Abstract
Background and goals Grapevine trunk diseases in the Columbia River Basin of eastern Washington include Cytospora dieback, Eutypa dieback, and Esca. Although some of the causal fungi are known (as Cytospora viticola, Eutypa lata, and Phaeomoniella chlamydospora, respectively), basic epidemiology is not. This makes it difficult to time management practices. The common assumption is that these pathogens infect through pruning wounds during the dormant season, as has been shown for causal fungi of some grapevine trunk diseases in California. As such, we evaluated fungicides for protecting wounds after pruning under eastern Washington conditions.
Methods and key findings In March 2019, 2020, and 2021, we evaluated the protection efficacy of pyraclostrobin + fluxapyroxad and thiophanate-methyl sprayed within three days of pruning at an established Vitis vinifera Chardonnay vineyard in Prosser, WA. Within two days of fungicide treatment, C. viticola, E. lata, or P. chlamydospora (2000 spores per wound) were inoculated separately onto spurs, and molecular-detection attempts were made five to eight weeks later (after budbreak). Compared to water-treated spurs, detection rates of C. viticola and P. chlamydospora from thiophanate-methyl-treated spurs were lower in all three study years. Detection rates of E. lata from thiophanate-methyl-treated spurs were lower in one year.
Conclusions and significance This suggests that dormant-season spray applications of thiophanate-methyl as a pruning-wound protectant can reduce grapevine spur infection by these pathogens. Little to no rain during the dormant season in eastern Washington may limit opportunities for disease spread, but winter injury to the permanent, woody structure of the vine may create additional infection courts.
Introduction
Grapevine trunk diseases impact vineyards worldwide. The fungal pathogens cause chronic infections in the permanent, woody structure of the vine. They can kill fruiting shoots, as is the case with the dieback-type trunk diseases (Gubler et al. 2013), or prevent fruit ripening, a symptom of Esca (Gramaje et al. 2018). Eradicating the causal pathogens from a vineyard is possible if done early in the infection process. A practice known as “trunk renewal” or “vine surgery” (Calzarano et al. 2004, Sosnowski et al. 2011) involves removing the entire vine canopy (typically the cordons and often including the trunk, and the fungal infections that go along with it) and retraining the vine from a presumably healthy base of the trunk. This is a labor-intensive and thus expensive approach, which takes a vine out of production for two years, while a new trunk and canopy are being retrained (Baumgartner et al. 2019). A more cost-effective approach to managing trunk diseases in the long term is to prevent the infections from happening in the first place (Kaplan et al. 2016). This can be done through annual practices that minimize the risk of pruning-wound infection, namely delayed pruning (Úrbez-Torres and Gubler 2011), double pruning (Weber et al. 2007), or applications of fungicides and other protectants to pruning wounds (Rolshausen and Gubler 2005, Rolshausen et al. 2010, Brown et al. 2021).
Although nursery stock can potentially be infected at the time of planting (Gramaje and Armengol 2011), the causal pathogens can also originate outside of a newly planted vineyard in the form of airborne spores, released after rain events or prolonged periods of high relative humidity (e.g., spores of the Eutypa dieback pathogen Eutypa lata [Carter 1991], and the Esca pathogens Phaeoacremonium minimum [Rooney-Latham et al. 2005] and Phaeomoniella chlamydospora [González-Domínguez et al. 2020]). The time it takes for one of these pathogens to complete its life cycle (from spore interception on a pruning wound, to spore germination, to host infection, to establishment of an internal wood infection, and finally to subsequent spore production on the infected host) can range from one year (e.g., Phomopsis dieback pathogen Diaporthe ampelina [Anco et al. 2012]) to several years (e.g., E. lata [Ramos et al. 1975]).
In growing regions with a Mediterranean climate, these spores are thought to spread primarily during the dormant season, given that most rain falls during this period. Spore-trapping experiments show a trend of numerous spore-dispersal events by a broad range of pathogenic species with rain throughout the entire dormant season (e.g., northern California [Úrbez-Torres et al. 2010, Fujiyoshi et al. 2021b], eastern and northern Spain [González-Domínguez et al. 2020], and the western Cape province of South Africa [van Niekerk et al. 2010]). Also in the dormant season of Mediterranean climates, the risk of vine infection is thought to be high, as the dormant-season practice of pruning creates wounds, which are susceptible infection courts for many of the causal fungi (e.g., E. lata [Petzoldt et al. 1981, Weber et al. 2007] and Botryosphaeria dieback pathogen Neofusicoccum parvum [Úrbez-Torres and Gubler 2011]). In western North America, most field trials of preventive pruning (e.g., delayed pruning [Úrbez-Torres and Gubler 2011]) or applications of fungicides after pruning (e.g., thiophanate-methyl and pyraclostrobin [Brown et al. 2021]) have been done in California. However, studies on spore dispersal and pruning-wound susceptibility from regions where the rainy season is not synchronous with the timing of dormant-season pruning suggest that spores are produced and dispersed over a longer period of time. For example, spores of Botryosphaeria dieback pathogens are trapped year-round in the maritime climate of New Zealand (Amponsah et al. 2009), and spores of Eutypella species (fungi in the same fungal family, Diatrypaceae, as E. lata) are trapped year-round in the desert climate of southern California (Úrbez-Torres et al. 2020). Without knowing which vine tissues are susceptible, when such tissues are at highest risk for infection in these climates, or if the trapped spores are actually infectious to such tissues, it is difficult to adapt the timing of practices originally developed for Mediterranean climates.
In a previous survey of Washington vineyards with trunk diseases, we identified Esca and Eutypa dieback (Travadon et al. 2022). A unique finding of that survey was the prevalence of the trunk disease Cytospora dieback, also known as Cytospora canker, and its causal pathogen Cytospora viticola, which was originally described as a new species from vineyards in the northeastern United States and southeastern Canada (Lawrence et al. 2017), and has since been reported from vineyards in the northern midwestern U.S. (Dekrey et al. 2022). Eastern Washington is the second largest U.S. producer of winegrapes and the largest producer of juice grapes (USDA NASS 2018). The climate characteristics of this semiarid steppe region are much colder and drier than that of the major grapegrowing areas in California, especially during the dormant season. While the dormant season is when most of this region’s 200 to 500 mm of annual precipitation falls, it often falls as snow (non-liquid form) and is accompanied by prolonged periods of freezing temperatures. Further, cold damage to vines during the dormant season can result in entire vineyards needing to be retrained from the base of the trunk, every 10 to 20 years.
Our goal was to identify effective protectants as fungicide applications against pathogens we identified in eastern Washington: C. viticola (Cytospora dieback), E. lata (Eutypa dieback), and P. chlamydospora (Esca) (Travadon et al. 2022). To our knowledge, no studies to date have tested fungicides against trunk diseases in Washington. As such, we evaluated fungicides previously shown to be effective against at least one of the pathogens. Without knowing the exact timing of pruning-wound susceptibility or spore dispersal in the cold, dry winter of the lower Columbia Basin of eastern Washington, we carried out the experimental steps based on the timing of dormant-season pruning.
Materials and Methods
Study vineyard in eastern Washington
A replicated field trial was conducted for three years, from 2019 to 2021, in an experimental vineyard in Prosser, Washington (Washington State University, Irrigated Agriculture Research and Extension Center). The vineyard was planted in 2011 with Vitis vinifera Chardonnay on its own roots. Planting materials were certified at the time of planting by the Washington State Department of Agriculture (https://agr.wa.gov/departments/plant-health). Spacing was 3 m between rows and 1.5 m between vines, at a density of ~1749 vines/ha (726 vines/acre). Vines are trained to a dual-trunk bilateral cordon, spur-pruned, and the canopy trained to a modified vertical-shoot positioned system. The vineyard is drip-irrigated, and the irrigation season runs from approximately April to October (per Sunnyside Valley Irrigation District allocations).
Prosser is located within the Yakima Valley American Viticultural Area of eastern Washington. The climate is a semiarid steppe, characterized by hot, dry summers and cold winters (USDA Cold Hardiness Zone 7a; https://plan-thardiness.ars.usda.gov/). The majority of the average 203 mm of annual precipitation falls between November and March. Weather data for this study (Figure 1) were recorded at an AgWeatherNet station (weather.wsu.edu; station code ‘Prosser.NE’), located ~500 m from the vineyard location.
Daily maximum temperature (solid line), minimum temperature (dotted line), and precipitation (columns) from 1 March to 1 June 2019 to 2021, in an experimental vineyard in Prosser, Washington. Arrows in each panel denote the dates of the following steps: 1) pruning, 2) application of fungicide treatments, 3) application of inoculation treatments, and 4) collection of spurs for pathogen detection.
Our experimental approach (Table 1) included the following steps: 1) prune all vines in data rows and buffer rows to spurs of 40 cm length; 2) apply fungicide treatments to data vines within three days after pruning; 3) carry out inoculation treatments of spurs on data vines within two days of fungicide treatments; and 4) collect spurs that received the inoculation treatments for pathogen detection, when shoots growing from those spurs were ~20 cm long. Pruning cuts were made horizontally, to prevent the droplet of inoculum from running off. The relatively long length of the retained spurs (40 cm) after pruning was intentional to create enough space to minimize the risk of infection progressing to the cordon from the pruning wounds, which are inoculated; it allowed us to remove inoculated tissue while still being able to leave sufficient segments of canes to allow normal canopy growth and development during the growing season. Also, the fungicide applications are more efficient and thorough when all the pruning wounds are at the same height in the canopy. The short incubation period between steps 3 and 4 (ranging from 39 to 56 days; Table 1) was also necessary to minimize the risk of disease spread from the inoculated spurs to the rest of the vineyard, which does not have a history of trunk diseases.
Timing of pruning, application of fungicide treatments and inoculation treatments, and spur-collection steps in the Prosser, Washington vineyard. Step 4 was carried out after budbreak, when shoots growing from inoculated spurs were ~20 cm long.
Fungicides applied after pruning
Fungicide treatments were as follows: a water-treated control, pyraclostrobin + fluxapyroxad (Merivon Xemium, BASF, EPA 7969-310; Fungicide [FRAC] groups Quinone outside inhibitor [QoI] + succinate-dehydrogenase inhibitor [SDHI]), and thiophanate-methyl (Topsin M, United Phosphorus, Inc., EPA CA-030001; FRAC group methyl benzimidazole carbamate [MBC]). Treatments were applied to pruning wounds at a spray volume equivalent of 935 L/ha, using a backpack sprayer (Solo Model 425) with an air induction spray nozzle (TeeJet AITXA 8002). The application rate of pyraclostrobin + fluxapyroxad (0.42 mL formulated product/L or 1.5 lbs/100 gal) was the maximum allowable rate on the manufacturer’s label. The application rate of thiophanate-methyl (1.8 g formulated product/L or 5.5 fl oz/100 gal) was recommended by the manufacturer for management of trunk diseases. During application, the spray nozzle was directed at each pruning wound and applied to run-off. Thorough coverage of spray applications was confirmed as 100% each year, using two, 2 cm2 pieces of water- and oil-sensitive paper (TeeJet) attached to the pruned spurs on one data vine per treatment per block.
Fungicide treatments (including the water-treated control) were applied in a randomized complete block design with three blocks, each of which consisted of one treated row separated by five buffer rows. Within each treated row (i.e., block), fungicide treatments (water-treated control, pyraclostrobin + fluxapyroxad, thiophanate-methyl) were distributed among eight-vine sets, and these fungicide treatments were randomized within the three blocks. For each eight-vine set, all eight vines were treated on both sides of the row, but the spurs did not receive inoculation treatments on the exterior cordons of vine 1 or vine 8 (i.e., 0.5 vine on either end of the eight-vine set was treated, but not subject to pathogen inoculation). On the remaining 14 cordon lengths (the total of 14 comes from the fact that there were two cordons per each of the eight vines, less the exterior cordons on both ends of the treated area), spurs of the central three vines (data vines) were randomly flagged for inoculation treatment, with five spurs per inoculation treatment per data vine (including the water-treated control). This gave a total of 540 spurs per year (3 blocks × 3 fungicide treatments × 4 inoculation treatments × 3 data vines per fungicide treatment × 5 spurs per inoculation treatment per data vine). The same eight-vine sets within the three blocks received the same fungicide treatments each year. Within data vines, however, inoculation treatments were randomized among spurs each year, as spurs are renewed annually in grape production systems.
Pathogens inoculated to pruning wounds after fungicide applications
Inoculation treatments were as follows: a water-inoculated control, C. viticola (Bent901), E. lata (ascospores collected from environmental stromata on the wood of Nerium oleander L., in Yolo County, California), and P. chlamydospora (Bent708). C. viticola (Bent901) and P. chlamydospora (Bent708) were originally isolated from vineyards in eastern Washington with symptoms of Esca and were shown to be virulent in greenhouse assays (Travadon et al. 2022). Water-inoculated controls (i.e., the spurs onto which water, rather than pathogen inoculum, was pipetted, in the eight-vine sets that were first sprayed with either water—the water-treated controls—or one of the fungicides) were included in the experimental design as a type of negative control, to determine background levels of local pathogens in the vineyard.
For C. viticola, inoculum consisted of spores (conidial) from pycnidia produced in culture on autoclaved grape wood (Lawrence et al. 2017). To induce development of pycnidia, one-year-old grape canes (~1 cm in diameter) were collected in the vineyard and cut into 5-cm-long segments. Wood segments were autoclaved in glass petri plates twice, 24 hrs apart, at 122°C for 25 min. Autoclaved wood segments were placed in petri plates (9 cm diam.), with two segments per plate, and autoclaved potato dextrose agar (PDA; Difco Laboratories) was poured to the level at which the segments were almost completely submerged. An agar plug from an actively growing culture on PDA was placed between the two wood segments, and plates were incubated at room temperature under natural lighting for four weeks. During the four weeks of incubation, when pycnidia appeared, mature pycnidia were crushed with a flame-sterilized probe in 1 mL of sterile, distilled water, the concentration was estimated with a hemocytometer, and then adjusted with sterile water to 1 × 105 spores/mL.
Because E. lata does not produce its infectious sexual spores (ascospores) in culture, ascospores were harvested from fruiting bodies (perithecia) collected in the field, from the infected wood (visibly covered in stromata, within which perithecia were embedded) of an N. oleander L., in Yolo County, California. To collect ascospores from perithecia, stromata were sliced with a sterile razor blade, to reveal and cut open the perithecial cavities, and a drop of sterile water was placed on the perithecia. Masses of ascospores were then collected with a sterile probe and transferred to 1 mL sterile water. The spore concentration was estimated with a hemocytometer, and then adjusted with sterile water to 1 × 105 spores/mL.
To produce spore (conidial) suspensions of P. chlamydospora, a liquid culture was first established by inoculating 10, 2 mm agar plugs from a seven-day-old culture on PDA to a 250-mL Erlenmeyer flask containing 100 mL of potato dextrose broth (Difco Laboratories). After incubation at 25°C and 150 rpm for five days, a hand-held disperser (IKA-ULTRA-TURRAX T8) was used to homogenize the culture (1 min, speed 5), and 100 µL of homogenate was spread onto each of three PDA plates (9 cm diam.). After 14 days, spores were harvested by pipetting 2 mL of sterile water onto the agar surface and filtering the suspension through two layers of sterile cheesecloth to remove fragments of aerial mycelium. The concentration was estimated with a hemocytometer, then adjusted with sterile water to 1 × 105 spores/mL.
Each year, inoculum (i.e., the spore suspensions described above) was prepared in Davis, CA on the day before inoculations, and shipped overnight to Prosser, WA. On the day of inoculation in the vineyard, inoculum viability for each pathogen was tested in the lab by plating it on PDA. For each inoculation treatment with a pathogen, inoculum was pipetted onto the cut surface of the pruning wounds of data vines (20 µL or 2000 spores per spur). For water-inoculated spurs, 20 µL of sterile water was pipetted onto the cut surface of the pruning wound. Incubation of the inoculation treatments in the field, between the time of inoculation and when spurs were collected for pathogen detection, ranged from 39 to 56 days (Table 1).
Detection of pathogens to measure fungicide efficacy
From spurs of each inoculation treatment, we collected the distal 15 cm of the spur wood, removing any shoots that had emerged. Spurs were shipped overnight to our lab in Davis, CA for pathogen detection. DNA-based detection was used for spurs inoculated with C. viticola, using published primers for genus-level detection of Cytospora species (Luo et al. 2017), as detailed below. Because of our past experience with low recovery of C. viticola in culture from inoculated grape in the greenhouse (Lawrence et al. 2017), we were hopeful for higher and especially more consistent data with DNA-based detection. DNA-based detection was also used for E. lata and P. chlamydospora, as both species have proven difficult to isolate in culture. Further, the species specificity against a range of other wood-colonizing fungi of grape has been tested for qPCR primers for E. lata (Pouzoulet et al. 2017, Fujiyoshi et al. 2021a) and P. chlamydospora (Pouzoulet et al. 2013). We have used these qPCR primers to detect E. lata and P. chlamydospora in the field (Brown et al. 2021, Fujiyoshi et al. 2021b). From the distal 15 cm of wood from each inoculated spur, the bark was first scraped off the surface with a flame-sterilized knife and the ~0.5 cm of dried wood at the cut surface of the pruning wound was cut away. A 2.5 cm section of wood from below this discarded 0.5 cm end was sealed in a pre-labeled glass vial and stored at −80°C. Wood samples were ground to a powder in chilled containers (Grinder MM400, Retsch) and stored at −80°C in 2 mL microcentrifuge tubes. For DNA extraction, 1 mL of extraction buffer (Tris-HCl 100 mM, EDTA 20 mM, NaCl 1.4 M, CTAB 2%, PVPP 2%, β-mercaptoethanol 0.5%, RNAse A 0.4% v/v [Qiagen]) was added to 100 mg of wood powder in the 2 mL tube. Tubes were briefly vortexed, 500 µL of chloroform-isoamyl-alcohol (24:1) was added, tubes were incubated on ice for 5 min, and then centrifuged (2300 g, 10 min, 4°C). The supernatant was transferred to a new tube and mixed with AP2 buffer and the rest of the manufacturer’s protocol for the DNeasy plant mini kit (Qiagen) was followed.
For qPCR, 1 µL of 1X DNA extraction was used as template in a 25 µL reaction volume consisting of 1X Brilliant SYBR Green q-PCR Master Mix (Stratagene), 150 nM per primer (Operon Biotechnologies), 30 nM ROX Reference Dye (Invitrogen), and sterile molecular biology-grade water (GIB-CO). All reactions were performed in 200 µL tubes in 96-well plates, in an Mx3000p Real-time PCR Thermal Cycler (Stratagene). The PCR program was as follows: initial denaturation step at 95°C for 3 min, 50 cycles of 20 sec at 94°C, followed by 20 sec at 65°C for both annealing and extension (62°C for C. viticola), and additional melting analysis. After the amplifications were completed, dissociation curves were obtained based on a standard protocol from manufacturer’s instructions, and the temperature of the peak of the curve was checked to confirm the correct PCR product. The threshold level for fluorescence was set arbitrarily within the log-linear phase of increase. Genomic DNA from pure cultures was used as positive controls. Amplification of target DNA was based on the dissociation temperature (81.5°C for C. viticola, 79.0-79.5°C for E. lata, 75.9°C for P. chlamydospora). Positive detections were samples crossing the threshold level by 40 cycles for C. viticola, and 45 cycles for E. lata and P. chlamydospora. Detection (%) for each data vine was the percentage of spurs positive for the inoculated pathogen, out of five inoculated spurs.
For DNA-based detection of C. viticola, we used Cytospora primers CtBTFF1 and CtBTFR1, which amplify a portion of the β-tubulin gene, and were originally developed for genus-level detection of Cytospora species from the tree crops walnut and plum (Luo et al. 2017). Luo et al. (2017) screened the primers against other genera of wood-colonizing fungi of walnut and plum, to demonstrate their specificity to the genus Cytospora. We further evaluated the specificity of the Cytospora primers within our study system by first screening Cytospora species known to be pathogenic to grape, using DNA of virulent isolates from our previous surveys of vineyards with trunk diseases (U.S. states from which isolates were collected are in parentheses; Supplemental Table 1): Cytospora species 1 (Washington [Travadon et al. 2022]), Cytospora vinacea (New Hampshire [Lawrence et al. 2017]), and C. viticola (California [Lawrence et al. 2017] and Washington [Travadon et al. 2022]). We also evaluated the Cytospora primers from all Cytospora species cultured from the water-inoculated (control) spurs, from which we made culture attempts each of the three study years, as detailed below. Species-level identity of the Cytospora isolates was confirmed by sequencing the rDNA internal transcribed spacer region (ITS) (White et al. 1990) and translational elongation factor 1-α (TEF) (Carbone and Kohn 1999), both of which have been shown to be informative for species delineation in the genus Cytospora, especially for some of the Cytospora species reported from grape (namely, C. vinacea and C. viticola) (Lawrence et al. 2017).
Although there were no vines with external symptoms of trunk diseases in the vineyard, our experimental design included water-inoculated (control) spurs on the data vines within each fungicide treatment, to determine the presence of local pathogens, originating possibly from asymptomatic vines in the vineyard or from symptomatic vines/alternate hosts outside the vineyard. Spores from such hosts may have infected spurs between the time of pruning and the collection of spurs (i.e., coinciding with the incubation period of the pathogens that were inoculated to the spurs). Groups of pathogens we were particularly interested in isolating from water-inoculated (control) spurs were as follows: 1) the three species inoculated as part of the experimental design (C. viticola, E. lata, and P. chlamydospora), the presence of which would be expected to contribute to the detection rates we measured; 2) causal fungi of Cytospora dieback, Eutypa dieback, and Esca, other than the three pathogens we inoculated to spurs; and 3) causal fungi of other trunk diseases, namely Botryosphaeria dieback and Phomopsis dieback. To check this “background level” of local pathogens, we attempted to culture fungi from all water-inoculated spurs on both fungicide-treated and water-treated data vines. From the distal 15 cm of wood from each water-inoculated spur, the bark was first scraped off the surface with a flame-sterilized knife and the ~0.5 cm of dried wood at the cut surface of the pruning wound was cut away. From the 2.5 cm section of wood from below this discarded 0.5 cm end, we cut 16 small (each ~5 mm3) pieces of wood from apparently healthy wood and discolored wood (if present). Wood chips were surface sterilized in 0.6% sodium hypochlorite (pH 7.2) for 15 sec, rinsed twice in sterile distilled water for 1 min, plated on two PDA dishes amended with tetracycline (1 mg/L, Sigma-Aldrich) per spur, and incubated at 23°C. At 3, 6, 10, 15, and 30 days (to accommodate fast- and slow-growing pathogens), subcultures were hyphal-tip purified to PDA for identification. Isolates were identified to the species level based on sequencing of ITS and TEF.
Statistical analyses
For each pathogen, separate analyses of variance (ANO-VAs) were used to determine the main effects of year and fungicide treatment, and their interaction on detection (%). Normality and homogeneity of variances were evaluated, and confirmed, using normal probability plots and Levene’s test, respectively. ANOVAs were performed using the MIXED procedure in SAS v. 9.4 (SAS Institute, Inc.), with year and treatment as fixed effects, and block and block interactions as random effects. Year was considered a repeated measure. Appropriate covariance models were selected for ANOVAs of each pathogen based on comparisons of information criteria (AIC, AICC, BIC), as specified in the REPEATED statement (unstructured for C. viticola and P. chlamydospora, autoregressive for E. lata). For significant main or interactive effects (p < 0.05), means were compared by Tukey’s test. When interactive effects were not significant, means for significant main effects were compared by Tukey’s tests. To aid in the presentation and interpretation of the detection rates, we also calculated efficacy (%) as 100 × (1 − [DetectionFungicide/DetectionControl]), where DetectionFungicide is detection (%) of the pathogen from fungicide-treated spurs and DetectionControl is detection (%) of the pathogen from water-treated spurs. In this way, a high detection rate corresponds to a low efficacy. Efficacies of the fungicides against the pathogens were calculated from detection rates, but efficacies were not analyzed statistically.
Results
Differences in detection and environmental variables among study years
To ensure that our experimental approach evaluated fungicide efficacy consistently among fungicide treatments and over time, we used the same procedures to prepare inoculum each year, then evaluated inoculum viability by inoculating it to PDA on the day of inoculation. However, spores of E. lata in 2019 (year 1) did not germinate on the day of inoculation, consistent with no detection of E. lata from the inoculated spurs (Table 2), which were collected 56 days later. Fortunately, E. lata inoculum was viable and inoculations were successful in 2020 and 2021 (p = 0.006 for year × fungicide treatment), with detection rates from the water-treated spurs of 40% and 83%, respectively (Table 2). Detection rates of C. viticola were consistently high in all study years, despite statistically significant variation over time (p = 0.03 for year). Detection rates of C. viticola from water-treated spurs ranged from a low of 91% in 2021 to a high of 98% in 2019. In contrast, detection rates of P. chlamydospora were much more variable (and statistically significant at p < 0.0001 for year) over time. Detection rates of P. chlamydospora from water-treated spurs ranged from a low of 29% in 2019 to a high of 98% in 2021.
DNA-based detection of pathogens inoculated to pruning wounds, after application of fungicide treatments, compared to a water-treated, inoculated control. Detection (%) is the percentage of spurs from which the pathogen was detected, out of the total number of inoculated spurs (95% confidence limits [CL] are shown in parentheses). Efficacy (%) is 100 × (1 − [DetectionFungicide/DetectionControl]). Fungicide treatments with detection rates higher than the water-treated control were completely ineffective; they were given an efficacy of 0, rather than the calculated negative value. Each value is the mean of three blocks (with three data vines per block and five spurs per pathogen per data vine). Detection rates within a year with different letters are significantly different (Tukey’s test, p < 0.05).
The timing of pruning at the start of the experiment corresponded to the time of pruning by growers in commercial vineyards. Because early March 2019 was cold and the vineyard was under snow (Figure 1), pruning and subsequent activities were later than in other years (pruning on 28 March 2019 and spur collection on 28 May 2019). That said, 2019 was characterized by the highest temperatures on the day of inoculation, during the week following inoculation, and during the entire incubation period (Table 3). In contrast, 2020 was characterized by the lowest temperatures during these same periods. Total precipitation during the incubation period was highest in 2019 (37.6 mm), but was extremely low in 2020 at 4.1 mm. There was no rain during the 2021 study period. Each year, on the day of inoculation, there was no precipitation (Figure 1), but relative humidity ranged from a low of 44.5% in 2019 to a high of 59.4% in 2020 (Table 3). Regardless, climate differences among study years did not seem to correspond to differences in detection rates of E. lata or P. chlamydospora from water-treated spurs.
Weather (total precipitation, maximum temperature, minimum temperature, and relative humidity) on the day of pathogen inoculation, during the week following inoculation, and during the entire incubation period (i.e., between inoculation treatment and collection of spurs for pathogen detection), in the Prosser, Washington vineyard.
Fungicide efficacy
Thiophanate-methyl was the most effective fungicide against C. viticola infection. Detection of C. viticola was lower for pruning wounds treated with thiophanate-methyl (p < 0.0001 for fungicide treatment) versus the water-treated control in all three study years (Table 2). Detection of C. viticola from pruning wounds treated with thiophanate-methyl ranged from 22 to 62%, which corresponded to efficacies ranging from 33 to 77% (Table 2). Detection of C. viticola was also lower for pruning wounds treated with pyraclostrobin + fluxapyroxad (p < 0.0001 for fungicide treatment); detection rates ranged from 71 to 87%, which corresponded to efficacies ranging from 7 to 27% (Table 2). The mean detection rates of C. viticola from water-treated spurs ranged from 91 to 98%, averaged across three blocks per year.
Thiophanate-methyl was the most effective fungicide against P. chlamydospora in two of three years. Detection of P. chlamydospora was significantly lower for pruning wounds treated with thiophanate-methyl (p = 0.04 for fungicide treatment) versus the water-treated control (Table 2). Nonetheless, in 2021, detection of P. chlamydospora from thiophanate-methyl-treated spurs was high at 93%; as such, the efficacy of thiophanate-methyl against P. chlamydospora was only 5% in 2021, compared to efficacies of 62% and 38% in 2019 and 2020, respectively. Pyraclostrobin + fluxapyroxad was not effective against P. chlamydospora; detection of P. chlamydospora from pruning wounds sprayed with pyraclostrobin + fluxapyroxad was in each year either close to, or higher than, that of the water-treated controls.
Thiophanate-methyl was the most effective fungicide against E. lata, but only in 2021 (p = 0.006 for year × fungicide treatment). Of the two study years when E. lata inoculations were successful (2020 and 2021), detection of E. lata was lower in 2021 for pruning wounds sprayed with thiophanate-methyl versus the water-treated control (mean detection rates of 24% versus 83%, averaged across three blocks per year), with a corresponding efficacy of 71% (Table 2). Detection of E. lata was intermediate in 2021 for pruning wounds sprayed with pyraclostrobin + fluxapyroxad (mean detection rate of 60%, averaged across three blocks per year, with a corresponding efficacy of 28%) (Table 2).
Pathogens and other fungi isolated from water-inoculated (control) spurs
Across the three years, we cultured 22 isolates, based on culture attempts from 405 total water-inoculated (control) spurs (45 water-inoculated spurs per fungicide treatment × three fungicide treatments × three years = 405 total water-inoculated spurs). These 22 isolates were identified as the following species (number of spurs per fungicide treatment are shown in parentheses): Cryptovalsa ampelina (nine water-treated spurs), Cytospora chrysosperma (one water-treated spur), Cytospora parakantschavelii (one water-treated spur), Cytospora parasitica (three water-treated spurs, one pyraclostrobin + fluxapyroxad-treated spur), Cytospora ulmicola (one pyraclostrobin + fluxapyroxad-treated spur), C. viticola (one water-treated spur, one thiophanate-methyl-treated spur), undescribed species Cytospora species 2 (one water-treated spur), and Diplodia seriata (one water-treated spur). Prior to the experiment, we found that in addition to C. viticola, the Cytospora primers used for detection of C. viticola also detected other Cytospora species known to be pathogenic to grape, based on positive amplification of isolates from our culture collection of Cytospora species 1 and C. vinacea (Supplemental Table 1). During the experiment, we found that the Cytospora primers detected isolates from the non-inoculated (control) spurs, namely C. chrysosperma, C. parakantschavelii, C. parasitica, Cytospora species 2, and C. ulmicola; the pathogenicity of these species on grape is not known. Altogether, the Cytospora primers detected nine of the 10 total Cytospora isolates we tested (Supplemental Table 1), with amplification products ranging in size from 85 to 93 bp. We assume therefore that some of our qPCR detections might represent a species of Cytospora other than C. viticola. However, given that Cytospora species other than C. viticola were isolated from only eight of the 405 water-inoculated spurs we made culture attempts from (2.2%), we assume their detection rate is negligible.
Discussion
The high efficacy of thiophanate-methyl against C. viticola and P. chlamydospora suggests this fungicide could minimize dormant-season pruning-wound infection by these pathogens. Based on current recommendations (Gubler et al. 2013), thiophanate-methyl is a grower standard fungicide treatment in California, but it was not registered for dormant-season use in Washington at the start of our study. Thiophanate-methyl was also the most effective fungicide against E. lata in one of two study years, which was the year in which the E. lata inoculations were most successful. Pyraclostrobin + fluxapyroxad was associated with lower detection rates of C. viticola than those of the water-treated controls in all three years, but its efficacy was below 27%. Our findings are novel with respect to C. viticola, a trunk-disease pathogen that has not been included in past studies on pruning-wound protectants. No studies have evaluated pruning-wound protectants for a Cytospora species virulent on grape, although studies on other Cytospora species that attack tree crops (e.g., peach and almond) report that thiophanate-methyl is effective against preventing their infection (Miller et al. 2019, Holland et al. 2021).
Thiophanate-methyl has been previously shown to have moderate to high efficacy against a broad range of trunkdisease pathogens: E. lata (Rolshausen et al. 2010), Botryosphaeria dieback pathogens (Botryosphaeria dothidea, Dothiorella viticola, D. seriata, and Lasiodiplodia theobromae [Rolshausen et al. 2010], D. seriata [Diaz and Latorre 2013, Martinez-Diz et al. 2021], Neofusicoccum luteum [Amponsah et al. 2012], and N. parvum [Brown et al. 2021]), Phomopsis dieback pathogen D. ampelina (Brown et al. 2021), and Esca pathogen P. chlamydospora (Diaz and Latorre 2013, Martinez-Diz et al. 2021). Most of these studies cited above relied on the application of thiophanate-methyl at an experimental scale and with spray bottles, whereas we used a backpack sprayer and timed the applications for when and how a grower would (i.e., soon after pruning). Our findings confirmed thiophanate-methyl could protect pruning wounds when applied with a backpack sprayer to each pruning wound to the point of run-off, from taxonomically diverse pathogens.
Pyraclostrobin has been tested in various formulations and was moderately effective against E. lata (e.g., in California [Rolshausen et al. 2010, Brown et al. 2021] and in Australia [Sosnowski et al. 2008, 2013, Ayres et al. 2017, 2022]), against Botryosphaeria dieback pathogens (e.g., B. dothidea, D. seriata, D. viticola, and L. theobromae in California [Rolshausen et al. 2010]), N. parvum in California [Brown et al. 2021], and N. luteum in Australia [Ayres et al. 2022]), and against P. chlamydospora (Rolshausen et al. 2010, Diaz and Latorre 2013). However, we did not see similar promising results; pyraclostrobin + fluxapyroxad had low efficacy against C. viticola, E. lata, and P. chlamydospora. When we consider variable efficacy of pyraclostrobin against the same pathogen species in different studies, differences could result from variable isolate virulence, differing formulations, concentrations, application methods, and/or inoculation methods. For example, we found low efficacy of pyraclostrobin, with P. chlamydospora spores inoculated to pruning wounds, after applying pyraclostrobin + fluxapyroxad as a liquid formulation via a backpack sprayer. In contrast, Diaz and Latorre (2013) found high efficacy of pyraclostrobin, with agar plugs from an actively growing culture of P. chlamydospora inoculated to pruning wounds, after applying pyraclostrobin as a paste formulation. To further compare and contrast studies on pyraclostrobin versus P. chlamydospora, Rolshausen et al. (2010) found high efficacy of pyraclostrobin, with P. chlamydospora spores inoculated to pruning wounds, after applying pyraclostrobin at 10 times the concentration we used. Our findings of low efficacy of pyraclostrobin against E. lata are consistent with those of other studies on grape (Sosnowski et al. 2013, Ayres et al. 2017) and almond (Holland et al. 2021).
The ideal environmental conditions for spore germination and subsequent infection of pruning wounds are not known for the pathogens we tested. In addition to freezing temperatures, other factors that may negatively impact the viability of the spore suspensions after they are pipetted onto pruning wounds include exposure to ultraviolet light and dry conditions caused by wind. As such, we tried not to inoculate on sunny, windy days that were either very hot or very cold. Each year, we inoculated with higher spore concentrations (2000 spores per spur) than would likely occur naturally, to achieve consistently high pathogen pressure across fungicide treatments and years. Nonetheless, this approach may not sufficiently compensate for the effects of poor environmental conditions at the time of pathogen inoculation and colonization in some years more than others; hence the variable detection rates we found from year to year for P. chlamydospora and E. lata. Higher detection rates of P. chlamydospora and E. lata in 2021 may be due to a combination of conditions that favored infection on the day of inoculation or during incubation—for example, no freezing temperatures and moderate relative humidity, and/or higher pruning-wound susceptibility. Variable detection rates from year to year for P. chlamydospora (2021 > 2020 and 2019) and E. lata (2021 > 2020) suggest there is annual variation in either inoculum viability in the laboratory or spore survival/pruning-wound susceptibility in the field. Conidia of P. chlamydospora were from a single isolate produced under the same laboratory conditions each year, whereas ascospores of E. lata had to be harvested from an infected oleander tree each year. The reliance on such environmental samples may explain the lack of viability (and the lack of infections in the field) of E. lata ascospores in 2019; this is one of the risks of having to use field-collected spores of E. lata for our research, as spore maturity and viability cannot be under our control.
Because we made culture attempts from all water-inoculated (control) spurs within each fungicide treatment, we were able to characterize the “background level” of local pathogens each year, albeit from isolates from a low proportion of samples (22 of 405 spurs). We identified C. viticola, which was one of our inoculation treatments (albeit on inoculated spurs), and other Cytospora species known to be pathogenic to grape: Cytospora species 1 (an undescribed species, which we have in our culture collection) and C. vinacea (Lawrence et al. 2017). Other species pathogenic to grape we identified included C. ampelina, which is in the same fungal family as E. lata (Diatrypaceae) and also causes dieback symptoms, and Botryosphaeria dieback pathogen D. seriata, which was identified in a previous survey of Washington vineyards (Holland et al. 2015). Although characterization of natural fungal infections of pruning wounds is rarely published (e.g., [Luque et al. 2014]) and could potentially be very site- and year-specific, this step helped us evaluate our DNA-based detection method of C. viticola. Certainly, we knew prior to the field trial that the qPCR primers were not species-specific. Indeed, the primers amplified five additional Cytospora species, which we identified along with C. viticola from a combined 2.2% of the 405 water-inoculated (control) spurs examined throughout the three years. The diversity of Cytospora species, many of which are of unknown pathogenicity to grape, suggests that Cytospora dieback may be caused by more species than just C. viticola.
The main benefit of DNA-based detection via qPCR over culture-based detection of the three pathogens was being able to store the samples (540 spurs each year) at −20°C for processing over a period of time. Our study coincided with the global COVID-19 pandemic, which limited our ability to process samples in the time-sensitive manner necessitated by culture-based detection; DNA-based detection allowed us to continue the study in consecutive years through lab-occupancy restrictions. We acknowledge, though, that this approach does have limits. For example, in this study, the consistently high detection rates of C. viticola, even from fungicide-treated spurs, may reflect detection of DNA from dead spores. It is also possible that in some cases we detected DNA of dead E. lata spores that traveled within xylem vessels below the surface of the pruning wound, as has been shown at depths of 5 to 8 mm after artificial inoculations under dry conditions, similar to inoculation conditions in this study (Carter 1960, Larignon 2010). However, we discarded the distal ~0.5-cm end of the spur and extracted DNA from the wood below. Further, in 2019, E. lata inoculum was not viable and we had 0% detection via qPCR. As such, it seems that our DNA-based detection method did not have a high rate of false positives (i.e., did not detect the DNA of dead spores of E. lata). Consequently, we assume that positive detection through qPCR reflects detection of the corresponding metabolically active pathogens.
Conclusion
Because we timed the fungicide applications in our study to closely follow the typical time of final pruning in commercial vineyards, we assume our findings on fungicide efficacy would be relevant if spores of C. viticola, E. lata, or P. chlamydospora are dispersed after pruning. We demonstrated that fresh pruning wounds are susceptible to infection by the three pathogens, albeit at higher spore concentrations than they may encounter naturally. Additionally, environmental conditions during the dormant season in eastern Washington are cold and dry, which may also limit disease pressure. Pruning wounds, however, may not be the only point of infection for these pathogens. Wounds to the permanent, woody structure of the vine that are created by winter injury may be an important infection court, in addition to or more so than pruning wounds. More work is needed on the susceptibility of such wounds and their natural healing process in eastern Washington to reveal the best time for preventive practices, like fungicide applications and the timing of pruning.
Supplemental Data
The following supplemental materials are available for this article in the Supplemental tab above:
Supplemental Table 1 Identity of Cytospora isolates from water-inoculated (control) spurs in the Prosser, Washington vineyard, in comparison to related Cytospora previously reported from grape.
Footnotes
This research was funded by grant 17-0728-001-SF to K. Baumgartner, R. Travadon, and M. Moyer from the USDA Multi-State Program, and was partially supported by the USDA, National Institute of Food and Agriculture’s Hatch project #1016563. For processing samples in the lab, we thank Paula Eschen and Alejandro Hernandez. Mention of trade names or commercial products is solely for the purpose of providing specific information and does not imply recommendation or endorsement by the USDA. USDA is an equal opportunity provider and employer.
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- Received March 2022.
- Accepted September 2022.
This is an open access article distributed under the CC BY 4.0 license.
References
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