 2020 and 2021 collections have been published and are available online. These results indicate a fungicide class for Botrytis management should not be used more than once in season and the Botrytis inoculum is potentially available throughout the growing season but from vineyard debris or nearby blackberries. They also indicate the benomyl resistance is not as stable as previously thought.
Powdery mildew is the dominant disease of grapevine. It infects all grape varietals and vines are typically treated 9-11 times over the growing season. Despite well-planned treatments, infection with powdery mildew can still occur and tolerance levels on grapes is very low. Many commonly used fungicides for powdery mildew control are becoming less effective as powdery mildews develop resistance and there is a demand for safer treatments. In this multi-year project we are developing a novel biological powdery mildew control. The process or technology is called Spray Induced Gene Silencing (SIGS), where the RNAi molecules are designed to target powdery mildew genes that are essential for infection development and growth. Using endogenous RNAi machinery, the designed long double-stranded (ds) RNA is processed into multiple small interfering RNAs (siRNAs) that result in cleavage of the powdery mildew target gene transcript and its degradation resulting in reduced powdery mildew growth and reproduction. In previous funding years, we developed and optimized the method for SIGS assay using CYP51 as target gene, which is a target for FRAC group-3 fungicides. Using our optimized multi-pronged method for selecting conserved powdery mildew genes likely to be critical to powdery mildew growth and reproduction, we screened numerous target genes for their ability to control powdery mildew when silenced using SIGS, and have filed a provisional patent on >100 of these targets. We developed two pathosystems – the Arabidopsis-G. orontii powdery mildew system for ease of use and the grapevine-E. necator powdery mildew system for commercial relevance. Effective SIGS targets identified in the Arabidopsis powdery mildew system were also effective in the grapevine powdery mildew system. Furthermore, effective SIGS targets identified in growth chamber/greenhouse studies with Arabidopsis and grapevine powdery mildew systems were also effective in limiting powdery mildew in vineyard trials (performed 1 this year at 2 sites). Our results suggest SIGS treatment could be used as a replacement for systemic fungicides in mid-growing season. Furthermore, the SIGS treatment had no impact on canopy or berry development, or berry chemistry. In order to increase the efficacy of SIGS for powdery mildew control, we explored multiplexing different gene targets, predicted to act in the same or different functional processes. For the combinations and dosages tested, we have not seen further reduction in the effectiveness of treatment in controlling mildew infections. We are now focusing on testing the formulations/delivery of the dsRNA to enhance stability, uptake, and systemic action. Preliminary experiments confirm the predicted systemic action of topical dsRNA, with reduced powdery mildew proliferation in unsprayed distal tissue. Once we have a good formulation/delivery method in hand, we may reexamine multiplexing SIGS targets. Our 2022-23 proposal focuses on assessing formulation/delivery methods for efficacy and systemic action in the greenhouse and field.
Eighteen blocks in 12 vineyards, located in five different counties, were identified as having clusters of vines characteristic of sudden vine collapse (SVC). The vines included eight scion varieties, two rootstock varieties, and one own-rooted. Leaf samples were collected from both symptomatic and asymptomatic vines within SVC clusters. In addition, asymptomatic vines outside of the SVC cluster were included as an indication of the overall infection status of the block. Samples were processed and tested by RT-qPCR for GLRaV-1, -2, -3, GVA and GVB. Eighty-seven percent of all vines were positive for GLRaV-3, 73% were positive for both GLRaV-3 and GVA, and 26% were positive for both GLRaV-3 and GVB. All GVA and GVB infections were co-infected with GLRaV-3. One vine was positive for GLRaV-1 but no GLRaV2 positive vines were detected. A comparison of GLRaV-3 and GVA infection rates in symptomatic versus asymptomatic vines within SVC clusters indicated that overall, the rates were slightly but significantly higher in symptomatic vines. Analysis at the block level indicated that this positive correlation was only significant in two out of 16 blocks due to the constraints of small sample size. Therefore, GLRaV-3/GVA co-infection rates in symptomatic versus asymptomatic vines were not significantly different in most of the blocks we sampled. A comparison of GLRaV-3/GVA infection rates in asymptomatic vines within and outside SVC clusters indicated that only two blocks had significantly higher co-infection rates in asymptomatic vines within clusters. Conversely, one block had significantly higher infection rates in asymptomatic vines outside the SVC cluster. These observations, coupled with the overall high percentage of vines positive for GLRaV-3 and GVA indicates that the blocks we selected for this study had high GLRaV-3/GVA co-infection rates despite the lack of disease symptoms outside SVC clusters. Fewer GVB positive vines were detected, and these infections were almost evenly divided between symptomatic and asymptomatic vines within SVC clusters, and between asymptomatic vines within and outside the SVC cluster. To track the progression of SVC in these 18 blocks over the next two years, a group of 300 SVC symptomatic and asymptomatic vines within SVC clusters were mapped by row and vine location. For the rootstock field trial, approximately 80 vines each of nine different rootstocks were propagated from dormant cuttings and chip-bud grafted with one bud from Pinot gris 09. These vines were transplanted into 1-gallon pots and are being kept in a FPS screenhouse until spring 2022. In addition, vines positive for GLRaV-3, GLRaV-3 and GVA, GLRaV-1 and GVA, and GLRaV-2 and GVB have been identified and analyzed by high throughput sequencing to verify their infection status. A field site has been identified at the UCD Armstrong Field Station and prepared for planting vines in the spring 2022.
A total of 20 vineyards from 10 counties across California were sampled during summer 2019. Cordon, trunk and root tissue samples were collected from mature vines using non-destructive methods in order to isolate, analyze and study endophytic bacterial communities between healthy looking and diseased vines exhibiting typical trunk disease symptoms. A collection of over 1,344 endophytic bacterial isolates was obtained and screened for their potential antifungal effect against the main GTD-causing pathogens in vitro. A first screening using Neofusicoccum parvum indicated that 24.7% of the collection caused over 40% of mycelial inhibition (333 isolates), and these were further tested against Diplodia seriata, Diaporthe ampelina and Eutypa lata. A subset of 90 bacterial isolates was selected by their biocontrol potential (higher inhibition percentages) against the four pathogens. Phylogenetic analyses showed that 70% correspond to Bacillus velezensis (65 isolates) whereas the remaining correspond to a broad range of Gram positive and Gram-negative bacteria, some of them known to secrete antifungal compounds. Different species are currently being tested in greenhouse experiments to elucidate their capability to colonize grapevines and protect them from trunk disease development. Furthermore, health status and trunk disease incidence were evaluated in vines that were treated with pesticides using vacuum infiltration in three commercial nurseries over the summer of 2019 and planted in the UC Davis Plant Pathology field station in October 2019. Even though the final evaluation of treated vines will be done at the end of 2021, preliminary field observations showed different levels of performance among treatments. Preliminary isolations from plant tissues showed that there were two predominant fungal groups: potentially pathogenic (Fusarium and Botryosphaeriaceae) and beneficial/plant protective (Trichoderma and Clonostachys).
While there are several disorders affecting grapevine berry development, Berry Shrivel (BS, also known as Sugar Accumulation Disorder, SAD) is a significant problem for grape growers in California, the West Coast, and internationally. BS is particularly problematic because it is difficult to diagnose, and all available evidence suggests that onset of the disorder is simultaneous with veraison. Although BS has been studied previously, several lagging questions remain; principally the potential existence of an etiological agent (i.e. pathogen), but also suggestions that only parts of a vine are affected, and how widespread the effects of sugar accumulation are in affected blocks. In the first project year, our team demonstrated that sugar accumulation of berries is uniformly lower in BS-affected blocks in relation to control blocks by several degrees Brix; similarly, anthocyanin content was reduced based on grape juice color. While preliminary, these observations suggest that BS is not a ‘rare and random’ occurrence in affected blocks, and that whole vine is affected rather than just sections of the vine. In addition, a bioinformatic analysis of publicly available data suggests that BS is not correlated with the presence of a particular RNA virus. This research is ongoing because the preliminary data are limited to RNA viruses and must be expanded to include DNA or other microbes. Altogether, in its first year, this project has provided important insights into the nature of the BS disorder and highlights the significant knowledge gaps that remain in our understanding of the impacts of this disorder.
Powdery mildews are widespread pathogens of grapevine that are difficult to control. Resistance has emerged against all current fungicides and health consequences associated with extensive use of sulfur are beginning to surface. Therefore, the grape industry is in great need of new methods for limiting powdery mildew disease. The potential of spray induced gene silencing (SIGS) in agricultural pest control has been recently realized. The method is also useful in characterizing gene function. The efficiency of SIGS has been demonstrated to control the growth of viral, fungal, insect and nematode pathogens in several plant host species. With the first year of AVF funding support, we showed that SIGS can also be effective in silencing powdery mildew genes, resulting in reduced powdery mildew growth and reproduction. We optimized dsRNA design, application method, dosage, timing of application and powdery mildew growth assessment for testing of SIGS against gene targets in both Golovinomyces orontii-Arabidopsis and Erysiphe necator-grapevine systems. With the continued support of AVF in Year 2, we screened powdery mildew genes prioritized to impact metabolic and regulatory pathways critical to powdery mildew colonization, growth, and reproduction. All selected G. orontii target genes had homologs in E. necator and are conserved among powdery mildews. dsRNA against individual target genes were designed, applied exogenously and the growth of powdery mildew was quantified. We had a 60% success rate in identifying efficacious novel targets, with reductions in powdery mildew proliferation ranging from 2- to 5-fold compared to the controls. The initial screening of target genes was done using the Arabidopsis-powdery mildew system as it was faster than grapevine. We then selected 6 genes that had shown significant reduction in G. orontii growth on Arabidopsis via SIGS and tested them in grapevine-powdery mildew system. SIGS against each of these six E. necator genes showed significant and reproducible reduction in powdery mildew growth and reproduction on grapevine. This year (Year 3),we refined our target prediction pipeline using multidimensional bioinformatic and modeling. Fifteen out of 16 novel gene targets tested, identified using this pipeline, showed significant reduction in powdery mildew growth in G. orontii-Arabidopsis system that increased our success rate of target prediction to 94% from 60% in the previous year. Five new grapevine powdery mildew genes were tested via SIGS and all of them successfully reduced powdery mildew infection. We are currently testing the effects of multiplexing SIGS 2 targets for further increase in SIGS effectiveness in controlling disease. Through the NSF-funded i-Corps customer discovery program, and interviews with 100 grapevine industry personnel, we learned of the needs and specific requirements for new powdery mildew control products. It appears our RNAi-based biopesticide has the potential to meet these needs. dsRNAs are biodegradable, flexible, and specific, with reduced negative environmental and health impacts compared with existing fungicides giving them excellent potential as future powdery mildew disease mitigation agents. We are optimizing this technology for effective powdery mildew control in the field, while testing new targets and multiplexing already tested targets. We completed one vineyard field trial in the 2020 growing season with Dr. Akif Eskalen and are conducting field tests at two locations this coming 2021 growing season.
In the 2020 field season, nine field sites from eight different vineyards (seven commercial vineyards and one research vineyard) in the Willamette Valley were sampled for Botrytis from June to September. Grape clusters, vineyard floor debris, and nearby wild blackberries were collected, incubated, and then visually accessed for Botrytis. Botrytis incidence on clusters over the field season ranged from about 3% to over 30%. Botrytis isolates generated were screened for fungicide resistance to Benomyl (FRAC 1), Iprodione (FRAC 2), Difenoconazole (FRAC 3), Boscalid (FRAC 7), Cyprodinil (FRAC 9), Trifloxystrobin (FRAC 11), and Fenhexamid (FRAC 17). Some level of tolerance was seen in all fungicide classes among isolates with tolerance to more than one fungicide class observed in 22% of the isolates. Monitoring for sources of inoculum was done by sampling dead grape rachis and cane tissue from vineyard floor and wild blackberries adjacent to the vineyard. Vineyard floor debris decreased as the season progressed but for field sites with wild blackberries (3 vineyard sites), Botrytis on blackberry flower parts and berries was found throughout the season and at one site increased dramatically at the end of the season near harvest after a significant rainfall. These results suggest that vineyard Botrytis resistance levels are of concern and should be continued to be monitored along with the changes of Botrytis inoculum to better time applications of fungicides and other integrative pest management tools.
This project focuses on the fundamental chemistries that GTD fungi causing Eutypa canker/dieback employ in infecting and causing damage to grapevines. We also are developing antioxidant/chelator (A/C) treatments for these GTD fungi which block their chemistries. Additionally, we are disseminating our findings on GTD causal systems and potential treatments to vineyard owners/managers and other parties. Our three Objectives for this two-year project are:
A) Understand the role of iron-binding compounds produced by the mixed-consortia fungi involved in Eutypa dieback, with particular focus on how these compounds generate hydroxyl radicals, the most damaging form of ‘free radical’ found in biological systems.
B) Use our enhanced understanding of Eutypa dieback to develop treatments and management strategies for the control of Eutypa GTD.
C) To disseminate and interpret our research results to vineyards personnel to allow a better practical understanding of how Eutypa dieback chemistries function in causing the disease, and disseminate results particularly related to treatments developed.
Findings to Date:
A New Causal Mechanism for Eutypa Dieback, which also has Potential for Involvement in Esca Disease: The three fungi in this study, Eutypa lata (Elata), Phaeomoniella chlamydospora (Pch), Phaeoacremonium minimum (Pmin) were all found to produce low molecular weight (LMW) polyphenolic compounds that reduce iron. Pch produced the greatest amount of polyphenolics and, in consortia growth with Pmin, also produced the greatest amount of iron reduction. The extracted LMW metabolites from these fungi were also found to nonenzymatically produce hydrogen peroxide. This previously unreported finding is important because hydrogen peroxide will react with reduced iron to generate highly destructive hydroxyl radicals. In other fungi, this type of chelator-mediated Fenton (CMF) reaction has been found to be responsible for aggressive damage and decay of plant tissue (wood cell walls). Our data provides the first evidence that Eutypa dieback, and also potentially esca disease, is not caused by enzymatic or toxin production by fungi. Rather, a CMF mechanism is active which causes both a canker and decay of grapevine wood. We postulate that as the canker/decay develops, fungal LMW metabolites may also diffuse to the foliage to produce end-stage scion/foliar symptoms. CMF mechanisms are known to be active in fungal attack of wood, causing common brown rot decay, but the mechanism has not previously been demonstrated to be causal in GTDs. Clear evidence for production of hydroxyl radicals suggests that CMF chemistry promoted by the pathogenic fungi is responsible for decay of the wood related to canker production in Eutypa dieback.
Development of Potential Treatments for Eutypa/Esca Based on Knowledge of New CMF Disease Mechanism: Laboratory testing of both A/Cs and biocontrol agents continues. However, one of the A/Cs (butylated hydroxyanisole or BHA) is a low-cost, food-safe antioxidant that shows suitability for potential treatments. BHA is showing positive results in completely preventing growth of all three fungal pathogens at relatively low concentrations (≥ 0.5 mM). We will continue testing and, although not part of AVF funded research, plan to conduct limited testing of BHA in pathogen-inoculated grapes in summer.