Evaluating alternative nursery practices to reduce graft union failure.

Graft failure can be caused by factors such as the poor formation of the graft union (due to problems with anatomical mismatching, poor grafting technique, adverse weather conditions, and poor hygiene), mechanical damage, and graft incompatibility.

Our objectives to address these issues are a) to evaluate the effect of the length of the fresh cut of rootstocks pre-grafting on graft union failure, b) to evaluate the effect of biological and chemical fungicides on dormant commercial propagation materials against saprophytes and fungal pathogens of grapevine trunk diseases.

Evaluation of the length of fresh cut of rootstocks pre-grafting on graft union failure.

On a grapevine nursery, dormant rootstock material was cut at three different lengths (¼ in, 1 in, and 2 in) and further treated with biological and synthetic fungicides by soaking the cuttings in the fungicidal solution for 10 minutes. Treated cuttings were grafted by the nursery standard protocol and transported to the laboratory for callus evaluation. Since grafting involved waxing of the graft union, it was not possible to evaluate the callusing of this tissue. However, the basal end of rootstocks were evaluated by level of circumferential coverage of the rootstock apex by callus on a visual scale (1=0% callus coverage, 2=50% callus coverage, and 3=100% callus coverage). Data was analyzed by comparing the effect of the fresh cut length on the controls alone (Fig. 1) and in combination with the fungicide treatments (Table 1) using one-way and two-way analyses of variance (ANOVA). On the first one, no significant differences were observed in callus formation between the three cut lengths (Fig. 1).

When the two factors were analyzed, the interaction between fungicide treatment and fresh cut length was significative, meaning that the effect of the fungicides depends on the length of the fresh cut. Therefore, the effect of the fungicide treatments was compared within each level of fresh cut (Table 1). The callus formation reached 90%, 100% and 86% in average in the controls previously cut at ¼, 1, and 2 inch, respectively, and no treatment improved it significantly. Differences were detected between certain treatments among the different levels of fresh cut. Under ¼-inch cuts, Topsin M and HP Fitor+Phosful yielded significantly higher callus formation than VitiSeal, OxiDate (1%) and Vintec. With 1-inch cuts, Vintec significantly increased the callusing than VitiSeal, P. chlororaphis, OxiDate (0.39%) and HP Fitor+Phosful. And with 2-inch cuts, HP Fitor+Phosful and OxiDate (0.39%) showed significantly higher callusing than B. velezensis, VitiSeal, OxiDate (1%) and Vintec.

From the Lab to the Field: Using Endophytic Bacteria as Biocontrol Agents Against Trunk Diseases in Vineyards.

Summary

Bacterial isolates selected from previous AVF grants (2019-2332 and 2021-2573) alongside other beneficial fungal isolates were investigated as potential biocontrol agents against grapevine trunk diseases pathogens. The biocontrol agents were applied as pruning wound protectants (i), as dipping treatments of propagation material prior grafting in nurseries (ii), and as soil drench applications (iii). Results of the second-year experiments are herein presented and discussed. Results showed that as pruning wound protectants, the synthetic fungicides and sealants significantly reduced the infection percentages by Neofusicoccum parvum when compared to the controls and other biocontrol treatments. From nursery trials, the biocontrol treatments with bacterial and fungal products improved the callus formation in graft union and rootstock, respectively. Furthermore, the different fungal-based fungicides (i.e. Vintec, Botector, among others) reduced the recovery of Botryosphaeriaceae and Fusarium pathogens from the graft union. Finally, the results from the soil drench trial showed that treatments with Trichoderma, Rhyme, and Serratia plymuthica reduced the recovery of Neofusicoccum parvum compared to the control, however they did not significantly reduce the lesion length. The treatments with Pseudomonas chlororaphis, and Trichoderma reduced the recovery of Eutypa lata compared to the control, whereas a different treatment based on Trichoderma, alongside Bacillus velezensis and Rhyme significantly reduced the lesion length. The treatments with bacterial endophytes (Bacillus velezensis and Pseudomonas chlororaphis) effectively reduced the recovery of Phaeoacremonium minimum up to 0%, however only the latter significantly reduced the lesion length. Altogether, these results indicate that biocontrol strategies have comparable reductions of the impact of grapevine trunk disease pathogens in field conditions and should be further studied for validation and development for the industry.

Sudden Vine Collapse

This project was planned to determine if co-infections of grapevine leafroll associated virus-3 (GLRaV-3) and grapevine virus A (GVA) lead to sudden vine collapse (SVC) on Freedom rootstock and to identify rootstocks that might be more resistant. In addition, we planned to determine if SVC was spreading within vineyards in a pattern consistent with the ecology of mealybugs, known vectors of GLRaV-3 and GVA. Eighteen blocks in 12 vineyards, located in five different counties, were identified in 2021 as having clusters of vines characteristic of SVC. 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. However, at the block level, this positive correlation was only significant in two out of 18 blocks. 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. Therefore, GLRaV-3 and GVA co- infections were not limited to SVC clusters. To determine if time was the missing factor in a positive correlation between SVC and co-infection by GLRaV-3 and GVA, we re-surveyed GLRaV-3/GVA asymptomatic vines in 2022 and 2023. None of these vines had developed SVC symptoms but only eight of the original 18 blocks were still in place in 2022 and three by 2023. Temporal and spatial analysis of SVC incidence in the remaining eight blocks indicated that SVC progressed in varying rates in five of the eight blocks from 2021-2022 before they were removed. In two of remaining three blocks, SVC incidence decreased from 2021-2023 and in the last block, yearly SVC incidence was variable. Spread occurred via a common dispersal mechanism, but at varying rates. SVC distribution within blocks was aggregated but not to the same extent as observed for GLRaV-3 epidemics. The rootstock field trial was planted in randomized blocks in the UCD Armstrong Field Station in summer 2022 and graft-inoculated in 2023 with dormant buds from vines positive for GLRaV-3, GLRaV-3 and GVA, GLRaV-1 and GVA, or GLRaV-2 and GVB, in addition to virus negative controls. A subset of graft-inoculated vines was tested by RT-qPCR in November 2023 to verify their virus infection status. The results indicated that the majority of these vines were positive for the viruses with which they were graft-inoculated.

Grape Powdery Mildew Reduction

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.

Field Trials of Biocontrol for Grapevine Trunk Disease

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.

Botrytis Bunch Rot: Who, Where, When, and What to Use

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.

Spray-Induced Silencing of Grape Powdery Mildew Genes to Reduce Powdery Mildew Growth

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.

The Role of Rootstocks and Single and Mixed Infections of Grapevine Leafroll Associated Virus-3 and Grapevine Virus A in Sudden Vine Collapse

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 GLRaV[1]2 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.

Controlling Grapevine Trunk Diseases in California

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).

Exploring the Etiology of Berry Shrivel Ripening Disorder: Considerations of Biotic and Abiotic Factors

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.