ORIGINAL ARTICLE

Inhibitory effect of chitinases isolated from Semillon grapes(Vitis vinifera) on growth of grapevine pathogens

Seiya Saito & Masanori Odagiri & Seiichi Furuya &

Shunji Suzuki & Tsutomu Takayanagi

Received: 5 May 2010 /Accepted: 29 November 2010 /Published online: 28 January 2011# Society for Plant Biochemistry and Biotechnology 2011

Abstract Characteristics and antifungal activity of chiti-nases in Semillon grapes were investigated. Chitinases wereisolated from the juice of Semillon grapes by chitin affinitychromatography. Native and SDS-PAGE analyses of thefraction showing chitin affinity (active fraction) demon-strated only the presence of protein bands of chitinases.Three types of class IV chitinases (chi-1a, chi-1b and chi-2)were purified from the active fraction. These chitinasesactively hydrolyzed chitin under acidic conditions (pH 4.0–4.5). The isoelectric points and the molecular weights ofchi-1a, chi-1b and chi-2 were 4.73, 4.60, and 7.87, and32.1 kDa, 31.6 kDa, and 29.0 kDa, respectively. The activefraction was found to inhibit Botrytis cinerea mycelialgrowth and the inhibitory effect was due to the activity ofchitinases. The active fraction inhibited twenty strains of B.cinerea collected from the experimental vineyard. Theeffect of chitinases was enhanced in media containing morethan 20% sugar. When the active fraction was tested onGlomerella cingulata, the growth inhibitory effect observedwas markedly less than that seen on B. cinerea.

Keywords Chitinase . Grapevine .Vitis vinifera . Botrytiscinerea .Glomerella cingulata

AbbreviationsNative-PAGE native polyacrylamide

gel electrophoresisSDS-PAGE sodium dodecyl sulphate

polyacrylamide gel electrophoresis

Introduction

Grey mould caused by the fungus Botrytis cinerea Persex Fr (anamorph of Botryotinia fuckeliana (de Bary)Whetz) is one of the major diseases affecting vineyardsworldwide (Dubos 2000; Elad et al. 2004). Grey mouldcauses serious damage in grape such as decreased yieldand quality. Ripe rot, which is caused by Glomerellacingulata (Stoneman) Spaulding and Schrenk (anamorphof Colletotrichum gloeosporioides (Penzig) Penzig andSaccardo), is another disease frequently occurring inJapan. The primary symptom of this disease is the rottingof ripe fruit at harvest. Wine grapes suffer huge damagefrom this disease. Because this disease can only becontrolled by preventive measures.

A number of strategies to resist fungal infection haveevolved in plants. One strategy is the production ofpathogenesis-related (PR) proteins (Linthorst 1991), suchas chitinase and β-1,3-glucanase. These two PR proteinsare believed to play an important role in the defenseagainst invading fungal pathogens because they areinduced in plants upon invasion or direct contact bypathogens and by a variety of abiotic stresses (Derckel etal. 1998; Elad 1997; Keulen et al. 2008; Renault et al.1996; Robert et al. 2002; Suo and Leung 2001; Trotel-Aziz et al. 2006; Uchibori et al. 2000). These enzymeshydrolyze chitin and β-1,3-glucan which are fungal cellwall components (Linthorst 1991) and exhibit antifungalactivity in vitro (Ano et al. 2000; Busam et al. 1997;Derckel et al 1996; Giannakis et al. 1998; Hong and Meng2003; Salzman et al. 1998; Van Sluyter et al. 2005;Uchibori et al. 2000). In addition, enhanced resistance tofungal pathogens has been demonstrated in transgenicplants overexpressing chitinase or β-1,3-glucanase, and asynergistic effect is observed when both enzymes are

S. Saito :M. Odagiri : S. Furuya : S. Suzuki (*) : T. TakayanagiLaboratory of Fruit Genetic Engineering, The Institute of Enologyand Viticulture, University of Yamanashi,Kofu, Yamanashi 400-0005, Japane-mail: suzukis@yamanashi.ac.jp

J. Plant Biochem. Biotechnol. (Jan–June 2011) 20(1):47–54DOI 10.1007/s13562-010-0025-2

present (Jach et al. 1995; Jongedijk et al. 1995; Mauch etal. 1988; Vellicoe et al. 2006; Yamamoto et al. 2000).

Multiple chitinase isozymes are found in plants andare divided into seven classes (classes I-VII) according tostructural properties (Collinge et al. 1993). Derckel et al.(1996) detected chitinase activity in grapevine leaves,roots, stems, and berries and found the highest activity inberries. Robinson et al. (1997) found that chitinase activityand class IV chitinase mRNA level increased precipitouslyin Shiraz grapes during ripening, whereas no β-1,3-glucanase activity was detected in berries at any stage ofdevelopment. Class I, III, and IV chitinases isolated fromgrapes showed antifungal activity (Ano et al. 2000; Busamet al. 1997; Derckel et al. 1996; Giannakis et al. 1998;Salzman et al 1998; Uchibori et al. 2000; Van Sluyter et al.2005).

In our preliminary research, we investigated chitinaseactivity in ten grape cultivars and found that Semillon grape(Vitis vinifera cv. Semillon) had the highest chitinaseactivity (unpublished data). Semillon is one of the majorgrape cultivars for making white wine. Semillon grapes area useful model in grape proteomics and have been used inthe study of the structure and role of proteins present ingrape and wines (Fukui et al. 2003). Interestingly, in spiteof the high chitinase activity in Semillon grapes, the grapesare relatively susceptible to grey mould (Dubos 2000). Theresistance of grapes to plant pathogens is presumed to becontributed by several factors. Therefore, to clarify therelationship between chitinase level and susceptibility tofungal pathogens in grapes, it is necessary to evaluate theinhibitory effect of chitinase exclusively on the growth offungal pathogens.

In this study we focused on the inhibitory effect ofboth chitinase and sugars in mature grapes on mycelialgrowth. Salzman et al. (1998) demonstrated that theantifungal activity of chitinase was enhanced by thepresence of glucose. Although both sugar content andchitinase activity increase in grape berries after véraison,there are few studies focusing on the synergistic effects ofchitinase and sugar on antimicrobial activity. Furthermore,far too many studies have not discussed whether therewould be a difference in chitinase sensitivity amongfungal strains.

The objectives of this study were; (I) to purify chitinasesfrom Semillon grapes; (II) to analyze their biochemicalproperties; (III) to assess the inhibitory effect of thechitinase active fraction on the mycelial growth of twograpevine pathogens in the presence of different sugarconcentrations, and (IV) to examine a difference ofsensitivity to active chitinase fraction among twenty wildtype strains of B. cinerea and seven wild type strains ofG. cingulata.

Materials and methods

Plant materials

Semillon grapes (Vitis vinifera) were harvested at the fullyripe stage from the experimental vineyard of the Universityof Yamanashi, Yamanashi, Japan.

Purification of chitinases

Grape berries were crushed and squeezed in a balloon press toobtain juice. The juice was clarified by centrifugation at 10000g and 4°C for 20 min. The protein fraction wasprecipitated from the supernatant by adding ammoniumsulfate (80% saturation). The precipitate was dissolved in100 mM potassium phosphate buffer (pH 7.0) and thendialyzed against the same buffer at 4°C. The dialysate wasloaded onto a chitin affinity column (10 mL) of ChitopearlBL-01 (Wako Chemicals, Osaka, Japan). After washing thecolumn with 500 mL of 100 mM potassium phosphate buffer(pH 7.0), chitinases were eluted with 0.1 M acetic acid (pH3.1) at a flow rate of 0.5 mL/min. The fraction showingchitinase activity (the active fraction) was dialyzed against10 mM sodium acetate buffer (pH 4.0) at 4°C, and loadedonto a cation-exchange column of HiTrap SP HP (5 mL)(GE Healthcare, Tokyo, Japan). After washing the columnwith 50 mL of the dialysis buffer, the enzymes were elutedwith a linear gradient of 0–0.5 M NaCl in the same buffer ata flow rate of 0.5 mL/min. Two peaks (peak I at 0.1 M NaCland peak II at 0.25 M NaCl) that exhibited chitinase activitywere obtained, and peak I fraction was designated as chi-1and peak II fraction, as chi-2.

Chi-1 fractions was dialyzed against 20 mM Tris-HClbuffer (pH 7.0) at 4°C, and then loaded onto an anion-exchange column of Resource Q column (6 mL, GEHealthcare). After washing the column with 60 mL of thedialysis buffer, the enzymes were eluted with a lineargradient of 0–0.5 M NaCl in the same buffer at a flow rateof 0.5 mL/min. Two peaks (peak Ia at 0.09 M NaCl andpeak Ib at 0.14 M NaCl) that exhibited chitinase activitywere obtained, and peak Ia fraction was designated aschi-1a and peak Ib fraction, as chi-1b.

Assay for chitinase activity

Chitinase activity was determined by measuring theincrease in reducing power of a reaction mixture, asdescribed previously (Ano et al. 2000; Uchibori et al.2000). A reaction mixture consisting of 1.0 mL of asubstrate solution (1.6% beta-chitin in 50 mM sodiumacetate buffer, pH 4.0) and 100 μL of the enzyme solutionwas incubated at 37°C for 1 h. The reaction was stopped by

48 J. Plant Biochem. Biotechnol. (Jan–June 2011) 20(1):47–54

adding 2 mL of 0.5 M potassium ferricyanide. After boilingfor 15 min, the solution was centrifuged at 10 000g at roomtemperature for 15 min, and the absorbance of thesupernatant at 420 nm was measured. The amount ofreducing groups was calculated on the basis of thecalibration curve made with N-acetyl-D-glucosamine. Oneunit (U) of the enzyme activity was defined as the amountof enzyme that catalyzed the liberation of reducing sugarequivalent to 1 μmol of N-acetyl-D-glucosamine from thesubstrate per minute under the above conditions.

Analysis of enzymatic characteristics

Native polyacrylamide gel electrophoresis (Native-PAGE)and sodium dodecylsulfate-polyacrylamide gel electrophore-sis (SDS-PAGE) were performed under basic conditionsaccording to the method of Laemmli with 14% slab gels.The gels were stained with Coomassie Brilliant Blue R-250.The N-terminal sequences of the purified enzymes wereanalyzed by a Procise 491 protein sequencer (PE Biosystems,Tokyo, Japan). The isoelectric points (pIs) of the purifiedenzymes were measured using Protean IEF Cell (Bio-Rad)with ReadyStrip IPG strips containing a carrier ampholyte ofpH 3.0-10.0 (Bio-Rad). Isoelectric focusing (IEF) wasperformed in accordance with the manufacturer’s instructions(Bio-Rad). Proteins on the gel were stained with IEF GelStaining Solution (Bio-Rad).

The effects of pH and temperature on the activity of thepurified enzymes were measured under standard assay

conditions at various pHs (0.2 M McIlavine buffer, pH 2.5–8.0) and temperatures (30–80°C), respectively. In order toinvestigate the stability of the enzyme at various pHs andtemperatures, enzyme solutions were pre-incubated in 0.2 MMcIlavine buffer (pH 2.5–8.0, 37°C for 5 min) and at 30–80°Cfor 5 min, respectively, and the residual activities of the treatedenzymes were assayed under standard assay conditions.

Assay for inhibitory effect on fungal mycelial growth

The inhibition of mycelial growth by chitinase and sugarswas determined using two grapevine pathogens, B. cinereaand G. cingulata. B. cinerea wild type strain YU0622 wascollected in 2006 from the experimental vineyard of theUniversity of Yamanashi in Japan (Saito et al. 2009)(Table 3). G. cingulata wild type strain NN005 was a giftfrom Dr. Hakuno Fumiaki (Nihon-Nouyaku, Osaka, Japan).Fungi were maintained on potato dextrose agar (PDA) for3–4 days. Fungal mycelial plugs (4 mm diameter) wereexcised from the edge of a colony growing actively onPDA plates and placed on PDA plates with different sugarconcentrations. The sugar concentration was varied from2.5 to 25.0% (w/v). Before measuring the diameter ofmycelial growth, the plates were incubated at 25°C in thedark for 3 days for B. cinerea and 6 days for G. cingulata.For each sugar concentration, sugarless plates were used ascontrol. At least three replicates were done for eachcondition. Relative mycelial growth rate was calculatedwith the following equation:

mycelial growth on control plate ðmmÞ �mycelial growth on tested plate ðmmÞmycelial growth on control plate ðmmÞ � 100ð%Þ

Dunnett’s multiple range test was used to assess thedifferences of the calculated mycelial growth rate, incomparison with that grown on control plate.

Unless otherwise stated, the active fraction obtainedby Chitopearl BL-01 affinity chromatography was usedfor all assays for the inhibitory effect on fungal mycelialgrowth. The active fraction was dialyzed against 10 mMpotassium phosphate buffer (pH 6.0) at 4°C and wassterilized by filtration. B. cinerea wild type strainYU0622 and G. cingulata wild type strain NN005 wereused for this test (Table 3). Both YU0622 and NN005were cultured on PDA plates at 25°C for 3–4 days. Theinhibitory effect of the enzymes was determined bymonitoring mycelial growth on the PDA plates. Auto-claved PDA medium was cooled to less than 40°C and thefilter-sterilized active fraction was added without or withglucose-fructose (1:1) solution adjusted to the concentra-

tion of 5.0–25.0% (w/v). The medium was spread on Petridishes (9 cm diameter). Fungal mycelial plugs (4 mmdiameter) were excised from the edge of colonies growingactively on the PDA plates and placed at the center of thePDA plates containing the active fraction in the concen-tration of 0.1 U/mL to 5.0 U/mL. The plates wereincubated at 25°C in the dark for 3–8 days prior to themeasurement of mycelial growth diameter. For each sugarconcentration, the plate containing the same amount ofsugar and boiled 5.0 U/mL of the active fraction was usedas control. At least three replicates were done for eachcondition. Relative mycelial growth rate was calculatedwith the same equation as that described above. Inhibitionof mycelial growth rate was calculated by subtractingrelative mycelial growth rate from 100%.

In order to investigate the sensitivities of various strainsof B. cinerea and G. cingulata to the active fraction, 20

J. Plant Biochem. Biotechnol. (Jan–June 2011) 20(1):47–54 49

strains of B. cinerea and 7 strains of G. cingulata were usedfor the assay for the inhibitory effect (Table 3). Fungalmycelial plugs were placed on PDA plates containing theactive fraction in the concentration of 0.1 U/mL to 5.0U/mL, as described above. Plates containing 5.0 U/mL ofboiled active fraction were used as control. The plates wereincubated at 25°C in the dark for 2 days.

Results

Purification and characterization of chitinasesfrom Semillon grapes

The protein fraction in Semillon grape juice was salted outwith ammonium sulfate (80% saturation). Several majorbands and many minor bands were detected on Native-PAGE and SDS-PAGE (Fig. 1a, lane 1; b, Lane 1).Fractions showing chitinase activity (active fraction) wereobtained by affinity chromatography on a Chitopearl BL-01column, resulting in two bands on both Native-PAGE andSDS-PAGE (Fig. 1a, lane 2; b, lane 2). Chitin affinity

chromatography of the protein fraction from the juiceshowed a high yield (81%) on chitinase activity (Table. 1).Cation-exchange chromatography (HiTrap SP HP) of theactive fraction yielded two protein peaks, arising from chi-1and chi-2. Chi-1 showed two protein bands on Native-PAGE and one band on SDS-PAGE (Fig. 1a, lane 3; b, lane3). On the other hand, chi-2 showed no protein band onNative-PAGE and one band on SDS-PAGE, suggesting thatchi-2 is a basic protein (Fig. 1a, lane 4; b, lane 4). Takentogether, these results suggest that the active fraction fromgrape berries contains one basic and two acidic chitinases,as reported previously (Takayanagi et al. 2004). Peak IIfractions that exhibited chitinase activity were collected aschi-2. SDS-PAGE revealed that the molecular weight ofchi-2 was 29.0 kDa (Fig. 1b, lane 4). pI of chi-2 wasestimated to be 7.87 by IEF (Table 2).

Chi-1 was rechromatographed on a Resource Q,resulting in two chitinase peaks (data not shown). Each ofthe peaks showed a homogeneous single band by Native-PAGE (Fig. 1a, lane 5 and b, lane 5; and Fig. 1a, lane 6 andb, lane 6). Fractions containing the two chitinase peakswere collected as chi-1a and chi-1b in the order of the

1 2 3 4 5 61 2 6

14.4

20.1

30

45

66

97

M 21 3 4 5 6(kD)a bFig. 1 Native PAGE and SDS

PAGE of chitinases. Native-PAGE (a) and SDS-PAGE (b) ofchitinases fraction in each puri-fication step. Gels were stainedwith Coomassie Brilliant BlueR250. Lane M, marker protein;lane 1, protein fraction obtainedby precipitation with ammoniumsulfate; Lane 2, chitinasefraction obtained by affinitychromatography; Lane 3, peak Ion cation-exchange chromatog-raphy; Lane 4, peak II on cation-exchange chromatography; Lane5, peak Ia fraction on anion-exchange chromatography, Lane6, peak Ib fraction on anion-exchange chromatography

Purification step Activity

Total (Units) Specific (U/mg) Yield (%)

Crude extract 25400 2.54 100

Ammonium sulfate precipitation 12600 1.26 48

Chitopearl BL-01 Chromatography 9840 16.4 39

Hi Trap SP HP Chromatography Peak I 5166 36.9 20

Peak II 2065 21.1 8

Resource Q Chromatography Peak Ia 1236 25.9 5

Peak Ib 3240 25.3 13

Table 1 Summary of the chiti-nase purification from Semillongrapes

50 J. Plant Biochem. Biotechnol. (Jan–June 2011) 20(1):47–54

retention time. The molecular weights of chi-1a and chi-1bwere estimated to be 32.1 and 31.6 kDa, respectively(Fig. 1b, lane 5 and lane 6). pIs of chi-1a and chi-1b wereestimated to be 4.73 and 4.60, respectively (Table 2).

The yields and the specific activities of the chitinases ateach purification step are summarized in Table 1. Thespecific activities of chi-1a, chi-1b, and chi-2 were 25.9U/mg, 25.3 U/mg, and 21.1 U/mg, respectively and theyields of chitinases were approximately 5%, 13%, and 8%,respectively (Table 1). The optimum pH for chi-1a and chi-2was 4.5 and that for chi-1b was 4.0 (Table. 2). Chi-1a andchi-b was stable between pH 3.0 and 8.0. Chi-2 was stablebetween pH 3.5 and 8.0 (Table. 2). The optimum tempera-ture for chi-1a and chi-1b was 60°C and that for chi-2 was50°C (Table. 2). All three chitinases were stable up to 50°C(Table. 2). The N-terminal amino acid sequences of chi-1a,chi-1b, and chi-2 were compared with that of another V.vinifera cultivar chitinase (Fig. 2). Except for the residue atposition 13 of chi-1b, the twenty amino acid residues fromthe N-terminus of each chitinase agreed with that ofVvChi4A, a class IV chitinase derived from Shiraz grapes(accession no. AAB65776) (Robinson et al. 1997) (Fig. 2).

Inhibitory effect on mycelial growth of grapevinepathogens

Fungal sensitivity to sugar was investigated by measuringmycelial growth on PDA in the presence of different sugarconcentrations. Figure 3 shows relative mycelial growth of B.cinerea and G. cingulata. Calculated relative mycelial growthrates were subjected to analysis of variance using Dunnett’smultiple range test. In comparison with the mycelial growthrate of the control plate, there was a significant difference in

growth rate for all plates with different sugar concentration,except that grown on the plate with 10% of sugar. In thepresence of low sugar concentration between 2.5 and 7.5%,the mycelial growth rates of both fungal pathogens weresignificantly higher than that of pathogens grown on PDAwithout sugar (Fig. 3). On the other hand, the mycelialgrowth rate was significantly lower in the presence of sugarconcentrations higher than 12.5% (Fig. 3).

The active fraction obtained by affinity chromatographywith Chitopearl BL-01 was used to assay for the inhibitoryeffect. The inhibitory effect of the active fraction on B.cinerea (YU0622) and G. cingulata (NN005) in thepresence of different sugar concentrations was evaluated.B. cinerea mycelial growth was inhibited by the activefraction and the inhibitory effect depended on the level ofchitinase activity (Fig. 4a and c). With 5.0 U/mL of theactive fraction, the percentage inhibition of mycelial growthreached approximately 50% regardless of the sugar con-centration (Fig. 4c). The percentage inhibition of mycelialgrowth on plates with sugar concentration higher than 20%was slightly higher than on plates without sugar. On thecontrary, the inhibition of mycelial growth on plates withsugar concentration lower than 10% was less than on plateswithout sugar, regardless of the concentration of the activefraction. The percentage inhibition of mycelial growth was

Table 2 Characteristics of three purified chitinases from Semillion cultivar (V. vinifera)

Molecular mass(kDa)

Specific activity(U/mg)

OptimumpH

Optimum Temperature(°C)

pHstability

Thermal stability*(°C)

pI

Chi-1a 32.1 25.9 4.5 60 3.0–8.0 <50 4.73

Chi-1b 31.6 25.3 4.0 60 3.0–8.0 <50 4.60

Chi-2 29.0 21.1 4.5 50 3.5–8.0 <50 7.87

* residue activity remains more than 80%

Chi-1bChi-2

Chi-1a

VvChi4A

Fig. 2 Comparison of N-terminal amino acid sequences of chitinases.N-terminal amino acid sequence of chitinases of Semillon cultivar wascompared with that of another Vitis vinifera cultivar, Shiraz(AAB65776). Identical residues are shown in white with a blackbackground

Rer

ativ

e m

ycel

ial g

row

th r

ate

(%)

Sugar concentration (%)

B. cinerea

G. cingulata

b b

a a

b bb b

b bb b

b bb b

b bb b

a a

120

100

80

60

40

20

00.0 2.5 5.0 7.5 10.0 12.5 15.0 17.5 20.0 22.5 25.0

Fig. 3 Relative mycelial growth rate of B. cinerea and G. cingulataon PDA plates at various sugar concentrations. Mean values ofmycelial growth diameters were measured after incubation at 25°C for3 days for B. cinerea and 6 days for G. cingulata, respectively. Errorbars represent SE (n=3). Letters above bars indicate significantdifference according to Dunnett’s multiple range test (P<0.01)

J. Plant Biochem. Biotechnol. (Jan–June 2011) 20(1):47–54 51

the highest (12.6%, mean value) in the presence of 25%sugar and 0.1 U/mg of the active fraction. The percentageinhibition of 5.0 U/mg of the active fraction in the presenceof 20% and 25% sugar was 52.7% and 52.2%, respectively.

The chitinase active fraction had much less of an effect onG. cingulata mycelial growth than B. cinerea. The highestpercentage inhibition of G. cingulata mycelial growth was5.8% (mean value) when 5.0 U/mg of the active fraction wasused in the presence of 15% sugar (Fig. 4b and c).

In order to examine the difference in sensitivity to theactive fraction among B. cinerea and G. cingulata strains,20 strains of B. cinerea and 7 strains of G. cingulata weretested (Table 3). The inhibition of mycelial growth in sevenstrains each of B. cinerea and G. cingulata is shown inFig. 5. No significant difference was observed among thestrains of both fungi (Fig. 5).

Discussion

In this study, three chitinases (chi-1a, chi-1b and chi-2) inSemillon grapes were purified and characterized. The N-

terminal amino acid sequences of the three chitinases werehighly homologous with that of class IV chitinase in Shiraz(Robinson et al. 1997) (Fig. 2). The pIs of chi-1a and chi-1b were very different from that of chi-2: chi-1a and chi-1bhad acidic pI and chi-2 had basic pI (Table 2). Derckel et al.(1996) detected several chitinases in grapevine stems,leaves, roots, and berries by a combination of IEF andactive staining, and found that acidic chitinases werepredominant in mature grape berries. Robinson et al.(1997) cloned class IV chitinase cDNAs from Shiraz grapesand found that the mRNA expression was high at weeks12–16 post-flowering. These results agree with our findingsthat the three grape chitinases of Semillon were classifiedinto class IV and that acidic chitinases (chi-1a and chi-1b)were more abundant than basic chitinases (chi-2).

B. cinerea mycelial growth was inhibited by the activefraction and the extent of inhibition depended on thechitinase activity (Fig. 4a and c). B. cinerea mycelialgrowth inhibition was also observed when sugar concen-tration was higher than 12.5% (Fig. 3). Inhibitory effect ofchitinase was enhanced in the presence of more than 20%sugar in medium (Fig. 4c). It is logical that the inhibitory

Concentration of affinity fraction (U/mL)

Inhi

bitio

n of

myc

elia

l gro

wth

rat

e (

%)

Control 0.1 5.02.51.00.50.25 (U/mL)

Control 0.1 5.02.51.00.50.25 (U/mL)

60

50

40

30

20

10

00.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0

BC25%

BC20%

BC15%

BC10%

BC5%

BC0%

GC15%

GC10%

GC5%

GC0%

a

b

c

Fig. 4 Inhibitory effect of theactive fraction at different con-centrations on B. cinerea and G.cingulata mycelial growth rate.a Inhibitory effect of the activefraction on B. cinerea in thepresence of 25% sugar. Boiledactive fraction (5.0 U/mL of theactive fraction was used ascontrol. b Inhibitory effect ofthe active fraction on G. cingu-lata in the presence of 15%sugar. Boiled active fraction(5.0 U/mL) was used as control.c Relative inhibition of mycelialgrowth of B. cinerea and G.cingulata at different sugar con-centrations. Boiled active frac-tion (5.0 U/mL) was used ascontrol. Mycelial growth wasmeasured after incubating at 25°C for 2–6 days for B. cinereaand for 5–7 days for G. cingu-lata. In the legend, BC repre-sents B. cinerea and GC, G.cingulata, followed by sugarconcentration (in %), respec-tively. Error bars represent SE(n=3)

52 J. Plant Biochem. Biotechnol. (Jan–June 2011) 20(1):47–54

effect of chitinase is enhanced in the presence of a highconcentration of sugar (Fig. 4c). This result is consistentwith the report by Salzman et al. (1998) demonstrating thatthe B. cinerea growth inhibition by grape chitinases wasenhanced by the presence of 1 M (18%) glucose.

Conventionally, antifungal assays of chitinase have beendetermined by measuring, using a microscope, the mycelialelongation grown on the medium distributed in a micro-tube, as in the case of Salzman et al. (1998). Otherwise,simply determined by paper disc method (Ano et al. 2000;Kim and Hwang 1996; Mauch et al. 1988), which didn’tshow any numerical data. In contrast, our method employedin this study allowed accurate measurements of theinhibitiory effect of chitinase. Only with this method wouldit be possible to analyze quantitatively effect betweenchitinase and other chemical compounds synthesized ingrape in order to understand the antifungal activities ofchitinase.

Twenty B. cinerea isolates collected in the vineyardwere examined to determine their sensitivity to the activefraction. The twenty B. cinerea strains were collectedover a three-year period at different grape developmentstages, and from various grape cultivars, and five strainswere collected from grapes exhibiting the noble rotsymptom. However, no difference in sensitivity tothe active fraction could be observed among the 20strains (Fig. 5a). The results suggest that regardless ofstrain, chitinase can uniformly affect B. cinerea mycelialgrowth.

The active fraction showed little inhibition of G.cingulata mycelial growth. Joosten et al. (1995) reportedthat chitinases and β-1,3-glucanase extracted from tomatodid not inhibit mycelial growth of Cladosporium fulvumeven at high concentrations. Ji and Kuć (1996) examinedthe antifungal activity of three chitinases purified fromcucumber leaves against C. lagenarium and found that twoof the chitinases inhibited neither C. lagenarium spore

Control 5.0 U/mL

YU0602

YU0720

YU0622

YU0617

YU0619

YU0715

YU0710

YU0602

YU0720

YU0622

YU0617

YU0619

YU0715

YU0710

YU0602

YU0720

YU0622

YU0617

YU0619

YU0715

YU0710

YU0602

YU0720

YU0622

YU0617

YU0619

YU0715

YU0710

GC001

NN005

GC017

GC002

GC015

GC023

GC019

GC001

NN005

GC017

GC002

GC015

GC023

GC019

GC001

NN005

GC017

GC002

GC015

GC023

GC019

GC001

NN005

GC017

GC002

GC015

GC023

GC019

Control 5.0 U/mLBotrytis cinerea

Glomerella cingulata

a

b

Fig. 5 Inhibitory effect of the active fraction on mycelial growth of B.cinerea and G. cingulata isolates. a Seven representative B. cinereaisolates are shown. Mycelial plug of each isolate was placed onto thePDA plate containing 5.0 U/mL of the active fraction. Boiled activefraction (5.0 U/mL) was used as control. Plates were incubated at 25°C for 36 h. b Seven G. cingulata isolates are shown. Mycelial plug ofeach isolate was placed onto the PDA media containing 5.0 U/ml ofthe active fraction. Boiled active fraction (5.0 U/mL) was used ascontrol. Plates were incubated at 25°C for 2 days

Table 3 B. cinerea and G. cingulata isolates used in this study

Strain Host cultivar Noble rot*

B. cinerea

YU0513 Cabernet sauvignon −YU0518 Semillon +

YU0532 Semillon +

YU0602 Merlot −YU0606 Merlot −YU0613 Chardonnay −YU0615 Chardonnay −YU0616 Chardonnay −YU0617 Chardonnay −YU0619 Koshu −YU0622 Syrah +

YU0707 Chardonnay −YU0710 Semillon −YU0715 Semillon −YU0716 Cabernet sauvignon −YU0720 Cabernet sauvignon −YU0723 Cabernet sauvignon −YU0737 Semillon +

YU0739 Semillon +

G. cingulata

NN005 Pio-ne −GC0601 Pinot noir −GC0602 Pinot noir −GC0615 Riesling −GC0617 Koshu −GC0619 Cabernet franc −GC0623 Chardonnay −

* Strains collected from grapes presenting noble rot symptom

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germination nor mycelial growth. Similarly, Kim andHwang (1996) investigated antifungal activities using crudeenzymes containing different chitinases extracted frompepper stem and revealed that they did not inhibit againstthe mycelial growth of Colletotrichum gloeosporioides.Together, these results suggest that plant chitinases do notalways show antifungal activity against fungal pathogens.Watanabe et al. (1986) reported that the outermost layer ofG. cingulata mycelium is covered with mannan (mainlyconsisting of D-mannose), which can not be hydrolyzedby chitinase. This could be one of the reasons why chitinaseshowed little or no inhibition of mycelial growth ofG. cingulata strains.

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