Original article

Antifungal effects of salicylic acid and other benzoic acid derivativestowardsEutypa lata: structure–activity relationship

Bénigne-Ernest Amborabéa, Pierrette Fleurat-Lessarda, Jean-François Cholletb,Gabriel Roblina,*

aLaboratoire de physiologie et biochimie végétales, université de Poitiers (CNRS, UMR 6161), station biologique de Beau-Site,25, faubourg Saint-Cyprien, 86000 Poitiers, France

bLaboratoire de synthèse et réactivité des substances naturelles, université de Poitiers (CNRS, UMR), 40, avenue du Recteur-Pineau,86022 Poitiers, France

Received 18 March 2002; accepted 24 June 2002

Abstract

Salicylic acid (SA) inhibited mycelial growth ofEutypa lata (Pers. Fr.)Tul. in a solid as well as in a liquid culture medium, in aconcentration-dependent manner, the threshold value being 0.1 mM. In conditions mimicking the plant environment (in particular, a pH nearthe apoplastic value, i.e. 5.5), 1 mM SA showed only fungistatic properties. However, modifications were observed in the structuralorganization of the mycelium at various levels (wall, mitochondria, vacuole and nucleus). A fungicidal effect was obtained at 2 mM orhigher concentrations and following this treatment, fungal filaments appeared empty. Antifungal efficiency of the molecule was increasedwhen the experimental pH was brought to more acidic values (pH 4). This observation was correlated with the increased capacity ofcompound uptake by the fungus. The in vitro antifungal effects of 57 benzoic acid derivatives were investigated onE. lata, in order to definea possible structure–activity relationship. A strategy leading to the synthesis of particular benzoic conjugates and integrating the dataobtained here is proposed to treat in planta againstE. lata, the causal agent of eutypa dieback, a severe disease of the grapevine and otherwoody plants resulting from an infection in the xylem of the host plant. © 2002 Éditions scientifiques et médicales Elsevier SAS. All rightsreserved.

Keywords: Eutypa lata; Eutypiosis; Phenolics; Salicylic acid; Uptake

1. Introduction

In certain plant diseases induced by fungi localized in thewoody tissues, curative treatments are difficult to carry out.A strategy may consist in identifying natural molecules withantibiotic properties which can be transported over longdistances in the plant towards the fungal target.

Considering their known chemical and biological prop-erties, phenolics might fulfill the requirements of such astrategy. Indeed, it has been shown in a wide variety ofhost/pathogen interactions that phenolic compounds mayplay a pivotal role in the plant resistance processes. Thus,phenolics such as many benzoic acid derivatives accumulate

in plants during the phase of infection[26]. These com-pounds result from the activation of the key enzyme,phenylalanine ammonia lyase, leading from phenylalanineto the formation of various derivatives[15]. Among benzoicacid derivatives, data in the literature show that salicylicacid (SA) may play a central role in plant disease resistance,particularly during systemic acquired resistance (SAR)[32].Thus, the level of SA increases several fold in tobacco andcucumber after pathogen infection[19,29]and this increaseis correlated with SAR[19,21]. Furthermore, transgenictobacco andArabidopsis plants that are unable to accumu-late SA due to the expression of the bacterialnahG gene(NahG plants) fail to develop SAR and exhibit increasedsusceptibility to an infection with virulent and avirulentpathogens[8,13].

The aim of this study was to check the impact of SA onmycelial growth ofEutypa lata (Pers. Fr.)Tul. This fungus isthe causal agent of eutypa dieback, a severe disease of the

Abbreviations: HEPES, N-[2-hydroxyethyl]piperazine-N'-[2-ethane-sulfonic acid]; MES, 2-[N-morpholino] ethanesulfonic acid; SA, salicylicacid; SAR, systemic acquired resistance

* Corresponding author.E-mail address: roblibio@club-internet.fr (G. Roblin).

Plant Physiol. Biochem. 40 (2002) 1051–1060

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© 2002 Éditions scientifiques et médicales Elsevier SAS. All rights reserved.PII: S 0 9 8 1 - 9 4 2 8 ( 0 2 ) 0 1 4 7 0 - 5

grapevine and many other woody fruit plants, such asapricots [3]. Ascospores of the fungus infect the xylemtissue through pruning wounds, and then colonize adjacentlignified parts of the plant [25] resulting in trunk canker,shoot inhibition and fruit abortion. Plants finally die sinceno curative treatments are available at present.

Although it has been suggested that SA has no directantifungal activity [22,27], different data have shown thatSA altered various levels in fungal development. Thus, SAaffected spore viability of Saccharomyces cerevisiae [31],reduced conidial germination and hyphae development ofSphaerotheca fuliginea [7], reduced and delayed germina-tion of blastospores of the entomopathogen Paecilomycesfumosoroseus [35] and inhibited sclerotial differentiationand growth in Sclerotium rolfsii and Sclerotina minor [14].Results presented in this paper show that SA undoubtedlypossesses fungicidal properties, resulting from considerabledisturbances observed in many compartments of the fungalcell. Moreover, we have shown that conditions of SAtransmembrane uptake by the mycelium constitute a majorcomponent of its lethal efficiency when exogenously ap-plied. In the course of this study, we also found that a weakmodification of the structural arrangement of the SA mol-ecule almost completely abolished its biological activity. Byusing phenolics, either naturally found in plants or chemi-cally synthesized, and presenting various modifications ofthe initial benzoic acid structure, we explored the relation-ship between the structure and the antifungal activity of themolecule. The data of this study are integrated in a strategyconcerning a mode of treatment against E. lata.

2. Results

2.1. Fungal growth inhibition by benzoic acid derivatives:specificity and pH effect

Results shown in Fig. 1A present the time course ofmycelial growth in our experimental conditions on solidmedium as a function of experimental pH. In all cases,mycelial development became visible on the first day afterinoculation and clearly measurable at the second day. Thedelay noted in this experimental approach is attributed toinitiation of growth in new hyphae after wounding madeduring the harvesting of the inoculum by a cork-borer.Measurements were made at least for 8 d until the experi-mental well was completely invaded by the hyphae. Thesepreliminary experiments showed that mycelial growth ofE. lata was slightly influenced by the experimental pHvalue. A maximal growth was noted in the range 5.0–7.5. Asmall hindering effect was observed at low pH values(4.0–4.5) and at high pH values (8.5) (Fig. 1B). This effectwas only visualized by a small slowing down of the growthrate. Nevertheless, the experimental well was nearly com-pletely invaded in 7 d by the hyphae in all pH conditionsjustifying that this experimental time lapse was chosen inthe expression of our experimental results.

Treatment with 1 mM benzoic acid delayed and hinderedthe mycelial growth (Fig. 2). An ortho-hydroxylation ofbenzoic acid (o-hydroxy benzoic acid = SA) increased theinhibition from 37% to 50% under the same experimentalconditions. The increased antifungal activity by thiso-hydroxylation was specific since m- and p-hydroxy-benzoic acids only induced a very weak inhibition (3% and6%, respectively). Furthermore, m- and p-hydroxylationmasked the inhibition observed with un-hydroxylated ben-zoic acid. It is noteworthy that complete growth inhibitionwas obtained following a 1 mM SA treatment when the pHof the culture medium was close to 4.0 (Fig. 2). The moredetailed data given in Fig. 3A show that efficiency of themolecule fell in treatments carried out at neutral and basicpH values. At pH 4.1, the threshold value of SA action wasnear 0.1 mM and a complete inhibition occurred at 0.5 mM(Fig. 3B).

Data in Fig. 4 show that mycelial growth in a liquidmedium maintained at pH 4.1 was altered in a very similarmanner to that observed on solid culture medium: thethreshold value was also 0.1 mM and total inhibition oc-curred at 0.5 mM, thus generalizing our previous observedeffects on the solid medium.

2.2. Fungicidal effects of SA

From the results given in Fig. 5, two main conclusionscan be drawn. Firstly, at pH 4.3, an addition of 2 mM SA orabove (10 mM) induced rapid and complete inhibition ofgrowth. Optical density went down in the flasks showingthat fungal cells aggregated. Secondly, at 12 d after replac-ing these small agregates in a new culture medium withoutSA, growth never started again, demonstrating that SA atthese concentrations presents fungicidal properties. By con-

Fig. 1. Effects of experimental pH on the mycelial development of E. lata.(A) Time course of mycelial growth on solid culture medium buffered atpH 4.0 (♦ ), 5.5 (• ), 6.5 (Á), 7.5 (▲) and 8.5 (▼). (B) recapitulative resultswith calculations made after a 6 d period. Mean ± SEM (SEM, standarderror); n = 18.

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trast, 1 mM SA only exhibited fungistatic activity since themycelium growth was restored after renewal of the culturemedium, although growth rate was slightly slower than inthe control. In this second part of the experiments, pH wasfixed at 5.5 in order to parallel the condition more likely tobe encountered in the neighbourhood of plant cells, whereapoplastic pH is considered to be close to 5.0. By comparingthe control sets in the two pH conditions, it can be noted that

mycelial growth was slightly faster at pH 5.5 than 4.3,showing thus the same characteristics as in solid medium(Fig. 1).

A similar fungicidal effect was obtained in experimentscarried out with solid medium. In a first phase, inoculumwas placed on a medium buffered at pH 4.1 containing1 mM SA. When transferred after 2 d in a second phase ona fresh medium buffered at pH 5.5, this inoculum developedas in control. Mycelial growth was completely inhibitedwhen the transfer was made after a 7 d treatment with SA(data not shown).

2.3. Structural modifications of the mycelium followingSA treatment

The previous results were supported by microscopicobservations (Fig. 6) on fungal samples grown in theconditions used in the physiological assays. In the control(Fig. 6A), filaments of 2 µm thick reached 50–100 µm long.The long central nucleus laying in the dense cytoplasm wassurrounded by abundant mitochondria and ribosomes. Largevacuoles with an opaque content occurred and many smalldroplets that are probably secretory vesicles, bordered theplasma membrane. A treatment with 1 mM SA at pH 5.5induced alterations noticeable by increased thickness anddecreased length of the filaments. This effect was clearlyvisible after a 10 d treatment (Fig. 6B). A more pronouncedeffect was seen after a 2 d treatment with 10 mM SA (Fig.6C). At this stage, the internal wall was lined by an opaquedeposit and the whole content of the filaments was modified.In particular, the periplasmic space was bordered by largeinfoldings of plasmalemma, groups of mitochondria withfew cristae and vacuoles occupied a large place. In theirregular shaped nucleus, opaque patches of chromatin werepresent. Treatment with 10 mM SA for 10 d dramaticallychanged the whole structure as most of the filaments

Fig. 2. Effects of benzoic acid and of its hydroxy-derivatives applied at1 mM on the time course of mycelial growth of E. lata. The solid culturemedium was buffered at pH 5.5, but an assay was also performed with SAat pH 4.1. C, control; m, m-hydroxybenzoic acid; p, p-hydroxybenzoicacid; B, benzoic acid; SA, salicylic acid. Mean ± SEM (SEM, standarderror); n = 24.

Fig. 3. Effects of SA on the mycelial growth of E. lata on a solid culturemedium (A) when used at 1 mM as a function of pH and (B) as a functionof concentration in a medium buffered at pH 4.1. Calculations made aftera 7 d treatment. Mean ± SEM (SEM, standard error); n = 24.

Fig. 4. Effect of SA as a function of concentration (expressed in mM) onthe time course of the mycelial growth of E. lata in a liquid culture mediumbuffered at pH 4.1 (initial weight of inoculum: 10 mg). The experiment wascarried out three times with similar results.

Fig. 5. Effect of SA applied at various concentrations on the time course ofthe mycelial growth of E. lata in a liquid culture medium buffered at pH 4.3(initial weight of inoculum: 25 mg). Concentrations of 1 mM (• ) and10 mM (▲) were applied at the beginning of the experiment (T = 0) whereas2 mM were applied at 1 d (a, i), 2 d (b, ▼) and 3 d (c, ♦ ) after the onset ofmycelial growth as indicated by black arrows. After 12 d (open arrow),aliquots of sets control (Á), 1 mM (• ),10 mM (▲) and set b (▼) wereinoculated into fresh culture medium (without SA), buffered at pH 5.5. Theexperiment has been carried out three times with similar results.

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appeared empty and limited by a highly altered wall bearinga filamentous deposit on its external side (Fig. 6D).

2.4. SA uptake by the mycelium

The effectiveness of an externally applied compounddepends on many parameters, including particularly, its

uptake by the cell. We therefore examined the modalities ofSA uptake by Eutypa mycelium. As shown in Fig. 7A,mycelium accumulated SA in a dose-dependent manner.Uptake was very rapid for 30 min whatever the concentra-tion and a plateau was reached after a 120 min treatment.SA uptake was strongly dependent on the pH of theincubation medium, decreasing considerably towards neu-

Fig. 6. Progressive alterations of mycelial filaments of E. lata grown in a liquid culture medium buffered at pH 5.5 in the presence of SA applied in variousconditions: (A) control; (B) 10 d treatment with 1 mM SA; (C) 2 d treatment with 10 mM SA; (D) 10 d treatment with 10 mM SA. gr, grana; m, mitochondria;N, nucleus; pe, external parietal zone; pi, internal parietal zone; V, vacuole; w, wall; Wb, Woronine body; *, dense vesicles near plasmalemma and tonoplast;arrow, enlarged periplasmic space; arrowhead, altered wall; bar = 1 µm.

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tral values (Fig. 7B). Compared to uptake at pH 4, residualvalues of 38%, 23% and 13% were, respectively, noted atpH 5, pH 6 and pH 7. This result can be correlated to thedegree of protonation of the carboxylate moiety of the SAmolecule. Indeed, calculations made by the ACD/log Dprogram give 92% of the R-COOH form at pH 4, 54% at pH5, 10% at pH 6 and 1% at pH 7. These data agreed with amechanism of weak acid trapping, involving mainly apassive diffusion through plasma membrane. After SAextraction from mycelium with 80% ethanol, analysis bythin-layer chromatography indicated that SA was not me-tabolized during the 120 min uptake.

2.5. Chemical structure–antifungal activity relationship

2.5.1. Effects of SA derivatives obtained by substitutionof various radicals on the benzene ring

The data of Fig. 8A show that the addition of a supple-mentary hydroxyl group in various positions on the benzenering (Table 1, nos. 2–5) suppressed the antifungal activitypresented by SA. The addition of two hydroxyl groups didnot significantly improve the effectiveness (Table 1, nos. 11,12). Addition of a methoxy- or a chlorine in the 3-positionresulted in a growth inhibition of about 50% (Table 1, nos.6, 7). A chlorine in 5-position was less active on its own(Table 1, no. 9) but reinforced the action when added witha chlorine in 3-position (Table 1, no. 13). Interestingly, the4-chloro derivative nearly completely inhibited mycelialgrowth (Table 1, no. 8) similar to SA (Table 1, no. 1).

2.5.2. Effects of derivatives obtained through substitutionof the 2-hydroxyl group

The results given in Fig. 8B underline both the impor-tance and specificity of an ortho-hydroxylation of thebenzene ring for the inhibition of mycelial growth. Indeed,substitution by methoxy or amino groups (Table 1, nos. 15,18) resulted in an inhibition of 50%. This inhibition wasreduced to near 25% following substitution by acetoxy(aspirin), acetyl or nitro groups (Table 1, nos. 16, 17, 19).An increasing inhibitory action, from 34% to 76% followedsubstitution by halogen atom in the series I > Br > Cl > F

(Table 1, nos. 21–24). The addition of a second chlorineatom reduced the biological activity of the 2-Cl derivative(Table 1, nos. 25–28).

2.5.3. Effects of benzoic acid derivatives obtainedby substitution of various groups on the benzene ring

In this set of experiments (Fig. 8C), the 2-position on thering was occupied by a hydrogen atom, and substitutionswere made at the other positions. Compared to SA, benzoicacid (Table 1, no. 29) is much less efficient and derivativesobtained through hydroxylation in meta- or para-position(Table 1, nos. 30, 31) also showed a reduced activity.Compared to these latter compounds, 3- and 4-methoxyderivatives (Table 1, nos. 32, 33) showed a similar activityto benzoic acid. Derivatives bearing two or three hydroxyl

Fig. 7. Characteristics of SA uptake by E. lata mycelium. (A) Time courseof SA uptake as a function of SA concentration expressed in mM. Theexperimental pH was fixed at 4.5. (B) Uptake of SA from 1 mM solutionas a function of experimental pH. Values represent uptake after 30 minincubation. Mean ± S.D. (S.D. = standard deviation); n = 3.

Fig. 8. Structure–antifungal activity of various phenolics assayed at 1 mMfinal concentration and at pH 4.1 on the mycelial growth of E. lata. (A)Effects of various SA derivatives. (B) Effects of benzoic acid derivativesobtained through a substitution of the 2-hydroxyl group on the benzenering. (C) Effect of benzoic acid derivatives obtained after substitution ofvarious groups on the benzene ring. (D) Effect of phenolic derivativesobtained though modifications of the aliphatic chain bearing the carboxylicacid function. Calculations were made after a 7 d treatment. Mean ± SEM(SEM, standard error); n = 18. Numbers of compounds used refer to theterminology in Table 1.

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Table 1Structure arrangements of the phenolics used in this study, values of log D and percentages of neutral form at pH 4.1

No. Compound R1 R2 R3 R4 R5 R6 log D Percentage ofneutral form

1 Salicylic acid COOH OH H H H H 0.93 90.32 2,3-Dihydroxybenzoic acid COOH OH OH H H H 0.62 82.03 2,4-Dihydroxybenzoic acid COOH OH H OH H H 0.76 87.24 2,5-Dihydroxybenzoic acid COOH OH H H OH H 0.44 75.45 2,6-Dihydroxybenzoic acid COOH OH H H H OH –0.54 21.66 2-Hydroxy-3-methoxybenzoic acid COOH OH OCH3 H H H 0.70 84.27 3-Chlorosalicylic acid COOH OH Cl H H H 1.35 95.48 4-Chlorosalicylic acid COOH OH H Cl H H 1.73 98.19 5-Chlorosalicylic acid COOH OH H H Cl H 1.94 98.710 5-Formylsalicylic acid COOH OH H H CHO H 0.80 86.711 2,3,4-Trihydroxybenzoic acid COOH OH OH OH H H 0.61 83.012 2,4,6-Trihydroxybenzoic acid COOH OH H OH H OH – 0.67 17.313 3,5-Dichlorosalicylic acid COOH OH Cl H Cl H 2.29 98.514 3,5,6-Trichlorosalicylic acid COOH OH Cl H Cl Cl 1.85 97.815 2-Methoxybenzoic acid COOH OCH3 H H H H 1.19 96.916 Acetylsalicylic acid (Aspirin) COOH OCOCH3 H H H H 0.48 79.917 2-Acetyl benzoic acid COOH COCH3 H H H H 0.52 88.918 2-Aminobenzoic acid COOH NH2 H H H H 1.14 99.119 2-Nitrobenzoic acid COOH NO2 H H H H –0.35 31.420 2-Mercaptobenzoic acid COOH SH H H H H 1.69 98.421 2-Fluorobenzoic acid COOH F H H H H 0.97 91.522 2-Chlorobenzoic acid COOH Cl H H H H 0.88 89.123 2-Bromobenzoic acid COOH Br H H H H 0.88 88.824 2-Iodobenzoic acid COOH I H H H H 0.90 89.325 2,3-Dichlorobenzoic acid COOH Cl Cl H H H 1.34 95.426 2,4-Dichlorobenzoic acid COOH Cl H Cl H H 1.37 95.927 2,5-Dichlorobenzoic acid COOH Cl H H Cl H 1.37 95.728 2,6-Dichlorobenzoic acid COOH Cl H H H Cl – 0.20 38.029 Benzoic acid COOH H H H H H 1.64 99.030 m-Hydroxybenzoic acid COOH H OH H H H 1.19 96.931 p-Hydroxybenzoic acid COOH H H OH H H 1.29 98.832 3-Methoxybenzoic acid COOH H OCH3 H H H 1.71 99.033 4-Methoxybenzoic acid COOH H H OCH3 H H 1.81 99.534 3-Chlorobenzoic acid COOH H Cl H H H 2.45 99.835 4-Chlorobenzoic acid COOH H H Cl H H 2.28 99.736 3,4-Dihydroxybenzoic acid COOH H OH OH H H 1.00 97.237 3,5-Dihydroxybenzoic acid COOH H OH H OH H 0.74 91.138 4-Hydroxy-3-methoxybenzoic acid COOH H OCH3 OH H H 1.17 98.139 3-Hydroxy-4-methoxybenzoic acid COOH H OH OCH3 H H 1.16 97.740 3,4-Dichlorobenzoic acid COOH H Cl Cl H H 2.91 99.941 3,5-Dichlorobenzoic acid COOH H Cl H Cl H 3.19 99.942 3,4,5-Trihydroxybenzoic acid COOH H OH OH OH H 0.71 94.043 Salicylaldehyde CHO OH H H H H 1.61 10044 2-Hydroxy-5-methoxybenzaldehyde CHO OH H H OCH3 H 1.52 10045 Salicylamide CONH2 OH H H H H 1.41 10046 o-Hydroxyhippuric acid CONHCH2COOH OH H H H H – 0.09 5747 2-Hydroxyphenylacetic acid CH2COOH OH H H H H 0.50 89.148 2,5-Dihydroxyphenylacetic acid CH2COOH OH H H OH H – 0.38 64.349 3-(2-hydroxyphenyl)propionic acid CH2CH2COOH OH H H H H 1.02 98.450 Phenylacetic acid CH2COOH H H H H H 1.29 98.151 3,4-Dihydroxyphenylacetic acid CH2COOH H OH OH H H 0.00 83.752 Hydrocinnamic acid CH2CH2COOH H H H H H 1.73 99.653 3,4-Dihydroxyhydrocinnamic acid CH2CH2COOH H OH OH H H 0.41 94.954 4-Phenylbutyric acid (CH2)3 COOH H H H H H 2.33 99.955 t-Cinnamic acid CH=CH–COOH H H H H H 1.99 99.456 Methyl salicylate COOCH3 OH H H H H 2.23 10057 Methyl-3-chlorobenzoate COOCH3 H Cl H H H 2.99 100

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groups (Table 1, nos. 36, 37, 42) had a low inhibitory effecton mycelial growth. Interesting results were obtained withchlorinated compounds (Table 1, nos. 34, 35, 40, 41) sincethe 3-chloro derivative induced a 90% growth inhibition,and the 3,5-dichloro compound completely suppressedmycelial development. Additional experiment with thislatter product showed that this result was obtained at aconcentration of 0.1 mM.

2.5.4. Effects of modifications on the aliphatic chainbearing the carboxylic acid function

Modification of the carboxylic acid function reduced thebiological activity of the original structure (Fig. 8D).Indeed, both salicylaldehyde (Table 1, no. 43) and salicyla-mide (Table 1, no. 45) only inhibited growth by no morethan 50% and 30%, respectively. Increasing the length ofthe carbon chain bearing the carboxylic acid function (Table1, nos. 47–49) dramatically reduced the efficiency of themolecule. The same result was observed with the benzoicacid derivatives (Table 1, nos. 9, 50–54). The introductionof a double bond in the aliphatic carbon chain hardlyaffected the molecule activity (compare in Table 1, nos. 55,52). Interestingly, lipophilic compounds without pH depen-dence, such as methyl esters of SA and 3-benzoic acid didnot show biological activity (Table 1, nos. 56, 57).

2.5.5. Octanol–water partition coeffıcient (log D)and biological activity of the compounds

The polarity or lipophilicity of a chemical, which is thebalance between water and lipid-soluble properties, is animportant criterion in determining the uptake pattern intoplant cells. The data plotted in Fig. 9 show that a strictlylinear relationship did not exist between the partitioncoefficient and biological efficiency for all 57 chemicalstested in this work. Although there was strong evidence thatbiological activity increased in most cases with increasinglog D, there were many discrepancies. Three areas can bedistinguished. The area (A) delimited by the regressionstraight line ± 10% regroups most of the compounds. In area(C), we found a series of compounds less active thanexpected. These compounds included a chloro derivativewith a high log D (Table 1, no. 9), hydroxy-derivatives withrelatively low log D (Table 1, nos. 2–5, 30, 37–39) andderivatives in which the aliphatic carbon chain was modi-fied (Table 1, nos. 45, 49, 54, 56, 57). On the other hand,several compounds showed a higher efficiency than ex-pected (area (B)). These included some halogeno-derivatives (Table 1, nos. 8, 23, 24, 34, 41) and verydistinctly SA (Table 1, no. 1).

3. Discussion

Following a pathogen attack, plants react by synthesizinga number of chemical defences in which the phenylpro-panoids occupy a central place. It has been shown in a widevariety of host/pathogen relationships that the phenolic

compounds may play a role in the resistance mechanisms ofhealthy plants. Their accumulation in infected tissues mayalso be implicated in the general mechanism of resistanceagainst pathogens [16]. More precisely, a large body ofevidence has accumulated suggesting a key role for SA inboth SAR signalling and disease resistance [28].

Our data demonstrate that SA acts directly on the fungaldevelopment in E. lata. This action appears to be specific tothe molecular arrangement, since a simple displacement ofthe hydroxyl group on the aromatic ring reduced theantifungal activity. This property has been further confirmedin a more extensive study devoted to the analysis of thestructure–activity relationship. A similar observation hadalso been made in the production of PR proteins in tobaccoplantlets following treatment with various benzoic acidderivatives [1].

The antifungal effect of SA was clearly observed whenapplied at a concentration of 1 mM, and at a pH similar tothat found in plant apoplast (i.e. about 5.5). A directfungicidal effect however needed higher concentrations (atleast 2 mM) in these experimental conditions, as demon-strated by physiological tests and microscopy observations.The latency period necessary to obtain this lethal effectsuggests that Eutypa may be able to induce a detoxifyingmechanism following SA application. SA can be degradedby a salicylate hydroxylase [37] or metabolically inactivatedthrough hydroxylation and/or conjugation reactions. Inplants, it is known that SA is rapidly conjugated to form�-O-d-glucosylsalicylic acid [11,20] following activation ofan SA-UDP-glucosyltransferase [36].

The resulting antifungal effects of SA can be ascribed tothe known impacts of the compound at various levels of thecell functioning. These effects have been visualized in ourstudy by the microscopic observations showing that SAalters many compartments of the fungal cells, particularlymitochondria. These data can be correlated in part to known

Fig. 9. Relationship between the inhibition of the mycelial growth ofE. lata and the octanol/water partition coefficients (as log D) determinedfor the various chemicals previously assayed and located by the numbers inTable 1.

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physiological impacts. Firstly, SA behaves as a decouplingagent [9,12,23,33] leading to a disruption of the transmembrane pH gradient across the plasmalemma and organellemembranes. This decoupling property would cause a loss ofthe energy charge of the cell. Secondly, SA directly affectscell respiration, redirecting electron flow from the cyto-chrome pathway into the cyanide-resistant alternative path-way. As a consequence, the cytochrome pathway would notbe fully utilized in SA-treated cells [17]. Thirdly, specificbinding of SA to a protein shown to be a catalase [5] wouldreduce the activity of the enzyme, promoting an accumula-tion of H2O2, which is toxic for many cell functions.

We have also shown that pH greatly affects the biologicalactivity of SA, since inhibition of mycelial growth wasconsiderably increased when experimental pH was broughtto acidic values (near 4). In this condition, a clear correla-tion has been drawn with the increased capacity of thecompound uptake by mycelium. SA has a pKa of 2.98 sothat the amount in the charged, dissociated form increasestowards a neutral pH. Cell membranes provide a goodbarrier to the diffusion of charged molecules, so that theresidual uptake at near neutral pH may indicate the presenceof specific carriers on the fungus cell membrane, playing thesame role as those noted in human colon adenocarcinomacell line [34].

In order to determine the most efficient compounds ofthis chemical family expressing antifungal properties, astudy dealing with a structure–activity relationship of vari-ous molecular arrangements was carried out. Following thisscreening, involving the determination of the antifungalefficiency of 57 compounds, some structural features haveemerged which affect the antifungal properties againstE. lata.

Few compounds match SA activity against E. lata devel-opment. This structure is particularly appropriate since asmall modification of the molecule dramatically reduced itseffects. The 2-hydroxyl group was the best substitution onthe benzene ring, but halogen atoms (iodine, in particular)can also be introduced in the molecule conferring in someinstances a high antifungal efficiency to the molecule. Thecarboxylic acid function was essential and must be borne bythe aromatic ring. Some chlorinated derivatives also showstrong activity and may be suitable compounds for anapplication to plants. In particular, 4-chlorosalicylic acid,3-chlorobenzoic acid and 3,5-dichlorobenzoic acid could beput forward as candidates. Chlorine atoms increase lipophi-licity and this property can be an advantage in the particularproblems we encounter in finding a curative treatment forE. lata. In fact, a critical phase is linked to the transport ofthe molecules from leaves towards the fungal target, local-ized in the xylem. The most polar and lipophilic derivativeswere poorly translocated from the petioles. It has thus beenobserved that the transport efficiency of substituted phenoxyacetic acids in barley and castor bean had an optimum oflog P between 1.5 and 2.2 [4,30]. In this aspect, SA and thethree chlorinated derivatives fulfill this requirement. The

next step in our study is now to determine the modalities ofthe in vivo transport of these selected molecules.

All these data have to be integrated in our introductoryproposal dealing with the use of SA as antifungal agent inwoody plant diseases. SA can be transported by plants sinceit was identified in phloem exudates following plant inocu-lation by pathogens [20,21,24,29]. A difficulty may arisefrom the fact that SA can also be toxic for plant cells,bearing in mind its general properties as a metabolicinhibitor. Thus, Rasmussen et al. [29] attributed chlorosisand stunting of inoculated leaves to the high concentrationof SA in the phloem tissues of the expanding leaves.Moreover, Abad et al. [1] showed that SA had a detrimentaleffect on the growth of in vitro tobacco plantlets. Therefore,in our curative perspectives, we have to find a conjugatedform of SA that can be applied to the plant, non-toxic in thisform to plant cells, transported by the plant conductingtissues towards the invading fungus. This conjugate has tobe absorbed by the fungus, in an optimal hypothesis throughspecific carriers and then dissociated to produce its maximalaction in situ. A type of compound involving conjugation ofan amino acid moiety to an active molecule was firstelaborated in our laboratory [6,10]. Such a protocol appliedto SA and the active chlorinated derivatives, taking intoaccount the results of the present study will fulfill most ofthe requirements developed above.

4. Methods

4.1. Fungal growth conditions

The strain of Eutypa lata Pers. Fr. Tul. & C. Tul. used inthese experiments was isolated from vineyard showingsymptoms of eutypiosis. The strain was grown on sterileculture medium solidified or not with agar (20 g l–1). Thegrowth medium was yeast nitrogen base minimal (YNBm,Difco, Detroit) at 1.7 g l–1, supplemented with glucose ascarbon source (10 g l–1) and proline as nitrogen source(5 g l–1).

4.2. Mycelium growth assays

In one type of assay, the effects of phenolics on mycelialgrowth were measured on cultures made in wells (36 mmdiameter), containing the solid medium (agar, YNBm,glucose, proline) in which the pH was fixed with anappropriate buffer (in a physiological range (4–8.5). Thus,10 mM MES/KOH were used in the range 5.0–6.5 and/or10 mM HEPES/KOH were used in the ranges 4.0–5.0 and7.0–8.5 in accordance with its two pKa values (3.6, 7.6). Theexperiments were carried out in the dark at 20 °C. Theinoculum consisted of a 3 mm diameter mycelial disk takenfrom a solid culture. The diameter of the mycelium wasmeasured daily in six identical wells for 8 d. This corre-sponds in the control sets to the complete invasion of thewell (expressed as 100% growth). In the experiments

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dealing with the chemical structure–antifungal activity rela-tionship, mycelial development was carried out on the solidmedium buffered at pH 4.1 by 10 mM HEPES, added byeach of the derivatives at 1 mM final concentration. In asecond type of assay, cultures were grown in 10 ml flasks inliquid medium maintained at pH 5.5 or 4.3 in buffers as usedabove. Flasks were inoculated with a known weight offiltered mycelium (generally 10 or 25 mg) taken from aliquid culture maintained in permanent agitation. Growthwas followed daily by measuring optical density in theflasks, 100% growth corresponding to a complete blockageof light transmission. It has been verified that a linearrelationship exists between optical density and fresh mate-rial weight in the course of the experiment. In each type ofassay, experiments were conducted at least three times. Themedia were autoclaved under 0.5 bars for 15 min. Com-pounds were added during cooling at 50 °C at the desiredconcentration from a 10 mM aqueous stock solution. Carewas taken to verify that the pH of the culture medium wasnot modified upon addition of the tested phenolics.

4.3. Microscopy

Eutypa mycelium was sampled from liquid cultures aftervarious treatment durations with SA. Mycelia were chemi-cally fixed to observe the effect of SA on the structure of thefungal filaments. Mycelium was fixed for 45 min at 25 °C in3% (v/v) glutaraldehyde buffered with 0.1 M phosphate(Sörensen buffer) at pH 6.8. Following washing and 1%(v/v) osmium tetroxide postfixation, dehydration occurredprogressively in ethanol series and impregnation was begunin propylene oxide (2 × 20 min). Until this step, a 5 mincentrifugation was necessary between each bath. We applieda first mixture of 2/3 propylene oxide and 1/3 resin(2 × 20 min), then a second mixture of 1/3 propylene oxideand 2/3 resin (2 × 40 min), before pure resin overnight.Semi-thin sections coloured with toluidine blue in boraxwere observed with a Zeiss (Axioplan) microscope. Thinsections, contrasted with uranyl acetate and lead citrate,were observed using a Jeol 100S microscope under 80 kV.

4.4. Uptake experiments

An aliquot of the liquid culture medium was centrifugedat 7 °C for 10 min at 3000 × g. The fungal pellet was twiceresuspended in the appropriate incubation medium andcentrifuged again. The final pellet was weighed and mixedin the incubation medium to give a mycelium density ofabout 60 mg ml–1. In 5 ml test tubes, 250 µl of this suspen-sion were added to an equal volume of incubation mediumcontaining SA at the indicated final concentration and18.5 kBq ml–1 [7-14C]SA (specific activity: 2.0 GBq m-mol–1). Uptake was rapidly initiated by vortexing. Eachexperimental point was determined on at least four replicatetubes maintained in a water bath at 25 °C. At the desiredtime, uptake was terminated by adding 2 ml of chilled

incubation medium in the test tube, vortexing and rapidlyfiltering under vacuum the content of the tube on Sartoriusfilters (ref. 13400 25 S) pre-wetted with 2.0 ml of the chilledincubation medium. The test tube was then rinsed with2.0 ml of this medium immediately added onto the filter. Azero time control (To) was determined in order to evaluatethe adsorbed radioactivity and for calculations, To wassubtracted from the experimental values. Filters were placedin 6 ml scintillation vials, dried in an oven at 55 °C for 1 h,and 4 ml of scintillation liquid (Ecolyte, ICN, Orsay) werethen added in each vial. Radioactivity was counted by liquidscintillation spectroscopy. In most cases, uptake experi-ments were carried out in an incubation medium composedof 250 mM sorbitol (to maintain an osmotic equilibrium),0.25 mM MgCl2 and 0.50 mM CaCl2, in which pH wasadjusted at 4.5 with 20 mM HEPES/KOH. This incubationmedium added with Mg and Ca ions was chosen for itsability to maintain membrane energization involved inoptimization of solute transport [2]. In experiments focusedon pH effects, 20 mM HEPES was used at pH 4 and 7 and20 mM MES was used at pH 5 and 6. It has been verifiedthat such a short-term experiment for 3 h in this absorptionmedium did not modify further development after transferof the mycelium on the solid growth medium. After a 3 htreatment in labelled SA, 500 mg mycelium was washedtwice by centrifugation (10 000 × g) in 30 ml cold bufferedmedium without SA. The last recovered pellet was groundin mortar and pestle in 1 ml 80% (v/v) ethanol added with1 g quartz. After centrifugation, the residue was extractedagain with 1 ml 80% ethanol and the combined extractswere evaporated under vacuum. Thin-layer chromatographywas used to identify SA on Merck silica gel 60F254 platesdeveloped with pentane–ethanol–ethyl acetate (8:1:1, v/v).In these conditions, the Rf value for SA visualized underUV light (254 nm) was 0.75. A 50 µl volume of labelledextract was deposited on plate. After migration, the platewas dried and 1 cm high silica gel areas were scrapped offalong the lane and put in scintillation vials with 4 mlEcolyte for counting.

4.5. Chemicals

Benzoic acid derivatives were purchased from Sigma-Aldrich Chimie (St Quentin Fallavier), and [14C]SA fromNew England Nuclear (Paris). Their chemical characteris-tics are given in Table 1. The given log D values have beencalculated with the log D Suite v.4.5 program (AdvancedChemistry Development, Toronto). log D corresponds to theoctanol–water partition coefficient calculated at a given pH,and thus takes into account the degree of dissociation of themolecules. Therefore, these calculations give a better rep-resentation than that of log P obtained experimentally andgiven in the literature [18]. Values in Table 1 were calcu-lated at a pH of 4.1 in order to fit with our experimentalconditions of mycelial growth.

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Acknowledgements

This work was supported by the Conseil Interprofes-sionel du Vin de Bordeaux. We thank Dr. Andrew King forimproving the English version.

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