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//-->FEMS Microbiology Letters 165 (1998) 65^70Biological control ofBotrytis cinereacausing grey mould diseaseof grapevine and elicitation of stilbene phytoalexin (resveratrol)by a soil bacteriumBernard PaulY*, Alphonsa Chereyathmanjiyil, Isaac Masih, Laurence Chapuis,êArmelle Beno|tèLaboratoire des Sciences de la Vigne, Institut Jules Guyot, Universite de Bourgogne, B.P. 138, 21004 Dijon, FranceDepartment of Zoology, St. Andrew's College, Gorkhpur, IndiaINRA, 71, avenue E. Bourleaux, B.P. 81, 33883 Villenave d'Ornon, FranceReceived 11 May 1998; revised 30 May 1998; accepted 8 June 1998AbstractBotrytis cinereaPers. 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 genusBacilluswasfound 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.z1998 Federation of European Microbio-logical Societies. Published by Elsevier Science B.V. All rights reserved.Keywords : Botrytis cinerea; Bacillus; Biological control; Resveratrol ; Elicitor; Antagonism1. IntroductionGrey mould disease caused byBotrytis cinereaPers. (=Botryotiniafuckelianade 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* Corresponding author.Tel.: +33 380-39-63-41; Fax: +33 380-39-62-65;E-mail: bpaul@u-bourgogne.frto 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 againstBotrytis cinereahave been attemptedby the utilisation ofTrichoderma[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 - 666B. Paul et al. / FEMS Microbiology Letters 165 (1998) 65^70cescens[2],Gliocladium roseumandPenicilliumsp.[17] andBacillus 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) ofVitis viniferaandVitis 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 methodsBotrytis cinereastrain 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 ofVitis viniferacultivar `Chardonnay'andVitis rupestriswere 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 was2.4U10Sml3Iwhile the bacterial concentration was170U10Tml3I. 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, 25Wlof each suspension was applied.Infection of both,Vitis viniferaandVitis 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 50Wlof 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 g3Ifresh weight. For HPLC anal-ysis 50Wlof each sample (5 mg fresh weight of theleaf) was used.2.1. HPLC analysisSamples were injected on a lichrocart Merck CIV(Merk-Clevenot Corp., Darmstadt, Germany) re-versed phase column (250U4 mm; 5Wm)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 oftrans-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.B. Paul et al. / FEMS Microbiology Letters 165 (1998) 65^7067Fig. 1.Botrytis cinerea.a: Normal hypha and conidiospores, b:bunch of normal conidia, c, d : normal conidiospores. Bar (a, b)40Wm,bar (c, d) 20Wm.2.2. Statistical analysisThe 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.Fig. 2.Botrytis cinereatogether with antagonist bacteria B-781.3. ResultsThe fungusBotrytis cinerea(BC 02) producesthick, cottony, grey coloured colonies on PDA.The mycelia were septate, branched, greyish in col-our, measuring up to 10Wmdiameter (Fig. 1a). Greycoloured conidiophores and conidia were formed inabundance (Fig. 1b). The conidia are elliptical andmeasure up to 10Wmin diameter (Fig. 1c^d).The antagonist bacterium B-781, identi¢ed asBa-cillussp. and maintained in our laboratory, producesthick, convex, viscous, opaque, creamish, not easilytransferable, colonies on PDA. The bacteria areGram-positive, rod shaped, endospore forming andmobile.WhenBotrytis 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 byBotrytis cinerea,the plants become poorly developed and eventuallyFig. 3.Botrytis cinerea.a, d: Fungal hypha showing constric-tions, b: hyphal swellings, c: abnormal conidiospores. Bar (a)40Wm,bar (b, c, d) 20Wm.68B. Paul et al. / FEMS Microbiology Letters 165 (1998) 65^70Fig. 4. Grapevine vitroplantsVitis rupestris(a, b) andVitis vinifera(c, d). a, c: Vitroplants infected withBotrytis cinerea,b, d: vitroplantsinfected withBotrytis cinerea+B-781.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 viniferaAll 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 fungusalone gave a maximum of 12.89Wgg3Ifresh weightof leaves, while the bacteria gave 6.07Wgg3Ifreshweight. The mixture BC 02+B-781 induced higherquantities of resveratrol, i.e. 78.3Wgg3Ifreshweight. Hence the elicitation was more signi¢cantin this case (P60.001) as compared to BC 02 orB-781 alone.3.2. Vitis rupestrisThe 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 byTable 1Elicitation of resveratrol (Wg g3Ifresh weight of leaves) byBotrytis cinerea(BC 02), bacteria (B-781), and the two together (BC 02+B-781)Vitis viniferaDaysWaterdBC 02B-781BC 02+B-78113.32.413.6924.042.9117.71312.896.0778.347.311.3326.4452.876.95Vitis rupestrisDaysWaterBC 02B-781BC 02+B-78118.631.9825.615.464.4735.6212.935.5341.6210.5631.0658.344.34For each species ofVitis,treatments designed by the same letter are not signi¢cantly di¡erent according tot-test(P60.001).B. Paul et al. / FEMS Microbiology Letters 165 (1998) 65^7069Fig. 5. Resveratrol elicitation by BC 02, B-781, and BC 02+B-781 onVitis viniferaandVitis rupestris.For each species ofVitis,histo-grams designed by the same letter are not signi¢cantly di¡erent tot-test(P60.001).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 (P60.001)from that induced by the bacteria. However it issigni¢cantly (P60.001) higher than that obtainedby BC 02 alone.4. DiscussionBotrytis cinereais 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 ofBotrytis cine-reaand 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 ofBotrytis 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. [ Pobierz całość w formacie PDF ]