Abstract
Background and goals Botryosphaeria dieback is a grapevine trunk disease responsible for significant economic losses to grape producers worldwide. There are currently no products registered in Canada to control grapevine trunk diseases, and sustainable production has become a focus of the Canadian grape and wine industry. Therefore, the objectives of this research were to evaluate locally sourced Trichoderma spp. as biocontrol of Botryosphaeria dieback fungi in the field, to compare Trichoderma spp. against commercial products registered in other countries, and to determine optimum temperatures for conidial germination of Trichoderma spp. found in British Columbia (BC).
Methods and key findings Detached cane assays under greenhouse conditions and field trials in a Merlot vineyard during two growing seasons assessed Trichoderma spp. from BC and commercial products as pruning wound protectants against Diplodia seriata and Neofusicoccum parvum over time. The Trichoderma-based treatments developed in this study provided pruning wound protection for up to 60 days after treatment and performed better or similarly to commercial products. Germination studies showed conidia of Trichoderma canadense germinated faster at lower temperatures than the other Trichoderma spp.
Conclusions and significance This represents the first study evaluating locally sourced Trichoderma spp. as protection against grapevine trunk disease fungi under field conditions in Canada, and shows the potential that these endophytic isolates have as pruning wound protectants over time. This study also screened commercial products to manage grapevine trunk diseases for the first time in Canada, providing key data to support product registration. The discovery of faster conidial germination of T. canadense at lower temperatures deserves further investigation, as this species could be better adapted and thus provide more effective pruning wound protection when applied at the colder temperatures experienced in BC during the pruning season.
- biological control
- Diplodia seriata
- grapevine trunk diseases
- Neofusicoccum parvum
- pruning wounds
- Trichoderma
- Vitis vinifera
Introduction
Botryosphaeria dieback is a grapevine trunk disease caused by several fungal species in the family Botryosphaeriaceae. It occurs wherever grapes are grown and is considered one of the main biotic factors reducing yields and shortening vineyard lifespan (Úrbez-Torres 2011). Botryosphaeriaceae spp. primarily infect grapevines via spores (conidia) through pruning wounds, but can also be introduced into vineyards with contaminated nursery stock (Gramaje and Armengol 2011, Hrycan et al. 2023). The most characteristic symptoms associated with Botryosphaeria dieback are the loss of spur positions and the presence of perennial, usually wedge-shaped, cankers in the vascular system of spurs, cordons, and/or trunks. Canker development through the grapevine framework limits transport of water and nutrients, causing vine dieback over time and eventual plant death, with accompanying economic loss to the grape and wine industry (Siebert 2001, Kaplan et al. 2016).
Currently, no product is available to eradicate Botryosphaeria dieback or any other grapevine trunk disease once grapevines are infected. Since pruning wounds are the primary point of infection every year, prophylactic strategies based on chemical treatment of pruning wounds, either by hand or spray application, provide the most effective control strategies against Botryosphaeria dieback (Rolshausen et al. 2010, Pitt et al. 2012, Ayres et al. 2017). Good control of Botryosphaeria dieback has been obtained using different active ingredients within the benzimidazole, phthalimide, and triazole families as pruning wound protectants in trials conducted in California (Leavitt 1990). Since then, more studies have resulted in registration of several chemical active ingredients in different countries (Gramaje et al. 2018). However, cumulative proof regarding the negative effects of some fungicides on animal, human, and environmental health have resulted in strict regulation of chemical usage and many active ingredients used to control grapevine trunk diseases being banned, phased out, or placed under re-evaluation (Decoin 2001, Pearson and Miller 2014). Additionally, the steady increase in organic cultivation and greater focus by the grape and wine industries on more sustainable production have created a demand for alternatives to chemicals and expanded biological control research on grapevine trunk diseases (Mesguida et al. 2023). Among the biological control agents investigated in grapevines and other crops, species in the genus Trichoderma are by far the most studied due to their strong generalist antifungal properties; their ability to induce plant immune responses; and their positive effects on plant growth, water retention, and nutrient forgaging (Woo et al. 2014, Compant and Mathieu 2016, Guzmán-Guzmán et al. 2019). As a result, over 60% of registered biocontrol products in agriculture are Trichoderma-based (Verma et al. 2007).
Trichoderma spp. have long been investigated as biocontrol agents against grapevine trunk diseases, including Botryosphaeria dieback, at the nursery level and as pruning wound protectants in vineyards (Gramaje et al. 2018). However, it is still challenging to draw conclusions about their efficacy due to contradictory results between available studies. For instance, Trichoderma atroviride strain SC1 (Vintec, Belchim Crop Protection) significantly reduced both incidence and severity of Diplodia seriata and Neofusicoccum parvum in nursery material and when establishing new vineyards (Berbegal et al. 2020). Similarly, there were significantly fewer Botryosphaeriaceae spp.-infected nursery plants after treatment with T. atroviride SC1 (Leal et al. 2023). Recently, SC1 efficiently protected grapevine pruning wounds against N. parvum in trials conducted in California (Travadon et al. 2023). However, the same SC1 strain, along with T. atroviride strain I-1237 (Esquive, Idai Nature S.L.), resulted in poor control of D. seriata when applied as a pruning wound protectant in mature vineyards in Spain (Martínez-Diz et al. 2021). In contrast, strain I-1237 significantly reduced Botryosphaeriaceae spp.-associated symptoms in vine trials conducted in France (Mounier et al. 2016).
Several factors may explain these inconsistencies. For instance, different antifungal capacity against the same Botryosphaeria dieback pathogen has been recorded not only among Trichoderma spp. but also between strains within the same species (Úrbez-Torres et al. 2020, Pollard-Flamand et al. 2022). Currently, most studies on Trichoderma control of grapevine trunk diseases are based on a small set of isolates of specific origin in the form of commercial products, some of which were originally developed and registered to control other diseases in crops other than grapevines (Gramaje et al. 2018). Recent research has focused more on identifying and evaluating native grapevine endophytic Trichoderma spp. and their potential as biocontrol agents against grapevine trunk diseases, with the objective of finding strains that are more effective and better adapted to the specific conditions of a region and host (Marraschi et al. 2019, Úrbez-Torres et al. 2020, Karličić et al. 2021, Kovács et al. 2021, Silva-Valderrama et al. 2021, Langa-Lomba et al. 2022, Pollard-Flamand et al. 2022). Overall, these studies have found up to 15 different Trichoderma spp. that provide promising results to control Botryosphaeriaceae spp. To date, field trials have primarily examined registered Trichoderma-based commercial products (Berbegal et al. 2020, Blundell and Eskalen 2022, Travadon et al. 2023). In addition, studies that screened naturally found endophytic Trichoderma spp. have only evaluated them under controlled conditions either in vitro in the laboratory or in planta in the greenhouse. To date, only one study has evaluated endophytically-found Trichoderma hamatum in field trials in California (Blundell and Eskalen 2022). Most studies have not completed evaluations over time under natural field conditions to determine how long a Trichoderma sp. can protect pruning wounds. This is important, since grapevine cultivar and/or environment at the time of application can affect the success of Trichoderma spp. colonization and thus, their efficacy as pruning wound protectants (Mutawila et al. 2011, 2016).
A recent study identified seven endophytic Trichoderma spp. from grapevines in British Columbia (BC) (Pollard-Flamand et al. 2022). Among these, three isolates of three different species, Trichoderma asperelloides (SuRDC-1442), T. atroviride (SuRDC-1440), and Trichoderma canadense (SuRDC-1422), showed high biocontrol activity as pruning wound protectants against Botryosphaeria dieback fungi when screened under controlled laboratory and greenhouse conditions. Since field evaluations are critical to determine the true potential of these Trichoderma spp. as biocontrol agents, the main objective of this study was to assess their biocontrol capacity under natural field conditions in a Merlot vineyard and to compare their performance against both chemical and biological commercial products registered in other countries. In addition, we further characterized these species for their ability to germinate at different temperatures to determine the best application time. Currently, no products are registered in Canada to control grapevine trunk diseases and thus, this study is an essential step toward providing the grape and wine industry with sustainable tools to manage these important diseases.
Materials and Methods
Trichoderma and pathogen inoculum preparation
Pure cultures of T. asperelloides (SuRDC-1442), T. atroviride (SuRDC-1440), T. canadense (SuRDC-1422), and the Botryosphaeria dieback pathogens D. seriata (SuRDC-1089) and N. parvum (SuRDC-1064) were retrieved from the Summerland Research and Development Centre (SuRDC) fungal collection in Summerland, BC. Spore (conidia) suspensions of Trichoderma spp. and pathogens were prepared as described (Pollard-Flamand et al. 2022). The conidial suspension of each Trichoderma isolate was adjusted to 1 × 106 conidia/mL with a haemocytometer before the solutions were combined in equal parts to obtain four different species-combination treatments, based on previous results (Pollard-Flamand et al. 2022), all with a final concentration of 1 × 106 conidia/mL (Table 1). D. seriata and N. parvum conidal suspensions were each adjusted to 1 × 105 conidia/mL using a haemocytometer. All suspensions were prepared fresh the same day of use and viability was confirmed by plating 50 μL of each Trichoderma sp. and fungal pathogen onto different potato dextrose agar (PDA; DIFCO) petri plates. Plates were incubated at 23°C in the dark for up to five days to confirm spore germination and fungal growth.
Pruning wound treatments used in this study.
Greenhouse detached cane assays
Three different detached cane assays, modified from Ayres et al. (2017), were conducted separately to evaluate the different Trichoderma treatments from BC and two different commercial products (Table 1) against D. seriata and N. parvum under controlled greenhouse conditions as described (Pollard-Flamand et al. 2022). All detached cane assays were conducted under the same conditions using the same greenhouse at different times. Chardonnay dormant canes were obtained from an experimental vineyard block at the SuRDC. Canes were pruned into two-node sections (~20 cm length) and placed through holes made in styrofoam trays floating on water tables, ensuring that the bottoms of the canes were submerged. All canes were pruned ~4 cm above the upper bud to simulate a fresh pruning wound. For each detached cane assay, each treatment included a total of 360 canes. Within three hours of pruning, 180 canes (pruning wounds) per treatment were treated with either 50 µL of 1 × 106 spores/mL (50,000 spores/wound) of the corresponding Trichoderma treatment or a commercial product at label rate (Table 1). For each treatment, 30 canes (three replicates of 10 canes) were challenged each at 24 hrs, seven days, and 21 days after treatment with 50 µL 1 × 105 conidia/mL (5000 spores/wound) of either D. seriata or N. parvum to determine how long the activity of Trichoderma or commercial products lasted on the pruning wound. Another 180 canes per treatment were left untreated as positive controls, which included 30 inoculated with 5000 spores/wound of D. seriata or N. parvum each at 24 hrs, seven days, and 21 days after pruning. An additional 90 canes per detached cane assay experiment were left untreated/uninoculated to serve as negative controls, with 30 canes each for 24 hrs, seven days, and 21 days, to determine whether natural background infections occured in the canes from the experimental vineyard or during the detached cane assays. Canes were completely randomized across the styrofoam trays and collected five weeks after each of the respective inoculation times.
Re-isolations from the canes were conducted on PDA amended with 1 mg/mL tetracycline (PDA-tet; Sigma-Aldrich) to assess the efficacy of the treatments as described (Pollard-Flamand et al. 2022). Plates were incubated at 23°C in the dark for up to 10 days. Plates were inspected visually and if a plate yielded either D. seriata or N. parvum, identified by the characteristic dark-olivaceous color of the mycelium colony, the corresponding cane was rated as colonized by the pathogen. Treatment efficacy was based on the mean percent recovery (MPR) of D. seriata and N. parvum from treated canes, and data are presented as mean percent disease control (MPDC) according to the formula: 100 × [1-(MPR treated canes / MPR control canes)]. Temperature (T) and relative humidity (RH) were monitored in the greenhouse over the duration of each detached cane assay using one Hygrochron I-button (IButtonLink, LLC). The binary (infected or uninfected) data produced from each detached cane assay experiment were fitted to a logistic regression: a general linear model specific for binomial data with a logit link. To determine the significance of the fixed effects of pathogen, time of inoculation, treatment, and their interactions, a three-way analysis of deviance was performed, which is ~χ2-distributed. This was followed by Tukey-Kramer’s honest significant difference (HSD) comparison post-hoc test to determine if there were significant statistical differences between the means across treatments for each pathogen and within each evaluation time. All statistical analyses were performed in R (R Core Team 2021).
Field trials
To evaluate and compare the Trichoderma treatments and commercial pruning wound protection products under natural conditions, a field trial was conducted at the SuRDC in an experimental vineyard of Merlot grafted onto 3309C rootstock for two consecutive growing seasons (2019 and 2020). The vineyard was planted in 2002 and vines in this block were trained in a bilateral cordon and spur-pruned, and shoots were placed in a vertical shoot-positioning system. Vine and row spacing were 3 m × 1.2 m and vineyard maintenance practices such as fertilization, irrigation, and disease and pest control were performed as standard for the region. Temperature, RH, and precipitation for the duration of the trial in both years were recorded using a HOBO (Onset Computer Corporation) weather station located in the middle of the experimental block. Each year, vines included in the trial were pre-pruned before budbreak, leaving four to five node canes (~40 cm length) so that neither D. seriata- nor N. parvum-inoculated pathogens had time to reach the final two spur-pruned buds left when collecting the canes for analysis. One day before treatment, all canes from each vine were pruned again ~2 to 3 cm above the last bud left when pre-pruned, so the surface of the pruning wound was horizontal for treatment and drop inoculation with pathogens via micropipette. All canes in each vine were then treated and inoculated. Pruning, inoculation, and collection dates for each year were as described (Table 2).
Pruning, treatment, inoculation, and sample collection dates for field trials conducted in 2019 and 2020 growing seasons.
Three of the four Trichoderma treatments (BC1, BC2, and BC3) used in the detached cane assays were selected and tested in the field by applying 50 µL aliquots of 1 × 106 spores/mL (50,000 spores/wound). Commercial products Gelseal, T-77, and VitiSeal were used as industry standard controls and were applied according to manufacturer instructions (Table 1). Trichoderma and commercial product treatments were applied within three hours of pruning (Table 2). In the 2019 field trial, immediately following treatment, the vineyard block experienced a heavy rain so the treatments were re-applied the following day. Pruning wounds were then challenged one, seven, 21, and 60 days posttreatment using 50 µL aliquots of 4 × 104 spores/mL suspensions (2000 spores/wound) of either D. seriata or N. parvum. Positive controls were untreated but inoculated with the same number of D. seriata or N. parvum spores one, seven, 21, and 60 days after pruning. Negative controls were pruned, left untreated, and exposed only to natural disease pressure to determine the base levels of pathogen incidence in the field. Treatments were arranged in a randomized block design to correct for variation within the vineyard. Each treatment × pathogen × timing combination was repeated on five vines and all canes per vine were treated. In total, 180 vines were used in the field trial and treatments were applied to the same vines from one year to the next. Five weeks after each inoculation time with the pathogen, a final two-bud spur pruning was conducted and all canes from each vine were collected. From each vine, 10 canes were selected and stored at 4°C until re-isolations were completed as described (Pollard-Flamand et al. 2022). The rest of the canes from each vine were discarded. Briefly, fungal re-isolations started by first shaving the bark around the pruning wound, then flame-sterilizing the surface of the cane with 95% ethanol, at which point 10 ~0.5 cm2 pieces of tissue were taken from the top of the pruning wound and plated on PDA-tet. Plates were incubated for up to 10 days at 23°C in the dark. If a plate yielded either D. seriata or N. parvum, the corresponding cane was rated as colonized by the pathogen. Treatment efficacy was based on the MPR of D. seriata and N. parvum from treated canes and data is presented as MPDC.
The binary (infected or uninfected) data produced in the field trials were statistically analyzed as described in the detached cane assays using R.
Optimum temperature for Trichoderma conidial germination
T. asperelloides (SuRDC-1442), T. atroviride (SuRDC-1440), and T. canadense (SuRDC-1422) isolates were selected to determine the optimum temperature for conidial germination by evaluating percent germination after five, eight, 10, 12, and 24 hrs incubation at 10, 15, 20, 25, and 30°C. Methods were adapted from Schubert et al. (2010) and Úrbez-Torres et al. (2010) and the following protocol was developed. Mycelial plugs (5 mm diameter) from an active growing colony of the respective Trichoderma isolates were plated on 90 mm PDA plates and grown on the benchtop at ~23°C for two days under laboratory lighting conditions next to a window that let in daylight to produce conidia. Plates were then parafilmed and placed again on the benchtop and incubated for 13 to 14 days. Conidial suspensions were made by adding 1 mL sterile distilled water amended with Tween 20 (~1 drop per 300 mL) and gently scraping the aerial mycelia to suspend conidia. The suspension was then filtered through a sterile cotton ball before it was serially diluted to a concentration of 1 × 105 conidia/mL using a hemocytometer. Petri plates (90 mm diameter) containing 20 mL PDA-tet were prepared and labeled the day before the start of the experiment and left in the laminar flow cabinet to dry overnight. All suspensions were made up at 1830 hrs the night before and stored in a 4°C refrigerator until plating the next morning. At 0530 hrs the next day, the suspension was vortexed before a treatment of one 10 µL drop of 1 × 105 conidia/mL suspension was plated in the center of each plate. All plating was completed within 30 minutes, at which point three plates per Trichoderma spp. were placed in each of five incubators set to the different temperatures. Two I-buttons were placed inside two separate treated plates and incubated with the other plates in each of the five incubators and set to measure absolute humidity and temperature every five minutes. Plates were left unsealed and removed at five, eight, 10, 12, and 24 hrs posttreatment and stored in the cold at 4°C for no longer than two hours while microscopy measurements were performed. To evaluate the percent germination of the plated conidia, 10 µL calcofluor (1000 mg/mL) was added to a glass cover slip before it was placed on top of the conidia for observation. The conidia were observed and photographed using a Zeiss Axio Imager.M2 with a Zeiss AxioCam MRm at 20× magnification with an excitation frequency of 365 nm observed through a DAPI filter. Photographs were taken using Zeiss ZenPro and used later to randomly measure 50 conidia from each replicate, resulting in 150 conidia counted per species × temperature × incubation time combination. Conidia were considered germinated when the germ tube length exceeded the diameter of the spore (Schubert et al. 2010). The entire experiment was repeated. The mean percent conidial germination and standard error were calculated for each experimental combination. The binomial germinated versus ungerminated data from each experiment was analyzed separately via general linear model with a logit link treating isolate, temperature, time, and all their interactions as fixed effects. Tukey-Kramer HSD multiple pairwise comparison tests were used to compare times within temperatures at the 5% significance level (Úrbez-Torres et al. 2010). All analyses were performed using R.
Results
Greenhouse detached cane assays
Average I-button temperature and humidity values were ~18°C and 40% RH over the duration of the experiments. The detached cane assays evaluating the local Trichoderma combinations and those evaluating the commercial products Gelseal and T-77 were analyzed separately, as they were conducted at different times (Tables 3 to 5). There was a high recovery of D. seriata and N. parvum in the untreated, inoculated positive controls from one to 21 days postpruning in all three detached cane assays (Tables 3 to 5). Recovery of D. seriata was generally lower across the experiment (53 to 87%) than N. parvum (73 to 100%). There was no recovery of either D. seriata or N. parvum from the negative controls in any of the three detached cane assays (data not shown). All Trichoderma treatment combinations (BC1, BC2, BC3, and BC4) were able to achieve between 93 and 100% MPDC against D. seriata and N. parvum from one to 21 days posttreatment (Table 3). The commercial Trichoderma-based product T-77 also provided a high level of control against both pathogens from one to 21 days. T-77 showed 84 to 93% MPDC against both pathogens one and seven days posttreatment and 100% and 91% MPDC against D. seriata and N. parvum, respectively, at 21 days posttreatment (Table 4). The wound-sealing, tebuconazole-based commercial fungicide Gelseal provided 100% MPDC against N. parvum from one to seven days posttreatment; however, the control dropped to 77% at 21 days posttreatment (Table 5). The Gelseal MPDC against D. seriata was significantly lower than against N. parvum at one (81%), seven (57%), and 21 (31%) days posttreatment.
Efficacy of Trichoderma isolates from British Columbia, applied immediately after pruning Chardonnay dormant canes, followed by inoculation of 5000 conidia of Diplodia seriata or Neofusicoccum parvum one, seven, and 21 days posttreatment in a detached cane assay experiment. Dpt, days posttreatment; MPR, D. seriata or N. parvum mean percent recovery; MPDC, mean percent disease control.
Efficacy of Trichoderma atroviride strain 77-b applied immediately after pruning Chardonnay dormant canes, followed by inoculation of 5000 conidia of Diplodia seriata or Neofusicoccum parvum one, seven, and 21 days posttreatment in a detached cane assay experiment. Dpt, days posttreatment; MPR, D. seriata or N. parvum mean percent recovery; MPDC, mean percent disease control.
Efficacy of tebuconazole (Gelseal) applied immediately after pruning Chardonnay dormant canes, followed by inoculation of 5000 conidia of Diplodia seriata or Neofusicoccum parvum one, seven, and 21 days posttreatment in a detached cane assay experiment. Dpt, days posttreatment; MPR, D. seriata or N. parvum mean percent recovery; MPDC, mean percent disease control.
Field trials
The first and repeated field trials evaluating three local Trichoderma treatments and three commercial pruning wound protection products were analyzed separately, since positive controls varied significantly between trials. In the 2019 field trial, analysis of deviance showed that the effects of pathogen and pathogen interacting with treatment were significant (p < 0.05; Table 6). The individual effects of time of inoculation; treatment; the two-way interaction of time of inoculation and treatment; and the three-way interaction of pathogen, time of inoculation, and treatment were highly significant (p < 0.001; Table 6). In the 2020 field trial, the individual effects of time of inoculation and treatment were significant (p < 0.001; Table 6).
Analysis of fixed effects of pathogen inoculated, time of inoculation, pruning wound treatment, and their interactions on mean percent recovery (MPR) of Diplodia seriata and Neofusicoccum parvum using two general linear models to analyze the original and repeat data separately.
In 2019, the MPR of D. seriata and N. parvum from the positive controls decreased as time between pruning and inoculation increased (Table 7). D. seriata and N. parvum MPR was greatest when pruning wounds were inoculated 24 hrs after treatment (90 and 82%, respectively) and lowest 60 days after treatment (24 and 21%, respectively). Botryosphaeriaceae spp. (Botryosphaeria dothidea and a Dothiorella sp.) were recovered only from 2% of the negative controls in the 24 hr and 21 day treatments (data not shown). In 2019, all treatments but one, VitiSeal on N. parvum 60 days after treatment, significantly reduced the MPR of both D. seriata and N. parvum (Table 7). Locally sourced Trichoderma spp. combination treatments and the commercial products T77 and Gelseal showed a high degree of pruning wound protection for up to 60 days after treatment against both pathogens, ranging from 75 to 100% MPDC for D. seriata and 81 to 100% MPDC for N. parvum (Table 7). Though not statistically different from BC1, BC2, T77, and Gelseal, treatment BC3 (T. atroviride + T. asperelloides) showed the greatest MPDC for up to 60 days for both pathogens. VitiSeal had the lowest MPDC when pruning wounds were challenged with D. seriata 24 hrs and 7 days after treatment and with N. parvum 24 hrs and 60 days after treatment. MPRs of both pathogens were not statistically different between Vitiseal and the other treatments for the other inoculation times after treatment.
Efficacy of Trichoderma isolates from British Columbia and commercial products applied immediately after pruning dormant canes, followed by inoculation of 2000 conidia of Diplodia seriata or Neofusicoccum parvum one, seven, 21, and 60 days posttreatment in a Merlot vineyard in 2019. Dpt, days posttreatment; MPR, D. seriata or N. parvum mean percent recovery; MPDC, mean percent disease control.
In 2020, the MPR of D. seriata and N. parvum from the positive controls also decreased as time between pruning and inoculation increased; however, the level of infection was overall lower than in 2019. The D. seriata and N. parvum MPR 24 hrs after treatment was 68 and 72%, respectively, and decreased to 20 and 24% 60 days after treatment. B. dothidea was also recovered at a low incidence (2%) from negative controls in the 24 hr, 21 day, and 60 day treatments. In 2020, all treatments significantly reduced the MPR of both D. seriata and N. parvum when compared to the positive controls (Table 8). All treatments, with the exception of Gelseal for D. seriata 60 days after treatment and VitiSeal 24 hrs after treatment for both pathogens, showed >80% MPDC for up to 60 days after treatment for D. seriata and N. parvum (Table 8). Trichoderma spp. treatment combinations and the commercial products T77 and Gelseal showed a high degree of pruning wound protection for up to 60 days after treatment against both pathogens, ranging from 75 to 100% MPDC for D. seriata and 81 to 100% MPDC for N. parvum (Table 8). Though not statistically different from BC1, BC2, T77, and Gelseal, treatment BC3 (T. atroviride + T. asperelloides) showed the highest MPDC for up to 60 days for both pathogens again in 2020. VitiSeal showed the lowest MPDC when pruning wounds were challenged with D. seriata 24 hrs and 7 days after treatment and with N. parvum 24 hrs and 60 days after treatment. MPR of both pathogens was not statistically different between Vitiseal and the other treatments for the rest of inoculation times after treatment.
Efficacy of Trichoderma isolates from British Columbia and commercial products applied immediately after pruning dormant canes, followed by inoculation of 2000 conidia of Diplodia seriata or Neofusicoccum parvum one, seven, 21, and 60 days posttreatment in a Merlot vineyard in 2020. Dpt, days posttreatment; MPR, D. seriata or N. parvum mean percent recovery; MPDC, mean percent disease control.
BC Trichoderma spp. treatments as well as T. atroviride from the commercial product T77 were reisolated from 100% of the treated canes in the detached cane assays and reisolated at a high frequency (68 to 100%) in both field trials for up to 60 days after treatment (data not shown).
Optimum temperature for Trichoderma conidial germination
Conidial germination of three Trichoderma spp. was evaluated after five, eight, 10, 12, and 24 hrs incubation at 10, 15, 20, 25, and 30°C. The data from the first and the repeat experiments did not meet statistical requirements to be combined, so were analyzed as two separate general linear models. The effects of isolate, time, and temperature were highly significant in both experiments (Table 9). The temperature at which 50% germination was reached in the shortest time was 30°C for all isolates in both experiments, with T. canadense, T. atroviride, and T. asperelloides reaching >50% germination at eight, 10, and 10 hrs, respectively (Figure 1). None of the isolates showed germination at 10°C over the 24 hr period of evaluation, although the conidia of each isolate did swell in size by the 24 hr evaluation. Each isolate exhibited germination after 24 hrs at 15°C incubation with a high germination rate of 87 and 91% for T. canadense and lower germination rates of 18 to 26% and 13 to 16% for T. atroviride and T. asperelloides, respectively. T. canadense was the only species showing germination (3 to 11%) after 12 hrs incubation at 20°C (Figure 1). All three species reached 100% germination after 24 hrs at 20°C. T. canadense germinated fastest (1 to 8%) at 25°C after eight hours, reaching 100% germination at 12 hrs. T. atroviride and T. asperelloides followed a similar trend at 25°C incubation and began germinating after 10 hrs, reaching 100% germination at 24 hrs in both experiments (Figure 1).
Analysis of fixed effects of isolate, temperature, time, and their interactions on conidial germination of three Trichoderma spp. using two general linear models to analyze the first and repeat data separately.
Effect of temperature on conidial germination of three (A, B, and C) Trichoderma spp. isolated from grapevines in British Columbia after five, eight, 10, 12, and 24 hrs incubation. The first (1) and repeat (2) experiments were analyzed separately. Means represent the percent conidial germination of 150 conidia and bars represent the standard error of the mean. Means within the same temperature with the same letter are not significantly different at the 0.05 level. Dotted lines indicate 50% germination.
Discussion
Most studies evaluating Trichoderma spp. as biocontrol agents against Botryosphaeria dieback fungi have focused on screening registered commercial products, with contradictory results (Fourie and Halleen 2006, Kotze et al. 2011, Pintos et al. 2012, Berbegal et al. 2020, Martínez-Diz et al. 2021, Blundell and Eskalen 2022, Leal et al. 2023, Travadon et al. 2023). The few studies available evaluating Trichoderma spp. naturally occurring in grapevines or other hosts for their effects against Botryosphaeria dieback fungi have only completed either in vitro studies in the laboratory and/or in planta under controlled greenhouse conditions (Marraschi et al. 2019, Silva-Valderrama et al. 2021, Úrbez-Torres et al. 2020, Kovács et al. 2021, Langa-Lomba et al. 2022, Pollard-Flamand et al. 2022). Since sustainable control of grapevine trunk diseases, including Botryosphaeria dieback, is a top priority for grape and wine industries around the world, evaluation of biocontrol agents under field conditions is critical to advance the development, implementation, and final registration of effective products. To date, very few studies have evaluated naturally-found endophytic Trichoderma spp. as pruning wound protectants against Botryosphaeria dieback fungi in field trials under natural conditions (Kotze et al. 2011, Blundell and Eskalen 2022). The current study demonstrates the high biocontrol activity achieved by different combinations of endophytic Trichoderma spp. isolated from grapevines in BC as pruning wound protectants against the Botryosphaeria dieback fungi D. seriata and N. parvum under field conditions. Our results agree with a report of a naturally found endophytic strain of T. hamatum from California, which showed similar or better control than commercial products as a pruning wound protectant against grapevine trunk disease-causing fungi in the field (Blundell and Eskalen 2022). Therefore, results from this study significantly contribute to an important, but incompletely explored, area of research.
The majority of commercialized Trichoderma-based products are comprised of a single species; however, several include combinations of Trichoderma spp. or Trichoderma with other beneficial microbes, which in some cases have resulted in higher levels of control than those products with a single species (Woo et al. 2014, Blundell and Eskalen 2022, Leal et al. 2023). In a previous detached cane assay under controlled greenhouse conditions, Trichoderma spp. used in this study were reported to have good biocontrol activity against D. seriata and N. parvum when applied individually (Pollard-Flamand et al. 2022). The detached cane assay conducted in the current study using either two (BC1, BC2, and BC3) or three (BC4) of these Trichoderma spp. combined in a treatment provided greater pruning wound protection against D. seriata and N. parvum than when used alone in previously reported detached cane assays conducted under the same conditions, suggesting that a combined-species treatment performs better than a single one. Detached cane assay results from treatments BC1 to BC4 also provided better pruning wound protection than the T. atroviride-based commercial product T-77 against both D. seriata and N. parvum. The detached cane assay also showed better pruning wound protection with the BC1 to BC4 treatments than with the tebuconazole-based commercial product Gelseal against D. seriata. Gelseal and BC1 to BC4 treatments showed similar pruning wound protection against N. parvum for up to seven days; however, while Gelseal control decreased significantly 21 days posttreatment, BC1 to BC4 treatments still provided 100% protection. Overall, the increase in MPDC from BC1 to BC4 was observed primarily when pruning wounds were inoculated with the pathogens one day posttreatment. Trichoderma spp. need time to colonize and get established in the pruning wound before providing pathogen control and thus, pruning wound protection is expected to be greater a few days after application, depending on the enviromental conditions and cultivar (Mutawila et al. 2016). The high pruning wound protection obtained with BC1 to BC4 treatments only one day after treatment could be a consequence of more favorable growing conditions for Trichoderma spp. to rapidly colonize the wound in a controlled greenhouse environment, with constant optimal T and RH for their growth. However, the BC Trichoderma isolates used in this study were selected based on their best performance among a total of eight isolates used in single treatments in previous detached cane assays (Pollard-Flamand et al. 2022). Therefore, we hypothesized that this greater pruning wound protection could also be a result of greater antifungal activity of these isolates in combination. It is also important to highlight that cuttings in the detached cane assay experiments were challenged with an amount of spores (5000 conidia/wound) that likely far exceed the natural pathogen pressure found in a pruning wound in a vineyard. Therefore, results from the detached cane assay probably underestimate the control activity that the commercial products T-77 and Gelseal may provide against natural inoculum pressure. The high MPDC obtained with BC1 to BC4 against such high inoculum pressure, from one to 60 days posttreatment, further supports the efficacy of these treatments as pruning wound protectants against Botryosphaeria dieback fungi. For the reasons above, though detached cane assays allowed rapid, inexpensive, high-throughput screening of pruning wound treatments in planta, they should be only considered as a step in the screening process, while final results must be drawn from field trials conducted under natural environmental conditions.
Susceptibility of pruning wounds to Botryosphaeriaceae spp. has been reported to be up to 12 and 16 weeks after pruning in California and Australia, respectively, with susceptibility decreasing significantly as the time between pruning and infection increases (Úrbez-Torres and Gubler 2011, Sosnowski et al. 2023). Results from the field trials conducted in this study showed similar results with pruning wounds being susceptible to D. seriata and N. parvum for up to 60 days, when pruning was done in April 2019 and May 2020. Infection of pruning wounds in the positive controls one day after treatment was greater in 2019 than in 2020 for both fungi. As a consequence of work restrictions due to the COViD-19 pandemic, the start of the trial was delayed for over a month in 2020, which could have influenced the susceptibility of the wounds (Mahoney et al. 2005). Nevertheless, the infection obtained in the positive controls in 2020 was sufficient for comparison with the treated wounds. Results from the field trials conducted during two growing seasons showed treatments comprised with a combination of two endophytic Trichoderma spp. from BC provided effective pruning wound protection against D. seriata and N. parvum for up to 60 days after treatment. Most notably, the BC Trichoderma treatments provided between 77 and 100% protection when wounds were infected one day after treatment. This result is of significance because, under optimum environmental conditions, pruning wounds can be infected by Botryosphaeria dieback fungi immediately after the cut is made (Úrbez-Torres and Gubler 2011). Epidemiological studies conducted in BC showed Botrysophaeriaceae spp. spores are present during the growing season, with high inoculum concentrations from early-spring to early-summer (O’Gorman et al. 2019). Sixty days after treatment, Trichoderma spp. from BC were still able to achieve good pruning wound protection (75 to 100%), providing control during the time with greatest inoculum in the environment. However, further studies should investigate for how much longer these Trichoderma-based treatments can provide pruning wound protection, since the presence of spores can extend until the fall under BC conditions (O’Gorman et al. 2019).
Mean percent recovery of D. seriata and N. parvum at all inoculation times was not statistically different between wounds treated with BC Trichoderma treatments BC1 and BC3 and the commercial products T-77 and Gelseal, which also provided effective pruning wound protection for up to 60 days in these trials. These results support previous reports on the efficacy of T-77 and Gelseal as pruning wound protectants against grapevine trunk disease-causing fungi (Kotze et al. 2011, Pitt et al. 2012, Sosnowski et al. 2013). Therefore, treatments based on endophytic Trichoderma found in BC provided similar control to commercial products registered in other countries. These results also support reports from previous field studies conducted in South Africa and California, in which treatments comprising endophytic Trichoderma performed similarly or better than registered chemical and biocontrol products (Kotze et al. 2011, Blundell and Eskalen 2022). These studies also showed the biocontrol activity of endophytic Trichoderma against other grapevine trunk disease fungi. Botryosphaeriaceae spp. have been reported to be the most prevalent fungi associated with cankers in BC; however, other fungi within the Diatrypaceae family and Diaporthe ampelina can also cause cankers in BC (Úrbez-Torres et al. 2014). Therefore, further studies should evaluate the potential biocontrol activity of BC Trichoderma treatments against these groups of fungi. Field trial results showed BC Trichoderma treatments T-77 and Gelseal performed better as pruning wound protectants than VitiSeal against D. seriata and N. parvum when wounds were infected one day after treatment. With the exception of seven days after treatment for D. seriata in 2019, there were no statistical differences on MPR of the pathogens between VitiSeal and the rest of the treatments for seven, 21, and 60 days posttreatment. These results suggest that VitiSeal may need several days to become fully active in the pruning wound, leaving the wounds exposed to infection at their most susceptible time (Úrbez-Torres and Gubler 2011, Sosnowski et al. 2023). Though our inoculum was reduced significantly from 5000 spores/wound in the detached cane assays to 2000 spores/wound in the field trials, this latter inoculum dose may still be significantly greater than natural infections occurring in the field and thus, the efficacy of each treatment could be underestimated. VitiSeal is registered against Eutypa dieback in California under the “natural plant oils” class (Adaskaveg et al. 2022). Further evaluation of VitiSeal is needed between 24 hrs and seven days after treatment to determine when exactly this product starts protecting the pruning wound against grapevine trunk disease fungi.
All three Trichoderma spp. from grapevines in BC showed conidial germination between 15 and 25°C after 24 hrs, but their optimum T (conidial germination reached 50% in the shortest time) was 30°C. This is the first determination of the effect of different temperatures on conidal germination in T. asperelloides and T. canadense. The optimum T for T. atroviride in this study agrees with previous results (Daryaei et al. 2016). T. canadense, a recently described species from grapevines from BC (Pollard-Flamand et al. 2022), was the only species to reach more than 50% conidial germination at 15°C after 24 hrs. Conidial germination results in this study were obtained under 100% RH, which may provide faster germination rates than would be observed in the field when applied to pruning wounds at potentially lower RH (Schubert et al. 2010). Our results may suggest a slightly better adaptation of T. canadense to lower T, which could be advantageous under BC climatic conditions. Grapevine pruning in BC can be conducted between December and April; however, most pruning is done late, from early March to mid-April, to avoid snow and delay budbreak to avoid early spring frosts, which overlaps with high inoculum presence of Botryosphaeriaceae spp. (O’Gorman et al. 2019). Accordingly, pruning wound protection should be optimized for BC conditions during this period. Historic average daily T for March and April in Summerland (BC), the location where the field trial was conducted, are 4.8 and 9.4°C, respectively (as found on the website https://www.canada.ca/en/services/environment/weather.html), which suggests daytime temperatures during April were more favorable for Trichoderma conidial germination and thus, colonization of the pruning wound. Treatments were conducted on 11 April 2019 (mean T 5.8°C) and 7 May 2020 (mean T 13.3°C). Mean T of the seven days following treatment was 8.1°C in 2019 and 14.4°C in 2020, which are both below the optimal T we report for rapid conidial germination. Although none of the isolates germinated at 10°C, the conidial swelling observed after 24 hrs suggests a potential for germination at this low T if evaluated over incubation times longer than 24 hrs. Further studies should investigate the potential latency period that these Trichoderma spp. could have under low T, so earlier application during winter would be possible. Colonization of pruning wounds by Trichoderma spp. is dependent on the climatic conditions at the time of pruning and the physiological state of the vines, with faster colonization found in vines at break of dormancy than in full dormancy (Mutawila et al. 2016).
This study successfully protected pruning wounds against D. seriata and N. parvum when Trichoderma treatments were applied in April and May under BC conditions. However, further research is needed to determine the efficacy of these treatments at different times, since grapegrowers in BC may need to protect pruning wounds earlier in the season. Though the BC4 treatment, comprised of a mix of all three Trichoderma spp., showed high biocontrol activity in the detached cane assay, there was no difference in MPDC between BC4 and the two-species combination in the detached cane assay. Thus, we only evaluated treatments with a combination of two species under field conditions. Future research should focus on better understanding the mode of action of these endophytic Trichoderma spp., with the end goal of developing a product containing any of the dual Trichoderma combinations examined in this study.
Conclusion
Greenhouse detached cane assays and field trials demonstrated the high control capacity that locally sourced Trichoderma-based treatments have as pruning wound protectancts against the Botryosphaeria dieback fungi D. seriata and N. parvum. Trichoderma treatments from BC provided high pruning wound protection from 24 hrs to 60 days after treatment. These results are important since these treatments can protect pruning wounds for a large part of the critical time during the season in which Botryosphaeria dieback inoculum is present in BC vineyards. Trichoderma treatments from BC provided better or similar pruning wound protection than other bicontrol and chemical products registered in other countries. This research has generated necessary data on the efficacy of commercial products available in other countries to control Botryosphaeria dieback fungi under BC conditions and thus, to support what would be the first registration of control products against grapevine trunk disease fungi in Canada. Optimum temperatures and time for conidial germination of the three Trichoderma spp. found in BC and used in the different treatments were obtained. These results provide information vital to further investigation of alternative application times to protect pruning wounds effectively by using these Trichoderma spp. under BC climatic conditions.
Footnotes
This research was supported financially by the British Columbia Wine Grape Council, the Canadian Grapevine Certification Network, and matching funds provided by Agriculture and Agri-Food Canada under the Canadian Agriculture Partnerships Funding Program. The authors thank Andermatt Canada, Omnia Specialities Australia Pty Ltd., and VitiSeal International LLC., USA for their support to complete field trials. The authors thank Cassandra Bruce for technical assistance in field trials and sample processing.
Pollard-Flamand J, Boulé J, Hart M and Úrbez-Torres JR. 2023. Biological control of Botryosphaeria dieback of grapevines in British Columbia, Canada. Am J Enol Vitic 74:0740034. DOI: 10.5344/ajev.2023.23052
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- Received July 2023.
- Accepted September 2023.
- Published online November 2023
- Copyright © His Majesty the King in Right of Canada, as represented by the Minister of Agriculture and Agri-Food Canada, 2023.