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%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%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.

Inception, Diagnosis, and Consequences of the Berry Shrivel Disorder

“Berry shrivel” (BS) has been used to describe a post-veraison disorder of table and wine grapes, and we have determined that the hallmark symptom of this disorder is a low Brix in berries but a green and healthy appearing rachis. This disorder contrasts to bunchstem necrosis (BSN), in which berries also shrivel, but bunches exhibit a necrotic rachis, and berries may exhibit a high or a low Brix, depending on the timing of the necrosis. This research has demonstrated that sugar accumulation in BS-affected berries is substantially slowed or stopped approximately 2 weeks prior to the appearance of shriveling, and we have suggested that the term ˜sugar accumulation disorder” (SAD) be used to describe this condition. Based on reduced sugar accumulation as a key element of SAD, crop thinning experiments were carried out in Sauvignon Blanc, Grenache and Cabernet Sauvignon vineyards at a total of 7 locations, and in one location thinning gave a significant reduction in SAD incidence. In 2008 however, the level of SAD was generally very low, with many of the monitored Cabernet Sauvignon vineyards showing 0%SAD in all thinning treatments. At the Oakville Experimental Vineyard (OEV, Napa) we have shown that the Brix of non-symptomatic berries on SAD affected vines is also substantially below the Brix of normally-developing berries, indicating that SAD effects all of the fruit on an affected vine. However, in two other North Coast vineyards (Juliana, Jordan), non-symptomatic berries on SAD affected vines had normal Brix, so it is possible that there is more than one cause of SAD. For the SAD affected vines at OEV we have been able to propagate the disorder, indicating that the disorder is carried in the wood source, and a key finding in the 2008 season was preliminary evidence from a number of grafting studies that SAD can spread from a wood source bud to the rest of the vine. This was the first year to obtain fruit from these grafts however, and this result must be confirmed with further study.

Inception, Diagnosis, and consequences of the Berry Shrivel disorder

Berry shrivel (BS) is a ripening disorder of unknown cause and sporadic appearance that has been increasingly observed in vineyards around California. BS has often been mistaken for another disorder, bunchstem necrosis (BSN), but we have found that the disorders can be distinguished by the condition of the rachis of affected clusters. The rachises on BS clusters are green and healthy looking, while BSN rachises are brown necrotic. BS fruit had less sugar, lower pH, and reduced coloration several weeks before visible shriveling of the fruit. These compositional differences are maintained throughout the ripening period. BS fruit stops sugar accumulation about two weeks prior to symptom appearance. The apparent rise in Brix of BS fruit after this point is due to dehydration of the fruit and concentration of existing solutes. BS also appears to be a vine phenomenon, as nonshriveled clusters on a vine with shriveled clusters are also compositionally different than fruit from a healthy vine. These ?likely to shrivel? (LTS) clusters have compositions intermediate between BS clusters and clusters from a healthy vine (control clusters). Nonshriveled clusters on a vine with BS clusters also stop, or significantly slow, sugar accumulation at the same time as BS fruit does.

Since the BS phenomenon appears to be due to phloem dysfunction, girdling treatments were set up at different developmental times in Davis and at the Oakville Experimental Vineyard (OEV) in Napa County. Artificially stopping sugar accumulation by girdling caused the berries to develop with reduced coloration and the berries eventually (3-4 weeks post girdling) began to shrivel.

There has been a trend at OEV for vines with a history of BS to be less water stressed (as determined by leaf water potential measurements) than control vines. To test the hypothesis that vine water status exacerbates or mitigates the incidence or severity of BS, an irrigation trial was set up in the Alexander Valley in Sonoma County. One irrigation treatment applied water at a rate that vastly exceeded the grower?s standard irrigation (approximately 5 gph as compared to 0.5 gph) and vines in the other treatment were not irrigated. While these treatments did affect the vine water status, they did not affect the BS incidence in the vines.

The hormone CPPU (Prestige®) was sprayed at two concentrations (2 ppm and 4 ppm) after set (at 8mm berry size) at two sites (OEV and Alexander Valley) in order to assess its effect on the incidence of BS and BSN. Neither of the hormone treatments, at either concentration, had an effect on the incidence of BS or BSN.

The BS disorder can be propogated by chip buds. Budding was done in 2003. Vines propagated with buds collected from vines with a history of BS had reduced sugar accumulation, shorter shoots after budbreak, and reduced weight per berry compared to vines propagated from healthy material. Wines were made with the addition of different amounts of BS and BSN fruit from the 2006 harvest (0%, 5% and 10% by weight). All of the wines fermented to dryness and will be used for difference testing and potentially descriptive analysis if differences can be detected.

Much of the data contained in this report has been submitted for publication to AJEV.

International Research Symposium on Syrah Disorder and Decline

The Syrah Vine Health Symposium provided an opportunity for virologists and other researchers from France, South Africa and the US to come together at UC Davis and present what they knew about the causes of abnormal Syrah vine growth to members of the California winegrape industry. It also provided scientists with an opportunity to observe symptoms in local Syrah vineyards, and to discuss differences in symptomology and focus on avenues of investigation that are most likely to provide an answer to the cause of Syrah Decline. Along with the symposium, a French journal article was translated to English; and recordings of seminar presentations as well as the symposium proceedings were posted on the internet.

Syrah (synonym Shiraz) is grown throughout California vineyard regions, with a total of 18,776 acres statewide (2006). Beginning in the late 1990?s, growers statewide began reporting abnormal vine growth in some plantings. In the North Coast, the appearance of abnormal graft unions and red leaves are observed on several clones of syrah on a range of rootstocks. In the Central Coast, beginning about 2001, an increasing number of plantings displayed reddish-brown leaves in mid-summer that appeared to be a blend of leafroll virus, potassium deficiency, salt toxicity and/or severe water stress ? a group of symptoms locally referred to as ?Syrah Disorder?.

Symptomology varies across regions; however a group of common symptoms include a swollen trunk just above or occasionally at the graft union; deep vertical cracks in the bark; distorted trunks; and closely spaced nodes along the trunk. When the bark is removed on such vines, deep necrotic pits are found in the wood just above the union as well as necrotic areas that sometimes involve half or more of the diameter of the trunk. Leaves on such vines turn fully red in late summer, and depending on severity of the wood necrosis, shoot growth is stunted. Symptoms are observed in vines as young as 3 years, and death results in severe cases.

Researchers in France, South Africa and Australia have focused their attention to vine health issues in Syrah. In France, symptoms were first reported in 1993 and currently all Syrah vineyards contain some degree of affected vines. Symptoms are referred to collectively as ?Syrah Decline? and are similar to those previously described as being common to some coastal vineyards in California. A formal program to research the cause of Syrah Decline was established in 2001 in ENTAV (L’Etablissement National Technique pour l’Amélioration de la Viticulture). In South Africa, Shiraz has been shown to be infected with a virus associated with symptoms collectively referred to as ?Shiraz Disease?. Similar symptoms have been reported by researchers in Australia. In earlier work, UC had identified a new strain of Rupestris Stem Pitting virus (RSPa-SY) in a symptomatic Syrah vine. In 2007, symptomatic and asymptomatic Syrah vines located in coastal and foothill regions in California were tested for the new strain, but no correlation was found between the virus and symptoms.

Expression of Anti-Microbial Genes in Transgenic Grapevines for Enhanced Disease Resistance

Grapevines are susceptible to numerous diseases harming both plants and profits. Transgenic grapevines that resist disease could provide improved disease control as well as economic benefits from the reduction in spray applications. Our overall goal has been to research and develop methods to create transgenic selections of elite cultivars with improved resistance to diseases. The transgenic strategy is especially appropriate for clonally-propagated crops, such as grapevines, where the grape industry is rooted in traditional European grapes with very high disease susceptibility. Eight different antimicrobial peptides (AMPs), which are small proteins known to be inhibitory to a range of bacteria and fungi, were tested to determine which might best provide resistance to bunch rot (Botrytis) and crown gall (Agrobacterium vitis). Two of these peptides, cecropin B and MSI-99, were most promising for controlling growth of A. vitis. A test was run to determine the effect of a combination of these two peptides on A. vitis growth, and their activity was additive. Cecropin B and Shiva 1 were tested against Botrytis cinerea, and were found to be no more effective than either PGL or ESF-39, which had been tested last year. The same eight AMPs were also tested for their effects on germination of powdery mildew conidia. These assays were technically difficult to carry out, but it was clear that none of the tested peptides were highly inhibitory to conidia germination. Some AMPs (e.g. ESF-39 and MSI-99) decreased conidia germination by approx. 50%at 10 µM and higher concentrations. Based on the results from peptide screening, new gene constructs for use in transformation were developed. Two constructs included MSI-99 and cecropin B downstream of a constitutive promoter, while the other two constructs placed these same genes downstream of wound- or pathogen-inducible promoters. Transformation of ‘Chardonnay’ is underway in order to test the potential of these constructs for the improvement of disease resistance.

Spider Mite Control in Vineyards

Three trials were conducted to evaluate the utility of oil sprays and other OMRI approved products as integrated controls of spider mites, leafhoppers and powdery mildew. The results of one trial in Tracy, CA show that JMS Organic Stylet Oil and to a lesser extent Trilogy, applied for powdery mildew control, significantly suppress leafhoppers and mites on grapevines. These two products also performed equal (Trilogy) or superior (Stylet Oil) to micronized sulfur as a powdery mildew treatment. No treatment effects on Brix or berry weight were detected. Also, it appeared that when Stylet Oil applications ceased at the end of June, leafhopper and mite populations remained suppressed until August. Stylet Oil season-long treatment in table grapes may not be acceptable due to the substantial effect on the berry bloom appearance. For table grape production ceasing Stylet Oil sprays at veraison will allow ample time for berries to regain their waxy bloom by harvest for mid to late harvest grapes. At a second location (Courtland, CA) spider mite densities were extremely low, however predator mites, which were abundant at this site, produced interesting observations, in that Stylet Oil (1%) did not appear to significantly affect them. A third trial (Lodi, CA) evaluated several OMRI approved miticides for the control of Pacific spider mite. Single treatments of Stylet Oil (1%and 2%), GC-Mite, and to a lesser degree M-pede appeared to suppress Pacific Mites for several weeks. Stylet Oil at 2%and GC-Mite caused substantial damage to the berry bloom, however Stylet Oil at 1%, Ecotrol EC and M-pede resulted in no bloom damage. Stylet Oil appears to be an excellent candidate alternative to sulfur for an integrated control program for mites, leafhoppers, and powdery mildew, offering superior control of all of these important pests relative to micronized sulfur. Our proposed research for 2007-08 will repeat the evaluation of season long Stylet Oil applications and its effects on mites, leafhoppers, powdery mildew, Brix, berry weight and juice pH. In addition, we will evaluate the utility of integrating several Stylet Oil (1%and 2%) sprays into a sulfur-based powdery mildew program.