Microbiol. Res. (1997) 152,413-420 Microbiological Research

Gustav Fischer Verlag

Supression of Botrytis cinerea causing grey mould disease of grape vine (Vitis vinifera) and its pectinolytic activities by a soil bacterium

Bernard PauJl, Irene Girardi, Tej BhatnagarJ, Philippe Bouchet2

J Institut Jules Guyot, Universite de Bourgogne, B.P. 138,21004 DIJON, France 2 Laboratoire de Cryptogamie, Facult€ de Pharmacie, Rue Cognac Jay, Reims, France

Accepted: September 28, 1997

Abstract

Botrytis cinerea was found to be pathogenic to grapevine, pro­ducing characteristic grey mould symptoms within 7 days of inoculation into vitroplants. An endospore forming bacterial strain (GI 070), Bacillus circulans, was found to be antagonist against this disease causing fungus. Under laboratory condi­tions the bacterial cultllre or its filtrate brings about complete suppression of the fungus in Petri-dishes and the grey mould symptoms on grapevine vitroplants. Pectinolytic activities (Polygalacaturonase, Pectin lyases) of Botrytis cinerea were also influenced by these bacteria. Details of the fungal para­site, its biological control and its pectinolytic activities are discussed in this mticle.

Key words: Botrytis cinerea - Bacillus circulans - antagonist - biological control - polygalacturonase - grape vine

Introduction

Grey mould disease caused by Botrytis cinerea (= Bo­tryotinia fuckeliana) causes heavy losses in table and wine grapes in many places around the world (Bulit and Dubos 1988; Fanizza et a!. 1995; Gessler and lermini 1985; McClellan and Hewitt 1973; Pearson and Riegel 1983). For controlling this disease in table or wine cultivars, growers depend mainly on chemical treat­ment. Together with the intensive agricultural practices, the chemical control of plant diseases developed rapidly after the 2nd World War. Soon it became the favourite and indispensable method for disease control. The first fungicide used against Botrytis cinerea during the 1960's belonged to the family of thirames and

Corresponding author-: B. Paul

phtalimides (Charnroux 1988). The benzimidazoles especially benomyl (C4HJSN403) appeared in 1969. Benzimadoles and dicarboximides are widely used fungicides (Elad 1994). These together with other fungicides decreased drastically the losses caused by Botrytis cinerea, which were 54% in 1979 against 20% in 1980 in the region of "loire atlantique" in France (Chamroux 1988).

Resistant forms of Botrytis cinerea, started appearing from 1970 onwards in France and throughout the world (Leroux and Gredt 1979). The development of resistant strains of Botrytis cinerea, to the fungicides has greatly reduced the effectivness of such chemical control. Resistance to the benzimidazoles and dicarboximides is quite frequent (Beever eta!' 1989, 1991; Elad etal. 1992; Hsiang and Chastagner 1992). Hence, the grey mould disease continues to be a challenge (Elad et al. 1988; Gullino and Garibaldi 1986; Pommer and Lorenz 1982).

Biological control has been attempted to control the spread of the grey mould disease caused by Botlytis cinerea. The natural bacterial flora found on the surface of the leaves of grape-vine has an inhibitory effect on the fungus (Blakeman and Fraser 1971). The inhibitory action of the extract of grapefruit BC-I000 was as important as benomyl or vinchlozoline at the concentra­tion of 1500ppm (Esterio eta!' 1992).

Successful expriments have been carried out with the antagonist fungus Trichodenna (Dubos et a!. 1982; Gullino and Garibaldi 1988; Gullino eta!' 1985). Other microorganisms used in biological control of this disease are Azotobacter sp. (Doneche and Marcantoni 1992). Serratia marcescens B2 inhibits the mycelial

Microbiol. Res. 152 (1997) 4 413

growth and the sporulation of Botrytis cinerea (Akutsu eta!' 1993). Gliocladium roseum and a species of Peni­cillium caused the suppression of conidia formation on strawberry leaves (Sutton and Peng 1993). Ulocladium atrum shows a competitive effect on Botrytis cinerea (Kohl etal. 1995). Serratia and Pseudomonas were ef­fective on cabbage infected by this fungus (Leifert etal. 1992).

Species of Bacillus are well known to be biological control agents. Six isolates of B. subtilis reduced Phytophthora cactorum infection on apple seedlings (Utkhede 1984). Isolate BACT-l of this bacterium showed significant antagonism in vitro against 17 fungi from soil (Utkhede 1992). Strain EBW-4 of the same species was reported to have considerable boilogical control potential against apple replant disease under orchard conditions in British Columbia (Utkhede and Smith 1992). Suppression of Pythium mamilla tum causing damping-off disease of cucumber seedlings was obtained by the application of the soil bacterium Bacil­lus mycoides (Paul et al. 1995). Bacillus brevis was utilised as control agent of Botlytis cinerea causing disease on protected chinese cabbage (Edwards and Seddon 1992). Bacillus cereus suppressed Phytophthora seedling diseases of alfalfa (Handelsman et ai. 1990), and the same bacteria were found to be antagonist to Pythium aphanidermatum causing cucumber fruit rot (Smith eta!' 1993).

Most Bacillus spp. produce antibiotics (Katz and Deamin 1977). A considerable number of antibiotics from the genus Bacillus have been identified, many of which are known to have antifungal activity (Berdy 1974). Recently a new antifungal antibiotic of the !turin group, Bacilliomycin Lc secreted by Bacillus subtilis has been purified (Eshita et al. 1995). A literature survey, however, reveals that very little work on the biological control of the grey mould disease of grapevine have been done using members of the genus Bacillus.

Products secreted by Botrytis cinerea cause cell necrosis. About ten enzymes and five toxins secreted by this fungus are described which contribute to host cell necrosis. Among all these secretions the most important for the cell necrosis are probably the polygalacturonases (Van der Cruyssen and Kamoen 1995).

In the present study we have found a soil bacterium belonging to the genus Bacillus (GI 070) to be antago­nist to Botrytis cinerea. In vitro studies reveal the complete suppression of the fungal mycelium, and inhibition of conidial germination in the presence of these bacteria. Microcuttings (vitroplants) of grape vine treated with bacterial extracts were resistant to the attack by Botrytis cinerea. A decline of the pectinolytic activi­ties of Botrytis cinerea (polygalacturonase, pectin lyase) in the presence of the antagonist bacteria has also been observed.

414 Microbio!. Res. 152 (1997) 4

Materials and methods

Two strains of Botrytis cinerea were used in this study; strain T4 was provided graciously by Dr. Brygoo, Y. of the Institut National de Recherche Agronomique of Versailles, France; and SPI was isolated at our institute. Most of the biological control experiments were carried out with the strain SPl, while the pectinolytic activities were carried out with both the strains. Fungal isolates were maintained on solid medium, ENTAV agar (Four­nioux and Bessis 1993).

The antagonistic bacterium (GI 070) was isolated by the senior author in the north of France. It is a rod shaped, Gram +ve, endospore forming organism belonging to the genus Bacillus. This was cultured on ENTAV agar and Trypcase Soy Broth (Biomerieux, France).

Antagonism between these two organisms was observed by placing them on the same agar plate and incubation in obscurity at 20-22°C, for seven days. To study the antifungal activity of the bacteria (GI 070), crude extract ACP (antifungal crude principle) was prepared by a fermentation process. Com steep glucose broth (Porter 197 5)(llitre) was inoculated with 200 ml bacterial pre culture (which was obtained by culturing the strain GI 070 on Trypcase soja broth for 24 h at 37°C). The culture was grown on a shaking incubator at 28 °C in batch culture condition for 72 hours. The fermentation product was extracted with an equal volume of ethyl acetate. The ethyl acetate fraction, slightly yellowish in colour, was separated. Since ethyl acetate was found toxic to Botrytis cinerea, it was evaporated to 11l0th volume. 70 ml of this concentrate were kept for further chemical studies, while the remain­ing 30 ml were further divided into two fractions of 20 and 10 ml. These were completely evaporated to remove all ethyl acetate and resuspended with equal volumes of ethanol and sterile distilled water respectively, to obtain the ACP-ethanol and ACP-water fractions.

50 III of Botrytis cinerea spore suspension (2.4 x 105

spores ml- I ) and increasing concentrations of the ACP­ethanol (0, 25, 50, 100, 150, 200, 300 and 400 Ill) were incorporated into 2 ml or ENTAV agar and were poured into sterile petri dishes (diam. 40 mrn). These were incubated at 20°C for 7 days and were observed at regu­lar intervals.

Since ethanol was found to be toxic to the vitroplants of both cultivars of Vitis vinifera, water fraction of ACP (ACP-water) was used to study infection control. Two months old vitroplants of the two cultivars of grapevine (Vitis vinifera) : Chardonnay and Pinot noir were cultured at our institute. The growth medium used for the two cultivars was ENTAV supplemented with myoinositol (lOOmg/I). The culture of vitroplants were done accord­ing to method described by Fourniox and Bessis (1993).

Fig.!. Botrytis cinerea and its antagonist bacteria. A. Nonnal mycelia and conidia of the fungus. B. Conidiophore bearing a bunch of conidia. C. Sporulated and vegetative forms of the bacterium OJ 070. All figures bar = 10 f.LIJ;l, unless otherwise stated.

Fifty III of spore suspension (2.4 x 105 ml-!) was mixed with different concentrations of ACP-water (0, 50, 100, 150,200 and 300 111). Thirty III of this mixture was placed on the undersurface of the 3 leaves per vitroplant. Thus 2 sets of six vitroplants were inoculated for each cultivar.

Both strains of Botrytis cinerea (~PI and T4) were used to study the pectinolytic activities (polygalacturon­ase and pectin lyase) of the fungus. Two sets of samples for each strain were prepared. The set SPI and T4 was prepared from actively growing fungal mycelium, while set SPI * and T4* was prepared from mycelium taken from the vicinity of the antagonist bacteria.

Polygalacturonase (PG) activity. A piece of agar block of ENTAV containing mycelium of Botrytis cinerea (strain T4 and SPI) was taken and inoculated into pectin broth (Yeast Nitrogen base 6.7 g, glucose 10 g, poly­galacturonic acid 109 in 11 phosphate buffer 50 mM, pH 5) in a 60 ml sterile capped bottle and placed on a rotary shaker for 7 days at 21°C. The broth was then filtered. PG activity of the fungus was determined by measuring the reducing groups released as described by Milner and Avigad (1967).

Pectin Lyase (PL) activity. This is assayed by measuring the unsaturated uronides and derivatives released in the pectin broth as described by Ayers et al. (1966), and

modified by Cooper and Wood (1975). Nine hundred III of the pectin fermentation broth was mixed with 600 III of thiobarbituric acid 0.04 M and 300 III of HCI IN. The mixture was then incubated at 100°C for 30 minutes. The pectin lyase activity was then obtained by meaSUr­ing the concentration of unsaturated uronides and derivatives by optical density of this preparation at 556nm: Concentration of unsaturated uronides and deri vati ves is calculated by using the law of Beer-Lambert:

DO = £.I.C

£ = 54178 mol-!.l.cm-! I=Icm

PG and PL activities were determined for both sets (SPI, T4; SPI*, T4*) of the two strains of Botrytis cinerea.

Results

The fungus Botrytis cinerea grows well on the ENTAV agar medium, producing thick, cottony, grey coloured colonies after five days of incubation. The optimal pH for the fungus was found to be 6.4, and its growth was optimal at 21°C (6.8 mm/day).

Microbial. Res. 152 (1997) 4 415

Fig. 2. Antagonism between the fungal and the bacterial strain. A. Inhibition of Botrytis cinerea by GI 070. B. zone of inhi­bition. C. non-gemlinatipg conidia within the inhibition zone (bar = 100 ~m). D. Fungal mycelium forming toruloid hyphal swellings in the zone of inhibition (bar = 10 ~m). E-F. Lysed conidiophore (arrow) near the zone of inhibition (E bar = 100 ~m, F bar = 40 ~m).

The fungal mycelium is septate, freely branched, greyish in colour, measuring up to 10 f.1m diameter (Fig. 1A). Grey coloured conidiophores and 'bunches of conidia' are formed in abundance. The conidia are spherical to slightly elliptical and measure up to 10 f.1m diam. (Fig.1B). The antagonist bacterium, strain GI070 grows well on ENTAV agar, producing thick, convey, viscous, opaque, hyaline to slightly whitish, not easily transferable, circular colonies which may grow upto 15 mm in diameter. The bacteria are 4-5 f.1m x 1-1.5 f.1m, Gram +, Catalase +, rod shaped, terminal spore forming and mobile (Fig.1C). The bacterium was identified as Bacillus circulans Jordan.

When Botrytis cinerea was grown with the antagonist bacteria (GI 070) on the same agar plate, a clear zone of inhibition appears around the bacterial innoculum after 7 days of incubation at 21°C, as shown in Fig. 2A. This zone of inhibition is persistant and remains constant even after 3 months of incubation. The conidia falling in the inhibition zone failed to germinate (Fig. 2C). No germination occurred even when these were taken out and reinoculated on a fresh ENTAV plate. Hyphae deve­loping at the edge of the inhibition zone showed marked morphological differepces; giving swollen, irregular,

416 Microbiol. Res. 152 (1997) 4

spherical, to toruloid hyphal bodies (Fig.2D). These hyphae do not produce the characteristic conidiophores and conidia, thus reducing the mycelial density. When reinoculated on fresh ENTAV agar, they form new, nor­mal colonies. The mycelium away from the inhibition zone produced conidiophores and conidia normally. Ho­wever, sometimes conidiophore lysis was also observed under which the conidiophore gets cut off as shown by arrows in Fig. 2 E and 2 F.

Experiments using increasing concentrations of ACP­ethanol in 2ml ENTAV agar and spore suspension showed complete inhibition of Botrytis cinerea from the fourth plate (100 f.11) onwards. No colonies were formed on all other agar plates (Fig. 3 A). Partial germination was observed on the third agar plate with 50 f.11 of ACP­ethanol (Fig.3B). The first agar plate without ACP­ethanol (0f.11) showed normal growth of Botrytis cine­rea.

Experiments with the vitroplants of Chardonnay and Pinot noir cultivars of Vitis vinifera showed that both are highly susceptible to fungal attack, producing the characteristic grey mould symptoms within 8-lOdays of inoculation (Figs. 4A and C). The first three sets of vitroplants of both cultivars (having 0, 50, 100 f.11 of

Fig.3. A. Suppresion of the fungus by different concentrations of the bacterial extract (0, 25, 50, 100, 150, 200 and 400 III of ACP in 2 ml agar). B. partial germination of fungal conidia 50 III concentration od ACP in 2 rn1 agar (bar = 100 11m).

Fig. 4. Grapevine cultivar Chardonnay. A. Untreated vitroplants with ACP. B. treated vitroplants with ACP. C. Grapevine cul­tivar Pinot nair untreated with ACP. D. treated with ACP.

ACP-water) were attacked by Botrytis cinerea while in the other three sets (having 150, 200, 300 III of ACP­water) the plants were healthy (Figs. 4B and D).

Polygalaturonase activity

T4 and SPI strains together with T4* and SPI * were tested for their PG activity according to Milner and Avigad's method (1976). The histograms in Fig. 5 indi­cate PG activity of SPI and T4 strains of Botrytis cine­rea, which are 6.03 and 1O.9Ilmoles/ml of reducing groups respectively, for a 7 day culture. For the same period the fungus taken from the antagonist bacterial vicinity SPI * and T4* the activity is decreased to 1.72 and 3.4llmoles/ml.

Pectin lyase activity

Pectin lyase activity was measured in a 7 day old culture of SPI, T4, SPI * and T4* by measuring unsaturated uronides and derivatives released in the pectin broth as

described by Ayers eta!' (1966) and modified by Cooper and Wood (1975). Fig. 6 shows this activity. SPI relea­ses 16.4 n moles/ml of unsaturated uronides and deri­vatives' while the SPI * releases none; T4 releases 19.3 n moles/ml of unsaturated uronoides and derivates, while T4* releases only 6.97 n moles/mI.

Discussion

Botrytis cinerea is a well known plant pathogen. Our results confirm that both strains (SPI and T4) are highly virulent to the two cultivars (Chardonnay and Pinot noir) of grapevine, producing the characteristic grey mould symptoms within 8-10 days after inoculation. However, in vitro studies reveal that the fungus can be contained by the use of an antagonist bacterium, Bacil­lus circulans (GI 070). Under the influence of the bacte­rial antagonist, the growth of the fungus is controlled, its sporulation suspended, its spore germination inhibited,

Microbiol. Res. 152 (1997) 4 417

sp1* ClI

~ Q) c: '0 (/) sp1

~ o m -o T4* (/) c:

.~

U5 T4

o 2 4 6 8 10 12

PG: IJmoles reducing groups I ml Fig.5. Polygalacturonase activity of 2 strains of Botrytis cinerea SPI and T4 (without bacterial influence) and SPI *, T4* (with bacterial influence) measured according to Milner and Avigad (1967) (/1 moles reducing groupes/ml medium).

sp1* ('II

~ Q) c: '(3 (/) sp1

~ o m ..-o T4* (/) c: ~

U5 T4

o 2 4 6 8 10 12 14 16 18 20

PL: nmoles insaturated uronides and derivatives Fig. 6. Pectin lyase activity of Botrytis cinerea. Strains SPI, T4 (without bacterial influence) and SPI *, T4* (with bacterial in­fluence) measured according to Cooper and Wood (1975) (nmoles unsaturated uronides and derivatives/mI).

its hyphal stmcture modified, and its conidiophore lysed. The modification in the hyphal stmcture was more critical than those observed by Akutsu eta!' (1993) on Botrytis spp. by Serratia marcescens. In repeated experiments the inhibitory zones produced by the anta­gonist bacteria were persistent upto three months and more.

GI 070 inoculated together with Botrytis cinerea on the leaves of the vitroplants however, has little controll­ing effect on the fungus. On the contrary, the bacterial extract (ACP) was fOl1.nd to be highly effective in the

418 Microbiol. Res. 152 (1997) 4

control of fungal development on vitroplants. This difference may be due to the more vigorous growth of the fungus which the bacteria cannot cope with.

Pectinolytic activities (PG, PL) were found in both strains of Botrytis cinerea. These activities were severely hampered by the antagonist bacteria. In the strain SP 1, pectin lyase activity was completely stopped, while in both cases PG activity was considerably decreased. The PG· activity is a key factor as far as the pathogenicity of Botrytis cinerea is concerned (VanderCmyssen and Kamoen 1995). Since this factor is influenced by the

bacteria, this may be one of the reasons for this anta­gonism.

Further studies on the enzymatic behaviour of Bo­try tis cinerea under the int1uence of these bacteria, the chemical nature of the active principle produced by the antagonist, and in vivo studies on several varities of grapevine have to be undertaken before intending the biological control of this aggressive plant parasite.

Acknowledgements

The authors would like to thank Professor C. Divies and Dr. H. Prevot (ENESBANA-Microbiologie) of the University of Burgundy for their help in the identification of the bacterial strain; to Professor A. Belarbi and Dr. A. Gainvors, of the University of Reims, for their kind cooperation in the accomplishment of this work, and to Dr. Y Brygoo of INRA Versailles for providing the fungal strains.

References

Akutsu, K, Hirata, A., Yamamoto, M., Hirayae, K, Okuyama, S., Hibi, T (1993): Growth inhibition of Botrytis spp. by Serratia marcescens B2 isolated from tomato phylloplane. Annals of the Phytopathological Society of Japan 59, 18-25.

Ayers, W. A., Papavizas, G. c., Diem, A. E (1966): Polyga­lacturonate trans-eliminase and polygalacturonase produc­tion by RhizoctoniG;.,solani. Phytopathology 56, 1006 -1011.

Beever, R. E., Larcy, E. P., Pak, H. A. (1989): Strains of Bo­try tis cinerea resistant to dicarboximide and benzimidazole fungicides in New Zealand Vineyards. Plant Pathology 38, 427-437.

Beever, R. E., Pak, H. A. (1991): An hypothesis to account for the behaviour of dicarbiximide-resistant strains of Botrytis cinerea in vineyards. Plant Pathology 40, 342-346.

Berdy, J. (1974): Recent developments qf antibiotic research and classification of antibiotics according to chemical structure. Adv. Appl. Microbiol. 18,309-406.

Blakeman, J. P., Fraser, A. K. (1971): Inhibition of Botrytis ci­nerea spores by bacteria on the surface of chrysanthemum leaves. Physiol. Plant Pathol. 1,45-54.

Built, J., Dubos, B. (1988): Botrytis bunch rot and blight. In: Compendium of grape diseases (Eds: Pearson, R. G., Goheen, A. C.). American Phytopathological Society Press. St-Paul, Minnesota. 13-15.

Chamroux,1. (1988): Botrytis, Ie coup de grace? Viti 117, 46-48.

Cooper, R. M., Wood, R. K. S. (1975): Regulation of synthe­sis of cell wall degrading enzymes by Verticillium albo­artum and Fusarium oxysporum f. sp. lycopersici. Physiol. Plant Pathol. 5, 135 -156.

Doneche, B., Marcantoni, G. (1992): Mise en evidence de 1'­inhibition de Botrytis cinerea par des bacteries telluriques. Possibilite de contr6le biologique de la pourriture grise. C. R. Acad. Sci. Pru1S 314 (III), 279-283.

Dubos, B., Jailloux, E, Bulit, J. (1982): L' antagonisme micro­bien dans la lutte contre la pourriture grise de la vigne. Bul­letin EPPO 12, 17L-175.

Eshita, S. M., Robelto, N. H., Beale. J. M., Mamiya, B. M .. Workman, R. E (1995): Bacilliomycin Lc' a new antibiotic of the Iturin group. Isolations, structures, and antifungal activities of the congeners. Antibiotics 48, 1240-1247.

Edwards, S. G., Seddon, B. (1992): Bacillus brevis as a bio­logical control agent against Botrytis cinerea on protected chinese cabbage. In: Recent Advances in Bouytis Research (Eds: Verhoeff, K., Malathrakis, N. E., Williamson, B.). Pu­doc Scientific Publishers, Wageningen 267-271.

Elad, Y (1994): Biological control of grape grey mould by Trichoderma harzianul1l. Crop protection 13, 35-38.

Elad, Y Shabi, E., Katan, T (1988): Negative growth resistance between enzimidazole and N-phenylcarbamate fungicides and control of Botrytis cinerea on grape. Plant Pathology 37,141-147.

Elad, Y, Yunis, H., Katan, T (1992): Multible fungicide re­sistance to benzimidazoles, dicarboximides and diethofen­carb in field isolates of Botrytis cinerea in Israel. Plant Pathology 41, 41-46.

Estel10, M. A., Auger, J. G., Vazquez, E., Reyes, M., Scheelje, E (1992): Efficacy of a grapefruit seed extract BC-l 000 for control of Botlytis cinerea in table grape in Chile. In: Recent Advances in Botrytis Research (Eds: Verhoeff, K, Malathrakis, N. E., Williamson, B.). Pudoc Scientific Publishers, Wageningen 211- 214.

Fanizza, G., Bisignano, v., Pollastro, S., Miazzi, M., Faretra, E (1995): Effects of polysaccharides from Botiytinia fucke­Zinia (Botrytis cinerea) on in vitm culture of table and wine grapes (Vitis vinifera). Vitis 34, 41-44.

Fournioux, J. C., Bessis, R. (1993): Use of carbon dioxide enrichment to obtain adult morphology of grapevine in vitm. Plant Cell, Tissue and Organ Culture 33, 51-57.

Gessler, c., Jermini, H. (1985): Role of flower infection of grape by Botrytis cinerea and consequences for the spraying schedule. Proceedings of the VIIIth Botrytis Symposium, Alba. Torino. Italy. Quaderni di vitucoltura ed Enologia dell' Universita di Torino 9, 245-250. ~

Gullino, M. L., Garibaldi, A. (1988): Biological and integrated control of grey mould of grapevine. Results in Italy. Bull. EPPO 18,9-12.

Gullino, M. L., Mezzalama, M., Gru'ibaldi, A. (1985): Bio­logical and integrated control of Botrytis cinerea.in Italy. Ex­perimental results and problems. Proceedings of the VIIIth Botrytis symposium, Alba. Torino. Italy. Quaderni di vitu­coltura ed Enologia dell' Universita di Torino 9, 299-308.

Handelsman, J., Raffel, S., Mester, E. H., Wunderlich, L., Grau, C. R. (1990): Biological control of damping off of alfaalfa seedlings with Bacillus cereus UW85. Appl. Envi­ronm. Microbiol. 56, 713-718.

Hsiang, T, Chastagner, G. A. (1992): Production and viability of sclerotia from fungicide-resistant and fungicide-sensitive isolates of Botrytis cinerea, B. elliptica and B. tulipae. Plant Pathology 41, 600-605.

Katz, E., Demain, A. 1. (1977): The peptide antibiotics of Bacillus: chemistry, biogenesis, and possible functions. Bacteriol. Rev. 41,449-474.

Kohl, J., Molhoeck, M. L., VanderPlas, C. H., Fokkema, N. J. (1995): Effects of Ulocladium atrum and other antagonists on sporulation of Botrytis cinerea on dead Lilly leaves exposed to field conditions. Phytopathology 85, 393-401.

Microbiol. Res. 152 (1997) 4 419

Leifert, C. , Sigee, D. c., Epton, H. A. S. (1992): Biological control of Botrytis cinerea on cold stored dutch white cabbage. In: Recent Advances in Botrytis Research (Eds: Verhoeff, K., Malathrakis, N. E., Williamson, B.). Pudoc Scientific Publishers, Wageningen 201-205.

Leroux, P. , Gredt, M. (1979): Phenomenes de resistance croisee negative chez Botrytis cinerea Pers. entre les fongi­cidefl bepzimidazoles et des herbicides carbamates. Phytia­trie-Phytopharmacie: 28, 79-86.

McClellan, W. D., Hewitt, W. B. (1973): Early Botrytis rot of grapes: time of infection and latency of Botrytis cinerea in Vitis vinifera L. Phytopathology 63, 1151-1157.

Milner, Y, Avigad, G. (1976): A copper reagent for the deter­mination of hexuronics acids and certain ketohexoses. Carbohydrate Res. 4, 359-361.

Paul, B., Romond, c., Bhatnagar, T. (1995): Biological control of Pythium mamillatum causing damping-off of cucumber seedlings by a soil bacterium, Bacillus mycoides. Microbiol. Res. ISO, 71-75.

Pearson, R. c., Riegel, D. G. (1983): Control of Botrytis bunch rot of lipening grapes: time of applications of the dicarbox­imide fungicide. Journal of Enology and Viticulture. 34, 167-172.

Pommer, E. H. , Lorenz, G. (1982) : Resistance of Botrytis

420 Microbial. Res. 152 (1997) 4

cinerea Pers. to dicarboximide fungicides. A literature re­view. Crop protection 1,221-230.

Porter, J. N. (1975): 'Cultural conditions for antibiotic produ­cing microorganisms. Methods in Enzymology. 63, 3-23.

Smith, K. P., Havey, M. J., Handelsman, 1. (1993): Suppres­sion of cottony leak of cucumber with Bacillus cereus strain UW85 . Plant Disease 77,139-142.

Sutton, J . c., Peng, G. (1993): Biocontrol of Botrytis cinerea in strawberry leaves. Phytopathology 81, 615-620.

Utkhede, R. S. (1984): Antagonism of isolated of Bacillus sub­tiIis to Phytophthora cactorum. Can. 1. Bot. 62,1032 -1035.

Utkhede, R. S. (1992) : Biological control of soilborne patho­gens of fruit trees and grapevine. Can. J. Plant Pathol. 14, 100-105.

Utkhede, R. S., Smith, E. M. (1992): Promotion of apple tree growht and fruit production by the EBW-4 strain of Bacil­lus subtilis in apple replant disease soil. Can. J. Microbiol. 38,1270-1273.

VanderCruyssen, G. , Kamoen, O. (1995): A proposal for biological control of Botrytis cinerea by means of a low-pa­thogenic Botrytis cinerea strain: In: Biological control of soil-borne and post-harvest pathogens (Eds: Orlikowski, L. B., Skrzypezak Cz. ). VI Conference of Polish Phytopatho­logical Society, Skierniewice 49-59.