Biological control of Botrytis cinerea causing grey mould diseaseof grapevine and elicitation of stilbene phytoalexin (resveratrol)

by a soil bacterium

Bernard Paul a;*, Alphonsa Chereyathmanjiyil a, Isaac Masih b, Laurence Chapuis c,Armelle Beno|êt a

a Laboratoire des Sciences de la Vigne, Institut Jules Guyot, Universiteè de Bourgogne, B.P. 138, 21004 Dijon, Franceb Department of Zoology, St. Andrew's College, Gorkhpur, India

c INRA, 71, avenue E. Bourleaux, B.P. 81, 33883 Villenave d'Ornon, France

Received 11 May 1998; revised 30 May 1998; accepted 8 June 1998

Abstract

Botrytis cinerea Pers. was found to be highly pathogenic to the grapevine plant, producing the characteristic grey mouldsymptoms within 7 days of inoculation on vitroplants. A bacterial strain, isolated from soil, belonging to the genus Bacillus wasfound to be an antagonist of this disease causing fungus. The fungal attack on the grapevine acts as an elicitor to theproduction of phytoalexines like resveratrol. This compound was also formed when the leaves of the grapevine vitroplants wereinoculated with the bacteria alone, and this activity was enhanced when a mixture of the pathogen and the antagonist bacteriawas applied. Since resveratrol in wine is considered to be beneficial to human health provided moderate consumption, thisbacteria can be used as a potential biological control agent as well as a biological elicitor of resveratrol. The article includes thedetails of the fungal parasite, its biological control and resveratrol elicitation. z 1998 Federation of European Microbio-logical Societies. Published by Elsevier Science B.V. All rights reserved.

Keywords: Botrytis cinerea ; Bacillus ; Biological control; Resveratrol ; Elicitor; Antagonism

1. Introduction

Grey mould disease caused by Botrytis cinereaPers. (=Botryotinia fuckeliana de bary) Whetz., is awell known disease and causes heavy losses of yieldin table and wine grapes in many places around theworld [6]. The quality of the wine is also a¡ected due

to the conversion of sugar into glycerol and gluconicacid and by producing enzymes catalysing oxidationof phenolic compounds such as stilbene phytoalex-ines [3].

Biological control agents are becoming increas-ingly interesting as alternatives to the use of chemicalfungicides which are proving hazardous to the envi-ronment as well as being responsible in bringingabout resistance to the disease [7]. Biocontrol experi-ments against Botrytis cinerea have been attemptedby the utilisation of Trichoderma [3], Serratia mar-

0378-1097 / 98 / $19.00 ß 1998 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved.PII: S 0 3 7 8 - 1 0 9 7 ( 9 8 ) 0 0 2 5 9 - 6

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* Corresponding author.Tel. : +33 380-39-63-41; Fax: +33 380-39-62-65;E-mail: bpaul@u-bourgogne.fr

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cescens [2], Gliocladium roseum and Penicillium sp.[17] and Bacillus circulans [15].

Phytoalexins are biologically active compoundsthat are produced in response to biotic or abioticstresses. In grapevines, such a response includes thesynthesis of a simple stilbene, resveratrol (trans-3,5,4P-trihydroxystilbene), and its glucoside, togetherwith the biosynthetically related compounds viniferinand pterostilbene [12]. Resveratrol has provoked anintense interest due to its presence in red wines whichis thought to confer protection against arteriosclero-sis, coronary heart diseases [12] and cancer [10]. Thisis particularly interesting in light of the `French par-adox' where Toulouse residents, who consumelargely the red wines, have a very low mortalityrate from cardiac diseases, despite a fat consumptionrate similar to that in the United States [16].

Botrytis cinerea (BC 02) was found to be highlypathogenic to the vitroplants (plants grown on sterilemedia in test tubes) of Vitis vinifera and Vitis rupes-tris. The fungus acts as an elicitor towards the for-mation of resveratrol as discovered elsewhere [13].Elicitation by bacteria is reported here for the ¢rsttime. The pathogenicity of the fungus, the inductionof the defence mechanism of grapevine by resveratrolproduction, and the biological control of the diseaseare discussed in this paper.

2. Materials and methods

Botrytis cinerea strain BC 02 (616) used in thisstudy was provided graciously by Dr. Y. Brygoo ofthe `Institut National de Recherche Agronomique' ofVersailles, France. The bacterial strain B-781 wasisolated from soil samples taken in Djibouti (Africa).Vitroplants of Vitis vinifera cultivar `Chardonnay'and Vitis rupestris were grown in our laboratory.Fungal and bacterial isolates were maintained onpotato dextrose agar (PDA), the bacterial isolateswere also cultured in nutrient broth.

Antagonism between the fungus and the bacteriumwas observed by placing both these organisms on thesame PDA plate and incubating at 20^22³C, for7 days. Conidial suspension for further experimentswas prepared from a 14 day old culture by using thetechniques of Bavaresco et al. [3]. The conidial con-

centrations were measured using a Mallasez cham-ber. Bacterial suspension of B-781 was obtainedfrom nutrient broth and their numbers calculatedby the `dilution plate method'. The conidial con-centration of all infection experiments here was2.4U105 ml31 while the bacterial concentration was170U106 ml31. Fifty microliters of these suspensionswas used to infect the leaves, while when the infec-tion was done with a mixture of bacterium and fun-gus, 25 Wl of each suspension was applied.

Infection of both, Vitis vinifera and Vitis rupestris,was done on 2 month old vitroplants which weregrown on MS (Murashige and Skoog) medium[14]. Four sets of six vitroplants were used in inocu-lation experiments for each cultivar. Three leaveswere infected in each vitroplant. Fungal spore sus-pension was placed on the under surface of theleaves of the ¢rst set of vitroplants, bacteria B-781were inoculated on the leaves of the second set, amixture of fungal conidia and bacteria were inocu-lated on the leaves of the third set, and the fourth setof vitroplants were inoculated with 50 Wl of steriledistilled water to act as control.

Leaves from the vitroplants were extracted at dif-ferent intervals. This was done in methanol accord-ing to a method described by Jeandet et al. [12]. Theextract was evaporated to dryness and redissolved in10 ml of methanol g31 fresh weight. For HPLC anal-ysis 50 Wl of each sample (5 mg fresh weight of theleaf) was used.

2.1. HPLC analysis

Samples were injected on a lichrocart Merck C18

(Merk-Clevenot Corp., Darmstadt, Germany) re-versed phase column (250U4 mm; 5 Wm) and ana-lysed isocratically with 40% acetonitrile/60% watereluent at a £ow rate of 0.6 ml/min using a Waterssystem comprising a Model W 717 sample injector, aModel W 996 photodiode array detector and a Mod-el W 474 £uorometer. Resveratrol was detected at308 nm [11]. For £uorometric detection, maximumexcitation wavelength was measured at 330 nm andemission at 374 nm. Identi¢cation of trans-resvera-trol in the leaf extracts was carried out by the com-parison of the retention time of pure resveratrol (Sig-ma) and that within the extracts.

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2.2. Statistical analysis

The di¡erences between treatments were tested forsigni¢cance by Fisher's test. The variance analysiswas performed with the aid of the STAT-ITCF stat-istical software.

3. Results

The fungus Botrytis cinerea (BC 02) producesthick, cottony, grey coloured colonies on PDA.The mycelia were septate, branched, greyish in col-our, measuring up to 10 Wm diameter (Fig. 1a). Greycoloured conidiophores and conidia were formed inabundance (Fig. 1b). The conidia are elliptical andmeasure up to 10 Wm in diameter (Fig. 1c^d).

The antagonist bacterium B-781, identi¢ed as Ba-cillus sp. and maintained in our laboratory, producesthick, convex, viscous, opaque, creamish, not easilytransferable, colonies on PDA. The bacteria areGram-positive, rod shaped, endospore forming andmobile.

When Botrytis cinerea (BC 02) was grown with theantagonist bacteria (B-781) on the same agar plate, aclear zone of inhibition appeared around the bacte-rial inoculum after 7 days of incubation (Fig. 2).This zone of inhibition is persistent and can attaina diameter of 20^25 mm. After about a month thezone is clearly marked by a blackish border indicat-

ing the presence of condensed mycelia and deformedconidia. Hyphae developing in the vicinity of theinhibition zone showed marked morphological di¡er-ences; giving at times, constrictions (Fig. 3a, d) andothers swollen, irregular, hyphal bodies (Fig. 3b).Conidia falling in the zone of inhibition failed togerminate (Fig. 3c).

Experiments with the grapevine vitroplantsshowed that, when inoculated by Botrytis cinerea,the plants become poorly developed and eventually

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Fig. 2. Botrytis cinerea together with antagonist bacteria B-781.

Fig. 3. Botrytis cinerea. a, d: Fungal hypha showing constric-tions, b: hyphal swellings, c: abnormal conidiospores. Bar (a)40 Wm, bar (b, c, d) 20 Wm.

Fig. 1. Botrytis cinerea. a: Normal hypha and conidiospores, b:bunch of normal conidia, c, d: normal conidiospores. Bar (a, b)40 Wm, bar (c, d) 20 Wm.

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die (Fig. 4a, c), while those inoculated with a mixtureof fungal conidia and the antagonist bacteria (BC02+B-781) were fully developed, vigorous and viable(Fig. 4b, d). Out of the four sets inoculated, only the¢rst set developed the grey mould symptoms. Res-veratrol was elicited in both species of grapevine.The quantity of resveratrol elicitation varied accord-ing to the number of days after infection and accord-ing to the nature of the inoculum (Table 1; Fig. 5).

3.1. Vitis vinifera

All the three types of inoculum (BC 02, B-781, andBC 02+B-781) gave the maximum yield of resvera-trol on the third day (Table 1; Fig. 5). The fungus

alone gave a maximum of 12.89 Wg g31 fresh weightof leaves, while the bacteria gave 6.07 Wg g31 freshweight. The mixture BC 02+B-781 induced higherquantities of resveratrol, i.e. 78.3 Wg g31 freshweight. Hence the elicitation was more signi¢cantin this case (P6 0.001) as compared to BC 02 orB-781 alone.

3.2. Vitis rupestris

The three types of inoculum (BC 02, B-781, andBC 02+B-781) induced the production of resveratrolas shown in Table 1, Fig. 5. Maximum elicitation bythe fungus (BC 02) was on the ¢rst day of inocula-tion, by the bacteria (B-781) on the third day, and by

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Fig. 4. Grapevine vitroplants Vitis rupestris (a, b) and Vitis vinifera (c, d). a, c: Vitroplants infected with Botrytis cinerea, b, d: vitroplantsinfected with Botrytis cinerea+B-781.

Table 1Elicitation of resveratrol (Wg g31 fresh weight of leaves) by Botrytis cinerea (BC 02), bacteria (B-781), and the two together (BC 02+B-781)

Vitis vinifera Vitis rupestris

Days 1�c� 2�bc� 3�a� 4�b� 5�c� Days 1�c� 2�c� 3�b� 4�a� 5�c�Water�d� 0 0 0 0 0 Water�c� 0 0 0 0 0BC 02�b� 3.3 4.04 12.89 7.31 2.87 BC 02�b� 8.63 5.61 5.62 1.62 0B-781�c� 2.41 2.91 6.07 1.33 0 B-781�a� 1.98 5.46 12.93 10.56 8.34BC 02+B-781�a� 3.69 17.71 78.3 26.44 6.95 BC 02+B-781�a� 0 4.47 5.53 31.06 4.34

For each species of Vitis, treatments designed by the same letter are not signi¢cantly di¡erent according to t-test (P6 0.001).

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the mixture (BC 02+B-781) on the fourth day. Thequantity of resveratrol induced by the mixture (BC02+B-781) is not signi¢cantly di¡erent (P6 0.001)from that induced by the bacteria. However it issigni¢cantly (P6 0.001) higher than that obtainedby BC 02 alone.

4. Discussion

Botrytis cinerea is a well known plant pathogenand is responsible for the grey mould disease ofgrapevine. Biological control of this fungus hasbeen reported in the past [5,6,9,15]. However mostof these studies were oriented towards the suppres-sion of the fungus. The resistance within the grape-vine plant was not considered in any of these studies.This is the ¢rst report that a bacterial biocontrolagent, B-781, arrests fungal growth and thus thegrey mould disease, while at the same time, the re-sistance of the grapevine is enhanced by elicitation ofphytoalexin (resveratrol).

Resveratrol, a stilbene phytoalexin is known to bean antifungal compound active against a number ofplant pathogens [1,4,8,13]. It is also known to beelicited by biotic and abiotic stresses in the grapevine

[13]. In our study the fungus (BC 02) elicited theformation of resveratrol in both species of grapevine.However, in vitro the fungus was completely sup-pressed by a soil bacterium, B-781. It also preventedthe appearance of grey mould symptoms on vitro-plants. When applied alone, the bacteria did not pro-duce any lesions or necrosis on the leaves, but eli-cited the formation of resveratrol. When inoculatedtogether with the fungal conidia, the production ofresveratrol increased considerably, while grey mouldwas suppressed.

For these reasons, the use of bacteria, B-781, isinteresting for future ¢eld trials and experimentsfrom three view points: suppression of Botrytis cine-rea and control of the grey mould disease, enhance-ment of the resistance of the grapevine plant, andaugmentation of resveratrol concentration in wine.Thus wine coming from a biologically controlledgrapevine, while free of Botrytis cinerea, retains anenhanced level of resveratrol, the component in wineshown to be bene¢cial to human health [10,12,16].

References

[1] Adrian, M., Jeandet, P., Veneau, J., Weston, L. and Bessis, R.

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Fig. 5. Resveratrol elicitation by BC 02, B-781, and BC 02+B-781 on Vitis vinifera and Vitis rupestris. For each species of Vitis, histo-grams designed by the same letter are not signi¢cantly di¡erent to t-test (P6 0.001).

B. Paul et al. / FEMS Microbiology Letters 165 (1998) 65^70 69

(1997) Biological activity of resveratrol, a stilbenic compoundfrom grapevines, against Botrytis cinerea, the causal agent forgrey mold. J. Chem. Ecol. 23, 1689^1699.

[2] Akutsu, K., Hirata, A., Yamamoto, M., Hirayae, K., Okuya-ma, S. and Hibi, T. (1993) Growth inhibition of Botrytis spp.by Serratia marcescens B2 isolated from tomato phylloplane.Ann. Phytopathol. Soc. Jpn. 59, 18^25.

[3] Bavaresco, L., Petegolli, D., Cantué , Fregoni, M., Chiusa, G.and Trevisan, M. (1997) Elicitation and accumulation of stil-bene phytoalexins in grapevine berries infected by Botrytiscinerea. Vitis 36 (2), 77^83.

[4] Dercks, W. and Creasy, L.L. (1989) The signi¢cance of stil-bene phytoalexins in the Plasmopara viticola-grapevine inter-action. Physiol. Mol. Plant Pathol. 34, 189^202.

[5] Dubos, B., Jailloux, F. and Bulit, J. (1982) L'antagonismemicrobien dans la lutte contre la pourriture grise de la vigne.Bull. EPPO 12, 171^175.

[6] Elad, Y. (1994) Biological control of grape grey mould byTrichoderma harzianum. Crop Protect. 13 (1), 35^38.

[7] Elad, Y., Yunis, H. and Katan, T. (1992) Multiple fungicideresistance to benzimidazoles, dicarboxymides, and diethofen-carb in ¢eld isolates of Botrytis cinerea in Israel. Plant Pathol.41, 41^46.

[8] Hoos, G. and Blaich, R. (1990) In£uence of resveratrol ongermination of conidia and mycelium growth of Botrytis ci-nerea and Phomopsis viticola. J. Phytopathol. 129, 102^110.

[9] Inbar, J., Menendez, A. and Chet, I. (1996) Hyphal interac-tion between Trichoderma harazianum and Sclerotinia sclero-tiorum and its role in biological control. Soil Biol. Biochem.28 (6), 757^763.

[10] Jang, M., Cai, L., Udeani, G.O., Slowing, K.V., Thomas,

C.F., Beecher, C.W.W., Fong, H.H.S., Farnsworth, N.R.,Kinghorn, A.D., Mehta, R.G., Moon, R.C. and Pezzuto,J.M. (1997) Cancer chemo-preventive activity of resveratrol,a natural product derived from grapes. Science 275 (5297),218^220.

[11] Jeandet, P., Bessis, R., Maume, B.F., Meunier, P., Peyron, D.and Trollat, P. (1995) E¡ect of ecological practices on theresveratrol isomer content of wine. J. Agric. Food Chem.43, 316^319.

[12] Jeandet, P., Breuil, A.C., Adrian, M., Weston, L., Debord, S.,Meunier, P., Mauma, G. and Bessis, R. (1997) HPLC analysisof grapevine phytoalexins coupling photodiode array detec-tion and £uorometry. Anal. Chem. 69 (24), 5172^5177.

[13] Langcake, P. and Pryce, R.J. (1976) The production of resver-atrol by Vitis vinifera and other members of the Vitaceae as aresponse to infection or injury. Physiol. Plant Pathol. 9, 77^86.

[14] Murashige, T. and Skoog, F. (1962) A revised medium forrapid growth and bioassays with tobacco cultures. Physiol.Plant 15, 473^496.

[15] Paul, B., Girard, I., Bhatnagar, T. and Bouchet, P. (1997)Suppression of Botrytis cinerea causing grey mould diseaseof grape vine (Vitis vini¢era) and its pectinolytic activities bya soil bacterium. Microbiol. Res. 152, 413^420.

[16] Seigneur, M., Bonnet, J., Dorian, B., Benchimol, D., Drouil-let, F., Gouverneur, G., Larrue, J., Crockett, R., Boisseau,M., Ribeèreau-Gayon, P. and Bricaud, H. (1990) E¡ect ofthe consumption of alcohol, white wine and red wine on plate-let function and serum lipids. J. Appl. Cardiol. 5, 215^222.

[17] Sutton, J.C. and Peng, G. (1993) Biocontrol of Botrytis cine-rea in strawberry leaves. Phytopathology 81, 615^620.

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