Pesticide Biochemistry and Physiology 81 (2005) 105–112

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PESTICIDEBiochemistry & Physiology

Antifungal activity and mode of action of Galla rhois-derivedphenolics against phytopathogenic fungi

Young-Joon Ahna,*, Hoi-Seon Leeb, Hong-Sik Oha, Heung-Tae Kimc,Yong-Hwan Leea

a School of Agricultural Biotechnology, Seoul National University, Seoul 151–742, Republic of Koreab Faculty of Biotechnology, College of Agriculture, Chonbuk National University, Chonju 561–756, Republic of Korea

c Department of Agricultural Biology, Chungbuk National University, Cheongju 360–763, Republic of Korea

Received 17 May 2004; accepted 17 October 2004Available online 10 December 2005

Abstract

Antifungal activity and target sites of methanolic extract and its constituents from the gall (Galla rhois) caused by theChinese sumac aphid, Schlechtendalia chinensis, on the nutgall sumac tree,Rhus javanica, were examined. In tests with sixphytopathogenic fungi using a whole plant bioassay, the gall extract exhibited good antifungal activity. The biologicallyactive constituents isolated from Galla rhois were characterized as the phenolics methyl gallate and gallic acid by spec-troscopic analyses. Methyl gallate was highly suppressive to Magnaporthe grisea, Botrytis cinerea, and Puccinia recond-

ita, whereas gallic acid exhibited good antifungal activity againstM. grisea and Erysiphe graminis. These two compoundswere ineffective against rice sheath blight caused by Rhizoctonia solani. Methyl gallate did not adversely affect conidialgermination (94%) but significantly inhibited appressorium formation (7%) of M. grisea. Moderate and significant inhi-bition of conidial germination (64%) and appressorium formation (5%) of M. grisea, respectively, were observed withgallic acid. In complementation tests with M. grisea, cAMP and 1,16-hexadecanediol restored significantly and slightlyappressorium formation inhibited by methyl gallate and gallic acid, respectively. These results indicate that methyl gal-late and gallic acid acted on a cAMP-related signaling pathway regulating appressorium formation in M. grisea.� 2004 Elsevier Inc. All rights reserved.

Keywords: Natural antifungal agent; Rhus javanica; Schlechtendalia chinensis; Galla rhois; Methyl gallate; Gallic acid; Conidialgermination; Appressorium formation inhibition; Target site; cAMP-related signaling pathway

0048-3575/$ - see front matter � 2004 Elsevier Inc. All rights reserve

doi:10.1016/j.pestbp.2004.10.003

* Corresponding author. Fax: +82 2 873 2319.E-mail address: yjahn@snu.ac.kr (Y.-J. Ahn).

1. Introduction

Galla rhois is the term used for the gall causedby the Chinese sumac aphid, Schlechtendalia

d.

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chinensis (Bell), on the nutgall sumac tree, Rhusjavanica L. (Anacardiaceae). This gall has beenwell recognized by medicinal, industrial, and agri-cultural interests as a source of tanning agents,ink, supplementary livestock feed, and naturaldrugs [1]. Fundatrix of S. chinensis moves to R.

javanica from Minum spp. such as M. vesicatum

Bescherelle in late April, and parasitizes the under-sides of immature leaves and tissues of R. javanicain May to June, resulting in gall formation [2].Interrelationships between R. javanica chemistryand S. chinensis biology have not yet been investi-gated, although the relative importance of bothnutritional and plant secondary substances indetermining host suitability to the aphids has beenwell described [3–5].

Correlations between aphid behavior and theproduction/accumulation of particular secondarysubstances, especially phenolics and tannins, inplants have been noted [4–6]. These compoundsare often toxic or deterrent to insects, patho-gens, and other organisms [4–7]. Galla rhoisformation has been considered as a defensemechanism of R. javanica against gall-formingsubstances excreted from the salivary glands ofS. chinensis [8]. During initial experiments forscreening antifungal agents from plants, a meth-anolic extract from Galla rhois had antifungalactivity against plant disease organisms andinhibited conidial germination and appressoriumformation, an infection structure, of Magnapor-

the grisea.This paper describes a glasshouse study aimed

at isolating antifungal principles from Galla rhoisactive against six economically important plantdisease organisms by using whole plant bioassays.Potential biochemical target sites of these chemi-cals were also investigated.

2. Materials and methods

2.1. Chemicals

cAMP and 1,16-hexadecanediol were purchasedfrom Sigma (St. Louis, MO). Tween 20 was sup-plied by Junsei Chemical (Tokyo, Japan). All otherchemicals were of reagent grade.

2.2. Phytopathogens tested

Six phytopathogenic fungi used in this studywere M. grisea, Rhizoctonia solani, Botrytis cine-

rea, Phytophthora infestans, Puccinia recondita,and Erysiphe graminis, which cause plant diseasesof many economically important crops [9]. Exceptfor P. recondita and E. graminis, they were rou-tinely maintained on potato dextrose agar slantsor V-8 agar slants, and kept for stock at 4 �C.

2.3. Isolation and identification of active principles

Air-dried mature Galla rhois (600 g) was pur-chased from Boeun medicinal herb shop (Kyung-dong Market, Seoul, Korea) and was pulverized.Antifungal principles from the crude methanolicextract were isolated according to the method de-scribed in the previous paper [10]. Briefly, the bio-active ethyl acetate fraction (10 g) waschromatographed on a silica gel (70–230 mesh,600 g, Merck, Darmstadt, Germany) column(5.5 cm · 70 cm), and successively eluted with astepwise gradient of chloroform/methanol (95/5,90/10, 80/20, 50/50, and 0/100). The two bioactive20/80 and 50/50 fractions were successively rechro-matographed on a silica gel column, using chloro-form/methanol. A preparatory HPLC (SpectraSystem P2000, Thermo Separation Products, SanJose, CA) was used for further separation of theconstituents. The column was a 7.8 mmi.d. · 300 mm lBondapak C18 (Waters, Milford,MA) using methanol/water at a flow rate of5.0 ml/min and detected at 280 nm. Finally, twophenolic compounds 1 and 2 were isolated from20/80 and 50/50 fractions, respectively.

2.4. Bioassays

Antifungal activities of Galla rhois-derivedmaterials against six phytopathogens were deter-mined by using a whole plant bioassay. In a preli-minary experiment, the number of days afterinoculation for damage rating was determined onthe basis of the time-course of symptom occur-rence as previously described [11]. Appropriateamounts of the test materials in 0.45 ml DMSOwere suspended in distilled water with Tween 20

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(250 mg/L). Each solution (45 ml) of the test mate-rials was sprayed onto pots planted with testplants on a turntable simultaneously. After evapo-ration in a glasshouse for one day, each pathogenwas inoculated onto treated test plants. Controlsreceived DMSO-Tween 20 solution.

In a test with rice blast (RCB) caused by M. gri-

sea, rice plants at the 2nd leaf stage (3 plants/pot, 3pots) were sprayed with each test solution. Treatedplants were inoculated with a suspension of conidiain distilled water (1 · 106 spores/ml) and kept in adew chamber (25 �C) for 1 day under 100% relativehumidity (RH). Treated and control plants werethen held in a growth chamber (26 ± 2 �C and85%RH) for 5 dayswhen disease severity was rated.

For rice sheath blight (RSB) caused by R. solani,each test solution was sprayed onto rice plants atthe 4th leaf stage (3 plants/pot, 3 pots). Plants wereinoculated by pouring inoculum at the base of therice plants. Inoculum was made by inoculatingmycelial plugs in wheat bran medium at 25 �C for7 days, and macerated at the rate of 500 g/L dis-tilled water with amixer. Treated and control plantswere held in a growth chamber (28 �C) for 10 days.

For cucumber gray mold (CGM) caused by B.

cinerea, cucumber plants at the 1st leaf stage (1plant/pot, 9 pots) were sprayed with each test solu-tion. Plants were inoculated with conidia(5 · 105 spores/ml) of B. cinerea and then placedin a dew chamber (20 �C) for 4 days.

For tomato late blight (TLB) caused by P. infe-

stans, each test solution was sprayed onto tomatoplants at the 2nd leaf stage (1 plant/pot, 9 pots).Plants were inoculated with a suspension of1 · 105 zoosporangia/ml. They were kept in adew chamber (18 �C) for 5 days when diseaseseverity was rated.

For wheat leaf rust (WLR) caused by P. recond-

ita, wheat plants at the 1st leaf stage (5 plants/pot,3 pots) were sprayed with each test solution. Plantswere sprayed with a suspension of uredosporescolonized on 2nd leaf and then placed in a moistchamber. One day after inoculation, plants wereheld in a growth chamber (22 �C and 70% RH).Disease severity was measured 10 days afterinoculation.

For barley powdery mildew (BPM) caused byE. graminis, barley plants with a fully expanded

first leaf (4 plants/pot, 3 pots) were sprayed witha suspension of each test material. Treated plantswere dusted with conidia of E. graminis formedon the primary leaf of barley and held in a growthchamber (20 �C and 50–60% RH). Disease severitywas rated 10 days after inoculation.

The control effect of the test materials on eachplant disease was evaluated with control value(CV) calculated by the formula CV(%) = [(A � B)/A] · 100, where A represents dis-ease area on untreated plants and B represents dis-ease area on treated plants. Disease area was basedon a percentage scale on a leaf or sheath, whichwas inoculated one day after test compoundapplication.

2.5. Conidial germination and appressorium

formation of M. grisea

The effects of each compound on conidial ger-mination and appressorium formation of M. gri-sea 70–15 were examined according to themethod of Lee and Dean [12]. The strain wasgrown on oatmeal agar (50 g/L) under continuousfluorescent light to promote conidiation at 25 �C.Conidia were harvested from a 7- to 10-day-oldculture. Conidia of M. grisea were harvested insterile distilled water and passed through four lay-ers of cheese cloth. Thirty-six microliters of conid-ial suspension (3 · 104 conidia/ml) with 4 ll ofeach compound were dropped on the hydrophobicside of GelBond (FMC BioProducts, Rockland,ME) and placed in a moistened plastic box. After24 h-incubation at 25 �C, inhibitory effect of eachcompound was determined. Each compound wasdissolved in DMSO at the final concentration of1000 ppm. Percentages of germinated conidia andgerminating conidia induced to form appressoriumwere determined from at least 100 conidia per rep-licate in five experiments with five replicates perthe isolated compound by microscopicexamination.

To reveal inhibition site on appressorium for-mation by each compound, complementation testswere performed by addition of effector chemicalsas previously described [12]. cAMP or 1,16-hex-adecanediol was added in final concentrations of10 mM and 1 lM, respectively.

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2.6. Statistical analysis

Control values and percentage inhibition ofconidial germination and appressorium formationwere determined and transformed to arcsinesquare-root values for ANOVA. Means were com-pared and separated by Scheffe test at the P = 0.05[13]. Means (SE) of untransformed data arereported.

Fig. 1. Structures of the phenolics methyl gallate (1) and gallicacid (2), antifungal principles of Galla rhois.

3. Results

3.1. Antifungal activity of Galla rhois

Using the whole plant bioassay, fractions fromthe methanolic extract of Galla rhois exhibited sig-nificant differences in the control values to sixplant disease organisms (Table 1). At a concentra-tion of 2000 ppm, the ethyl acetate fractionshowed good antifungal activity against RCB,CGM, TLB, WLR, and BPM but not againstRSB. No antifungal activity against all the plantdisease organisms was observed with the hexane,chloroform, and water fractions.

3.2. Identification of active principles from Galla

rhois

Whole plant bioassay-guided fractionation ofGalla rhois extract afforded two active principles.The isolates were identified by spectroscopic

Table 1Effectiveness of Galla rhois-derived materials on various plant diseas

Materiala Control value (%) (SE)b

RCBc RSB CGM

Methanol 100 a 0 a 76 (1.5) aHexane 0 b 0 a 0 bChloroform 0 b 0 a 0 bEthyl acetate 100 a 0 a 82 (2.1) aWater 0 b 0 a 0 b

a Treated with 2000 ppm test solution.b Mean within a column followed by the same letter are not sign

transformed to arcsine square-root before ANOVA. Means (SE) of uc RCB, rice blast caused by Magnaporthe grisea; RSB, rice sheath b

caused by Botrytis cinerea; TLB, tomato late blight caused by Phy

recondita; and BPM, barley powdery mildew caused by Erysiphe gram

analyses, including EI-MS, and 1H- and 13C-NMR, and by direct comparison with publishedliterature [10]. Their chemical structures were eluci-dated as methyl gallate 1 and gallic acid 2 (Fig. 1).

3.3. Antifungal activity of Galla rhois phenolics

Antifungal activity of gallic acid and its methylester against plant disease organisms was deter-mined (Table 2). Methyl gallate at 500 ppm gave100, 100, and 63% control values against RCB,WLR, and CGM, respectively but was ineffectiveagainst RSB, TLB, and BPM. At 125 ppm methylgallate, 63 and 84% control values against RCBand WLR were obtained, respectively. Gallic acidat 500 ppm resulted in 81 and 82% control valuesagainst RCB and BPM, respectively, but showedno antifungal activity against RSB, CGM, TLB,and WLR. This compound revealed moderateactivity against RCB and BPM at 125 ppm.

es, using a whole plant bioassay

TLB WLR BPM

92 (1.2) a 94 (1.0) b 71 (1.5) b0 b 0 c 0 c0 b 0 c 0 c94 (1.0) a 100 a 80 (1.5) a0 b 0 c 0 c

ificantly different (P = 0.05, Scheffe test). Control values werentransformed data are reported.light caused by Rhizoctonia solani; CGM, cucumber gray moldtophthora infestans; WLR, wheat leaf rust caused by Puccinia

inis.

Table 3Effects of Galla rhois-derived methyl gallate (MG) and gallic acid (GA) on conidial germination and appressorium formation by M.

griesa 70–15 and restoration of appressorium formation inhibited by these compounds in the presence of cAMP or 1,16-hexadecanediol (HDD)

Treatment Conidial germination (%) (SE)c Appressorium formation (%) (SE)c

MG onlya 94.3 (1.3) b 6.7 (1.2) b+ cAMP 97.7 (0.3) ab 98.0 (0.0) a+ HDD 98.7 (0.3) a 98.3 (0.3) a

DMSOb 97.0 (0.6) ab 95.0 (1.2) a

GA onlya 64.0 (5.0) b 5.3 (0.9) c+ cAMP 92.0 (0.6) a 40.7 (1.5) b+ HDD 93.3 (0.3) a 44.3 (1.8) b

DMSOb 97.3 (0.3) a 97.3 (0.3) a

a Treated with 1000 ppm in final concentration.b As a control, DMSO was treated at 1% (v/v).c Mean within a column followed by the same letter are not significantly different (P = 0.05, Scheffe test). Control values were

transformed to arcsine square-root before ANOVA. Means (SE) of untransformed data are reported.

Table 2Effectiveness of Galla rhois-derived methyl gallate (MG) and gallic acid (GA) on six plant diseases, using a whole plant bioassay

Compound Conc. (ppm) Control value (%) (SE)a

RCBb RSB CGM TLB WLR BPM

MG 1000 100 a 0 a 81 (1.2) a 44 (0.6) a 100 a 0 d500 100 a 0 a 63 (0.3) b 0 b 100 a 0 d250 87 (2.2) b 0 a 48 (1.7) c 0 b 85 (0.9) b 0 d125 63 (1.5) c 0 a 42 (1.0) d 0 b 84 (0.9) b 0 d

GA 1000 100 a 0 a 0 e 42 (1.2) a 0 c 87 (1.5) a500 81 (1.2) b 0 a 0 e 0 b 0 c 82 (1.5) a250 68 (1.5) c 0 a 0 e 0 b 0 c 73 (2.3) b125 62 (2.3) c 0 a 0 e 0 b 0 c 62 (2.1) c

a Mean within a column followed by the same letter are not significantly different (P = 0.05, Scheffe test). Control values weretransformed to arcsine square-root before ANOVA. Means (SE) of untransformed data are reported.

b For explanation, see 1.

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3.4. Effects of Galla rhois phenolics on conidial

germination and appressorium formation of

M. grisea

The effects of gallic acid and its methyl ester onconidial germination and appressorium formationof M. grisea are recorded in Table 3. Methyl gal-late at 1000 ppm did not adversely affect conidialgermination (94%) but significantly inhibitedappressorium formation (7%) of M. grisea (Fig.2). Gallic acid exhibited moderate (64%) andstrong (5%) inhibition of conidial germinationand appressorium formation of M. grisea, respec-tively. It has been known that modulation of intra-cellular cAMP concentration regulatesappressorium formation in M. grisea [12].

To reveal the action site of methyl gallate andgallic acid, complementation tests were conductedby the addition of cAMP or 1,16-hexadecanediolin the presence of the compounds (Table 3). BothcAMP and 1,16-hexadecanediol significantly andslightly restored appressorium formation inhib-ited by methyl gallate and gallic acid, respectively(Fig. 2).

4. Discussion

Induction of phenolics in response to aphidfeeding has been described [4–6]. Plant responsesto the feeding of chewing insects by increasedproduction of phenolics, which are often identi-

Fig. 2. Appressorium formation of M. grisea. (A) Appressoriaformed on inductive surface, (B) inhibition of appressoriumformation by methyl gallate, and (C) restoration of appresso-rium formation by addition of cAMP in the presence of methylgallate.

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fied as condensed tannins, have been extensivelystudied and well documented [5,14–16]. Plantgalls represent a neoplastic growth reaction of

the plant to an attack by a foreign organismand are often related in some way to the feedingactivity and nutritional physiology of organism[17]. The study of plant galls has gained consid-erable significance in the investigations on the eti-ology of animal and human cancers as well as inthe development of anticancer agents [17]. Be-cause of this, much concern has been focusedon the determination of the distribution, nature,and practical use of plant gall-derived chemicalsubstances that have biological activities. Gallarhois is a product of the interspecific associationbetween R. javanica and S. chinensis. It is rich ingallotannin (50–80%) such as penta-m-digalloyl-b-glucoside as well as in gallic acid and m-digallicacid [1,10]. Tannins and phenolics including gal-lic acid are found to be toxic to pathogens andother organisms [5,7,10,18]. It has been shownthat the methanolic extract of Galla rhois hasgrowth-inhibiting effects on harmful human intes-tinal bacteria such as Clostridium perfringens andEscherichia coli [10].

In the present study, the methanolic extract ofGalla rhois exhibited good antifungal activityagainst several plant pathogenic organisms. Theantifungal principles were identified as the pheno-lics methyl gallate and gallic acid. Responses variedaccording to compound and disease organism.Methyl gallate exhibited potent antifungal activityagainst RCB, CGM, andWLR, whereas gallic acidexhibited strong activity against RCB and BPM.The differences in the antifungal activities betweenthe two compounds may be a function of transportor metabolism rather than a different mode of ac-tion as mentioned below. The exact action remainsto be proven. Gallic acid and methyl gallate appearto be good candidates as naturally occurring con-trol agents for plant pathogens. Methyl gallateand gallic acid are known to possess growth-inhib-iting activity against C. perfringens and E. coli

without adversely affecting the growth of lacticacid-producing bacteria, with the activity beingmore pronounced by methyl gallate than with gal-lic acid [10].

For the development of new types of fungicidesand effective control of fungicide resistantpathogens, novel target sites of fungicide actionhave been extensively studied [19]. Additionally,

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certain plant natural products have been found tobe highly effective against fungicide-resistantmicroorganisms [20–22]. For example, naturalcompounds such as cinnamaldehyde and salicylal-dehyde are effective against four strains of Fusa-rium sambucium resistant to the syntheticfungicide thiabendazole [21]. Appressorium for-mation in many plant pathogenic fungi is prerequi-site to penetrate host plants successively. Recently,a novel strategy to identify antifungal compoundsas target-site specific on appressorium formationhas been developed inM. grisea [23]. In the currentstudy, methyl gallate did not adversely affectconidial germination but significantly inhibitedappressorium formation of M. grisea. On the con-trary, moderate and significant inhibition of conid-ial germination and appressorium formation of M.

grisea, respectively, were obtained with gallic acid.In a complementation test, both cAMP signifi-cantly and 1,16-hexadecanediol slightly restoredappressorium formation inhibited by methyl gal-late and gallic acid. These results indicate thatmethyl gallate and gallic acid acted on a cAMP-re-lated signaling pathway regulating appressoriumformation in M. grisea. There has been no suchmode of action associated with commerciallyavailable fungicides. We are proposing the inhibi-tion of appressorium formation as a novel targetsite developing for new types of antifungal agentsfor the control of rice blast disease.

Acknowledgment

This work was supported by the Ministry ofEducation and Human Resources Developmentfor Brain Korea 21 Project of the Korean Govern-ment to Y.J.A.

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