Biological control of Botrytis cinerea causing grey mould disease

of 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, France

b

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 mould

symptoms within 7 days of inoculation on vitroplants. A bacterial strain, isolated from soil, belonging to the genus Bacillus was

found to be an antagonist of this disease causing fungus. The fungal attack on the grapevine acts as an elicitor to the

production of phytoalexines like resveratrol. This compound was also formed when the leaves of the grapevine vitroplants were

inoculated with the bacteria alone, and this activity was enhanced when a mixture of the pathogen and the antagonist bacteria

was applied. Since resveratrol in wine is considered to be beneficial to human health provided moderate consumption, this

bacteria can be used as a potential biological control agent as well as a biological elicitor of resveratrol. The article includes the

details 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 cinerea

Pers. (=Botryotinia fuckeliana de bary) Whetz., is a

well known disease and causes heavy losses of yield

in table and wine grapes in many places around the

world [6]. The quality of the wine is also a¡ected due

to the conversion of sugar into glycerol and gluconic

acid and by producing enzymes catalysing oxidation

of phenolic compounds such as stilbene phytoalex-

ines [3].

Biological control agents are becoming increas-

ingly interesting as alternatives to the use of chemical

fungicides which are proving hazardous to the envi-

ronment as well as being responsible in bringing

about resistance to the disease [7]. Biocontrol experi-

ments against Botrytis cinerea have been attempted

by 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

FEMSLE 8265 30-7-98

* Corresponding author.

Tel.: [phone]; Fax: [phone];

E-mail: [e-mail]

FEMS Microbiology Letters 165 (1998) 65^70

cescens [2], Gliocladium roseum and Penicillium sp.

[17] and Bacillus circulans [15].

Phytoalexins are biologically active compounds

that are produced in response to biotic or abiotic

stresses. In grapevines, such a response includes the

synthesis of a simple stilbene, resveratrol (trans-

3,5,4P-trihydroxystilbene), and its glucoside, together

with the biosynthetically related compounds viniferin

and pterostilbene [12]. Resveratrol has provoked an

intense interest due to its presence in red wines which

is thought to confer protection against arteriosclero-

sis, coronary heart diseases [12] and cancer [10]. This

is particularly interesting in light of the `French par-

adox' where Toulouse residents, who consume

largely the red wines, have a very low mortality

rate from cardiac diseases, despite a fat consumption

rate similar to that in the United States [16].

Botrytis cinerea (BC 02) was found to be highly

pathogenic to the vitroplants (plants grown on sterile

media 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 ¢rst

time. The pathogenicity of the fungus, the induction

of the defence mechanism of grapevine by resveratrol

production, and the biological control of the disease

are discussed in this paper.

2. Materials and methods

Botrytis cinerea strain BC 02 (616) used in this

study was provided graciously by Dr. Y. Brygoo of

the `Institut National de Recherche Agronomique' of

Versailles, France. The bacterial strain B-781 was

isolated 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 on

potato dextrose agar (PDA), the bacterial isolates

were also cultured in nutrient broth.

Antagonism between the fungus and the bacterium

was observed by placing both these organisms on the

same PDA plate and incubating at 20^22³C, for

7 days. Conidial suspension for further experiments

was prepared from a 14 day old culture by using the

techniques of Bavaresco et al. [3]. The conidial con-

centrations were measured using a Mallasez cham-

ber. Bacterial suspension of B-781 was obtained

from nutrient broth and their numbers calculated

by the `dilution plate method'. The conidial con-

centration of all infection experiments here was

2.4U10

5

ml

31

while the bacterial concentration was

170U10

6

ml

31

. Fifty microliters of these suspensions

was 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 were

grown on MS (Murashige and Skoog) medium

[14]. Four sets of six vitroplants were used in inocu-

lation experiments for each cultivar. Three leaves

were infected in each vitroplant. Fungal spore sus-

pension was placed on the under surface of the

leaves of the ¢rst set of vitroplants, bacteria B-781

were inoculated on the leaves of the second set, a

mixture of fungal conidia and bacteria were inocu-

lated on the leaves of the third set, and the fourth set

of vitroplants were inoculated with 50 Wl of sterile

distilled 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]. The

extract was evaporated to dryness and redissolved in

10 ml of methanol g

31

fresh weight. For HPLC anal-

ysis 50 Wl of each sample (5 mg fresh weight of the

leaf) was used.

2.1. HPLC analysis

Samples were injected on a lichrocart Merck C

18

(Merk-Clevenot Corp., Darmstadt, Germany) re-

versed phase column (250U4 mm; 5 Wm) and ana-

lysed isocratically with 40% acetonitrile/60% water

eluent at a £ow rate of 0.6 ml/min using a Waters

system comprising a Model W 717 sample injector, a

Model W 996 photodiode array detector and a Mod-

el W 474 £uorometer. Resveratrol was detected at

308 nm [11]. For £uorometric detection, maximum

excitation wavelength was measured at 330 nm and

emission 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.

FEMSLE 8265 30-7-98

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

66

2.2. Statistical analysis

The di¡erences between treatments were tested for

signi¢cance by Fisher's test. The variance analysis

was performed with the aid of the STAT-ITCF stat-

istical software.

3. Results

The fungus Botrytis cinerea (BC 02) produces

thick, cottony, grey coloured colonies on PDA.

The mycelia were septate, branched, greyish in col-

our, measuring up to 10 Wm diameter (Fig. 1a). Grey

coloured conidiophores and conidia were formed in

abundance (Fig. 1b). The conidia are elliptical and

measure 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, produces

thick, convex, viscous, opaque, creamish, not easily

transferable, colonies on PDA. The bacteria are

Gram-positive, rod shaped, endospore forming and

mobile.

When Botrytis cinerea (BC 02) was grown with the

antagonist bacteria (B-781) on the same agar plate, a

clear zone of inhibition appeared around the bacte-

rial inoculum after 7 days of incubation (Fig. 2).

This zone of inhibition is persistent and can attain

a diameter of 20^25 mm. After about a month the

zone is clearly marked by a blackish border indicat-

ing the presence of condensed mycelia and deformed

conidia. Hyphae developing in the vicinity of the

inhibition zone showed marked morphological di¡er-

ences; giving at times, constrictions (Fig. 3a, d) and

others swollen, irregular, hyphal bodies (Fig. 3b).

Conidia falling in the zone of inhibition failed to

germinate (Fig. 3c).

Experiments with the grapevine vitroplants

showed that, when inoculated by Botrytis cinerea,

the plants become poorly developed and eventually

FEMSLE 8265 30-7-98

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.

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

67

die (Fig. 4a, c), while those inoculated with a mixture

of fungal conidia and the antagonist bacteria (BC

02+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, and

BC 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 g

31

fresh weight

of leaves, while the bacteria gave 6.07 Wg g

31

fresh

weight. The mixture BC 02+B-781 induced higher

quantities of resveratrol, i.e. 78.3 Wg g

31

fresh

weight. Hence the elicitation was more signi¢cant

in this case (P 6 0.001) as compared to BC 02 or

B-781 alone.

3.2. Vitis rupestris

The three types of inoculum (BC 02, B-781, and

BC 02+B-781) induced the production of resveratrol

as shown in Table 1, Fig. 5. Maximum elicitation by

the fungus (BC 02) was on the ¢rst day of inocula-

tion, by the bacteria (B-781) on the third day, and by

FEMSLE 8265 30-7-98

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

Table 1
Elicitation of resveratrol (Wg g

31

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

0

BC 02

…b†

3.3

4.04

12.89

7.31

2.87

BC 02

…b†

8.63

5.61

5.62

1.62

0

B-781

…c†

2.41

2.91

6.07

1.33

0

B-781

…a†

1.98

5.46

12.93

10.56

8.34

BC 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 (P 6 0.001).

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

68

the mixture (BC 02+B-781) on the fourth day. The

quantity of resveratrol induced by the mixture (BC

02+B-781) is not signi¢cantly di¡erent (P 6 0.001)

from that induced by the bacteria. However it is

signi¢cantly (P 6 0.001) higher than that obtained

by BC 02 alone.

4. Discussion

Botrytis cinerea is a well known plant pathogen

and is responsible for the grey mould disease of

grapevine. Biological control of this fungus has

been reported in the past [5,6,9,15]. However most

of 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 biocontrol

agent, B-781, arrests fungal growth and thus the

grey mould disease, while at the same time, the re-

sistance of the grapevine is enhanced by elicitation of

phytoalexin (resveratrol).

Resveratrol, a stilbene phytoalexin is known to be

an antifungal compound active against a number of

plant pathogens [1,4,8,13]. It is also known to be

elicited by biotic and abiotic stresses in the grapevine

[13]. In our study the fungus (BC 02) elicited the

formation of resveratrol in both species of grapevine.

However, in vitro the fungus was completely sup-

pressed by a soil bacterium, B-781. It also prevented

the 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 inoculated

together with the fungal conidia, the production of

resveratrol increased considerably, while grey mould

was suppressed.

For these reasons, the use of bacteria, B-781, is

interesting for future ¢eld trials and experiments

from three view points: suppression of Botrytis cine-

rea and control of the grey mould disease, enhance-

ment of the resistance of the grapevine plant, and

augmentation of resveratrol concentration in wine.

Thus wine coming from a biologically controlled

grapevine, while free of Botrytis cinerea, retains an

enhanced level of resveratrol, the component in wine

shown to be bene¢cial to human health [10,12,16].

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FEMSLE 8265 30-7-98

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 (P 6 0.001).

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

69

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