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Keywords:

  • Botrytis cinerea;
  • Bacillus;
  • Biological control;
  • Resveratrol;
  • Elicitor;
  • Antagonism

Abstract

  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2Materials and methods
  5. 3Results
  6. 4Discussion
  7. References

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.


1Introduction

  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2Materials and methods
  5. 3Results
  6. 4Discussion
  7. References

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 affected due to the conversion of sugar into glycerol and gluconic acid and by producing enzymes catalysing oxidation of phenolic compounds such as stilbene phytoalexines [3].

Biological control agents are becoming increasingly interesting as alternatives to the use of chemical fungicides which are proving hazardous to the environment as well as being responsible in bringing about resistance to the disease [7]. Biocontrol experiments against Botrytis cinerea have been attempted by the utilisation of Trichoderma[3], Serratia marcescens[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-3,5,4′-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 arteriosclerosis, coronary heart diseases [12] and cancer [10]. This is particularly interesting in light of the ‘French paradox’ 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 rupestris. The fungus acts as an elicitor towards the formation of resveratrol as discovered elsewhere [13]. Elicitation by bacteria is reported here for the first 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.

2Materials and methods

  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2Materials and methods
  5. 3Results
  6. 4Discussion
  7. References

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 20–22°C, 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 concentrations were measured using a Mallasez chamber. Bacterial suspension of B-781 was obtained from nutrient broth and their numbers calculated by the ‘dilution plate method’. The conidial concentration of all infection experiments here was 2.4×105 ml−1 while the bacterial concentration was 170×106 ml−1. Fifty microliters of these suspensions was used to infect the leaves, while when the infection was done with a mixture of bacterium and fungus, 25 μl 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 inoculation experiments for each cultivar. Three leaves were infected in each vitroplant. Fungal spore suspension was placed on the under surface of the leaves of the first set of vitroplants, bacteria B-781 were inoculated on the leaves of the second set, a mixture of fungal conidia and bacteria were inoculated on the leaves of the third set, and the fourth set of vitroplants were inoculated with 50 μl of sterile distilled water to act as control.

Leaves from the vitroplants were extracted at different intervals. This was done in methanol according to a method described by Jeandet et al. [12]. The extract was evaporated to dryness and redissolved in 10 ml of methanol g−1 fresh weight. For HPLC analysis 50 μl of each sample (5 mg fresh weight of the leaf) was used.

2.1HPLC analysis

Samples were injected on a lichrocart Merck C18 (Merk-Clevenot Corp., Darmstadt, Germany) reversed phase column (250×4 mm; 5 μm) and analysed isocratically with 40% acetonitrile/60% water eluent at a flow 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 Model W 474 fluorometer. Resveratrol was detected at 308 nm [11]. For fluorometric detection, maximum excitation wavelength was measured at 330 nm and emission at 374 nm. Identification of trans-resveratrol in the leaf extracts was carried out by the comparison of the retention time of pure resveratrol (Sigma) and that within the extracts.

2.2Statistical analysis

The differences between treatments were tested for significance by Fisher's test. The variance analysis was performed with the aid of the STAT-ITCF statistical software.

3Results

  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2Materials and methods
  5. 3Results
  6. 4Discussion
  7. References

The fungus Botrytis cinerea (BC 02) produces thick, cottony, grey coloured colonies on PDA. The mycelia were septate, branched, greyish in colour, measuring up to 10 μm diameter (Fig. 1a). Grey coloured conidiophores and conidia were formed in abundance (Fig. 1b). The conidia are elliptical and measure up to 10 μm in diameter (Fig. 1c–d).

image

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

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The antagonist bacterium B-781, identified as Bacillus 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 bacterial 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 indicating the presence of condensed mycelia and deformed conidia. Hyphae developing in the vicinity of the inhibition zone showed marked morphological differences; 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).

image

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

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image

Figure 3. Botrytis cinerea. a, d: Fungal hypha showing constrictions, b: hyphal swellings, c: abnormal conidiospores. Bar (a) 40 μm, bar (b, c, d) 20 μm.

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Experiments with the grapevine vitroplants showed that, when inoculated by Botrytis cinerea, the plants become poorly developed and eventually 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 first set developed the grey mould symptoms. Resveratrol was elicited in both species of grapevine. The quantity of resveratrol elicitation varied according to the number of days after infection and according to the nature of the inoculum (Table 1; Fig. 5).

image

Figure 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.

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Table 1.  Elicitation of resveratrol (μg g−1 fresh weight of leaves) by Botrytis cinerea (BC 02), bacteria (B-781), and the two together (BC 02+B-781)
Vitis viniferaVitis rupestris
  1. For each species of Vitis, treatments designed by the same letter are not significantly different according to t-test (P<0.001).

Days1(c)2(bc)3(a)4(b)5(c)Days1(c)2(c)3(b)4(a)5(c)
Water(d)00000Water(c)00000
BC 02(b)3.34.0412.897.312.87BC 02(b)8.635.615.621.620
B-781(c)2.412.916.071.330B-781(a)1.985.4612.9310.568.34
BC 02+B-781(a)3.6917.7178.326.446.95BC 02+B-781(a)04.475.5331.064.34
image

Figure 5. Resveratrol elicitation by BC 02, B-781, and BC 02+B-781 on Vitis vinifera and Vitis rupestris. For each species of Vitis, histograms designed by the same letter are not significantly different to t-test (P<0.001).

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3.1Vitis vinifera

All the three types of inoculum (BC 02, B-781, and BC 02+B-781) gave the maximum yield of resveratrol on the third day (Table 1; Fig. 5). The fungus alone gave a maximum of 12.89 μg g−1 fresh weight of leaves, while the bacteria gave 6.07 μg g−1 fresh weight. The mixture BC 02+B-781 induced higher quantities of resveratrol, i.e. 78.3 μg g−1 fresh weight. Hence the elicitation was more significant in this case (P<0.001) as compared to BC 02 or B-781 alone.

3.2Vitis 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 first day of inoculation, by the bacteria (B-781) on the third day, and by the mixture (BC 02+B-781) on the fourth day. The quantity of resveratrol induced by the mixture (BC 02+B-781) is not significantly different (P<0.001) from that induced by the bacteria. However it is significantly (P<0.001) higher than that obtained by BC 02 alone.

4Discussion

  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2Materials and methods
  5. 3Results
  6. 4Discussion
  7. References

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 suppression of the fungus. The resistance within the grapevine plant was not considered in any of these studies. This is the first report that a bacterial biocontrol agent, B-781, arrests fungal growth and thus the grey mould disease, while at the same time, the resistance 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 suppressed by a soil bacterium, B-781. It also prevented the appearance of grey mould symptoms on vitroplants. When applied alone, the bacteria did not produce any lesions or necrosis on the leaves, but elicited 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 field trials and experiments from three view points: suppression of Botrytis cinerea and control of the grey mould disease, enhancement 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 beneficial to human health [10, 12, 16].

References

  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2Materials and methods
  5. 3Results
  6. 4Discussion
  7. References
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