Introduction

Young vine decline, comprised by the grapevine trunk diseases black foot and Petri disease, is caused by several taxonomically unrelated xylem-colonizing fungi, including Phaeomoniella chlamydospora. These fungi induce vascular necrosis leading to poor grapevine vigour, lower fruit yield, and eventual grapevine mortality within the first few years after planting (Gramaje & Armengol, 2011). Young vine decline fungi have been shown to be prevalent in both asymptomatic and symptomatic wood of ready-to-plant nursery material, with significant variation in pathogen abundance between grapevines, different parts of the grapevine, and among nurseries (Berlanas et al., 2020; Billones-Baaijens et al., 2013; Hrycan et al., 2023; Maldonado-González et al., 2020). In addition, many young vine decline fungi are commonly found in asymptomatic grapevine tissue in mature grapevines in vineyards (Gainza-Cortés et al., 2020; González & Tello, 2011; Nerva et al., 2019).

Most grapevines harbor young vine decline fungi, and if their presence always resulted in symptom development and grapevine mortality, viticulture would not exist in its current form today. Studies conducted in British Columbia (BC) showed that approximately 8 % of young grapevines were symptomatic for young vine decline across the province (Úrbez-Torres et al., 2014), yet up to 100 % of ready-to-plant nursery grapevines sold in Canada are infected with young vine decline fungi (Hrycan et al., 2023). It has been hypothesized that some Petri disease fungi may act as latent pathogens, living as endophytes within the grapevine microbiome until a trigger in the form of grapevine stress occurs, resulting in a transition to a pathogenic lifestyle (Hrycan et al., 2020; Sieber, 2007).

Several observations have led to the hypothesis of an interaction between grapevine stress and disease development in grapevines affected by grapevine trunk diseases. For instance, Esca symptoms, in which Pch is thought to be involved, can vary yearly, and that variation has been correlated with climatic conditions (Marchi et al., 2006; Péros et al., 2008; Surico et al., 2000). Esca foliar symptom development has also been correlated with higher internal necrosis (Maher et al., 2012). A combination of pathogen infection level and grapevine stress is hypothesized to increase pathogen growth in the grapevine, leading to an increase in internal necrosis and mortality (Hrycan et al., 2020; Sieber, 2007). Investigations into Esca symptom development and water stress found that Esca foliar symptoms were inhibited in water stressed grapevines (Bortolami et al., 2021a), contrary to the hypothesis that grapevine stress results in symptom development and grapevine mortality. However, Esca is a complex disease with two forms, characterized as chronic foliar symptom development, which can vary year to year, and apoplexy, resulting in grapevine wilting and sudden collapse (Gramaje et al., 2018). Esca-related apoplexy has been associated with high temperature and low rainfall conditions (Surico et al., 2006). The differing conditions leading to chronic and apoplectic symptoms of Esca suggest the mechanisms for chronic symptom development may be different than apoplexy, which could be the result of grapevine stress. Like the apoplectic form in Esca, Petri disease in younger grapevines can result in sudden grapevine collapse (Gramaje & Armengol, 2011). Both diseases share some of the same fungi, which could respond similarly to stress in both young and older grapevines.

The effect of water stress on Pch growth in grapevines has been a research focus. Phaeomoniella chlamydospora can grow at osmotic water potentials as low as -8.3 MPa on potato dextrose agar, indicating the fungus is able to grow under severe water deficit (Whiting et al., 2001). Xylem sap and nutrient concentrations increase in infected grapevines subjected to water stress (Lima et al., 2017), which may provide the fungus with increased carbohydrates under water stress conditions. Phaeomoniella chlamydospora can also impact grapevine-water relations. ‘Cabernet-Sauvignon’ and ‘Zinfandel’ vines infected with Pch and subjected to water stress displayed lower leaf water potential compared with non-infected water stressed grapevines, indicating that these fungi may increase susceptibility to water stress (Edwards et al., 2007a; Edwards et al., 2007b). Effects of Pch on grapevine-water relations may be due to the formation of tyloses in the xylem, which can impact stem hydraulic conductivity and may affect grapevine survival during periods of stress (Bortolami et al., 2021b). In combination, these studies indicate Pch can grow during periods of severe drought, increase grapevine susceptibility to water stress, and may gain access to increased resources for pathogen growth. While conditions for pathogen growth may improve under water stress, the implications of Pch abundance on grapevine health are not well understood.

Long-term or severe water stress may also limit grapevine defense responses to pathogen infection. Plant defense against biotrophic plant pathogens is thought to be mediated by salicylic acid pathways while defense against necrotrophic plant pathogens is activated by jasmonic acid pathways (Glazebrook, 2005). In grapevines subjected to long-term water stress, jasmonate content was slightly decreased (Zhan et al., 2023). Water stress may also induce stomatal closure, limiting photosynthesis, and under severe water stress, xylem embolism may occur (Gambetta et al., 2020), which may create more favourable conditions for pathogen growth and limit the amount of carbohydrates available to produce defense compounds.

Arbuscular mycorrhizal (AM) fungi are root symbionts that mitigate abiotic and biotic stresses in plants, including grapevine (Trouvelot et al., 2015). Under drought conditions, AM-colonized grapevines maintain greater leaf water content, chlorophyll content, and root and shoot dry weights (Ye et al., 2022). Colonization of AM fungi may also increase jasmonic acid levels in the roots (Hause et al., 2007), which may improve plant defense against necrotrophic pathogens. It has been shown that AM fungi can reduce black foot disease severity in grapevines, suggesting a potential use in disease management (Petit & Gubler, 2006). However, recent work has reported increased internal necrosis and abundance of black foot disease fungi in mycorrhizal grapevines compared to non-mycorrhizal grapevines (Holland et al., 2019; Vukicevich et al., 2018), thus further investigation is required to understand the benefits or detriments of AM colonization in grapevine trunk disease infected grapevines.

Understanding how grapevine stress factors affect the growth of young vine decline fungi is essential in limiting the impact of the disease in vineyards, especially as water stress management in vineyards has other goals that need to be considered. Previous studies on Pch infection of grapevines have primarily focused on foliar symptom development, lesion length and phenotypical measurements to record the effect of water stress on disease development. Droplet Digital PCR™ technology has been used to quantify Pch in grapevines (Hrycan et al., 2023) and can be used to help further our understanding of how pathogen abundance affects grapevine health. Accordingly, the main objectives of this study were to investigate if water stress increases Pch abundance and disease development in both the greenhouse and field, and to determine if AM fungi can mitigate water stress under greenhouse conditions, thus reducing disease development.

Materials and methods

1. Greenhouse experiment

1.1. Preparation of inoculated, rooted grapevines

Dormant ‘Merlot’ canes were collected in February 2020, from an experimental vineyard block located at the Summerland Research and Development Centre (SuRDC), British Columbia, Canada. The canes were placed in bags and stored in the dark at 2.5 °C until used. Phaeomoniella chlamydospora isolate SuRDC-1070, obtained from grapevines showing Petri disease symptoms in BC and kept in the SuRDC plant pathology fungal collection was used for this study (Úrbez-Torres et al., 2014). Mycelial plugs from the isolate were retrieved from the collection and plated onto potato dextrose agar (PDA; Sigma-Aldrich, St Louis, MO) Petri plates and incubated at 22 °C in the dark for three weeks until mycelial colony developed. Sterile distilled water (2 mL) amended with a drop of Tween 80, a non-ionic surfactant to reduce clumping of spores, was pipetted onto the fresh mycelial colony and gently scraped with a sterile spoonula. The resulting spore suspension was vortexed and adjusted to 25,000 (25k), 5,000 (5k), and 1,000 (1k) spores per 10 µL with a haemocytometer. Viability of Pch was determined by plating a 10 µL suspension from each inoculum concentration in triplicate immediately following the vacuum inoculation procedure. Plates were incubated at 22 °C in the dark for seven days until fungal colonies were observed.

A total of 280 ‘Merlot’ canes were pruned to three nodes (approximately 22 cm in length) and fifty-six canes were allocated to each of the five inoculum treatments: 25k, 5k, 1k, a water inoculated control which contained Tween 80 as in spore suspensions, and a non-inoculated control. Each vine was vacuum inoculated by pipetting 10 µL of allocated inoculum suspension onto the freshly cut base of each cutting and then applying vacuum at the other end of the cane for 1 s. Vacuum pressure was 71.5 mmHg measured with a manometer (Model 06-664-19, Fisher Scientific, Waltham, MA). Dormant canes were transferred to the greenhouse and rooted in five 2 L buckets per treatment containing 750 mL of water, changed twice weekly. Canes were placed in separate buckets by treatment. Buckets were placed on rooting mats set to 20 ℃ for six weeks. Upon budbreak, greenhouse lights were turned on and the greenhouse temperature was set to 20 ℃.

On March 31, 2020, twenty-eight rooted cuttings from each treatment were randomly selected for planting. Rooted cuttings were planted in 3.78 L pots containing 3,000 g of pasteurized soil and with saucers underneath to retain water. The loamy sand y soil was collected from a field at SuRDC and pasteurized twice in a LANSA soil pasteurizer (Model LA, Johnson Machine Company LTD, Burlington, Ontario) set at a temperature reaching 72 to 88 ℃ (Trevors, 1996) following manufacturer’s instructions. Half of the pots (14) were amended with 0.25 g of AGTIV specialty crops powder (Premier Tech Growers and Consumers, Quebec, Canada) containing 3,000 Rhizophagus irregularis spores following manufacturer’s instructions.

1.2. Experimental design and grapevine health measurements

The 5 x 2 x 2 factorial combination of treatments was arranged in a completely randomized design and consisted of five levels of inoculum: 25,000 (25k), 5,000 (5k), and 1,000 (1k) Pch spores, water-inoculated (WIC), and non-inoculated (NIC); two levels of AM fungi: AM inoculated (AM+), and non-inoculated (AM-); and two levels of irrigation: fully irrigated (NWD) and 50 % irrigation (WD). Leaf water potential measurements were used to adjust irrigation volume based on WD measurements to maintain a minimum leaf water potential of -1.2 to -1.4 MPa and NWD above -1.0 MPA. There were seven replicate pots of each of the 20 treatment combinations with 140 grapevines total. In 2020, newly emerging shoots were removed to retain one shoot. As a result of pandemic restrictions during 2020, irrigation treatments were not applied and the potted grapevines were watered with 500 mL twice weekly until July 10, and three times a week onward until mid-September when water was gradually reduced to 50 mL three times a week in preparation for dormancy. Grapevines were placed in cold storage in the dark at 2.5 °C on December 18, 2020, and returned to the same greenhouse compartment on March 5, 2021, and equally watered until start of irrigation treatments on June 18, 2021. In 2021, two shoots were retained, and the grapevines were watered every second day starting June 18. NWD grapevines received 500 ml at each irrigation event from June 18 to August 14; 400 ml from August 18 to August 28; and 300 ml from September 1 until experiment concluded on September 13, 2021. WD grapevines were watered on the same days as NWD grapevines but with one-half the volume. All grapevines were fertilized with the same amount of 20-20-20 NPK fertilizer bi-weekly.

Leaf water potential, leaf chlorophyll content, and soil matric potential were measured in 2021. Mid-day leaf water potential was measured bi-weekly between 1:00 pm to 3:00 pm from June 29 to September 9, 2021, with a pressure chamber (Model 610, PMS Instrument Co. Corvallis, OR). Leaf chlorophyll content was measured between 9:00 am to 11:00 am bi-weekly from May 25 to September 9, 2021, with a leaf chlorophyll meter (SPAD 502 Plus, Spectrum Technologies, Aurora, IL). Soil matric potential was measured using soil tensiometers (Model T5, Meter Group, Pullman, WA) inserted in the soil of six each of NWD and WD pots randomly distributed in the greenhouse. Measurements were taken every 15 minutes and averaged daily from June 8 to September 9, 2021. On December 18, 2020, and at the end of the experiment in 2021, the number of grapevine nodes were counted, and grapevines were pruned to retain two buds before the prunings were weighed as a measure of growth. Root weight was measured at the end of the experiment. The prunings and roots were dried in an environmental chamber (Model 217502, Hotpack, Anaheim, CA) at 65 °C until dry weights were consistent over a 24 h period.

1.3. Trunk measurements

Total basal surface area (whole cross-section) and necrosis measurements were conducted upon conclusion of the experiment at one cm from the base of each grapevine as described in Hrycan et al. (2023) with the following alterations: Images were set at 8-bit colour and a threshold analysis was conducted in Fiji ImageJ version 1.53t. Threshold numbers were set values for the degree of shading from black to white (0-255). Black wood discolouration (Black necrosis) was set at 5-45, brown wood discolouration (Brown necrosis) was set at 46-80, and black and brown wood discolouration (total necrosis) was 5-80 (Figure 1).

1.4. Phaeomoniella chlamydospora absolute quantification

In order to verify presence of Pch in the grapevine and possible Pch growth between inoculation and planting, three grapevines from each treatment were randomly selected 24 h after vacuum inoculation and at the time of planting (six weeks post-inoculation). Grapevines were cut into 2 cm sections from 0-18 cm from the base. At the end of the experiment, a 2 cm section of wood was cut 2 cm above the base. Individual sections were surface sterilized via submersion in 70 % ethanol for 30 s, 10 % bleach for 1 min, and sterile water 2 x 1 min washes. Sections were cut in half longitudinally, finely chopped with a razor blade, and 0.2 g were placed in Qiagen DNeasy PowerSoil DNA extraction tubes (Qiagen, Hilden, Germany). The other half of each section was finely cut and plated on PDA amended with 0.1 mg/mL tetracycline hydrochloride (PDA-tet) to re-isolate Pch as described in Hrycan et al. (2023). A Qubit 1.0 Fluorometer dsDNA HS Qubit assay (Thermofisher, Waltham, MA) was used to calculate DNA concentration. Quantification of Pch was conducted with a Droplet Digital™ PCR (ddPCR) system (Bio-Rad Laboratory, Hercules, CA) using ITS primers (Martín et al., 2012) with the addition of a probe (Hrycan et al., 2023). Reactions and analysis were conducted as described in Hrycan et al. (2023). Pch abundance quantification results were standardized via calculation of gene copies/ng of DNA (Hrycan et al., 2023). Pch abundance analysis was conducted on grapevine sections at time of inoculation, time of planting (Figure S1) and at the end of the experiment.

1.5. Arbuscular mycorrhizal fungal colonization

Fresh roots were collected from one replicate of each treatment at the end of the experiment. Roots were frozen in liquid nitrogen, crushed with a pestle and mortar, and 0.2 g of crushed root material was placed in Lysing Matrix A (MP Biomedicals Irvine, CA) tubes and extracted using the Qiagen DNeasy Powerlyzer Powersoil kit according to manufacturer’s specifications. The Qiagen DNeasy Powerlyzer Powersoil kit was used due to manufacturer discontinuation of the DNeasy Powersoil kit. DNA concentration was calculated using a Qubit 1.0 fluorometer as previously described and ddPCR was conducted on each sample to determine presence or absence of the AM fungi using primers and probe described in Kokkoris et al. (2019). Black Sheaffer ink root staining was conducted to visually confirm presence of AM fungi via microscope (Vierheilig et al., 1998).

2. Field experiment

2.1. Grapevine material, inoculum preparation and vacuum inoculation

Dormant ‘Merlot’ and dormant ‘SO4’ canes were collected from vineyard blocks at SuRDC in February 2020 and stored in bags in the dark at 2.5 °C. Mycelial plugs containing Pch (SuRDC-1070) obtained from the SuRDC Plant Pathology fungal collection were plated, incubated, and used to create spore suspensions containing 25k and 2,500 (2.5k) spores as previously described. Canes were pruned to retain three nodes (22 cm) and 10 µl aliquots were vacuum inoculated (Rooney & Gubler, 2001) from the base with WIC, 2.5k, or 25k as previously described. Phaeomoniella chlamydospora viability was determined by plating 10 µL of each suspension in triplicate onto PDA as previously described.

Inoculated dormant ‘SO4’ canes, inoculated ‘Merlot’ canes and non-inoculated ‘Merlot’ canes used for grafting were dipped in 10 % bleach for 1 s. ‘SO4’ canes were entirely disbudded while inoculated ‘Merlot’ canes were disbudded below the top bud. Non-inoculated ‘Merlot’ canes were cut to retain one bud and were omega grafted onto ‘SO4’ grapevines. The grafted and non-grafted grapevines were then dipped in Stim-Root rooting hormone (Premier Tech Home and Garden, Brantford, ON) and layered in bins containing moist perlite, separated by treatment, and stored at room temperature in the dark for four weeks. On April 6, 2020, callused grapevines were removed from perlite, the top bud was dipped in wax, and planted in individual cartons measuring 35 x 8.5 x 8.5 cm containing 50 % perlite and 50 % Sunshine Mix #4 (Sun Gro Horticulture, Agawam, MA). The grapevines were placed in the greenhouse at 20 ℃ and watered evenly 3x per week. The rooted grapevines were planted on July 9, 2020, in an experimental field plot at the SuRDC.

2.2. Experimental design

The experiment was conducted in a field site at the SuRDC containing loamy sand y soil with row x grapevine spacing of 2.5 m x 0.93 m. Treatments were allocated in a blocked split-plot design as follows: Each block consisted of twelve vines and each block was replicated eight times. Whole-plot treatments of fully irrigated (NWD, no water deficit) or 50 % irrigation (WD, water deficit) were randomly applied to half of each block. The six vine-treatment combinations of vine type: self-rooted or grafted, and inoculum: WIC, 2.5k, and 25k were randomly allocated within each irrigation whole-plot. NWD plots had two Toro 0.5 GPH pressure compensating emitters (Toro, Bloomington, MI) on each vine while WD plots had one emitter per vine. Grapevines were irrigated Mon-Wed-Fri with equal timing between treatments with the duration of each irrigation run adjusted according to leaf water potential measurements. Grapevines were fertilized each spring equally with 15 g of 14-14-14 slow-release fertilizer placed under each grapevine. Grapevines were shoot thinned to one shoot in 2020, and three shoots in 2021 and 2022. The grapevines were removed in the spring of 2023.

2.3. Grapevine physiology and growth

No measurements were taken in 2020 due to pandemic restrictions. Mid-day leaf water potential was measured with a pressure chamber (Model 610, PMS Instrument Co. Corvallis, OR) in 2021 and 2022. In 2021, mid-day leaf water potential was measured on one leaf per grapevine on September 9. In 2022, leaf water potential was measured bi-weekly on Tuesday or Thursday from July 19 to August 16, and then weekly until September 15.

Leaf chlorophyll content was measured on ten leaves per grapevine and averaged, on August 17 and September 8 in 2021 due to poor growth in 2021, weekly from July 11 to September 26 in 2022, and on May 29 in 2023 with a chlorophyll meter (SPAD 502 Plus, Spectrum Technologies, Aurora, IL). Pruned shoots were collected and node numbers were counted each growing season. The shoots were dried at 65 °C and weighed as previously described. Development of young vine decline-like foliar symptoms and grapevine mortality were recorded each year. On May 30, 2023, grapevines were uprooted and placed in cold storage in the dark at 2.5 ℃ for analysis. Roots were cleaned in fresh water, removed from the grapevine, and dried at 65 ℃ until weights were consistent over two days.

2.4. Trunk measurements

Total basal surface area (entire cross-section) and necrosis measurements were taken 1 cm from the base as previously described. Trunk diameter was measured below the primary bud of the initial cane with a digital caliper (Model CD-6” C, Mitutoyo Corp. Japan).

2.5. Phaeomoniella chlamydospora absolute quantification

To verify presence of Pch, three grapevines from each treatment were randomly selected at time of planting, 13 weeks post-vacuum inoculation. The Qiagen DNeasy Powersoil kit was used for DNA extractions and DNA concentration and Pch abundance was analyzed as previously described. At the end of the experiment, grapevines were drilled at 0-1 cm, 2-4 cm, and 4-6 cm locations from the base using a sterile 1/8” drill-bit for re-isolation of Pch. 0.1 g of wood obtained from each drilling was used, placed in Lysing Matrix A tubes, frozen in liquid nitrogen, and macerated in a FastPrep-24™ at 6.0 m/s x 40 s x3. DNA was extracted using the Qiagen DNeasy Plant Mini kit according to manufacturer’s instructions. The DNA extraction kit was changed due to manufacturer discontinuation of the Qiagen DNeasy Powersoil kit. The DNA concentration and ddPCR were conducted as previously described. Each grapevine section was quantified separately and the sum of copies/ng DNA for 0-1, 2-4, and 4-6 cm was used for analysis to compare Pch abundance between grapevines.

2.6. Statistical analysis

For the greenhouse experiment, a three-way analysis of variance (ANOVA) was used to assess individual main-factor and interaction effects of Pch inoculum concentration, irrigation level and AM inoculation on necrosis, pruning and root weights, node number, chlorophyll, leaf water potential and soil matric potential. Tukey’s Honest Significant Difference test set at 5 % significance was used for post-hoc comparison of inoculum level x treatment combinations. Residuals were tested for normality with the Shapiro-Wilk test. An ANOVA was used to assess the effects of WIC and NIC on all measurements. Spearman’s coefficients of correlation were used to assess relationships between inoculum level and response variables.

Field experiment data (pruning weights, node number, chlorophyll, leaf water potential) were analyzed with a linear mixed effect ANOVA model considering the blocked split-plot design and fit with REML with the package nlme. Fixed effects were irrigation treatment, inoculum and grafting treatment (self-rooted or grafted), while block was considered as a random effect. Irrigation treatments were designated whole-plot experimental units, and the inoculum x rootstock vine treatment combinations were sub-plot experimental units nested within whole-plots. Significance was tested with a Type 3 sums of squares. Residuals were inspected visually. Tukey’s Honest Significant Difference test set at 5 % was conducted on significant interactions using the emmeans package in R Version 4.3.0 (Lenth, 2023; Pinheiro & Bates, 2000; R Core Team, 2023). Correlations between inoculum level and other response variables were assessed using a Spearman correlation coefficient. A chi-squared test was used to determine effects of grafting treatment (self-rooted vs. grafted), inoculum, and irrigation treatment on proportions of grapevines expressing symptoms and proportional grapevine mortality.

Results

1. Greenhouse experiment

1.1. Grapevine physiology and growth measurements

In 2021, leaf chlorophyll content differed with irrigation x date (p < 0.001). Leaf chlorophyll content was 4.1 % and 2.5 % higher in WD than NWD grapevines on July 29 and September 9 respectively (Table S1), while no other treatment effects were observed. Mid-day leaf water potential differed based on irrigation treatment (p < 0.001), date (p < 0.001), and irrigation treatment x date (p < 0.001). Mid-day leaf water potential was lower in WD than NWD grapevines at each time point (Figure 2A) and overall (WD: -1.9 MPa; NWD: -1.32 MPa), but no effects of inoculum (p = 0.817) or AM fungi were observed. (p = 0.709). Irrigation treatment x AM had no effects (p = 0.547). Mean leaf water potential of all measurement dates was NWD/AM-: -1.30 MPa, NWD/AM+: -1.34 MPa, WD/AM-: -1.90 MPa, WD/AM+: -1.89 MPa. Soil matric potential differed based on irrigation x date (p < 0.001), with soil matric potential lower in response to WD treatment (-19.75 kPa) compared with NWD (-7.58 kPa) overall. Soil matric potential daily average was lower in response to WD treatment compared with NWD over time, reaching a minimum of -72.35 kPa (Figure 2B) in the soil of WD treated pots.

Pruning weights differed based on irrigation x year (p < 0.001), with dry pruning weight reduced by 10.4 % due to WD treatment in 2021. Node number also differed with irrigation x year (p < 0.001) with a 22.4 % reduction in node number in WD treated grapevines. Node number also different based on irrigation x AM inoculation (p = 0.047) with mean node number higher in NWD/AM+ grapevines and NWD/AM- than both WD/AM+ and WD/AM- grapevines in 2021 (Table S2). Dry root weight differed based on irrigation treatment (p < 0.001) at the end of the experiment with WD treated grapevine dry root weight 17.7 % lower than in NWD treated grapevines (Table S2). Inoculation with AM had no effect on pruning weight, node number, or root weight in either year.

27 % of AM+ grapevines had roots colonized whereas no AM- grapevines were colonized with AM fungi. Visual confirmation via compound microscope confirmed the presence of AM fungi in the roots.

1.2. Quantification of Phaeomoniella chlamydospora

Phaeomoniella chlamydospora was not detected with ddPCR at 2-4 cm from the base at time of inoculation; however, re-isolations confirmed the presence of Pch in 100 % of inoculated grapevines. Abundance of Pch at 2-4 cm from the base was correlated with inoculum levels (Spearman r = 0.91), while WIC and NIC grapevines were negative for Pch. Re-isolations confirmed the presence of Pch at time of planting in 100 % of inoculated grapevines while the fungus was not isolated from WIC or NIC grapevines. At the end of the experiment, Pch was not detected in WIC or NIC grapevines with ddPCR or traditional plating, but it was isolated from 96 % of 25k, 93 % of 5k, and 90 % of 1k grapevines. Initial inoculum quantity was also correlated with Pch abundance at the end of the experiment (Spearman r = 0.63). Mean Pch abundance for 1k was 3,300 copies/ng, 5k was 5,614 copies/ng, 25k was 4,996 copies/ng. WD treatment and AM fungi had a positive effect on pathogen growth in Pch inoculated grapevines (Table 1); Pch abundance was greater in WD/AM+ grapevines than in WD/AM-, NWD/AM+ and NWD/AM- grapevines (Figure 3).

1.3. Trunk measurements

Basal necrosis measurements indicated main-factor effects of inoculum and irrigation on black and brown necrosis, total necrosis, and basal surface area (Table 1). Coefficients of correlation between inoculum level and black necrosis (p < 0.001), brown necrosis (p < 0.001), total necrosis (p = 0.002), and basal surface area (p = 0.027) were 0.53, -0.67, 0.29 and 0.21, respectively, showing a correlation between Pch inoculated and WIC/NIC grapevines (Table 2). Black necrosis, total necrosis, and basal surface area were impacted by irrigation level (Table 1). Black and total necrosis were greater in grapevines containing Pch and in WD grapevines, while grapevines containing Pch had less brown necrosis compared with WIC grapevines (Table 2). Basal surface area was greater in NWD grapevines compared with WD grapevines regardless of inoculum level (Table 2).

2. Field experiment

2.1. Grapevine physiology and growth measurements

Leaf chlorophyll content was affected by inoculum x grafting treatment (p = 0.022) and irrigation treatment (p = 0.013) in 2021. Spearman correlation r was -0.27 for the effect of inoculum on grafted leaf chlorophyll content in 2021 and was not significant for self-rooted (p = 0.481). NWD grapevines showed overall a 4.8 % lower leaf chlorophyll content than WD grapevines. In 2022, grafting treatment had main-factor effects (p = 0.003) on leaf chlorophyll and was 2.7 % greater in self-rooted than grafted. Inoculum had no impact on leaf chlorophyll in 2022 or 2023 (Table S3).

Mid-day leaf water potential was affected by irrigation treatment (p = 0.020) in 2021. In 2022 the irrigation x date (p = 0.003) and irrigation x grafting treatment (p = 0.001) interactions affected mid-day leaf water potential. Mid-day leaf water potential was lower in WD grapevines (-0.85 MPa) than NWD grapevines (-0.79 MPa) on September 9, 2021, and overall, in 2022 (WD = -1.11 MPa, NWD = -0.93 MPa) according to a repeated measures ANOVA. In 2022, WD grapevines had lower leaf water potential on August 25 and September 6 (Figure 2C). Mean leaf water potential did not differ between self-rooted (-0.84 MPa) and grafted (-0.80 MPa) grapevines in 2021 but self-rooted leaf water potential (-0.99 MPa) was greater than grafted (-1.11 MPa) in 2022.

Dry pruning weights (p < 0.001) and node numbers (p = 0.002) had main factor effects of grafting treatment x year. Self-rooted dry pruning weight was 41.4 % lower than grafted in 2021, 44.5 % in 2022, and 64.7 % in 2023 (Table S4). Self-rooted node number was 30.2% lower in 2022 (self-rooted: 26.6, grafted 38.1), and 38.8 % lower in 2023 (self-rooted: 42.9. grafted: 70). Dry root weight differed due to grafting treatment (p = 0.007) and was 45 % lower in self-rooted than grafted upon conclusion of the experiment, but irrigation treatment and inoculum had no effect (Table S4).

2.2. Quantification of Phaeomoniella chlamydospora

Phaeomoniella chlamydospora abundance was correlated with inoculum at time of planting (r = 0.73). However, ddPCR analysis showed WIC grapevines to be infected with Pch at time of planting. 25k abundance was 69,253 ± 15,370, 2.5k was 22,055 ± 9,967, WIC was 8,685 ± 5,758. Re-isolations confirmed the presence and viability from the first 0-6 cm of Pch in 66 % of 25k and 2.5k grapevines, and 0 % of WIC grapevines.

Phaeomoniella chlamydospora abundance was impacted by initial inoculum only (p < 0.001; r = 0.46). At the end of the experiment, 25k grapevines contained 65,605 ± 14,669 copies/ng, 2.5k contained 44,227 ± 9,889 copies/ng and WIC contained 21,167 ± 4,233 copies/ng.

2.2. Trunk measurements

Black necrosis was positively correlated with inoculum for self-rooted (p = 0.036; r = 0.40), grafted (p < 0.001; r = 0.54), and for mean of self-rooted and grafted (p = 002; r = 0.37) [Table 4]. Brown necrosis was not different between treatments (Table 4). Total necrosis was positively correlated with inoculum for self-rooted (p = 0.021; r = 0.43), grafted (p = 0.07; r = 0.43), and mean of self-rooted and grafted (p < 0.001; r = 0.43) [Table 4]. Basal surface area was negatively correlated with inoculum for the mean of self-rooted and grafted (p = 0.033; r = -0.26), while trunk diameter was not different between treatments (Table 4). Total necrosis was higher in self-rooted compared with grafted and basal surface area was higher in self-rooted than grafted.

2.3. Grapevine symptom development and mortality

Grapevine mortality was observed in the winter of 2021, followed by young vine decline-like symptom development and grapevine mortality in the summer of 2022 and grapevine mortality in the winter of 2022. In 2021, grapevine mortality was greater in self-rooted grapevines inoculated with 25k and 2.5k compared with 100 % survival of WIC grapevines (Table 5). In 2021, 100 % of grafted grapevines survived. In 2022, WIC grapevines under 50% irrigation had higher mortality compared with fully irrigated WIC grapevines (Table 5). In 2022 and cumulatively, 50 % irrigation treatment increased mortality compared with fully irrigated grapevines (2022: 47 % compared with 13 %; Cumulatively: 62 % compared with 28 %) in self-rooted grapevines but not for grafted grapevines (Table 6). Across all years, self-rooted grapevine mortality was 46.2 % which was greater than grafted grapevine mortality of 18 %.

Six grapevines were symptomatic for young vine decline in 2022 (Figure 4). Leaf water potential and leaf chlorophyll content were measured on symptomatic leaves due to lack of asymptomatic leaves in symptomatic grapevines. The leaf water potential of symptomatic grapevines averaged -0.71 MPa compared with -1.04 MPa in asymptomatic grapevines. Leaf chlorophyll content readings were lower in symptomatic grapevines, averaging 18.81 in SPAD units compared with 27.25 in asymptomatic grapevines in 2022. Three of the six symptomatic grapevines died prior to the conclusion of the experiment in 2023, when most grapevine mortality occurred, thus these three grapevines were excluded from overall grapevine health and Pch abundance analysis. The three grapevines that died were 1) self-rooted WIC/WD, 2) grafted 2.5k/WD, and 3) grafted WIC/WD. Pch abundance was 1) 33,700, 2) 11,384, and 3) 17,466 copies/ng DNA. For the three grapevines mean dry shoot weight was 2.82 g, mean node number was 32, and mean dry root weight was 11.05 g. Mean black necrosis was 25.8 %, brown necrosis was 42.2 %, and total necrosis was 68 %. Mean basal surface area was 129.04 mm.

Discussion

This is the first study to investigate the interactive effects of grapevine water stress and Phaeomoniella chlamydospora inoculum levels on Petri disease development in grapevines. The results of this study suggest water stress influences Pch growth and grapevine mortality within the first few years after planting.

The main objective of this study was to understand whether water stress increases Pch growth within the grapevine. Phaeomoniella chlamydospora can grow under severe water deficit and may have better access to carbohydrates under drought conditions (Lima et al., 2017; Whiting et al., 2001), indicating water stress may favor pathogen growth and colonization (Hrycan et al., 2020). Recently, in grapevines naturally infected with Pch, water stress reduced microbiome diversity and increased Pch abundance (Leal et al., 2024). In our greenhouse experiment, Pch abundance was greater in inoculated grapevines under 50 % deficit irrigation, confirming the results of Leal et al. (2024). However, in our study, Pch abundance was higher only when AM fungi were also present. Arbuscular mycorrhizal fungi can increase grapevine growth and improve resistance to abiotic and biotic stress factors (Trouvelot et al., 2015; Ye et al., 2022). It was hypothesized that AM fungi would reduce grapevine water stress and decrease disease development in grapevines infected with Pch. However, AM treatment had no impact on plant growth measurements in this study and our results indicate AM fungi are beneficial to Pch growth under grapevine water stress. These findings are in accordance with Holland et al. (2019) and Vukicevich et al. (2018) who observed an increase in fungal abundance and necrosis in grapevines infected with black foot fungi and colonized by AM fungi. In our study, no link between AM fungal colonization and increased internal necrosis was observed in Pch inoculated grapevines, however, due to the shorter length of the greenhouse experiment, it may not have been enough time for greater Pch abundance to result in greater internal necrosis.

The mechanism behind the influence of AM colonization and drought on Pch fungal abundance is unclear, but an interactive effect of AM fungi and drought on grapevine defenses may explain Pch growth. Water stress alone and in combination with AM fungi has been reported to increase salicylic acid production in other hosts (Choudhary & Senthil-Kumar, 2022; Sanchez-Romera et al., 2018). An increase in salicylic acid, which is antagonistic to jasmonic acid, involved in induced systemic resistance (Thakur & Sohal, 2013), could result in reduced grapevine resistance against necrotrophic pathogens (El Rahman et al., 2012). Additionally, Pch showed increased access to carbohydrates in the xylem under water stress (Lima et al., 2017), which could further promote pathogen growth in grapevines with suppressed induced systemic resistance. If Pch is a latent pathogen, increased Pch growth under water stress conditions will reach the colonization threshold in which grapevine mortality occurs sooner (Hrycan et al., 2020). Overall, our greenhouse Pch abundance results support the hypothesis of increased Pch growth under grapevine water stress, but other factors may be involved in Pch growth, as combined water stress and AM+ treatment increased Pch abundance but water stress alone did not. It is unclear whether greater Pch abundance is detrimental to grapevine health, but pathogen abundance has been correlated with symptom development in other hosts such as Swiss Needle Cast disease in Douglas-Fir and brown stem rot of soy (Impullitti et al., 2009; Manter et al., 2003).

In contrast to the greenhouse experiment, Pch abundance was not impacted by water stress under natural field conditions. Differences between findings in the greenhouse and field may be due to the much higher levels of water stress obtained in the greenhouse compared with field grapevines. In the field, mid-day leaf water potential indicated mild to moderate grapevine water stress under the WD irrigation regime compared with the NWD, while in the greenhouse, mid-day leaf water potential indicated high to severe grapevine water stress under the WD irrigation regime (Deloire et al., 2020). While Pch abundance was not affected by water stress in the field, initial inoculum quantity influenced Pch abundance at the end of the experiment. In both the greenhouse and field, Pch abundance was positively correlated with initial inoculum level. However, Pch abundance also varied widely among individual grapevines. Variation in Pch abundance is in accordance with the findings of Hrycan et al. (2023) in ready-to-plant nursery material, however, while variation in nursery material could be attributed to natural infection, it is unclear why we obtained such high Pch variation in both greenhouse and field grapevines inoculated with known Pch spore quantities, but may be the result of variations in spore distribution within the grapevine in nursery grapevines and vacuum inoculated grapevines. Further work is required to understand the underlying cause of this variation between grapevines. The microbiome of individual grapevines plays a role in protection of wood against pathogens (Xiong & Yang, 2023). Variation in the microbiomes of individual grapevines could similarly influence Pch establishment and growth within the grapevine, but further investigation is required to determine if the individual grapevine microbiome is the cause of this variation in Pch abundance.

Infection of Pch in the WIC grapevines in the field was observed at time of planting suggesting infection occurred during the propagation process. Infection was not observed in greenhouse grapevines, but the propagation process was different between the greenhouse and field and contamination or natural infection may have spread during the callusing stage which was not completed for the greenhouse experiment.

A second objective of the present study was to determine whether initial Pch abundance had an impact on internal necrosis within the grapevine after planting. Previous work has reported no correlation between internal necrosis and pathogen abundance in ready-to-plant nursery grapevines (Hrycan et al., 2023). In both greenhouse and field experiments, Pch abundance was correlated with internal necrosis. In the greenhouse and field, black and total necrosis were positively correlated with inoculum level. Brown necrosis in the greenhouse was negatively correlated with inoculum level, while in the field inoculum level had no effect on brown necrosis. However, internal necrosis was similar between Pch inoculated grapevines, but was higher than WIC/NIC grapevines. Recent nursery surveys have reported a correlation between necrosis and individual nurseries and rootstocks rather than pathogen abundance or infection (Carbone et al. 2022; Gramaje & Armengol, 2011; Hrycan et al., 2023; Lade et al., 2022). However, previous studies have primarily investigated total necrosis without separating black necrosis from browning of the wood (Hrycan et al., 2023). Necrosis from both greenhouse and field grapevines suggests browning of the wood should be excluded from pathogen necrosis analysis for Pch.

In the field, black necrosis and total necrosis were positively correlated with inoculum level, while basal surface area negatively correlated with inoculum level, however, differences were primarily observed between WIC/2.5k and 25k. Water stress alone was mild to moderate and unlikely to induce grapevine death, instead, increased necrosis and lower basal surface area could have impacted grapevine symptom development and mortality in combination with water stress (Maher et al., 2012; Oliva et al., 2014). In addition, grapevine water stress was higher in the greenhouse than the field, and stress induced higher necrosis in the greenhouse, potentially due to xylem cavitation (Gambetta et al., 2020). Interestingly, different effects were observed in self-rooted grapevines on basal surface area, increasing in the greenhouse and having no effect on the self-rooted grapevines in the field experiment. Phaeomoniella chlamydospora infection can result in xylem blockage due to necrosis or tylose formation (Bortolami et al., 2021b), which the grapevine could respond to by the growth of new xylem vessels as observed in other host responses to xylem dysfunction after water stress (Gauthey et al., 2022). A decrease of basal surface area in field grapevines could be due to increased Pch growth in the field over the longer-term.

Increased internal necrosis and decreased basal surface area could have influenced symptom development and grapevine mortality. Foliar symptoms developed in the field in August and September of 2022, primarily in 25k and 2.5k inoculated grafted grapevines, leading to sudden vine collapse, a characteristic symptom of severe Petri disease (Gramaje et al., 2018). Symptoms included reddening of the leaves, lower leaf chlorophyll content, and greater leaf water potential in 2022, indicating leaf dysfunction, expected of sudden vine collapse. In older grapevines with Esca, in which Pch is also involved, water stress has been correlated with the inhibition of symptoms (Bortolami et al., 2021a), in contrast to the findings of this study. However, Esca is a separate and complex disease, and has two forms, chronic and apoplectic (Gramaje et al., 2018), and symptom development in this study was followed by sudden vine collapse, like the Esca apoplectic form. Chronic Esca symptom development may be unrelated to internal necrosis, as observed in other grapevine trunk diseases (Travadon et al., 2013; Sosnowski et al., 2011) and instead could be due to leaf vascular occlusions (Bortolami et al., 2023). On the other hand, Esca apoplexy has been associated with high temperatures and lack of rainfall (Surico et al., 2006).

In our study, self-rooted grapevine mortality was higher in WD than NWD grapevines. In contrast, symptom development and grapevine mortality did not occur in the greenhouse, which is unexpected due to the greater Pch abundance observed in WD Pch inoculated grapevines in the greenhouse. However, the greenhouse experiment was shorter in time than the field experiment and it was conducted under controlled conditions in pasteurized soil, which may have resulted in more favourable conditions for grapevine survival due to exposure to fewer external factors than field grapevines. Irrigation treatment was applied for a longer period in the field and external abiotic and biotic stress factors in the field may have weakened the grapevines further compared with the greenhouse. The findings of this study suggest Pch abundance is correlated with symptom development, which has been observed in other diseases in other hosts (Impullitti et al., 2009; Manter et al., 2003). Phaeomoniella chlamydospora has often been isolated from asymptomatic grapevines and nursery material, indicating it can have a limited impact on grapevine growth (Gainza-Cortes et al., 2020; Nerva et al., 2019).

Variations in rootstock susceptibility to Pch infection have been previously reported (Eskalen et al., 2001), but rootstock tolerance to infection under stress has not been investigated. In this study, self-rooted grapevines had lower dry pruning weight, node numbers, dry root weight, higher Pch abundance, total necrosis, and overall grapevine mortality than grafted grapevines, suggesting self-rooted has higher susceptibility to Pch infection compared with grafted rootstock under natural field conditions. However, this study found grafted grapevines had lower leaf water potential compared with self-rooted indicating a higher level of stress in grafted, which may have been due to the increased growth of grafted grapevines. Murolo and Romanazzi (2014) observed increased disease incidence in ‘SO4’ compared with ‘1103P’ rootstock, which is thought to be more drought tolerant, but our results indicate grapevine water stress may not be the only factor. Xylem diameter can also play a role in pathogen abundance and grapevine susceptibility to Pch (Pouzoulet et al., 2017). In grapevines infected with Phaeoacremonium minimum, another Petri disease associated fungus, smaller xylem diameter was associated with lower pathogen abundance (Ramsing et al., 2021). Xylem diameter in combination with grapevine water stress should be investigated to determine how pathogen growth and symptom expression are impacted.

Several Petri disease fungi have been hypothesized to be latent pathogens, in part due to isolating them from both asymptomatic grapevine wood tissue and their ability to grow under low osmotic water potentials, which affect nutrient levels in the xylem (Lima et al., 2017; Hrycan et al., 2020; Whiting et al., 2001). This study revealed a correlation between water stress and AM fungal inoculation with increased Pch abundance in the greenhouse. In the field, all grapevines contained Pch and overall, water stressed grapevines had greater mortality than non-stressed grapevines. These results support the hypothesis that Pch is a latent pathogen and increased Pch abundance leads to mortality in young grapevines under stress (Hrycan et al., 2020; Sieber, 2007). To our knowledge, this is the first study to induce young vine decline-like foliar symptom expression, and sudden vine collapse, with a link between Pch abundance and grapevine mortality within the first few years after planting. However, the underlying mechanisms for symptom development and Pch growth remain unclear. Increased initial Pch inoculation was correlated with an increase in internal necrosis which could lead to heightened risk of severe grapevine water stress. Grapevines under severe stress could upregulate anthocyanin biosynthesis genes resulting in anthocyanin production in the leaves, and sudden vine collapse during periods of high stress (Dabravolski & Isayenkov, 2023; Gramaje et al., 2018; Haider et al., 2017). This study suggests that grafted grapevines may be less susceptible to symptom expression and mortality compared with self-rooted in natural environments, but further investigation into the factors’ influencing susceptibility is required to further understand the relationship between Pch infection, grapevine water stress, and self-rooted or grafted grapevine susceptibility on symptom expression and grapevine mortality.

Acknowledgements

This research was funded by the British Columbia Wine Grape Council (BCWGC), The Canadian Grapevine Certification Network (CGCN-RCCV), and Agriculture and Agri-Food Canada (AAFC) under the Canadian Agricultural Partnership (CAP) program.