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

Kateřina Štůsková, Vincenzo Mondello, Eliška Hakalová, Dorota Tekielska, Florence Fontaine, Aleš Eichmeier

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## To cite this version:

Kateřina Štůsková, Vincenzo Mondello, Eliška Hakalová, Dorota Tekielska, Florence Fontaine, et al. "Phenolic compounds inhibit viability and infectivity of the grapevine pathogens Diplodia seriata, Eutypa lata, Fomitiporia mediterranea, and Neofusicoccum parvum." Phytopathologia Mediterranea, 2023, 60 (2), pp.307-319. 10.3625/phyto-14716. hal-05222842

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**Citation:** K. Štůsková, V. Mondello, E. Hakalová, D. Tekielska, F. Fontaine, A. Eichmeier (2023) Phenolic compounds inhibit viability and infectivity of the grapevine pathogens *Diplodia seriata*, *Eutypa lata*, *Fomitiporia mediterranea*, and *Neofusicoccum parvum*. *Phytopathologia Mediterranea* 62(2): 307-319.
doi: 10.36253/phyto-14716

**Accepted:** August 20, 2023

**Published:** September 15, 2023

**Copyright:** ©2023 K. Štůsková, V. Mondello, E. Hakalová, D. Tekielska, F. Fontaine, A. Eichmeier. This is an open access, peer-reviewed article published by Firenze University Press (http://www.fupress.com/pm) and distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

**Data Availability Statement:** All relevant data are within the paper and its Supporting Information files.

**Competing Interests:** The Author(s) declare(s) no conflict of interest.

**Editor:** José R. Úrbez Torres, Agriculture and Agri-Food Canada, Summerland, British Columbia, Canada.

**ORCID:**

KS: 0000-0002-5778-8439

VM: 0000-0003-4005-0494

EH: 0000-0002-5433-8993

DT: 0000-0002-2863-5812

FF: 0000-0003-2918-8812

AE: 0000-0001-7358-3903

Research Papers – 12th Special issue on Grapevine Trunk Diseases

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

KATEŘINA ŠTŮSKOVÁ<fnref n="1" />, VINCENZO MONDELLO<fnref n="2" />, ELIŠKA HAKALOVÁ<fnref n="1" />, DOROTA TEKIELSKA<fnref n="1" />, FLORENCE FONTAINE<fnref n="2" />,*, ALEŠ EICHMEIER<fnref n="1" />,*

**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 mediterranea* 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⁻<fnref n="1" />, fungicidal at 2,500 µg mL⁻<fnref n="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⁻<fnref n="1" /> for thymol and 750 µg mL⁻<fnref n="1" /> for eugenol. In the grapevine wound protection tests, thymol was effective against *N. parvum* at 360 µg mL⁻<fnref n="1" />, but eugenol was not.

**Keyword.** Grapevine trunk diseases, thymol, eugenol, epigallocatechin-3-O-gallate, mycelium growth, spore germination, wound protection.

## 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-

<footnote n="1">¹Mendeleum – Institute of Genetics, Mendel University in Brno, Valtická 334, 691 44, Lednice, Czech Republic</footnote>

<footnote n="2">²Unité Résistance Induite et Bioprotection des Plantes USC INRAE 1488, Université de Reims Champagne–Ardenne, Moulin de la Housse, Bâtiment 18, Reims, France</footnote>

<footnote>*Corresponding authors. E-mail: florence fontaine@univ-reims.fr; ales.eichmeier@mendelu.cz</footnote>
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).

GTDs include the six diseases, the ESCA complex, Eutypa dieback, Botryosphaeria dieback, and Phomopsis dieback occurring especially in mature vineyards, and Petri disease and black foot in young grapevines (Fussler et al., 2008; Bertsch et al., 2009; Hofstetter et al., 2012; Fontaine et al., 2016; Gramaje et al., 2018; Mondello et al., 2018a). Effective management of GTDs is difficult, especially after sodium arsenite was banned, due to its human and animal toxicity (Mondello et al., 2018b). Because complete eradication is not possible, GTD control is primarily focused on disease prevention and alle viation (Urbez-Torres, 2011).

In addition to chemical control attempts (Mondello et al., 2018a), recent studies have focused on developing sustainable management strategies against GTD fungi (Compant and Mathieu, 2016). Many organic extracts, such as chitosan, Evernia prunastri lichen extract, garlic extract, lemon peel extract, vanillin, and propolis, have been shown to inhibit GTD pathogens such as Botry osphaeria dothidea, Diaporthe ampelina Diplodia seriata, Eutypa lata, Ilyonectria macrodidyma, Phaeoacremonium minimum, and Phaeomoniella chlamydospora (Cobos et al., 2015, Mondello et al., 2018b).

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 prod ucts are considered more environmentally friendly than synthetic pesticides (Raveau et al., 2020). The use of bio logical 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 agricul ture in line with sustainable development.

The present study aimed to evaluate the potential of phenolic compounds for natural resource protection of grapevines. Phenolic compounds are secondary metabo lites ubiquitous in most plant tissues (de la Rosa et al., 2019). Eugenol (C₁₀H₁₂O₂; 2-methoxy-4-(2-propenyl) phenol) is a major component of clove oil (Syzygium aromaticum, Myrtaceae), and is also occurs basil, cin namon, lemon balm, and nutmeg (Dable-Tupas and Egbuna, 2022). It is an allyl chain-substituted guaiacol, clear, light yellow, and greasy liquid, that is slightly solu ble in water and soluble in organic solvents (Pramod et al., 2010). The antifungal effects of eugenol have been demonstrated on Aspergillus spp. and Cladosporium spp. (Abbaszadeh et al., 2014), and inhibition of the patho-

gens Botrytis cinerea (Hastoy et al., 2023; Wang et al., 2010), Fusarium oxysporum f. sp. vasinfectum (Abd- Elsalam and Khokhlov, 2015), and Rhizoctonia solani (Zhao et al., 2021).

The polyphenolic compound (-)-epigallocatechin-3-O-gallate, C₂₂H₁₈O₁₁ (EGCG), is the main catechin detected in green tea (Camellia sinensis (L.) Kunze). This compound is an ester of epigallocatechin and gallic acid (Nagle et al., 2006), and has antiviral (Calland et al., 2012; Steinmann et al., 2013; Kaihatsu et al., 2018), anti- bacterial (Kanagaratnam et al., 2017; Lee et al., 2017), and antifungal effects (Li et al., 1999; Navarro-Martinez et al., 2006; Park et al., 2006) which have been demon- strated in *in vitro* studies.

Thymol (C₁₀H₁₄O; 2-isopropyl-5-methylphenol) is the main monoterpene phenol present in essential oils isolated from plants in Lamiaceae (Thymus, Ocimum, Origanum, and Monarda). Thymol can also be isolated from other plants in Verbenaceae, Scrophulariaceae, Ranunculaceae and Apiaceae (Marchese et al., 2016). Thymol had marked *in vitro* inhibitory activity against the phytopathogens Alternaria spp., Botrytis sp., Fusari- um spp., Phytophora capsici (Kordali et al., 2008), Colle- totrichum acutatum, Lasiodiplodia theobromae (Num- paque et al., 2011), and Rhizoctonia solani. In *in planta* tests, thymol promoted the emergence of cucumber seedlings (Chauhan et al., 2017).

Based on current knowledge of antifungal activities of the phenolic substances, the compounds eugenol, thy- mol, and EGCG were tested *in vitro* and *in planta* (euge- nol and thymol) against GTD fungi. Diplodia seriata, Eutypa lata, Fomitiporia mediterranea, and Neofusicoc- cum 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 sustain- able management of GTDs in nurseries and vineyards. Inhibitory effects on mycelium growth and spore germi- nation of the three phenolic compounds were tested *in vitro*, and then *in planta* as pruning wound treatments of grapevine cuttings.

MATERIALS AND METHODS

Phenolic substances and chemicals

The phenolic compounds and chemicals used in this study are listed in Table 1.

In the *in vitro* tests where the effects of phenolic sub- stances on the growth of fungal mycelia were assessed, eugenol was used at concentrations of 1.5 µL mL⁻¹ (effec- tive concentration (e.c.) 1,500 µg mL⁻¹) and 2.5 µL mL⁻¹
<table><caption>**Table 1.** Phenolic compounds and chemicals used in this study.</caption><thead><tr><th>Name</th><th>Form</th><th>Chemical formula</th><th>Stock solution concentration (mg mL<sup>-1</sup>)</th><th>Concentration used (µg mL<sup>-1</sup>)</th><th>Supplier</th></tr></thead><tbody><tr><td>Ethanol absolute</td><td>liquid</td><td>C<sub>2</sub>H<sub>6</sub>O</td><td>-</td><td>2,250.0<br>13,500.0<br>18,000.0<br>375.0</td><td>Charbonneaux Bradant, France</td></tr><tr><td>Eugenol (100%)</td><td>liquid</td><td>C<sub>10</sub>H<sub>12</sub>O<sub>2</sub></td><td>-</td><td>750.0<br>1,500.0<br>2,500.0</td><td>Carl Roth GmbH & Co. KG, Karlsruhe, Germany</td></tr><tr><td>Epigallocatechin-3-O-gallate (EGCG)</td><td>powder</td><td>C<sub>22</sub>H<sub>18</sub>O<sub>11</sub></td><td>1</td><td>7.5<br>45.0<br>7.5</td><td>Merck KGaA, Darmstadt, Germany</td></tr><tr><td>Thymol</td><td>powder</td><td>C<sub>10</sub>H<sub>14</sub>O</td><td>1</td><td>45.0<br>90.0<br>180.0<br>360.0</td><td>Carl Roth GmbH & Co. KG, Karlsruhe, Germany</td></tr><tr><td>Benomyl. 1-(butylcarbamoyl)-1H-1,3-benzimidazol-2-yl methylcarbamate</td><td>powder</td><td>C<sub>14</sub>H<sub>18</sub>N<sub>4</sub>O<sub>3</sub></td><td>10</td><td>20.0</td><td>Sigma Aldrich, Saint Louis, MO, USA</td></tr></tbody></table>

<table><caption>**Table 2.** Details of the four selected grapevine trunk pathogens examined in this study.</caption><thead><tr><th>Pathogen</th><th>Isolate</th><th>Geographical origin</th><th>Sampling date</th><th>Grape cultivar</th><th>Accession number</th><th>Literature source</th></tr></thead><tbody><tr><td>Diplodia seriata</td><td>Ds 99-7</td><td>Perpignan, France</td><td>1998</td><td>Syrah</td><td>MSZU00000000</td><td>Robert-Siegwald et al., 2017</td></tr><tr><td>Eutypa lata</td><td>Bx1.10</td><td>Gironde, Bordeaux, France</td><td>1990</td><td>Cabernet-Sauvignon</td><td>-</td><td>Péros and Berger, 1994</td></tr><tr><td>Fomitiporia mediterranea</td><td>CO36</td><td>Saint-Preuil, France</td><td>1996</td><td>Ugni-Blanc</td><td>-</td><td>Laveau et al., 2009</td></tr><tr><td>Neofusicoccum parvum</td><td>Bt 67</td><td>Portugal</td><td>-</td><td>Aragonez</td><td>CBS140888</td><td>Rego et al., 2009</td></tr></tbody></table>

(e.c. 2,500 µg mL⁻¹), according to Chauhan *et al.* (2017). For thymol, a stock solution of 1.0 mg mL⁻¹ was first prepared using 70:30 water:absolute ethanol solvent (Scorallik *et al.*, 2012). Thymol was used at concentrations of 7.5 µL mL⁻¹ (e.c. 7.5 µg mL⁻¹) and 45 µL mL⁻¹ (e.c. 45 µg mL⁻¹) (Chauhan *et al.*, 2017). Effects of ethanol on fungal growth were assessed at of 2.25 µL mL⁻¹ (e.c. 2,500 µg mL⁻¹) and 13.5 µL mL⁻¹ (e.c. 13,500 µg mL⁻¹). An EGCG stock solution of 1.0 mg mL⁻¹ was prepared in water (Bartosikova and Necas, 2018). The tested EGCG concentrations were 7.5 µL mL⁻¹ (e.c. 7.5 µg mL⁻¹) and 45 µL mL⁻¹ (e.c. 45 µg mL⁻¹) (Chauhan *et al.*, 2017).

In the *in vitro* tests for effects on spore germination, thymol solution was prepared at 1.0 mg mL⁻¹ in 5% ethanol and then diluted with sterile deionized water to obtain final concentrations of 7.5, 45, 90, 180, or 360 µg mL⁻¹. Eugenol solutions were prepared in 1% DMSO at final concentrations of 375, 750, 1,500, or 2,500 µg mL⁻¹. To exclude inhibitory effects of ethanol and DMSO on spore germination, concentrations of 18,000 µg mL⁻¹

of ethanol and 1% of DMSO were tested. Benomyl (1-(butylcarbamoyl)-1H-1,3-benzimidazol-2-yl methylcarbamate; Sigma Aldrich) at 20 µg mL⁻¹ was used as a synthetic fungicide for comparisons.

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.

### Fungal isolates

The selected pathogens are shown in Table 2. These fungi were isolated from grapevine wood, and the isolates are available at the collection at Unité Résistance Induite et Bioprotection des Plantes, Université de Reims Champagne-Ardenne, France. Four GTD fungi were tested. These were *D. seriata* Ds 99-7 (Larignon *et al.*,
<table><caption>**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.</caption><thead><tr><th>Phenolic compound</th><th>Concentration (µL mL<sup>-1</sup>)</th><th>Amounts of stock solution per 20 mL PDA</th><th>Effective concentration (µg mL<sup>-1</sup>)</th><th>Pathogen</th><th>Number of repetitions</th><th>Total</th></tr></thead><tbody><tr><td rowspan="4">Ethanol</td><td>2.25</td><td>45 µL</td><td>2,250.0</td><td>Diplodia seriata</td><td>4</td><td>8</td></tr><tr><td rowspan="3">13.5</td><td rowspan="3">270 µL</td><td rowspan="3">13,500.0</td><td>Eutypa lata</td><td>4</td><td>8</td></tr><tr><td>Fomitiporia mediterranea</td><td>4</td><td>8</td></tr><tr><td>Neofusicoccum parvum</td><td>4</td><td>8</td></tr><tr><td rowspan="4">Eugenol</td><td>1.5</td><td>30 µL</td><td>1,500.0</td><td>Diplodia seriata</td><td>4</td><td>8</td></tr><tr><td rowspan="3">2.5</td><td rowspan="3">50 µL</td><td rowspan="3">2,500.0</td><td>Eutypa lata</td><td>4</td><td>8</td></tr><tr><td>Fomitiporia mediterranea</td><td>4</td><td>8</td></tr><tr><td>Neofusicoccum parvum</td><td>4</td><td>8</td></tr><tr><td rowspan="4">EGCG</td><td>7.5</td><td>150 µL</td><td>7.5</td><td>Diplodia seriata</td><td>4</td><td>8</td></tr><tr><td rowspan="3">45.0</td><td rowspan="3">900 µL</td><td rowspan="3">45.0</td><td>Eutypa lata</td><td>4</td><td>8</td></tr><tr><td>Fomitiporia mediterranea</td><td>4</td><td>8</td></tr><tr><td>Neofusicoccum parvum</td><td>4</td><td>8</td></tr><tr><td rowspan="4">Thymol</td><td>7.5</td><td>150 µL</td><td>7.5</td><td>Diplodia seriata</td><td>4</td><td>8</td></tr><tr><td rowspan="3">45.0</td><td rowspan="3">900 µL</td><td rowspan="3">45.0</td><td>Eutypa lata</td><td>4</td><td>8</td></tr><tr><td>Fomitiporia mediterranea</td><td>4</td><td>8</td></tr><tr><td>Neofusicoccum parvum</td><td>4</td><td>8</td></tr><tr><td rowspan="4">Control</td><td>-</td><td>-</td><td>-</td><td>Diplodia seriata</td><td>4</td><td>4</td></tr><tr><td>-</td><td>-</td><td>-</td><td>Eutypa lata</td><td>4</td><td>4</td></tr><tr><td>-</td><td>-</td><td>-</td><td>Fomitiporia mediterranea</td><td>4</td><td>4</td></tr><tr><td>-</td><td>-</td><td>-</td><td>Neofusicoccum parvum</td><td>4</td><td>4</td></tr><tr><td colspan="4">Total Petri dishes</td><td></td><td></td><td>140</td></tr></tbody></table>

2001; Robert-Siegwald *et al.*, 2017), *E. lata* Bx1.10 (Péros and Berger, 1994; Laveau *et al.*, 2009), *F. mediterranea* CO36 (Laveau *et al.*, 2009) and *N. parvum* Np Bt 67 (Rego *et al.*, 2009; Reis *et al.*, 2016).

### 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™, 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 EC₅₀ 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 EC₅₀ 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 × 10⁴ 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-year-old 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 dominating 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 $%_{col.} = x \times 100/n$, and the percentage of treatment efficiency was calculated as $%_{ef.} = y \times 100/n$, 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%).

## RESULTS

### 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 anti-fungal 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.

EC₅₀s for phenolic compounds with statistically significant inhibitory activity were calculated using linear regression analyses (Table 5). For eugenol, the EC₅₀ were

<table><caption>**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).</caption><thead><tr><th>Substance</th><th>Concentration (µL mL<sup>-1</sup>)</th><th>Effective concentration (µg mL<sup>-1</sup>)</th><th>Diplodia seriata</th><th>Eutypa lata</th><th>Fomitiporia mediterranea</th><th>Neofusicoccum parvum</th></tr></thead><tbody><tr><td rowspan="2">Ethanol</td><td>2.25</td><td>2,250.0</td><td>0%</td><td>-</td><td>-</td><td>2%</td></tr><tr><td>13.5</td><td>13,500.0</td><td>0%</td><td>-</td><td>-</td><td>19%</td></tr><tr><td rowspan="2">Eugenol</td><td>1.5</td><td>1,500.0</td><td>98%*</td><td>99%*</td><td>100%*</td><td>99%*</td></tr><tr><td>2.5</td><td>2,500.0</td><td>100%*</td><td>100%*</td><td>100%*</td><td>100%*</td></tr><tr><td rowspan="2">EGCG</td><td>7.5</td><td>7.5</td><td>0%</td><td>9%</td><td>11%</td><td>8%</td></tr><tr><td>45.0</td><td>45.0</td><td>0%</td><td>3%</td><td>0%</td><td>6%</td></tr><tr><td rowspan="2">Thymol</td><td>7.5</td><td>7.5</td><td>12%*</td><td>4%</td><td>23%*</td><td>16%*</td></tr><tr><td>45.0</td><td>45.0</td><td>89%*</td><td>67%*</td><td>74%*</td><td>88%*</td></tr></tbody></table>

<table><caption>**Table 5.** Linear regression analyses, and determination of the EC₅₀ concentrations, for two phenolic compounds (eugenol or thymol) with statistically significant inhibitory activities when tested against four grapevine trunk pathogens.</caption><thead><tr><th>Treatment</th><th>Pathogen</th><th>Regression equations</th><th>R<sup>2</sup></th><th>EC<sub>50</sub> (µg mL<sup>-1</sup>)</th></tr></thead><tbody><tr><td rowspan="4">Eugenol</td><td>Diplodia seriata</td><td>y = 41.707x + 10.139</td><td>0.8492</td><td>960.0</td></tr><tr><td>Eutypa lata</td><td>y = 41.758x + 10.379</td><td>0.8435</td><td>950.0</td></tr><tr><td>Fomitiporia mediterranea</td><td>y = 42.061x + 10.438</td><td>0.8440</td><td>940.0</td></tr><tr><td>Neofusicoccum parvum</td><td>y = 41.571x + 8.544</td><td>0.8659</td><td>1,000.0</td></tr><tr><td rowspan="4">Thymol</td><td>Diplodia seriata</td><td>y = 2.0062x - 1.0627</td><td>0.9977</td><td>25.45</td></tr><tr><td>Eutypa lata</td><td>y = 1.5565x - 3.3607</td><td>0.9469</td><td>34.28</td></tr><tr><td>Fomitiporia mediterranea</td><td>y = 1.5472x + 5.0982</td><td>0.9728</td><td>29.02</td></tr><tr><td>Neofusicoccum parvum</td><td>y = 1.9466x + 0.6071</td><td>0.9961</td><td>25.37</td></tr></tbody></table>
<figure><img src="image_3.png" /><figcaption>**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.</figcaption></figure>

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.

<figure><img src="image_4.png" /><figcaption>**Figure 2.** Absorbances measured for *Neofusicoccum parvum* spore suspensions treated with different phenolic compounds each at two concentrations, or chemicals (benomyl or DMSO).</figcaption></figure>

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
<figure><img src="image_5.png" /><figcaption>**Figure 3.** Efficiency (%) of grapevine wound protection from *Neofusicoccum parvum* infection, with: A) thymol, and B) eugenol.</figcaption></figure>

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 anti-fungal 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 concentration

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 *Can-
dida 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 cat-
echin 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 EC₅₀ determined for eugenol in the present study was greater than those established for other pathogens. The EC₅₀s ranged from 940 µg mL⁻¹ for *F. medi-terranea* to 1,000 µg mL⁻¹ for *N. parvum*, which contrast with the published EC₅₀ of 3.6 µg mL⁻¹ for *Rhizoctonia solani* (Zhao *et al.*, 2021). Lower EC₅₀ 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* (EC₅₀ = 65.07 µg mL⁻¹; Wang *et al.*, 2010) and *B. dothidea* (EC₅₀ = 114.43 µL mL⁻¹/120.08 µg mL⁻¹; Li *et al.*, 2021).

The EC₅₀ values determined for thymol in the pre-
sent 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 EC₅₀
of 38.52 µg mL⁻¹ for the six fungi. This is similar to the
EC₅₀ observed here for *E. lata* (34.28 µg mL⁻¹). The thy-
mol EC₅₀ for the remaining pathogens tested in the pre-
sent 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 indi-
cates that eugenol can exhibit diverse efficacy for sup-
pressing conidium germination, depending on the fungal
species. Conidium germination of *Diplodia seriata* and
*N. parvum* was completely inhibited by eugenol at con-
centration 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 EC₅₀s 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 inhib-
ited 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 report-
ed 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* coloniza-
tion 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 chla-
mydospora* 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.
## CONCLUSIONS

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*.

## ACKNOWLEDGEMENTS

This research was funded by: Support of Multidisciplinary Research Study Programmes for Young Scientists, Project No. CZ.02.2.69/0.0/0.0/16_018/000233 3, Project No. 8J22FR013; Programme, Promoting the Mobility of Researchers and Workers in the Framework of International Cooperation in R&D between Czech Republic and France, student Project Nos. 48048RD and student Project No. IGA-ZF/2021-SI2004. The academic chaire Maldive also supported the experiments conducted in the laboratory Résistance Induite et Bioprotection des Plantes, Reims.

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