Phenolic compounds inhibit viability and infectivity of the grapevine pathogens Diplodia seriata , Eutypa lata , Fomitiporia mediterranea, and Neofusicoccum parvum

Summary. Many fungal pathogens are associated with grapevine trunk diseases (GTDs), which cause important yield and economic losses in grape production. There are no effective control methods against GTDs once plants are infected, so research is aimed at preventive measures to avoid infections in nurseries and vineyards. Inhibitory activities of the phenolic compounds eugenol, epigallocatechin–3–O–gallate (EGCG) and thymol against the GTD fungi Diplodia seriata, Eutypa lata, Fomitiporia mediterra-nea and Neofusicoccum parvum were assessed in vitro , and in planta as grapevine pruning wound treatments. Greatest inhibition of pathogen mycelium growth was observed with eugenol (fungistatic at 1,500 µg mL –1 , fungicidal at 2,500 µg mL –1 ). No inhibitory activity against GTD fungi was observed with EGCG. Minimum concentrations with in vitro inhibitory effects on D. seriata and N. parvum spore germination were 360 µg mL –1 for thymol and 750 µg mL –1 for eugenol. In the grapevine wound protection tests, thymol was effective against N. parvum at 360 µg mL –1 , but eugenol was not.


INTRODUCTION
Grapevine trunk diseases (GTDs) are major threats to the sustainability of vineyards, and are widespread in all wine-producing countries (Songy et al., 2019).As no curative treatments for these diseases is available, infected grapevine plants must be replaced, resulting in financial losses of approx.$1.5 billion annually (Fischer et al., 2019).In the Czech Republic, damage caused by GTDs is estimated at CZK 150 million p.a. (approx.€ 6 million) (Baránek et al., 2017).In the 2000s, GTDs reduced potential wine produc-tion in France by 13% (Bruez et al., 2013).More than 130 species of fungi in 34 genera have been associated with GTDs, and are the largest group of pathogenic fungi that infect one host species (Gramaje et al., 2018).
With the increasing implementation of integrated pest management (IPM) systems, natural products are of considerable importance.These substances are obtained from biological sources and include essential oils (phenolic compounds), and natural plant protection products are considered more environmentally friendly than synthetic pesticides (Raveau et al., 2020).The use of biological agents instead of synthetic pesticides is strongly encouraged in Europe by Directive 2009/128/EC, which aims to reduce the pesticide use and thus bring agriculture in line with sustainable development.
Based on current knowledge of antifungal activities of the phenolic substances, the compounds eugenol, thymol, and EGCG were tested in vitro and in planta (eugenol and thymol) against GTD fungi.Diplodia seriata, Eutypa lata, Fomitiporia mediterranea, and Neofusicoccum parvum were selected for these experiments.The aim of this study was to determine whether selected phenolic substances have inhibitory effects against these selected GTD fungi that could be utilized for sustainable management of GTDs in nurseries and vineyards.Inhibitory effects on mycelium growth and spore germination of the three phenolic compounds were tested in vitro, and then in planta as pruning wound treatments of grapevine cuttings.

Phenolic substances and chemicals
The phenolic compounds and chemicals used in this study are listed in Table 1.
The lowest concentrations that inhibited spore germination were selected to evaluate the potential protective effects of the phenolic compounds on pruning wounds.Thymol solution was used at 360 µg mL -1 , and eugenol solution was used at 750 µg mL -1 with the addition of 1% DMSO.

In vitro assessments of inhibition of mycelium growth
Inhibitory effects of the selected substances against the four GTD fungi were tested in vitro using the potato dextrose agar (PDA; Difco TM , Becton, Dickinson and Company) supplemented with different concentrations of phenolic compounds (Table 3).The enriched media were pipetted onto Petri dishes (20 mL per dish).After solidification, a mycelium plug (5 mm diam.)taken from the edge of a 7-d-old colony of the assessed GTD fungus was placed into the centre of the prepared Petri plate.The plates (four replicates per treatment) were then incubated at 25°C in the dark and observed until the mycelium of the control treatment plates reached the plate edges, or for a maximum of 9 days post inoculation (dpi).Two orthogonal diameters of each fungi colony were measured, and the mean colony diameter was calculated.The mycelial plugs that did not show any growth in the enriched PDA were transferred onto nonamended PDA to verify fungistatic or fungicidal effects of the tested phenolic compounds.The same method was used to test effects of ethanol on the growth of D. seriata and N. parvum, because ethanol was used to prepare the thymol stock solutions.
Statistical analyses of the data obtained were carried out using Statistica 12 CZ StatSoft Prague CZ.Analysis of variance (ANOVA) was performed (at P = 0.01), and post hoc tests (Tukey's HSD tests: P = 0.01) were then applied.The percentage inhibition was calculated according to the formula I=[(r c -0.5)-(r t -0.5)]/ [(r c -0.5)×100], where I = percentage of inhibition, r c = average of the measured values for the experimental controls, and r t = average of the measured values for the treated condition.The value 0.5 cm was subtracted as the disc size (Chauhan et al., 2017).
The EC 50 were determined for the phenolic compounds that gave statistically significant inhibition.Concentrations and percentage inhibition values were used for linear regression analyses.Regression equations were determined, from which the EC 50 value for each compound was calculated.This experiment was carried out twice.

In vitro assessments of inhibition of spore germination
Based on the results from the mycelium growth assessments, thymol and eugenol were selected for in vitro treatments of D. seriata and N. parvum spore suspensions.Eutypa lata and F. mediterranea were discarded due to the lack of sporulation of both fungi in laboratory conditions, difficulty in collecting E. lata ascospores from infected grapevine wood, and the low germination rates of F. mediterranea basidiospores (Živković et al., 2012;Moretti et al., 2021).
Isolates of D. seriata Ds 99-7 and N. parvum Bt 67 were maintained on PDA at 25°C.Pycnidia of D. seriata were observed after an approx.4 week incubation period.For N. parvum, conidia were obtained according to Úrbez-Torres and Gubler (2009).Mycelial plugs from 7-to 10-day-old cultures were placed on 2% water agar containing sterile pine needles.Petri dishes were incubated at 25°C in a 12 h light:12 h dark cycle, until pycnidia were formed.Pycnidia of the two isolates were collected separately, and were then each crushed in sterile demineralized water in a 1.5 mL sterile microcentrifuge tube, using a sterile plastic pestle.Each spore suspension was filtered through sterile cheesecloth, and the spore concentrations were adjusted with potato dextrose broth (PDB, Difco Laboratories) to 2 × 10 4 conidia mL -1 using haemacytometer spore counts.
In the first phase of testing, inhibitory effects of thymol and eugenol on D. seriata spore germination were assessed at the same concentrations that were used in the in vitro tests on mycelium growth, thymol at 7.5 and 45 µg mL -1 , and eugenol at 1,500 and 2,500 µg mL -1 .The fungicide benomyl at 20.0 µg mL -1 , and water with 1% DMSO were also tested, respectively, as positive and negative experimental controls.In the second phase of testing against D. seriata, double concentrations of thymol (90, 180 and 360 µg mL -1 ) and half doses of eugenol (375 and 750 µg mL -1 ) were used.The effects of ethanol (at 18,000 µg mL -1 ), which was used for the preparation of thymol solutions, and 1% DMSO, which was used to increase the dispersion of eugenol in water, were also assessed.For N. parvum, the two greatest concentrations of thymol (180 and 360 µg mL -1 ), and the two lowest concentrations of eugenol (375 and 750 µg mL -1 ) were tested.Effects of benomyl (20.0 µg mL -1 ) were also assessed.Spore suspension (100 µL) was mixed with 100 µL of each tested solution in each well of 96-well microplates, and these were incubated at 25°C with shaking (180 rpm) in the dark.Optical density measurements were carried out at 0 and 72 h at 600 nm wavelength, using a spectrophotometer (MP96 Safas).This experiment was carried out twice.

In planta treatment of grapevine wounds with phenolic compounds
Evaluation of the potential protective effects of phenolic compounds on grapevine wounds was carried out using the method described by Lecomte and Dewasme (2004), with some modifications.Cuttings from Vitis vinifera 'Chardonnay' plants were collected from 15-yearold pruned canes growing in established vineyards, after winter pruning (January).The three-node long segments were cut and kept in a cold chamber at 4°C for 1 month.These segments were then surface sterilized with 0.05% cryptonol (8-hydroxyquinoline sulfate), rinsed with tap water, and placed back at 4°C until used.The cuttings were cut into 1-node segments, surface sterilized by wiping with cotton wool swabs of 70% ethanol, and the tips were each trimmed to 1 cm lengths with scissors, to produce fresh wounds.The cuttings were then placed vertically in plastic boxes containing moistened sand to maintain high humidity.On each fresh wound spray applications were made with approx.650 µL of thymol solution (360.0 µg mL -1 ) or eugenol solution (750 µg mL -1 with 1% DMSO).For experimental controls, the same volumes of water containing 1% DMSO were used.Each plastic box was covered with a lid, and the samples were incubated at 25°C for 24 h.Subsequently, 50 µL of spore suspension of N. parvum or D. seriata in sterile deionized water (prepared as described above), containing approx.200 conidia were pipetted onto each wound, and the samples were left at room temperature until the conidium suspension drops were absorbed.Plastic boxes (one for each tested condition) were covered with lids and incubated at 25°C for 7 d.Ten cuttings were used for each treatment.
After incubation, each cutting was removed from the sand, and the surface was sterilized by cotton wool wiping with 70% ethanol.The cutting tip was briefly sterilized in the flame, and the bark was removed using a sterile scalpel.Five wood chips (approx. 2 mm thick) were cut using sterilized scissors that were flamed before each cut.The five wood chips (levels) cut from the same cane were placed onto one Petri dish containing PDA, noting their position with regard to the inoculated wound zone, and the dishes were then incubated at 25°C for 5 d.
Development of N. parvum mycelium was evaluated on every wood chip, to determine how deeply the germinating conidia of the pathogens were able to colonize the woody tissues of each cane, and, inversely, the efficacy of phenolic compound treatment to prevent pathogen colonization.The percentage of colonized chips was calculated using the formulae % col.= x × 100/n, and the percentage of treatment efficiency was calculated as % ef .= y × 100/n, where % col.= colonization percentage, x = number of chips in which pathogen mycelium growth was observed, % ef .= efficiency percentage, y = number of chips in which no mycelial growth was observed, and n = 10 (the number of replicates).The efficiency percentages were calculated for each level of wood section (1 to 5).The test results were considered valid if the experimental controls gave at least 60% colonized chips in the first two wood levels.The treatments were considered efficient if the colonized chips did not exceed 30% in the first two wood levels (= efficiency at > 70%).

In vitro inhibitory effects on mycelium growth
The results of the in vitro assays are reported in Table 4.Among the studied phenolic compounds, eugenol and thymol inhibited all of the tested GTD fungi, while no significant inhibition was detected from the EGCG treatments.
Eugenol at both concentrations had strong antifungal activity.Complete growth inhibition (100%) was observed for all four GTD pathogens after treatment with 2,500 µg mL -1 , due to an ascertained fungistatic effect, and only a slightly lower effect was observed at 1,500 µL mL -1 , where D. seriata growth was reduced by 98%, E. lata by 99%, F. mediterranea by 100%, and N. parvum by 99%.
Thymol at a concentration of 45 µg mL -1 inhibited the growth of all the tested pathogens, with greatest activity (89%) against D. seriata and least (67%) against E. lata.Concentration of 7.5 µg mL -1 thymol was less effective and reduced the mycelium growth of three of the four pathogens, by 23% for F. mediterranea, 16% for N. parvum and 12% for D. seriata.Ethanol, regardless of concentration, did not affect (P > 0.01) growth of D. seriata or N. parvum growth, so ethanol did not affect the efficacy of thymol solution.For E. lata and F. mediterranea, the effect of ethanol was not tested due to the low percentages of thymol inhibition found for the four fungi.
EC 50 s for phenolic compounds with statistically significant inhibitory activity were calculated using linear regression analyses (Table 5).For eugenol, the EC 50 were from 940 to 1,000 µg mL -1 , and for thymol, were from 25.4 to 34.3 µg mL -1 , depending on the pathogen.

In vitro effects of phenolic compounds on conidium germination
Thymol and eugenol, at the same concentrations as those for the mycelium inhibition assays, were used to evaluate their activities against conidia of D. seriata.Eugenol at 1,500 or 2,500 µg mL -1 prevented germination of conidia (Figure 1A).Thymol at 7.5 µg mL -1 did not inhibit spore germination, while at 45 µg mL -1 conidium germination was reduced.Similar results were obtained for the fungicide benomyl at 20 µg mL -1 , which also suppressed D. seriata conidium germination.
To determine the minimum effective concentrations against D. seriata conidia, the eugenol concentration was decreased, and the thymol concentration increased.Eugenol completely inhibited spore germination at 750 µg mL -1 , while thymol was similarly efficient at 375 µg mL -1 (Figure 1B).Conidia treated with ethanol at 18,000 µg mL -1 germinated at a similar rate as the control, showing that ethanol did not affect the thymol assay, even at the greatest thymol concentration used.
For treatments of N. parvum conidia, thymol and eugenol concentrations were selected based on results obtained for D. seriata.No increased absorbance was observed for thymol at 360 µg mL -1 , or eugenol at 375 or 750 µg mL -1 , indicating inhibition of conidium germination (Figure 2).For N. parvum, the fungicide benomyl completely inhibited conidium germination.
Water plus 1% DMSO had no effect on the germination of D. seriata or N. parvum conidia, indicating that use of DMSO to facilitate formation eugenol emulsions did not affect the results (Figure 1 A and B, and Figure 2).

In planta treatments of grapevine wounds with phenolic compounds
Thymol at 360 µg mL -1 or eugenol at 750 µg mL -1 were assessed on grapevine canes as wound protection agents against N. parvum and D. seriata.The results for N. parvum can be considered valid, as the colonization  of wood at the first two levels exceeded 60% in the controls (Figure 3 A and B).In contrast, D. seriata did not reach the minimum of colonized fragments in untreated wood under the same conditions as N. parvum, despite repetition of the experiment (observed colonization rates were 35% in the first experiment and 40% in the second).The thymol treatment reduced N. parvum colonization to 30% in the first three wood chips, which confirms efficiency of this compound against N. parvum.In contrast, eugenol showed maximum efficacy of 60%; so it is unlikely to be an effective grapevine wound treatment for against N. parvum.

DISCUSSION
Previous studies have confirmed the antimicrobial effects of phenolic compounds produced and purified from plants.Substances that inhibit fungi, bacteria, or viruses include eugenol, thymol, and EGCG (Li et al., 1999;Kordali et al., 2008;Wang et al., 2010;Calland et al., 2012;Kanagaratnam et al., 2017;Hakalová et al. 2022;Hastoy et al., 2023), and these were selected for the present study.The in vitro tests demonstrated the antifungal activity of thymol and eugenol towards mycelium growth and conidium germination of the tested GTD fungi.In addition, thymol was also effective for pruning wound protection against N. parvum.
These results confirm previous reports demonstrating that eugenol and thymol reduced mycelium growth of fungal plant pathogens.Eugenol limited in vitro growth of the postharvest apple pathogens Phlyctema vagabunda, Penicillium expansum, Botrytis cinerea and Monilinia fructigena by 73% to 90% at a concentra-tion of 1,000 µg mL -1 , depending on the pathogen species and incubation temperature, and 100% at 2,000 µg mL -1 for all these fungi and at 4 and 20°C (Amiri et al., 2008).This is similar to the present study results where greater than 98% growth decreases were obtained after treatment with 1,500 µg mL -1 eugenol, and complete inhibition with 2,500 µg mL -1 .Lower concentration (30 µg mL -1 ) of eugenol added to liquid medium inhibited growth of Botrytis fabae by 73% (Oxenham et al., 2005).
The efficiency of thymol for suppression of GTD pathogen mycelium growth in the present study was comparable to or greater than that of other pathogenic fungi, according to some reports.Numpaque et al. (2011) showed that thymol, at 50 µg mL -1 , reduced mycelium growth of Lasiodiplodia theobromae, a pathogen associated with grapevine dieback, by approx.25%.The present study measured approx.90% decreases for D. seriata and N. parvum at 45 µg mL -1 of thymol.Complete inhibition of L. theobromae growth was achieved only after treatment with 150 µg mL -1 .The same concentration completely inhibited the mycelium growth of Fusarium moniliforme, Rhizoctonia solani, and Sclerotinia sclerotiorum (Müeller-Riebau et al., 1995).Zhang et al. (2019) reported 100% effectiveness of thymol at 65 µg mL -1 against B. cinerea.
For EGCG (concentrations 7.5 and 45.0 µg mL -1 ), no inhibitory effects against the tested GTD fungi were measured.EGCG has exhibited antifungal activity in other studies, at concentrations similar to those used in the present study experiment.Li et al. (1999) measured an EGCG MIC of 12.5 µg mL -1 against the human pathogen Cryptococcus neoformans.This result was different from the MIC determined by Matsumoto et al. (2012) (64 µg mL -1 ) for the same species.Only 0.31 µg mL -1 EGCG was sufficient to inhibit the growth of Candida glabrata, while for C. albicans and C. parapsilosis, 5 µg mL -1 was necessary (Chen et al., 2015).Hirasawa and Takada (2004) reported that antifungal activity of catechin was pH dependent.pH strongly influenced EGCG efficiency against C. albicans strains, as demonstrated by yeast growth reduction of 90% from 2,000 µg mL -1 at pH 6.0, and from 15.6 to 250 µg mL -1 at pH 7.0.
The EC 50 determined for eugenol in the present study was greater than those established for other pathogens.The EC 50 s ranged from 940 µg mL -1 for F. mediterranea to 1,000 µg mL -1 for N. parvum, which contrast with the published EC 50 of 3.6 µg mL -1 for Rhizoctonia solani (Zhao et al., 2021).Lower EC 50 values than those for the fungi examined in the present study were also reported for B. cinerea, at 36.62 µg mL -1 ) by Wang et al. (2010) and 240 and 263 µg mL -1 by Hastoy et al. (2023), depending on the isolate.Other Botryosphaeriaceae pathogens were found to be sensitive to lower eugenol concentrations than D. seriata and N. parvum, such as Botryosphaeria kuwatsukai (EC 50 = 65.07 µg mL -1 ; Wang et al., 2010) and B. dothidea (EC 50 = 114.43µL mL -1 /120.08 µg mL -1 ; Li et al., 2021).
The EC 50 values determined for thymol in the present study were 40 times less than those for eugenol, and are similar to those reported in other studies.Ding et al. (2023) evaluated thymol activity against the postharvest blueberry pathogens Aspergillus niger, Neopestalotiopsis sp., Alternaria alternata, Penicillium sp., Cladosporium xylophilum and B. cinerea, which indicated a mean EC 50 of 38.52 µg mL -1 for the six fungi.This is similar to the EC 50 observed here for E. lata (34.28 µg mL -1 ).The thymol EC 50 for the remaining pathogens tested in the present study was from 25.37 µg mL -1 for N. parvum to 29.02 µg mL -1 for F. mediteranea, which correspond to reports for Fusarium graminearum (26.3 µg mL -1 ; Gao et al., 2016) and for F. oxysporum (26.4 µg mL -1 ; Liu et al., 2022).
Compared to other research, the present study indicates that eugenol can exhibit diverse efficacy for suppressing conidium germination, depending on the fungal species.Conidium germination of Diplodia seriata and N. parvum was completely inhibited by eugenol at concentration of 750 µg mL -1 .Abbaszadeh et al. (2014) tested eugenol against propagules of important plant and food pathogens, and determined MIC values from 350 µg mL -1 for Aspergillus ochraceus to 500 µg mL -1 for Alternaria alternata, while Wang et al. (2010) indicated that 40,000 µg mL -1 did not inhibit conidium germination of B. cinerea.The concentration of eugenol was greater (2,000 µg mL -1 ) than the EC 50 s determined in the present study for D. seriata and N. parvum, and reduced germination of Phytophthora vagabunda and P. expansum by less than 60% (Amiri et al., 2008).Thymol had greater efficacy than eugenol, and at 360.0 µg mL -1 , thymol completely inhibited germination of N. parvum and D. seriata conidia.A lower thymol concentration (100.0 µg mL -1 ) was sufficient to reduce F. oxysporum spore germination by 80% (Liu et al., 2022), and 100% inhibition was achieved by Oh et al. (2022) for Botrytis aclada conidia.Their study also reported that Aspergillus awamoris conidia were resistant to thymol, and only treatment with 500 µg mL -1 completely inhibited germination.These results indicate that the two tested GTD fungi are susceptible to thymol at similar concentrations to those of other investigated fungi.
Of the two pathogens inoculated onto grapevine wounds, only N. parvum colonized the wood of control canes at sufficient depths (70% in the first two wood levels) to give valid results.Diplodia seriata colonization was only 40% at maximum, which could be due to reduced aggressiveness of the isolate assessed, or to low conidium viability.Grapevine wound treatments with eugenol against N. parvum were not effective.
No previous studies have focused on the application of eugenol to wood wounds for management of fungal pathogens.Thymol treatment (360 µg mL -1 ) was efficient against N. parvum.According to Jankura (2012), thymol at 500 µg mL -1 inhibited in planta Phaeomoniella chlamydospora on the ends of woody grapevine segments.
Commercial antifungal preparations containing phenolic compounds are approved in some European countries.For example, BIOXEDA (clove (Syzygium aromaticum) essential oil), which can be used as a fungicide or bactericide for apple and pear storage pathogens.PREV-AM and ESSEN' CIEL (sweet orange (Citrus sinensis) essential oil) is approved for use against mealybug, potato blight, powdery mildew and tobacco thrips on vegetables, fruit, ornamental crops, and tobacco.BIOXEDA has certified approval in Cyprus, France, Greece, and Spain.PREV-AM is approved in Belgium, France, German, Italy, Romania, and Spain.ESSEN' CIEL is approved for use in France, Italy, and Spain.The use of these products can therefore lead to reductions in the use of chemical pesticides in agriculture (Raveau et al., 2020).According to Torre et al. (2014), BIOXEDA can also be used in vineyards to protect against downy mildew (Plasmopara viticola).Application of the product to grapevine leaves reduced symptom incidence by 30%.According to Lasorello et al. (2018), applying PREV-AM in vineyards reduced incidence of powdery mildew (Erysiphe necator) by 92% on leaves and 79% on grapes.Applications of preparations based on phenolic substances that inhibit grapevine pathogens is therefore likely to reduce application of chemical pesticides, especially in vineyards with ecological management systems.
This study examined the antifungal effects of three phenolic compounds on the GTD fungi D. seriata, E. lata, F. mediterranea, and N. parvum.In the in vitro mycelial growth tests, the inhibitory effects were demonstrated for eugenol and thymol, but no impacts of EGCG on mycelium growth were detected.Based on the inhibitory effects observed for eugenol and thymol against GTD fungi, their efficacy was further evaluated by treating conidium suspensions and grapevine pruning wounds under controlled conditions.Protection with thymol at 360 µg mL -1 was effective against conidium germination of these fungi in vitro, and against N. parvum development on pruning wounds.Eugenol at 750 µg mL -1 , prevented conidium germination in vitro but did not prevent development of N. parvum on pruning wounds.These results show that thymol has potential for use in vineyards as a natural agent for use against the Botryosphaeriaceae pathogen N. parvum.

Figure 1 .
Figure 1.Absorbances measured for Diplodia seriata conidium suspensions treated with different phenolic compounds or chemicals (benomyl or DMSO).A) Tests of different concentrations of thymol and eugenol in in vitro tests for effects on conidium germination.B) Tests of doubled concentrations of thymol and halved concentrations of eugenol.

Figure 2 .
Figure 2. Absorbances measured for Neofusicoccum parvum spore suspensions treated with different phenolic compounds each at two concentrations, or chemicals (benomyl or DMSO).

Table 1 .
Phenolic compounds and chemicals used in this study.

Table 2 .
Details of the four selected grapevine trunk pathogens examined in this study.

Table 3 .
Details of the phenolic compounds assessed in this study for their effects on grapevine trunk diseases pathogens.The tests were carried out in PDA culture plates.

Table 4 .
Mean percent (%) inhibition of mycelial growth of four grapevine trunk disease pathogens measured in in vitro tests of phenolic compounds.(* indicates treatments giving differences (P = 0.01) from experimental controls).

Table 5 .
Linear regression analyses, and determination of the EC 50 concentrations, for two phenolic compounds (eugenol or thymol) with statistically significant inhibitory activities when tested against four grapevine trunk pathogens.