# Effects of fludioxonil on Botrytis cinerea and on grapevine defence response

Anne Noelle A. N. Petit, Nathalie N. Vaillant-Gaveau, Anne Sophie A. S. Walker, Pierre P. Leroux, Fabienne F. Baillieul, Marie-Laure M.-L. Panon, Christophe C. Clément, Florence F. Fontaine

<figure><img src="image_1.png" /></figure>

## ► To cite this version:

Anne Noelle A. N. Petit, Nathalie N. Vaillant-Gaveau, Anne Sophie A. S. Walker, Pierre P. Leroux, Fabienne F. Baillieul, et al.. Effects of fludioxonil on Botrytis cinerea and on grapevine defence response. Phytopathologia Mediterranea, 2011, 50 (1), pp.130-138. 10.14601/Phytopathol_Mediterr-9049. hal-01000560

HAL Id: hal-01000560

https://hal.science/hal-01000560

Submitted on 29 May 2020

HAL is a multi-disciplinary open access
archive for the deposit and dissemination of sci-
entific research documents, whether they are pub-
lished or not. The documents may come from
teaching and research institutions in France or
abroad, or from public or private research centers.

L'archive ouverte pluridisciplinaire HAL, est
destinée au dépôt et à la diffusion de documents
scientifiques de niveau recherche, publiés ou non,
émanant des établissements d'enseignement et de
recherche français ou étrangers, des laboratoires
publics ou privés.

<figure><img src="image_2.png" /></figure>
# Effects of fludioxonil on *Botrytis cinerea* and on grapevine defence response

ANNE-NOËLLE PETIT<fnref n="1" />, NATHALIE VAILLANT-GAVEAU<fnref n="1" />, ANNE-SOPHIE WALKER<fnref n="2" />, PIERRE LEROUX<fnref n="2" />, FABIENNE BAILLIEUL<fnref n="1" />, MARIE-LAURE PANON<fnref n="3" />, CHRISTOPHE CLÈMENT<fnref n="1" /> and FLORENCE FONTAINE<fnref n="1" />

**Summary.** Botrytis bunch rot of grapes is mainly controlled by applying fungicides at three crop stages: the end of flowering (BBCH 68), bunch closure (BBCH 77) and the beginning of veraison (BBCH 81). The phenyl-pyrroles derivative fludioxonil is among the most effective fungicides registered to control *Botrytis cinerea*. Its effectiveness was investigated in relation to spray timing, fungicide resistance and defence responses of grapevine. Frequencies of *B. cinerea* strains which were resistant to fungicides were evaluated at harvest. The frequencies of resistant phenotypes were similar in all treatments except for a class of multidrug resistant strains (MDR 1) whose frequency increased after fludioxonil applications. None of the treatments tested induced defence responses in flowers/herries after fungicide application, suggesting that fludioxonil effectiveness was not related to a stimulation of plant defence processes. The standard program of three fungicide applications provided the best control of *B. cinerea* in the Champagne region in comparison with a single treatment of fludioxonil at any of the crop stages tested.

**Key words:** fungicide resistance, grey mould, treatment efficacy.

## Introduction

*Botrytis cinerea* (teleomorph *Botryotinia fuckeliana*) is a widespread fungal pathogen, responsible for the grey mould disease, and Botrytis bunch rot of grapevine causes severe damage in vineyards around the world (Bulit tableand Dubos, 1988). In the vineyards in Champagne, France, *B. cinerea* is especially feared by grape growers because of considerable economic losses related to this pathogen. Depending on the year, incidence of *Botrytis* bunch rot can reach 15–25% of bunches infected (Panon *et al.*, 2006). In addition, wines prepared from infected grapes usually exhibit organoleptic defaults, such as oxidation of the colour or the oc-

currence of typical aromatic notes (“mouldy”, “rotten”) which are not appreciated by consumers, and alteration of foaming properties (Bocquet *et al.*, 1995; Marchal *et al.*, 2001; Cilindre *et al.*, 2007, 2008).

In association with cultural methods of disease control, use of chemical fungicides against *B. cinerea* remains the main way to reduce the incidence and severity of bunch rot. Several classes of fungicides are available (Leroux *et al.*, 2002). A standard fungicide application program consisting of three preventive applications of fungicide was recommended until 2006 in the Champagne region: at the end of flowering (BBCH 68), at bunch closure (BBCH 77) and at the beginning of berry ripening (veraison, BBCH 81) (Meier *et al.*, 2001). In most cases, the fungicides consisted of fenhexamid (at BBCH 68), fludioxonil (at BBCH 77) and pyrimethanil (at BBCH 81). Fludioxonil is among the most effective fungicides registered for con-

<footnote n="1">¹Laboratoire de Stress, Défenses et Reproduction des Plantes, URVVC-SE UPRES EA 2069, Université de Reims Champagne-Ardenne, UFR Sciences Exactes et Naturelles, Moulin de la Housse, B.P. 1039, 51687 Reims Cedex 2, France</footnote>

<footnote n="2">²INRA, UMR 1290 BIOGER CPP, Route de St Cyr, 78000 Versailles, France</footnote>

<footnote n="3">³Comité Interprofessionnel du Vin de Champagne, 5 rue Henri Martin, BP 135, 51204 Epernay Cedex, France</footnote>

<footnote>Corresponding author: F. Fontaine
Fax: +33 (0)3 2691 3427
E-mail: florence fontaine@univ-reims.fr</footnote>
trol of *B. cinerea* since it inhibits spore germination, germ-tube elongation and mycelium growth (Hänßler and Pontzen, 1999). Investigations on the mode of action of this chemical have suggested that it increases the glycerol content in the fungus, leading to perturbation of the osmoregulation potential (Pillonel and Meyer, 1997; Liu *et al.*, 2008).

The chemical control of Botrytis bunch rot is impeded in vineyards by the development of resistant strains of *B. cinerea* to several classes of fungicides. Shortly after the introduction of benzimidazoles and dicarboximides, several cases of resistance were recorded in European vineyards (Leroux *et al.*, 1999, 2002). Natural resistance to fenhexamid (phenotype Hyd R1 and Hyd R2) was detected in populations of *B. cinerea* in many vineyards, even before this fungicide was introduced for crop protection (Suty *et al.*, 1999). No resistance was detected to fludioxonil in French vineyards, whereas strains of other fungi (e.g. *Stemphylium vesicarium* [Alberoni *et al.*, 2010] and *Alternaria brassicicola* [Avenot *et al.*, 2005]) exhibiting resistance to this fungicide were detected. In some *B. cinerea* laboratory mutants, cross-resistance was observed between phenylpyrroles (e.g. fludioxonil) and dicarboximides, but this was never detected in field isolates (Leroux, 2004; Liu *et al.*, 2008). In addition to the specific resistance, multi-resistant strains of *B. cinerea* were detected in French vineyards, and especially in the Champagne vineyards, in the late 1990s. These strains differ from multiple resistant ones which accumulate various target alterations. On the contrary, the resistance mechanism is monogenic and was related to multidrug resistance (MDR), as had been observed for human pathogens. MDR strains are cross-resistant to a variety of fungicides belonging to different chemical families. This kind of resistance is not determined by target gene alterations but rather by over-expression of drug transporters located in fungal cell membranes (Kretschmer *et al.*, 2009). The development of these resistant strains may influence fungicide effectiveness if they propagate in field populations at significant frequencies.

In addition to their toxicity against pathogens, some fungicides can indirectly act by changing plant physiology (Prudet, 1994) or stimulating plant defence responses (Garcia *et al.*, 2003). In grapevine, inducible defence mechanisms were

characterized in berries following *B. cinerea* infection. They consist in (i) induction of gene expression encoding pathogenesis-related (PR) proteins (Bézier *et al.*, 2002), (ii) enhancement of chitinase and glucanase activity (Derckel *et al.*, 1998), and (iii) increase of stilbene phytoalexins formed via the phenylpropanoid/polymalonate pathway (Jedandet *et al.*, 1995). If fludioxonil treatment may be involved in the activation of these defence mechanisms to resist *B. cinerea* infection, the fungicide may increase its effectiveness against *B. cinerea* development. Although defence responses following fludioxonil application have been studied in grapevine leaves (Petit *et al.*, 2009a), there is no information available on effects in flowers or berries, although fungicide spraying against *B. cinerea* is essentially directed to the grapevine reproductive organs.

Fludioxonil was applied at different grapevine growth stages in the Champagne region of France during 4 years. Our objective was to examine fluidioxonil effectiveness in relation to spray timing, fungicide resistance and defence responses. Therefore, we evaluated consequences of fludioxonil treatment on the frequency of *B. cinerea* strains which were resistant to fungicides. Defence was also quantified in grapevine reproductive organs following fludioxonil application, focusing on (i) expression of various genes coding for PR proteins and phenylalanine ammonia-lyase (PAL), the first enzyme of the phenylpropanoid/polymalonate pathway; and (ii) chitinase activity.

## Materials and methods

### Field trials and experimental design

Experiments were conducted in an experimental vineyard located in Loisy-en-Brie, in the Champagne region (France). This vineyard has a history of severe bunch rot every year. Grapevines (*Vitis vinifera* L. cv. Pinot Meunier), grafted on 41B rootstock and trained according to the Chablis method, were planted in 1986.

All field experiments were conducted using formulated products. Fludioxonil was formulated as the commercial fungicide Geoxe (50% a.i.; Bayer) and was applied at 1 kg ha⁻¹, at the BBCH stages 68, 77 or 81. Each treatment applied individually was compared to the standard program, which consisted of three applications of fungicide:
fenhexamid (50% a.i. Teldor-Syngenta; at BBCH 68), fludioxonil (at BBCH 77) and pyrimethanil (at BBCH 81). Fludioxonil was used at a rate of 1 kg ha⁻¹ while pyrimethanil (Scala-BASF, 400 g l⁻¹ a.i.) was used at a rate of 2.5 L ha⁻¹. Fenhexamid was applied at 1.5 kg ha⁻¹. Fungicides were sprayed on both sides of vines with a hand-operated backpack sprayer (250 L ha⁻¹). Non-sprayed grapevines were used as controls. Individual treatment plots (control, standard and three fludioxonil treatments) were arranged in a randomized complete block design with four replications. Each replication consisted of at least twelve grapevines. Each treated row was bordered by two unsprayed buffer rows to minimize drift of fungicide from outside the trial.

## Disease assessment

During each harvest from 2002 to 2007, bunch rot infection was evaluated on two clusters per grapevine *i.e.* about 25 clusters per replication and a total of 100 clusters. Incidence of bunch rot was calculated as the percentage of infected clusters (showing at least one rotten berry with typical symptoms). In addition, disease severity was assessed as the percentage of symptomatic berries (area rotten and/or sporulating with *Botrytis*) in each cluster.

## Characterization of *B. cinerea* populations

Field populations of *B. cinerea* were isolated from diseased berries at harvest. A minimum of 20 infected berries with sporulating *B. cinerea* per treatment plot were randomly collected. Berries were suspended in 15 ml of sterile water, without surfactant, and vigorously shaken. The phenotypes were characterized according to Leroux *et al.* (1999): the bulk conidium suspension was adjusted to 300,000 conidia mL⁻¹ with the aid of a haemocytometer and then 300 µL were used directly to inoculate 55 mm diameter Petri dishes containing agar medium amended with doses of various fungicides previously shown to discriminate the various phenotypes (Leroux *et al.*, 1999). Microscopic observations at ×100 magnification of a minimum of 100 conidia per treatment were carried out after 24 h and 48 h to determine the proportion of germinated conidia with long germ tubes (representing at least 50% of the length of conidia in experimental control, i.e. on-amended medium. This was to evaluate the frequency of resistance to anilinopyrimidines (Ani R1), benzimidazoles and phenylcarbamates (Ben R1 and Ben R2), dicarboximides (Imi R1), hydroxyanilides (Hyd R1 and Hyd R3) as well as multidrug resistant (MDR) strains (phenotypes MDR 1 and MDR 2, distinguished respectively

<table><caption>Table 1. Phenotypes of sensitivity towards several fungicides described in field populations of *B. cinerea*; analysis according to germ tube elongation. Phenotypes were classified according to the resistance levels (RL) calculated for the various fungicides (RL=LC₅₀ resistant strain/LC₅₀ sensitive strain); HS, hypersensitive (RL<0.5); S, sensitive (0.5<RL<2); LR, low resistance (2<RL<10); MR, moderate resistance (10<RL<25); HR, high resistance (RL>25).</caption><thead><tr><th rowspan="2">Fungicide families</th><th colspan="8">Phenotypes of sensitivity</th></tr><tr><th>Ben R1</th><th>Ben R2</th><th>Ani R1</th><th>Imi R1</th><th>Hyd R1</th><th>Hyd R3</th><th>MDR 1</th><th>MDR 2</th></tr></thead><tbody><tr><td>Benzimidazoles</td><td>HR</td><td>HR</td><td>/</td><td>/</td><td>/</td><td>/</td><td>LR</td><td>LR</td></tr><tr><td>Phenylcarbamates</td><td>HS</td><td>HR</td><td>/</td><td>/</td><td>/</td><td>/</td><td>LR</td><td>LR</td></tr><tr><td>Dicarboximides</td><td>/</td><td>/</td><td>/</td><td>MR</td><td>/</td><td>/</td><td>LR</td><td>LR</td></tr><tr><td>Phenylpyrroles</td><td>/</td><td>/</td><td>/</td><td>S</td><td>/</td><td>/</td><td>MR</td><td>S/LR</td></tr><tr><td>Anilinopyrimidines</td><td>/</td><td>/</td><td>MR/HR</td><td>/</td><td>/</td><td>/</td><td>LR/MR</td><td>LR/MR</td></tr><tr><td>Hydroxyanilides</td><td>/</td><td>/</td><td>/</td><td>/</td><td>LR</td><td>MR/HR</td><td>S/LR</td><td>LR/MR</td></tr></tbody></table>
by their higher resistance to fludioxonil and fenhexamid (Leroux *et al.*, 1999) (Table 1).

## Grapevine defense responses

### RNA extraction and Real-time PCR analysis

In 2006 and 2007, one apparently non-infected inflorescence/cluster per plant from eight plants treated with fludioxonil or untreated was collected 24 h after fungicide spraying at stages BBCH 68, 77 or 81. They were immediately frozen in liquid nitrogen then stored at -80°C. Flowers/berries were separated from each bunch stem (Jackson and Coombe, 1995) and were then ground in liquid nitrogen to a fine powder. For flowers, a 100 mg aliquot of powder was used for total RNA extraction and homogenized in extraction buffer (Plant Purification RNA Reagent, Invitrogen), according to the manufacturer's instructions. For berries, total RNA was extracted according to the method of Davies and Robinson (1996). Each RNA pellet was resuspended in 20 µL of RNase-free water and quantified by absorbance at 260 nm. RNA was stored at -80°C until use for RT-PCR.

A 150 ng aliquot of total RNA was reverse-transcribed using M-MLV reverse-transcriptase (Invitrogen) according to the manufacturer's pro-

tocol. PCR conditions were as described in Bézi-er *et al.* (2002). The reaction was carried out in duplicate in a GeneAmp 5700 sequence detection system (Applied Biosystems) using the following thermal profile for 40 cycles: 15 s at 95°C (denaturation) and 1 min at 60°C (annealing/extension). The copy number for each sample was calculated according to Petit *et al.* (2009a). The induction factor following fludioxonil treatment was calculated: the results were normalized using the gene EF1α as an endogenous control and data were expressed as -fold change relative to the control samples (untreated flowers/berries). Expression of four defence-related genes encoding class IV chitinase (*Chi4C*), β-1,3-glucanase (*GLUC*), class 6 pathogenesis-related protein (*PR6*) and phenylalanine ammonia-lyase (*PAL*) were tracked (Table 2).

### Chitinase extraction and activity

As described above for RNA extraction, in 2006 and 2007, one inflorescence/cluster per plant from eight plants treated with fludioxonil or untreated was collected 24 h after fungicide spraying at stages BBCH 68, 77 or 81. Flowers/berries were separated from each bunch stem. Protein extraction was performed according to Petit *et al.* (2009a)

<table><caption>Table 2. Defence-related genes analyzed by real-time RT-PCR. The mRNA copy number of each sample was calculated from the standard curve using its Ct value and corrected by normalization against EF1α mRNA (Terrier *et al.*, 2005).</caption><thead><tr><th>Gene</th><th>Encoding</th><th>Primer sequence</th><th>Accession No.</th></tr></thead><tbody><tr><td>EF1-α</td><td>Elongation factor 1-alpha</td><td>Sense 5' GAA CTG GGT GCT TGA TAG GC 3'<br>Antisense 5' AAC CAA AAT ATC CGG AGT AAA AGA 3'</td><td>BQ799343</td></tr><tr><td>Chi4C</td><td>Class IV chitinase</td><td>Sense 5' TCG AAT GCG ATG GTG GAA A 3'<br>Antisense 5' TCC CCT GTC GAA ACA CCA AG 3'</td><td>AY137377</td></tr><tr><td>Gluc</td><td>β-1,3-glucanase</td><td>Sense 5' TCA ATG GCT GCA ATG GTG C 3'<br>Antisense 5' CGG TCG ATG TTG CGA GAT TTA 3'</td><td>AF239617</td></tr><tr><td>PR6</td><td>Class 6 pathogenesis-related protein</td><td>Sense 5' AGT TCA GGG AGA GGT TGC TG 3'<br>Antisense 5' GCA CTA GGG TCC GTG TTT GGG TCG ACG 3'</td><td>AY156047</td></tr><tr><td>PAL</td><td>Phenylalanine ammonia-lyase</td><td>Sense 5' TCC TCC CGG AAA ACA GCT G 3'<br>Antisense 5' TCC TCC AAA TGC CTC AAA TCA 3'</td><td>X75967</td></tr></tbody></table>
and then chitinase activity was assayed using a commercial blue enzyme substrate, CM-chitin-RBV solution (Loewe Biochemica) according to Magnin-Robert *et al.* (2007). Measurements were conducted in triplicate. Results were expressed in mg min⁻¹ g⁻¹ fresh weight (FW).

## Statistical analysis

Values of disease incidence and severity represent means of data from 2002 to 2007. Results of gene expression and chitinase activity represent means of replicates performed over 2 years. To determine whether values of control plants and plants of treatment plots were significantly different, analysis of variance (ANOVA) followed by a Student's *t* test were used. Differences at *P*<0.05 were considered as statistically significant.

## Results

### Effectiveness of fungicide treatments

In control plants, mean disease incidence was

close to 75% and decreased to 35% when plants were treated with the standard reference program (Figure 1). Incidence decrease was not significant when fludioxonil was applied at stages BBCH 68 and 81 but declined to about 25% for fungicide applied at stage 77. Mean bunch rot severity was close to 27% in control plants and significantly reduced by 4-fold after treatment with the standard reference program (Figure 1). After fludioxonil application, severity was reduced similarly whatever the stage of application and was close to 25%.

### Sensitivity to fungicides

Similar frequencies of benzimidazole (Ben R1 and Ben R2), anilinopyrimidine (Ani R1), dicarboximides (Imi R1) and hydroxyanilides-resistant strains (Hyd R1 and Hyd R3) were found from control and treated plants, whatever the stage of treatment (Table 3). A high frequency of Ben R1 strains was observed varying between 42.5 and 53.3%. Proportions of Ben R2, Ani R1, Imi R1, Hyd R1 and Hyd R3 strains were lower, varying between 0 and 20.0%.

<figure><img src="image_3.png" /><figcaption>Figure 1. Mean severity (a) and incidence (b) of grey mould on grape berries at harvest, following different fungicide treatments. The effectiveness of fludioxonil is compared according to stage of application: end of flowering (BBCH stage 68), bunch closure (BBCH stage 77), or veraison (BBCH stage 81). Means with the same letter were not significantly different (*P*<0.05) as determined by the Student's *t* test.</figcaption></figure>
<table><caption>Table 3. Percentage of resistance towards anilinopyrimidines (Ani R1), benzimidazoles and phenylcarbamates (Ben R1 and Ben R2), dicarboximides (Imi R1) or hydroxyanilides (Hyd R1 and Hyd R3) in *B. cinerea* strains, in control plants, in treated plants with fludioxonil at stages 68, 77 or 81, or with reference program. Means represent data from 2002 to 2007. No significant differences were found (*P*<0.05).</caption><thead><tr><th rowspan="2">Phenotypes</th><th rowspan="2">Control</th><th colspan="3">Fludioxonil-treated at stage</th><th rowspan="2">Standard program</th></tr><tr><th>BBCH 68</th><th>BBCH 77</th><th>BBCH 81</th></tr></thead><tbody><tr><td>Ani R1</td><td>0.0a<sup>a</sup></td><td>10.0a</td><td>0.0a</td><td>2.5a</td><td>0.0a</td></tr><tr><td>Ben R1</td><td>43.3a</td><td>53.3a</td><td>51.7a</td><td>42.5a</td><td>50.0a</td></tr><tr><td>Ben R2</td><td>2.5a</td><td>7.5a</td><td>7.5a</td><td>3.3a</td><td>2.0a</td></tr><tr><td>Imi R1</td><td>19.2a</td><td>5.0a</td><td>20.0a</td><td>6.7a</td><td>10.0a</td></tr><tr><td>Hyd R1</td><td>1.7a</td><td>1.7a</td><td>5.0a</td><td>1.0a</td><td>2.7a</td></tr><tr><td>Hyd R3</td><td>0.0a</td><td>0.0a</td><td>10.0a</td><td>0.0a</td><td>0.0a</td></tr><tr><td>MDR1</td><td>4.0a</td><td>16.0a</td><td>13.3a</td><td>20.8a</td><td>12.0a</td></tr><tr><td>MDR2</td><td>15.0a</td><td>23.3a</td><td>15.0a</td><td>14.2a</td><td>19.2a</td></tr></tbody></table>

The trend observed was for a higher frequency of MDR after fludioxonil treatment compared to control plants. Similarly, an increase was noticed after the standard reference program application. MDR1 increase was at maximum with a factor 5.2 in fludioxonil-treated plants compared to control plants whereas the increase was only by a factor 1.5 for MDR2 strains.

## Grapevine defence responses

No significant modification in expression of *PAL*, *LOX*, and genes encoding PR proteins (*Chi4C*, *GLUC* and *PR6*) was observed in flowers (stage BBCH 68) or berries (stages BBCH 77 and 81) following fludioxonil treatments (data not shown). Basal level of chitinase activity was 1.1 and 1.4 mg min⁻¹ g⁻¹ FW in control plants at stages BBCH 68 and 77, respectively (Figure 2), and was weakly higher in berries at stage BBCH 81 (1.8 mg min⁻¹ g⁻¹ FW). Following fludioxonil treatments, a significant 60% increase in chitinase activity was occurred only after treatment at stage BBCH 81.

## Discussion

Our results showed that the standard program of three fungicide applications provided the best control of *B. cinerea* in the Champagne region in

comparison with a single treatment of fludioxonil at any of the crop stages tested and in each of the years studied. Single applications of fludioxonil therefore are specifically adapted at a given vine growth stage, while a significant reduction of both disease severity and incidence was demonstrated when fenhexamid was applied at stage BBCH 68 (Petit *et al.*, 2010). This indicates that application at BBCH 68 is decisive for the most effective control of grey mould disease (Nair *et al.*, 1995; Jermi ni *et al.*, 1986; Pezet and Pont, 1986). In addition, fludioxonil seems to have a greater effect on disease severity than on disease incidence, indicating that this fungicide may act by diminishing the size of fungal infection foci rather than in reducing the number of foci. Selection pressure exerted by fungicides on *B. cinerea* strains and defence responses of grapevine to fungicides were then tested to evaluate potential interactions between these factors and effectiveness of fungicide treatments.

High frequencies of Ben R1 strains were recorded in each year of this study despite the absence of selection pressure. Benzimidazoles were developed at the end of 1960's and the use of these compounds rapidly induced development of highly resistant strains, particularly in locations of intensive use such as in the Champagne region (Leroux and Clerjeau, 1985). These strains generally exhibited the mutation E198A in the gene en-

<footnote><sup>a</sup> Values followed by the same letter are not statistically different at the 5% level by ANOVA followed by a Student's t test.</footnote>
coding β-tubulin (Leroux *et al.*, 2002). These high proportions of Ben R1 strains, despite the absence of contemporary selection pressure, may indicate that the biological cost of this previous mutation is reduced (Johnson *et al.*, 1994).

Conversely, the frequency of Ben R2, AniR1, Imi R1, Hyd R1 and Hyd R3 phenotypes was low or zero in most cases, including the standard reference fungicide program. The phenotype Ben R2, which is simultaneously resistant to benzimidazoles and phenylcarbamates, is generally determined by the mutation F200Y in the β-tubulin gene (Leroux *et al.*, 2006). Low frequencies of this phenotype in our trials, as well as in the Champagne region, may indicate that the mutation F200Y induces a high fitness penalty. The low frequencies ImiR1 and AniR1 phenotypes confirm that their resistance was not significantly selected by the dicarboximide application and the pyrimethanil treatment applied at stage BBCH 81 respectively. Imi R1 resistance is conferred by alterations within the *Bos1* gene and the most frequent mutation is I365R/S/G (Leroux *et al.*, 2002; Cui *et al.*, 2004). AniR1 resistance, the mechanism of which is still unclear (Fritz *et al.*, 2003), was often described as unstable, probably because of a fitness penalty (Leroux *et al.*, 2004). Resistance to hydroxyanilides, low frequency of resistant strain (Hyd R1) and low specific resistance (Hyd R3) confirm that no significant efficacy loss has been recorded with fenhexamid (Leroux *et al.*, 2002).

For MDR strains, MDR1 increase was greater

than MDR2 in fludioxonil-treated plants. Indeed, MDR1 stains exhibited higher levels of resistance to fludioxonil (Table 1) and were probably better selected by fludioxonil, as already reported by Walker *et al.* (2006). Nevertheless, even if the highest frequencies of MDR phenotypes were recorded after fludioxonil application at stage BBCH 68, efficacy of the fungicide was below that achieved with the standard reference program, but was still better than the control and acceptable because frequency of MDR1 and associated resistant levels do not lead to great efficacy loss. In the context of this study, this could indicate that limiting the use of fungicides in French vineyards (to a maximum of one treatment by class of mode of action) is sufficient to reduce bunch rot significantly when combined with cultural control measures, and when specific resistance leading to high resistant levels is not likely to get selected.

Defence responses of grapevine reproductive organs were evaluated following fludioxonil application at the three tested vine growth stages. Several fungicides used to control bunch rot, such as benzimidazoles, strobilurins or triazoles, stimulate the increase of PR gene expression and the accumulation of PR proteins, the increase in PAL activity or the accumulation of phenolics in various crops (Siefert *et al.*, 1996; Garcia *et al.*, 2003; Pasquer *et al.*, 2005). In grapevine, it was observed that application of dicarboximide fungicides in vineyards indirectly acted against *B. cinerea* by changing plant physiology. These fun-

<figure><img src="image_4.png" /></figure>

Figure 2. Mean chitinase activity in control and fludioxonil-treated flowers (BBCH stage 68) or berries (BBCH stages 77 and 81) of grapevine, 4 days after treatment. Data are means ± standard errors (n = 16). Asterisks indicate significant differences (*P*<0.05) between control and treated plants, as determined by the Student's *t* test.
gicides acted positively by conservating berry in-
hibition against *B. cinerea* and delayed skin de-
structuring. These changes led to better protection
of grapevine against *B. cinerea*. Nevertheless, our
results showed that no increase of the defence
processes tested (changes in the level of gene ex-
pression and chitinase activity) was observed in
flowers at stage BBCH 68 and in berries at stage
77 following fludioxonil application as well as fol-
lowing fenhexamid application (Petit *et al.*, 2010).
Only an increase in chitinase activity was noticed
in berries at stage 81. Reduction of bunch rot was
not significant after fludioxonil treatment at stage
81, suggesting that fungicide effectiveness was
not related to an activation of defence responses in
grapevine. The lack of response of chitinase activ-
ity in reproductive organs at flowering or bunch
closure following fungicide treatment might be ex-
plained by a poor capacity to induce their defence
mechanisms contrary to berries at latest stages.
Indeed, although multiple defence responses were
induced in berries at later stages to UV-C irradia-
tion, no significant induction of defence responses
was observed in flowers (Adrian *et al.*, 2000; Bais
*et al.*, 2000; Petit *et al.*, 2009b).

**Literature cited**

Adrian M., P. Jeandet, A.C. Douillet-Breuil, L. Tesson and R. Bessis, 2000. Stilbene content of mature *Vitis vinifera* berries in response to UV-C elicitation. *Journal of Agriculture and Food Chemistry* 48, 6103–6105.

Alberoni G., M. Collina, Lanen C, Leorux P. and A. Brunel-
li, 2010. Field strains of *Stemphylium vesicarium* with
a resistance to dicarboximide fungicides correlated with
changes in a two-component histidine kinase. *European
Journal of Plant Pathology* 128, 171–184.

Avenot H., P. Simoneau, B. Iacomi-Vasilescu and N. Bataillé-Simoneau, 2005. Characterization of mutations in the two-component histidine kinase gene AbNIK1 from *Alternaria brassicicola* that confer high dicarboximide and phenylpyrrole resistance. *Current Genetics* 47, 234–43.

Bais A.J., P.J. Murphy and I.B. Dry, 2000. The molecular regulation of stilbene phytoalexin biosynthesis in *Vitis vinifera* during grape berry development. *Australian Journal of Plant Physiology* 27, 425–433.

Bézier A., B. Lambert and F. Baillieul, 2002. Study of defense-related gene expression in grapevine leaves and berries infected with *Botrytis cinerea*. *European Journal of Plant Pathology* 108, 111–120.

Bocquet F., D. Moncomble and M. Valade, 1995. Etat sanitaire de la vendange et qualité des vins. *Le Vigneron Champenois* 7/8, 15–23.

Bulit J. and B. Dubos, 1988. Botrytis bunch rot and blight. In: *Compendium of Grape Diseases*, (Pearson R.C., Goheen A.C., ed.), APS Press, St Paul, Minnesota, USA, 13–14.

Cilindre C., A.J. Castro, C. Clément, P. Jeandet and R. Marchal, 2007. Influence of *Botrytis cinerea* infection on Champagne wine proteins (characterized by two-dimensional electrophoresis/immunodetection) and wine foaming properties. *Food Chemistry* 103, 139–149.

Cilindre C., S. Jégou, A. Hovasse, C. Schaeffer, A.J. Castro, C. Clément, A. Van Dorsselaer, P. Jeandet and R. Marchal, 2008. Proteomic approach to identify champagne wine proteins as modified by *Botrytis cinerea* infection. *Journal of Proteome Research* 7, 1199–1208.

Cui W., R.E. Beever, S.L. Parkes and M.D. Templeton, 2004. Evolution of an osmosensing histidine kinase in field strains of *Botryotinia fuckeliana* (*Botrytis cinerea*) in response to dicarboximides fungicide usage. *Phytopathology* 94, 1129–1135.

Davies C. and S.P. Robinson, 1996. Sugar accumulation in grape berries. Cloning of two putative vacuolar invertase cDNAs and their expression in grapevine tissues. *Plant Physiology* 111, 275-283.

Derckel J.P., J.C. Audran, B. Haye, B. Lambert and L. Legendre, 1998. Characterization, induction by wounding and salicylic acid, and activity against *Botrytis cinerea* of chitinases and β-1,3-glucanases of ripening grape berries. *Physiologia Plantatum* 104, 56–64.

Fritz R., C. Lanen, F. Chapeland-Leclerc and P. Leroux, 2003. Effect of the anilinopyrimidine fungicide pyrimethanil on the cystathionine β-lyase of *Botrytis cinerea*. *Pesticide Biochemistry and Physiology* 77, 54–65.

García P.C., R.M. Rivero, J.M. Ruiz and L. Romero, 2003. The role of fungicides in the physiology of higher plants: implications for defense responses. *The Botanical Review* 69, 162–172.

Hänßler G. and R. Pontzen, 1999. Effect of fenhexamid on the development of *Botrytis cinerea*. *Pflanzenschutz-Nachrichten Bayer* 52, 158–176.

Jackson D.I. and B.G. Coombe, 1995. Early bunchstem necrosis—a matter of nomenclature. *American Journal of Enology and Viticulture* 46, 579–580.

Jeandet P., R. Bessis, M. Sbaghi and P. Meunier, 1995. Production of the phytoalexin resveratrol by grapes as a response to Botrytis attacks in the vineyard. *Journal of Phytopathology* 143, 135–139.

Jermini M., G. Jelmini and C. Gessler, 1986. La lutte contre le *Botrytis cinerea* du Merlot au Tessin: le rôle des infections latentes. *Revue Suisse de Viticulture, d'Arboriculture et d'Horticulture* 18, 161–166.

Johnson K.B., T.L. Sawyer and M.L. Powelson, 1994. Frequency of benzimidazole-resistant and dicarboximide-resistant strains of *Botrytis cinerea* in western Oregon small fruit and snap bean plantings. *Plant Disease* 78, 572–577.

Kretschmer M., M. Leroch, A. Mosbach, A.S. Walker, S. Fillinger, D. Mernke, H. Schoonbeek, J.M. Pradier, P. Leroux, M. De Waard and M. Hahn, 2009. Fungicide-driven evolution and molecular basis of multidrug re-
sistance in field population of grey mould fungus *Botrytis cinerea*. *PLoS Pathogens* 5(12), e1000696.

Leroux P. and M. Clerjeau, 1985. Resistance of *Botrytis cinerea* and *Plasmopara viticola* to fungicides in French vineyards. *Crop Protection* 4, 137–160.

Leroux P., F. Chapeland, D. Desbrosses and M. Gredt, 1999. Patterns of cross-resistance to fungicides in *Botryotinia fuckeliana* (*Botrytis cinerea*) isolates from French vineyards. *Crop Protection* 18, 687–697.

Leroux P., R. Fritz, D. Debieu, C. Albertini, C. Lanen, J. Bach, M. Gredt and F. Chapeland, 2002. Mechanisms of resistance to fungicides in field strains of *Botrytis cinerea*. *Pest Management Science* 58, 876–888.

Leroux P., 2004. Chemical control of *Botrytis* and its resistance to chemical fungicides. In: *Botrytis: Biology, Pathology and Control*. (Elad Y., Williamson B., Tudzynski P., Delen N., ed.), Kluwer Academic Publishers, Dordrecht, The Netherlands, 195–222.

Leroux P., M. Gredt, A.S. Walker and M.L. Panon, 2006. Botrytis de la vigne et neuf sortes de fongicides, distribution des souches résistances en Champagne. *Phytoma, La Défense des Végétaux* 599, 31–35.

Liu W., P. Leroux and S. Fillinger, 2008. The HOG1-like MAP kinase Sak1 of *Botrytis cinerea* is negatively regulated by the upstream histidine kinase Bos1 and is not involved in dicarboximide- and phenylpyrrole-resistance. *Fungal Genetics and Biology* 45, 1062–1074.

Magnin-Robert M., P. Trotel-Aziz, D. Quantinet, S. Bia gianti and A. Aziz, 2007. Biological control of *Botrytis cinerea* by selected grapevine-associated bacteria and stimulation of chitinase and β-1,3 glucanase activities under field conditions. *European Journal of Plant Pathology* 118, 43–57.

Marchal R., I. Tabary, M. Valade, D. Moncomble, L. Viaux, B. Robillard and P. Jeandet, 2001. Effects of *Botrytis cinerea* infection on Champagne wine foaming properties. *Journal of the Science of Food and Agriculture* 81, 1371–1378.

Meier U., 2001. Grapevine. In: *Growth Stages of Mono- and Dicotyledonous Plants*. (Meier U., ed.) *BBCH Monograph: Federal Biological Research Centre for Agriculture and Forestry*. Berlin, Germany, Blackwell Wissenschafts-verlag, 93–95.

Nair N.G., S. Guilbaud-Oulton, I. Barchia and R. Emmet, 1995. Significance of carry over inoculum, flower infection and latency on the incidence of *Botrytis cinerea* in berries of grapevines at harvest in New South Wales. *Australian Journal of Experimental Agriculture* 35, 1177–1180.

Panon M.L., L. Panigai, A.S. Walker and P. Leroux, 2006. Les nouveautés concernant la pourriture grise en quelques points. *Le Vigneron Champenois* 127, 26–36.

Pasquer F., E. Isidore, J. Zarn and B. Keller, 2005. Specific pattern of changes in wheat gene expression after treatment with three antifungal compounds. *Plant Molecular Biology* 57, 693–707.

Petit A.N., G.Wojnarowiez, M.L. Panon, F. Baillieul, C. Clément, F. Fontaine and N. Vaillant-Gaveau, 2009a. Botritycides affect grapevine leaf photosynthesis without inducing defense mechanisms. *Planta* 229, 497–506.

Petit A.N., F. Baillieul, N. Vaillant-Gaveau, L. Jacquens, A. Conreux, P. Jeandet, C. Clément and F. Fontaine, 2009b. Low responsiveness of grapevine flowers and berries at fruit set to UV-C irradiation. *Journal of Experimental Botany* 60, 1155–1162.

Petit A.N., N. Vaillant-Gaveau, A.S. Walker, P. Leroux, F. Baillieul, M.L. Panon, C. Clément and F. Fontaine, 2010. Determinants of fenhexamid effectiveness against grey mould on grapevine: respective role of spray timing, fungicide resistance and plant defences. *Crop Protection* 29(10), 1162-1167.

Pezet R. and V. Pont, 1986. Infection florale et latence de *Botrytis cinerea* dans les grappes de *Vitis vinifera* (var. Gamay). *Revue Suisse de Viticulture, d'Arboriculture et d'Horticulture* 18, 317-322.

Pillonel C. and T. Meyer, 1997. Effect of phenylpyrroles on glycerol accumulation and protein kinase activity of *Neurospora crassa*. *Pesticide Science* 49, 229-236.

Prudet S., 1994. *Contribution à l'Etude du Rôle du Complexe Pelliculaire dans la Résistance de la Baie de Raisin à Botrytis cinerea Pers., Agent de la Pourriture Grise de la Vigne*. Thèse de doctorat de l'Université de Bordeaux II, Bordeaux, France.

Siefert F., M. Thalmair, C. Langebartels, H. Jr Sandermann and K. Grossman, 1996. Epoxiconazole-induced stimulation of the antifungal hydrolases chitinase and β-1,3-glucanase in wheat. *Plant Growth Regulation* 20, 279-286.

Suty A., R. Pontzen and K. Stenzel, 1999. Fenhexamid - Sensitivity of *Botrytis cinerea*: Determination of baseline sensitivity and assessment of the risk of resistance. *Pflanzenschutz-Nachrichten Bayer* 52, 149-161.

Terrier N., D. Glissant, J. Grimplet, F. Barrieu, P. Abbal, C. Couture, A. Ageorges, R. Atanassova, C. Léon, J.P. Renaudin, F. Dédaldéchamp, C. Romieu, S. Delrot and S. Hamdi, 2005. Isogene specific oligo arrays reveal multifaceted changes in gene expression during grape berry (*Vitis vinifera* L.) development. *Planta* 222, 832–847.

Walker A.S., I. Ilisescu, L. Bill, E. Fournier, M.L. Panon and P. Leroux, 2006. Dynamique et évolution de la résistance aux fongicides au sein des populations de pourriture grise (*Botrytis cinerea*) en Champagne. *AFPP - 8eme Conférence Internationale sur les Maladies des Plantes*, 5-6 Décembre 2006, Tours, France, 593-606.

Accepted for publication: February 11, 2011