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.

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

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.

Understanding and managing the trunk disease Esca

Summary: Vine surgery (also known as trunk renewal) has been shown to be effective against
Eutypa dieback, but trunk pathogens cause mixed infections. Vineyards are thus likely to be
affected by more than one trunk disease. Furthermore, Eutypa dieback is not even the most
common trunk disease in California. It is important to also test the efficacy of vine surgery
against Esca because infections by two common Esca pathogens, Phaeomoniella chlamydospora
and Phaeoacremonium minimum, can occur at the base of the trunk, either during the
propagation process in the nursery or potentially by infections through roots after being planted
in the vineyard. To accomplish this objective, we established a replicated field trial in a
Sauvignon blanc vineyard in Lake County, CA. Vine surgery was done on vines with leaf
symptoms on shoots growing from only one cane, the idea being that the infection might be
restricted to that portion of the vasculature. In February 2018, we chain-sawed such vines above
the graft union and noted the presence of wood symptoms in 98 total data vines, which were
located among three experimental blocks in the vineyard. The vast majority of the 98 data vines
had wood symptoms, although they were restricted to less than 10% of the cross-section of the
trunk. This observation is important and is not so discouraging because a previous study of vine
surgery, combined with annual fungicide treatments of the cut trunk, showed that a higher
proportion of vines with wood symptoms on less than 20% of the trunk cross-section had normal
vigor and yields 8 years later, compared to vines with more advanced wood symptoms. In the 3rd
year after vine surgery (2020), the data vines will be fully trained and (assuming they are
healthy) are likely to be producing normal yields. After all, this is the goal of vine surgery, to
return vines to productivity and faster than would be possible with a replant. In January 2020,
we will submit a proposal to evaluate the progress of vine surgery, based on presence/absence of
leaf symptoms, vine growth, yields, and juice quality. The second objective of our study was to
identify the Basidiomycete wood-rotting fungi that cause Esca. For many years, there was an
untested assumption that these fungi infected vines after they were already infected by
Ascomycetes Phaeomoniella chlamydospora or Phaeoacremonium species. Our pathogenicity
tests show the wood-rotting fungi are pathogens when inoculated to vines alone. Because the
Basidiomycetes are assumed to be secondary pathogens, management practices have not been
evaluated against them. The most widespread Basidiomycete was Fomitiporia polymorpha.
This species was previously reported from one vineyard in California, so our finding expands its
range within the state. We described a new Basidiomycete species Inonotus vitis, which
represents the first report of Inonotus on grape in the Americas. Pathogenicity studies will be
established in January 2019 to evaluate whether I. vitis is pathogenic. If it is, then it might make
sense to include this species (along with F. polymorpha) in evaluations of pruning-wound
protectants.

Deep sequencing for trunk disease diagnostics

The aim of this multi-year project is to develop rapid and cost-effective diagnostic methods
for detection, identification, and quantification of trunk pathogens in asymptomatic and
symptomatic grape wood. Healthy vines are essential for the successful establishment and
sustainability of all grape production systems. Since wood pathogens may remain
asymptomatic in young, non-stressed vines, propagation material may contain latent fungal
infections and may become symptomatic after planting and serve as a source of inoculum
for further infections of potentially clean plants. Methods of virus detection and eradication
have been crucial in ensuring that the material in germplasm repositories and clean plant
programs is free of known viruses. There remains much to be developed in terms of fungal
pathogen detection. Our laboratories have developed comprehensive genomic information
on several ascomycetes associated with the most common and aggressive trunk diseases,
which provides the unprecedented opportunity for the implementation of new sequencingbased
diagnostic tools that take advantage of Next Generation Sequencing (NGS)
technologies. By allowing the testing of mother plants in foundation blocks and propagation
material in nurseries, we expect that the applications of deep sequencing diagnostics will
help establish a certification program for trunk pathogen-free germplasm and reduce the
amount of trunk pathogens introduced into vineyards at planting as well as the incidence of
young vine decline. Deep-sequencing diagnostics will also help identify disease-causing
organisms associated with diseased vines in older vineyards.
In the 1st year of the project (2015 – 2016) we collected diseased wood material from
commercial vineyards and characterized the associated fungal pathogen species using
traditional methods, such as morphological and sequence-based identification of purified
fungal colonies. We used these samples to determine how effective ITS-sequencing, metagenome
sequencing and meta-transcriptome sequencing approaches are in identifying and
quantifying pathogenic species directly in planta. Data simulations allowed us to determine
what mapping algorithm was the most specific and sensitive in detecting trunk pathogens
both qualitatively and quantitatively. All NGS methods we tested were in agreement with
traditional diagnostic methods, but also allowed us to detect simultaneously multiple
pathogen species with no need of hands-on sample culturing and colony purification.
Additionally, unlike traditional diagnostics, which are strictly qualitative, NGS approaches
allowed us to determine the relative abundances of the different infecting species. This
work was published in Molecular Plant Pathology (Morales-cruz et al., 2017). Among all
methods tested, ITS-seq is still the most cost-effective until library preparation costs for
RNA and DNA-seq do not decline significantly. For this reason, ITS-seq was chosen for
further protocol optimization to improve sensitivity and specificity for diagnostics purposes.
In the second year of the project (2016-2017), we (a) confirmed that NGS allows the
detection with high specificity of actively infecting pathogens when vines are experimentally
infected with individual pathogen strains; (b) established that NGS detection is quantitative
and allows to differentiate between diseased and healthy vines; (c) developed a protocol for
testing dormant cuttings and started testing cuttings provided by a commercial nursery. In
the 2016-2017 funding cycle, we also developed a new DNA extraction protocol that
reduced the time required for processing and the amounts of sample, reagents and waste.
In the 3rd and 4th year of the project, our effort focused on the development and
optimization of a new set of optimized primers for ITS-seq designed specifically to target
the ITS of grapevine trunk pathogens. The primers as well as the method are publicly
available and described in a peer-reviewed article published in December 2018 (Moralescruz
et al., 2018).
In summary, in these four years we have:
1. Applied NGS to trunk pathogen diagnostics and demonstrated that NGS provides
qualitatively and quantitatively accurate simultaneous identification of multiple trunk
pathogens directly from grapevine wood samples (Morales-Cruz et al., 2017 Mol
Plant Pathol).
2. Developed a new protocol with optimized diagnostic markers for NGS ITS-seq diagnostics of trunk diseases, which is publicly available and described in detail in
Morales-Cruz et al. (2018; BMC Microbiology).

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%A. erythroneurae and 13%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%, 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.