Fenhexamid Resistance in the Botrytis Species Complex , Responsible for Grey Mould Disease

The three major fungal pests of grapevine, powdery and downy mildew and grey mould are mostly controlled through the application of fungicides. Some of those are particularly active against the grey mould agent Botrytis cinerea. The panel of fungicides authorized in France comprise since many years anilinopyrimidines, benzimidazoles, dithiocarbamates, dicarboximides, phenylpyrroles and pyridinamines. Lately, the panel has been completed by fenhexamid, a sterol biosynthesis inhibitor (SBI) and the pyridine boscalid, a succinate dehydrogenase inhibitor (SDHI). In addition, a biofungicide based on the Bacillus subtilis strain QST 713 (Serenade) has been authorized in 2010 against grey mould in vineyards. The compounds authorized against grey mould in French vineyards are listed in Table 1.


Introduction 1.1 Chemical control of grey mould in French vineyards
The three major fungal pests of grapevine, powdery and downy mildew and grey mould are mostly controlled through the application of fungicides.Some of those are particularly active against the grey mould agent Botrytis cinerea.The panel of fungicides authorized in France comprise since many years anilinopyrimidines, benzimidazoles, dithiocarbamates, dicarboximides, phenylpyrroles and pyridinamines.Lately, the panel has been completed by fenhexamid, a sterol biosynthesis inhibitor (SBI) and the pyridine boscalid, a succinate dehydrogenase inhibitor (SDHI).In addition, a biofungicide based on the Bacillus subtilis strain QST 713 (Serenade) has been authorized in 2010 against grey mould in vineyards.The compounds authorized against grey mould in French vineyards are listed in Table 1.
Since grey mould may infect grapevine from flowering until harvest, optimal protection needs to be obtained during this period.Nowadays up to three treatments per season are recommended in vineyards, corresponding to the stages A-C (A: flower cap falling -B: bunch closure -C: veraison).To reduce pesticide applications along with the general trend of reduction of chemical inputs in agriculture chemical treatments against grey mould are positioned according to epidemiological and meteorological parameters.Grey mould not only affects quantity of harvest but also the wine quality.Therefore, treatments also depend on the economic value of the wine.The number of treatments is variable between regions and years according to the factors cited above.
In those regions with regular applications of anti-Botrytis fungicides, especially in the Northern regions, resistant strains have been selected which can ultimately lead to treatment failure.In order to reduce the risk of specific resistance development each anti-Botrytis mode-of-action is limited to one application/season in France since the 90's, involving alternations of different chemical families to combat grey mould in the vineyards.For further explanations, see text.

Fenhexamid: A single mode of action
Fenhexamid is a narrow spectrum fungicide (Rossenbloich and Stuebler, 2000) belonging to the chemical family of hydroxyanilides inhibiting sterol biosynthesis (SBI) (Debieu et al., 2001).Bayer CropScience has introduced it to the French fungicide market more than 10 years ago as a specific anti-Botrytis.It is principally used in vineyards, but also on tomato and strawberry cultures up to two treatments per season (Couteux & Lejeune, 2003).Fenhexamid differs from other known SBIs (allylamines, azoles, imidazoles and morpholines/amines) by the enzymatic step that it inhibits.Sterols are essential lipid compounds found in all eukaryotes.They are principally localized to cytoplasmic or endomembranes, although the latter are composed predominantly of phospholipids.Sterols participate in membrane permeability and rigidity, in the formation of lipid rafts and, eventually, in post-translational control of membrane proteins (Hac-Wydro et al., 2007;Yeagle, 1990;Epand, 2008;Gilbert, 2010;Paila & Chattopadhyay, 2010).The major fungal sterol is ergosterol and specific to this kingdom.Therefore, many attempts to develop targets for antifungal treatments have been orientated towards the ergosterol biosynthesis pathway.Noticeably, all SBIs used against fungal diseases target enzymatic steps that are conserved among all kingdoms and not fungal specific.
The major class of SBIs used in agriculture represented by azoles, imidazoles and triazolinethions inhibits the 14-demethylase enzyme therefore called DMIs (demethylation inhibitors), amines inhibit the ∆14-reductase and/or the ∆8∆7-isomerase.The youngest SBI used against grey mould, fenhexamid, blocks the demethylation at C4 of sterols.This important process involves three different enzymes: Erg25 (C4-methyle oxidase), Erg26 (C3dehydrogenase, C4-decarboxylase) and Erg27 (3-ketoreductase).The first two enzymes eliminate methyl groups at C4 leading to a keto group at C3.The Erg27 enzyme then reduces this keto group to a hydroxyl (Gachotte et al., 1999) leading to a functional sterol.
This last step is the target of fenhexamid.Its mode-of-action has been uncovered in 2001, by the characterization of sterol composition of fenhexamid treated B. cinerea strains.Debieu and co-workers, observed an accumulation of compounds with a keto group at C3, suggesting the inhibition of the 3-ketoreductase step (Debieu et al., 2001).Since this enzymatic step is conserved in all euascomycetes, one may expect fenhexamid to be a large spectrum fungicide as are the DMIs instead of its narrow spectrum (see paragraph 2.3).

Resistance to fenhexamid in the Botrytis species complex
The activity spectrum of a new fungicide is generally established by biological tests on diverse fungal species allowing distinguishing sensitive and tolerant species.While naturally resistant -or tolerant species -are insensitive to a given fungicide prior to its introduction and defining the fungicide's spectrum of activity, sensitive fungal species can evolve and select less susceptible -or resistant -isolates.Natural polymorphism or randomly occurring mutations may confer a certain level of resistance to some strains.Through the application of the corresponding fungicide, these isolates are preferentially selected and their proportion within the population will increase (selection pressure of the fungicide).The differentiation between natural and acquired resistance can sometimes lead to the identification of new fungal species; e.g.Also in the case of grey mould, it became clear that the species later on named Botrytis pseudocinerea a close relative of B. cinerea, was naturally resistant to fenhexamid at the stage of mycelial growth (Fournier et al., 2005, Walker et al., 2011).Later, resistant isolates were selected from the B. cinerea sensu stricto populations, initially sensitive to fenhexamid.Until now, four different fenhexamid resistance categories could be identified among grey mould populations (Table 2): HydR1, corresponding to the naturally resistant species B. pseudocinerea and the B. cinerea phenotypes MDR, HydR2 and HydR3 (Leroux et al, 2002).
For the phenotypes HydR1 (B.pseudocinerea) and HydR2 (B.cinerea), high to moderate resistance levels respectively, are observed nearly exclusively at the level of mycelial growth, whereas the other phenotypes display similar resistance levels at both developmental stages, germ tube elongation and mycelial growth.In addition, B. pseudocinerea (HydR1) presents increased sensitivities to other fungicides, such as DMIs, fenproprimorph, fenpropidine as well as to some SDHIs (Leroux et al., 2002).Only few HydR2 strains have been isolated and to our knowledge, they have not yet been found in French vineyard populations.On the opposite, HydR3 phenotypes are regularly found since 2003.This category has been subdivided into two sub-classes, HydR3 -or HydR3 + with moderate or high resistance levels respectively.
MDR2 (and MDR3) phenotypes display weak to moderate resistance levels (resistance factor <15) to various unlinked mode-of-actions including fenhexamid, SDHIs, dicroboximides, anilinopyrimidines etc.This multidrug resistance (MDR) is linked to the over expression of a membrane transporter leading to increased drug-efflux outside the fungal cell (Kretschmer et al., 2009).Anti-resistance strategies involving systematic alterations of different mode-ofactions seem to favor the selection of these MDR phenotypes instead of specific resistances, in particular in regions with more than one treatment per season such as the Champagne wine region.

Target alterations cause reduced affinity of fenhexamid and are responsible for resistance
In agriculture, acquired resistance towards agrochemical products is frequently conferred by target site modifications leading to decreased inhibitor affinities.Target overproduction is rarely found.Pesticide detoxification is a mechanism particularly important for insecticide resistance involving diverse enzymes such as hydrolases, cytochrome P450 monooxygenases, glutathione-S-transferases or glycosyl transferases.In rare cases, resistance results from nonactivation of pro-pesticides.Concerning antifungal compounds, the principal resistance mechanisms are target site modifications and increased efflux, in the agronomical field as well as in the medical field (Ma & Michaelides, 2005;Sanglard et al., 2009).
With respect to the B. cinerea HydR phenotypes, target site modifications were found in the HydR3 (+ and -) isolates, but not in HydR2 phenotypes (Albertini & Leroux, 2004;Fillinger et al., 2008).In B. pseudocinerea the fenhexamid target encoding gene, erg27 shows a high degree of polymorphism compared to the sensitive B. cinerea allele.Its implication in fenhexamid resistance is described in paragraph 3.1.
Other erg27 mutations have been found in fenhexamid resistant strains selected after chemical mutagenesis (G23S, C53R, T63I, K73E, H105Y, K159N, L195S, T273A, S310P, I397V, I411V, H423R, A452P, Q495R and C516R) (Saito et al., 2010).However, their involvement in resistance to fenhexamid remains to be shown.We analyzed the involvement of the natural HydR3 mutations in fenhexamid resistance by site directed mutagenesis.The erg27 allele -encoding 3-ketoreductase -was replaced in a sensitive strain by the erg27 allele of a HydR3 strain by homologous recombination.Two mutations identified from HydR3 -strains (L195F et V309M) in addition to the three HydR3 + alleles were studied.The fenhexamid sensitivity profiles of the generated mutants were similar to those from the field strains (Table 3).Indeed the transgenic erg27L195F or erg27V309M mutants display low to moderate resistance levels according to the developmental stage, whereas the transgenic HydR3 + mutants (F412S, F412I, F412V) present high resistance levels at both growth stages, as do the highly resistant HydR3 + field strains.In addition, the inhibition of 3-ketoreductase activity by fenhexamid is reduced in the artificial mutants (Billard et al., unpublished), strongly suggesting a decrease in fenhexamid's affinity to its target 3-ketoreductase in HydR3 mutants.These results prove the direct correlation between the amino acid substitution and the specific resistance to fenhexamid in the B. cinerea HydR3 + and HydR3 -phenotypes.In addition they show target site modification as sole resistance mechanism in these isolates.
In contrast to HydR3 phenotypes, neither target site modification nor over expression seem to be responsible for fenhexamid resistance in HydR2 phenotypes.The only hint towards the potential resistance mechanism is an observed synergy between fenhexamid and DMIs suggesting fenhexamid metabolization involving a cytochrome P450 monooxygenase enzyme.Which mechanism precisely is at work in HydR2 strains needs yet to be identified.
We have modeled the B. cinerea Erg27 protein after alignment with the peptide sequences of various dehydrogenases/reductases whose crystal structures have been established.The homology-based model presented in Fig. 2 monitors the position of the HydR3 mutations relative to the substrate-and cofactor binding sites.None of the mutations are in the vicinity of these docking sites, although it cannot be excluded that the L195F and N369D replacements modify the Erg27 protein structure leading to different substrate-and cofactor affinities.Mutations of F412 however, potentially interfere with the helical structure of the transmembrane helix.

Why fenhexamid has a narrow spectrum?
Fenhexamid is the sole SBI with a narrow antifungal spectrum.Its activity is restricted to close relatives of B. cinerea, e.g.Botrytis and Monilinia spp., Sclerotinia sclerotiorum (Rosslenbroich & Stuebler, 2000).We therefore raised the question if 3-ketoreductase of ascomycetes naturally resistant to fenhexamid is insentitive to inhibition exerted by fenhexamid.The alignment of Erg27 to orthologous peptide sequences show that both 3ketoreductase sequences, those from B. cinerea and S. sclerotiorum, harbor three additional fragments compared to the other fungal proteins, including one extension of 34 amino acids (Albertini & Leroux, 2004; Fillinger, unpublished; Fig. 2).In addition, we have shown by biochemical assays that 3-ketoredcutase activity of insensitive fungal species, is barely inhibited by fenhexamid, suggesting that fenhexamid has a strong affinity towards 3ketoreductase of Botrytis related species only (Debieu et al., submitted).In how far the above mentioned protein extensions and/or specific residues of the B. cinerea Erg27 protein sequence are involved in this affinity remains to be established.

The HydR3 + resistance acquisition entails a cost in controlled conditions
The fitness corresponds to the capacity of one individual (strain) to survive among others under the same conditions.In the absence of selective pressure exerted by the application of fungicides, the relative fitness determines the persistence of a resistant isolate among a natural fungal population.Acquiring a fungicide resistance linked to a high fitness cost is a clear disadvantage for the fungal strain, as it is counter selected in absence of the corresponding fungicide from a mixed population with sensitive and resistant strains.In natural grey mould populations, this seems to be the case for the phenylpyrrol fungicides for which no (or only few) specifically resistant strains were detected up to now (Leroux et al, 2002;Moyano et al., 2004).The efficacy of such fungicides can be maintained over long periods.On the opposite, resistant mutations that are not associated to a fitness cost, may threaten the "life-time" of the corresponding fungicide.Indeed, such kind of resistances without associated fitness costs were obtained after few years only with the strobilurins used against cereal fungal pathogens or -on different crops and different pathogens -with the benzimidazoles.Resistance against either or both fungicides is generalized in many pests.
Concerning fenhexamid, the frequency of the HydR3 + strains of the greatest concern in practice, is in progression since 2003 and has reached now non-negligible levels, e.g.27% in Champagne.This increase remains weak in regions despite annual applications and not comparable to the rapid generalization of strobilurin resistance.This evolution indicates that probably an effective anti-resistance strategy combined to a potential fitness cost linked to high levels of fenhexamid resistance limits the progression of HydR3 + frequencies among grey mould populations.
In order to evaluate the fitness cost linked to the HydR3 + phenotypes, we measured divers physiological parameters under controlled conditions.The general problem for fitness measurements in natural B. cinerea strains is the phenotypic variability between strains.Different growth parameters cannot be easily correlated to resistance due to different genetic backgrounds.We circumvented this problem by the construction of HydR3 + (F412S, F412I and F412V) mutants using site directed mutagenesis.The strains were isogenic except for the erg27 allele providing the ideal material for fitness analyses.Particular attention was paid to temperature and nutritional factors in the measurement of the fitness parameters as well as the quantification of developmental stages important for survival and dispersion (sclerotia and conidia respectively).Statistically significant decreases were observed in the mutants for mycelial growth -especially in extreme conditions (restricted nutrients at low temperatures), for conidia-and sclerotia-production -the last parameter especially at low temperatures, and for mycelial outgrowth from frozen sclerotia.Theses results obtained with isogenic strains, like those reported by Ziogas et al (2003) and Saito et al. (2010) for chemically resistant mutants of B. cinerea, indicate that the acquisition of high-level specific resistance to fenhexamid is associated with a decrease in fitness (Billard et al., submitted).Our data show that in vitro pathogenicity is not affected (measured on detached bean leaves), whereas stages of the fungal life cycle during which there is no selection pressure seem to be the most affected.In laboratory conditions, a fitness cost of the HydR3 + resistance was observed in isogenic mutants at different phases of the asexual cycle (in red) of B. cinerea.However, the pathogenicity of these mutants, measured on beans, remained unchanged from the parental strain sensitive to fenhexamid (green).
These findings for isogenic erg27 F412 mutants suggest that the HydR3 + phenotype does not persist in the absence of the fungicide and that this phenotype has a moderate impact on the efficacy of fenhexamid treatments for controlling grey mould disease in vineyards.However long-term studies in vineyards are required to confirm these laboratory observations.

Fenhexamid monitoring in fields: A molecular approach
Monitoring of this resistance is a crucial area of research where our knowledge of the field distribution, evolution and impact of fungicide resistance depends on.In most cases, the degree of sensitivity of fungal populations to one or more fungicides is assessed by biological methods.Kianianmomeni et al., 2007).However, one limitation of this method concerns the nonspecific amplification of alleles, which may affect precision.This limitation does not generally hinder detection of the polymorphism, but it may affect quantification capacity, particularly for mutated alleles with a low abundance.
Taking these results into account, we investigated the development of a new technique for quantifying, with a high precision, the three different erg27 alleles from the HydR3 + phenotype.The underlying DNA polymorphism is the modification of the TTC codon encoding the F412 residue, which is converted into a TCC (serine), GTC (isoleucine) or ATC (valine) (Fillinger et al., 2008) codon.The best result was obtained with a non multiplexed method combining four allele-specific MGB Taqman ® probes and four mismatched specific primers.This technique was named the Allele Specific Probe and Primer Amplification Assay (ASPPAA PCR), (Fig. 4; Billard et al., submitted).
The sensitivity of ASPPAA PCR is sufficiently good to quantify a SNP at a rate of 1% in a DNA pool.In the future, the multiplexing, in the same run, of the analysis of several polymorphisms at different genomic loci with probes picked up in different fluorophore channels is conceivable and would be expected to decrease the time required for monitoring, and its cost, significantly.
The principle disadvantage of these molecular quantification methods is that they do not allow to detect emerging resistances in contrast to biological methods.One alternative molecular method to be considered is the HRM (high resolution melt curve) analysis (Pasay et al., 2008).HRM offers a fast and convenient method of assessing the presence of mutations without sequencing in a short (< 400 bp) defined genomic region.Using this tool, the identification of new mutations in the erg27 gene is possible.

Natural resistance to fenhexamid in Botrytis pseudocinerea
The genus Botrytis (Ascomycota) contains 22 highly specialised species and one hybrid.A multiple-gene gene genealogy study recently showed that this genus could be subdivided into two categories, one consisting of Botrytis species acting as pest on monocots, and the other containing Botrytis species acting as parasites on eudicots (Staats et al., 2005).Within this second category, Botrytis cinerea has the widest host range, being able to infect more than 220 types of eudicot, including grapevine and many fruit and vegetable crops.However, B. cinerea has recently been shown to be a complex of two sibling species living in sympatry.B. cinerea sensu stricto is the predominant species (Fournier et al., 2005).The other species, called Botrytis pseudocinerea, has been found at low frequency in French populations.This species is morphologically indistinguishable from B. cinerea sensu stricto, but Walker et al (2011) established several molecular markers to distinguish both species.In addition B. pseudocinerea has a different pattern of fungicide susceptibility (phenotype HydR1), displaying natural resistance to fenhexamid and hypersensitivity to fenpropidin and edifenphos (Leroux et al, 2002).
In contrast to the B. cinerea fenhexamid resistant phenotypes (HydR2 and HydR3), B. pseudocinerea was resistant to fenhexamid prior to its introduction, therefore displaying a natural resistance to fenhexamid.

Role of the natural target polymorphism in fenhexamid resistance
The genetic polymorphism of B. pseudocinerea compared to B. cinerea is also visible on the 3ketoredcutase encoding gene erg27.Twelve codon modifications lead to amino acid replacements in the Erg27 protein (N93V, D146L, I211V, I215V, M218T, V234A, I235V, D261G, S264T, P269L, A285T and Q354K) (Albertini & Leroux, 2004).None of theses substitutions corresponds to any of the HydR3 mutations.In order to evaluate the impact of the B; pseudocinerea erg27 allele in fenhexamid resistance we replaced the erg27 wt allele in a sensitive B. cinerea strain by that of a HydR1 "resistant" B. pseudocinerea strain.The gene replacement strategy is explained in Fig. 5.

Characterization of an unusual resistance mechanism towards fungicides: Detoxification
Indications about the second mechanism were obtained more than ten years ago by two studies.Suty and co-workers (1999) performed fenhexamid metabolization assays on B. cinerea and B. pseudocinerea and observed that naturally resistant species displayed a metabolization pattern different from B. cinerea suggesting fenhexamid degradation as possible resistance mechanism.These results were supported by the synergy between DMIs and fenhexamid observed only for B. pseudocinerea (Leroux et al., 2002).Together these results suggested for B. pseudocinera an enzyme(s) similar to Cyp51 (the target of DMIs) to degrade fenhexamid and therefore conferring resistance to fenhexamid.
We have searched the B. cinerea genome database (Amselem et al., 2011) (http://urgi.versailles.inra.fr/Species/Botrytis)for cytochrome P450 proteins similar to Cyp51 and compared their expression profiles between B. cinerea and B. pseudocinerea.The candidate gene showing the highest similarity to cyp51 (19 % protein identity, 37 % protein similarity) showed increased expression in B. pseudocinerea (Billard et al., unpublished).According to the nomenclature of P450s, after phylogenic analysis against all P450s of the ascomycete Aspergillus nidulans (Kelly et al., 2009) the corresponding gene was named cyp684.We created cyp684 deletion mutants in a B. pseudocinerea strain through a gene replacement strategy.The ∆cyp684 mutants showed a 200 fold reduction in fenhexamid resistance and a simultaneous reduction in DMI-fenhexamid synergy (Table 4,  In B. pseudocinerea however the cyp684 deletion abolishes the hypersensitivity to the phosphothiolate edifenphos in addition to the loss of fenhexamid resistance (   www.intechopen.com These compounds need to be modified in order to become active.In the case of edifenphos, the active metabolite is produced by the cleavage of a phosphor-sulfur bridge (Uesugi, 2001).As for B. pseudocinerea, a strong synergy was observed between phosphorothiolates and DMIs.Moreover negative cross-resistance between phosphorothiolates and phosphoroamidates in M. oryzae allowed the identification of the biochemical reactions involved (Uesugi & Takenake, 1992 ; Fig. 7).The enzymes involved are yet unknown, but negative cross resistance between BPA and edifenphos on one hand, and the synergy between edifenphos and DMIs on the other, suggest the involvement of a cytochrome P450 accepting as a substrate edifenphos as well as BPA.The comparable situation observed in B. pseudocinerea is in favor of a comparable enzymatic reaction involved in the detoxification of fenhexamid.We are currently analyzing the metabolites produced by B. pseudocinerea from fenhexamid in order to unravel the reaction mechanism.

Conclusions
We and others have identified and characterized various phenotypes displaying reduced sensitivity to fenhexamid among natural grey mould populations.In the case of the phenotypes with low resistance at the spore germination stage (HydR1, HydR2, MDR2 et MDR3 phenotypes), Petit et al., 2010 have shown these have only a limited impact on fenhexamid's efficacy in artificial inoculation studies and in vineyards studies.In the same line, monitoring started before the introduction of fenhexamid, show that the variations in frequencies of B. pseudocinerea -naturally resistant to fenhexamid -in grey mould populations, seem to be independent of selection by fenhexamid.B. cinerea and B. pseudocinerea are found in sympatry on the same hosts, but clearly differ in their phenology, demonstrating differences in ecological niche (Walker et al., 2011): although present at low frequencies (0-15 %), B. pseudocinerea is predominantly found in spring populations and has only a reduced impact on grey mould epidemics.
Finally, the HydR3 phenotype comprises strains with moderate (HydR3 -) to high (HydR3 + ) resistance levels at all growth stages.This resistance is conferred by target site modifications.HydR3 + strains are the predominant strains resistant to fenhexamid in French vineyards and those of greatest concern in practice, because they threaten the sustainability of fenhexamid in combating grey mould.Frequencies of HydR3 + strains are slowly but steadily increasing in French vineyard populations.Even if their mean frequencies stayed moderately low (e.g.27 % in Champagne in 2010, Fig. 8), their proportion within same plots has strongly increased in the last years.Although one can observe over 50 % highly resistant strains at the end of the season after fenhexamid treatment, our multi-annual monitoring survey did not reveal sites with such important frequencies over several years.In addition, we are not aware of any loss-of-efficacy observed for fenhexamid due to the selection of resistant populations.Several hypotheses can be put forward to explain the situation: (1) the use of fenhexamid limited to one application per year (as for other botryticides), ( 2) the fitness cost of the HydR3 + mutations and (3) the predominance of MDR strains at least in Champagne (55 % mean frequencies).Indeed, strains combining MDR (multiple drug resistance) and specific resistance to fenhexamid are rarely found.We have developed a molecular tool allowing the rapid detection and quantification of the three major HydR3 alleles from a mixture of B. cinerea spores.(ASPPAA PCR).This tool will make large scale field assays realistic to evaluate the percentage of HydR3 + strains among populations subjected to different fungicide pressures in order to precise the best strategy for fenhexamid treatment and the best state for its application.Moreover, this method may monitor frequencies of HydR3 + strains after stopping fenhexamid treatments and therefore allows to analyze the persistence of such resistance phenotype under real conditions.

Fig. 1 .
Fig. 1.Protein structure and functional domains of the 3-ketoreductase Erg27 of B. cinerea.The active site catalyzing the formation of a hydroxyl group on position C3 of sterols is located near the transmembrane domain allowing the anchorage of the enzyme to the endoplasmic reticulum.The position of the mutations detected in HydR3 -resistant isolates are indicated by black arrows and concerning HydR3 + isolates by a red one.

Fig. 2 .
Fig.2.Predicted 3D structure of the 3-ketoreductase (Erg27) of Botrytis cinerea based on the alignement with Salutaridine Reductase From Papaver Somniferum(Higashi et al., 2011).The cofactor (NADHP) binding site and the active sites are highlighted in cyan and purple respectively.The green ribbon represents the transmembrane domain.The mutated residues responsible for specific fenhexamid resistance (HydR3) are highlighted in red.The blue circle designates the extension of Botrytis and Sclerotinia Erg27 proteins compared to other fungal orthologs.

Fig. 3 .
Fig. 3. Life cycle of Botrytis cinerea.In laboratory conditions, a fitness cost of the HydR3 + resistance was observed in isogenic mutants at different phases of the asexual cycle (in red) of B. cinerea.However, the pathogenicity of these mutants, measured on beans, remained unchanged from the parental strain sensitive to fenhexamid (green).

Fig. 4 .
Fig. 4. Principle of ASPPAA PCR for quantitative SNP detection.The high robustness of this new allele quantification method using real-time PCR is caused by the specificity of amplification of the mutation by allele specific probes and primers present on both DNA strands.

Fig. 5 .
Fig. 5. Gene replacement strategy of the erg27 allele by that of B. pseudocinerea in a sensitive strain of B. cinerea.The hph gene conferring resistance to hygromycine was used as transformation selection marker.The transformants harboring the B. pseudocinerea allele instead of the B. cinerea allele showed a slightly increased resistance to fenhexamid (RF<10) compared to the parental B. cinerea strain (Billard et al., unpublished).Those results indicate that the major part of fenhexamid resistance in B. pseudocinerea is conferred by a mechanism independent of the target.
Fig. 6).Although the B. pseudocinerea ∆cyp684 mutants do not reach B. cinerea sensitivity levels, theses results show that the cytochrome P450 encoding gene cyp684 is responsible for the biggest part of B. pseudocinerea's natural resistance to fenhexamid.Natural polymorphism between the B. cinerea/B.pseudocinerea cyp684 orthologues may account for the different phenotypes.It remains to be shown if cyp684 overexpression and/or differential enzyme parameters are involved.

Fig. 6 .
Fig. 6.Joint action between fenhexamid (vertical) and prochloraz or tebuconazole (horizontal strip) on the B. pseudocinerea B900 strain and a B900∆cyp684 deletion mutant.The curves around paper crosses indicate strong synergism between chemicals (in the left panel).In the B. pseudocinerea ∆cyp684 mutant growth inhibition is typical of independent actions (right panel).

Table 1 .
Active substances approved against grey mould of grapevine in France and recommended stages of applications (French National Botrytis Note -Vigne 2010).

cinerea B. pseudocinerea HydR1 EC50 a HydS Resistance Factor HydR2 HydR3 - HydR3 + MDR2
Oculimacula yallundae and O. acuformis: the second species is naturally resistant to triazole SBIs.Another example is Rhizoctonia solani which is subdivided into different anastomosis groups some of which are tolerant to pencycuron, an anti-microtubular agent(Campion et al., 2005).

Table 3 .
Sensitivity to fenhexamid of transgenic mutants.LR=low resistance; MR=moderate resistance; HR=high resistance These bioassays conducted, in vitro or in vivo, have been miniaturized (i.e.microtiter plate methods), but nonetheless consume considerable resources and remain time-consuming.When the molecular mechanisms of resistance are known (e.g.target mutation, target over expression, increased drug efflux), and particularly when the underlying DNA polymorphisms (single-nucleotide polymorphism (SNPs), deletions or insertions) have been defined, various molecular methods can be used to monitor antimicrobial resistance.The principal methods for quantifying resistance are based on realtime PCR (polymerase chain reaction) technology.Generally, studies have been made on isolated and purified strains using qualitative methods like RFLP, AS PCR or Real time PCR Sierotzki et al., 2000)ce in Botrytis cinerea Banno et al., 2008, Venturia inequaelis Koenraadt et al., 1992, Rhyncosporium secalis Wheller et al., 1995, Helminthosporium solani McKay et al., 1998; Strobilurines resistance on Erisiphe graminis Baümler et al., 2003, Plasmopara viticola Furuya et al., 2009, Alternaria spp.Ma & Michailides, 2003, Mycosphaerella graminicola Fraaije et al., 2007, Mycosphaerella fijiensisSierotzki et al., 2000)and on the opposite some of them on populations, where resistance allele is quantify in gDNA pools by using Real time PCR technologies (Benzimidazole resistance in Sclerotinia sclerotiorum Chen et al., 2009 ; Strobilurins resistance on Blumeria graminisFraaije et al., 2002 and Pyrenophora teres

Table 6 )
suggesting that Cyp684 could have a similar enzyme activity on fenhexamid as on edifenphos.Phosphorothiolates (e.g.edifenphos) are profungicides, nearly exclusively used against the rice-blast disease caused by Magnaporthe oryzae.