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. 2009 Dec;5(12):e1000696.
doi: 10.1371/journal.ppat.1000696. Epub 2009 Dec 18.

Fungicide-driven Evolution and Molecular Basis of Multidrug Resistance in Field Populations of the Grey Mould Fungus Botrytis Cinerea

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Fungicide-driven Evolution and Molecular Basis of Multidrug Resistance in Field Populations of the Grey Mould Fungus Botrytis Cinerea

Matthias Kretschmer et al. PLoS Pathog. .
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Abstract

The grey mould fungus Botrytis cinerea causes losses of commercially important fruits, vegetables and ornamentals worldwide. Fungicide treatments are effective for disease control, but bear the risk of resistance development. The major resistance mechanism in fungi is target protein modification resulting in reduced drug binding. Multiple drug resistance (MDR) caused by increased efflux activity is common in human pathogenic microbes, but rarely described for plant pathogens. Annual monitoring for fungicide resistance in field isolates from fungicide-treated vineyards in France and Germany revealed a rapidly increasing appearance of B. cinerea field populations with three distinct MDR phenotypes. All MDR strains showed increased fungicide efflux activity and overexpression of efflux transporter genes. Similar to clinical MDR isolates of Candida yeasts that are due to transcription factor mutations, all MDR1 strains were shown to harbor activating mutations in a transcription factor (Mrr1) that controls the gene encoding ABC transporter AtrB. MDR2 strains had undergone a unique rearrangement in the promoter region of the major facilitator superfamily transporter gene mfsM2, induced by insertion of a retrotransposon-derived sequence. MDR2 strains carrying the same rearranged mfsM2 allele have probably migrated from French to German wine-growing regions. The roles of atrB, mrr1 and mfsM2 were proven by the phenotypes of knock-out and overexpression mutants. As confirmed by sexual crosses, combinations of mrr1 and mfsM2 mutations lead to MDR3 strains with higher broad-spectrum resistance. An MDR3 strain was shown in field experiments to be selected against sensitive strains by fungicide treatments. Our data document for the first time the rising prevalence, spread and molecular basis of MDR populations in a major plant pathogen in agricultural environments. These populations will increase the risk of grey mould rot and hamper the effectiveness of current strategies for fungicide resistance management.

Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Isolation frequencies of B. cinerea MDR strains from French and German wine-growing regions.
(A) Appearance of MDR strains in the Champagne. While MDR1 and MDR2 strains, named initially AniR2 and AniR3, respectively, were first detected in 1994 ,, MDR3 strains have been observed since 2001. (B) Frequency of MDR strains in the German Wine Road region.
Figure 2
Figure 2. Differential fungicide accumulation by B. cinerea sensitive and MDR strains.
(A) Kinetics of fludioxonil (14C-labeled) accumulation by germinated spores of sensitive strain B05.10 (square) and of MDR1 strains D04.375 (circle) and D04.104 (triangle). Addition of carbonyl cyanide m-chlorophenylhydrazone (CCCP, 10 µM) after 90 min led to a net influx of the fungicide into the cells (dashed lines). (B) Accumulation of 14C-fludioxonil and 14C-bitertanol by sensitive and MDR strains. Samples were taken 10 min (shaded bars) and 60 min (white bars) after addition of labeled fungicide. The following strains were analyzed (from left to right): B05.10, D06.6-15 (sensitive); D06.5-16, D04.375 (MDR1); D06.2-6, D06.6-5 (MDR2); D06.7-33, D06.7-39 (MDR3). Significant differences of values (10 min) to those of sensitive strain B05.10 are indicated: n.s.: not significant; * p<0.05; ** p<0.01; *** p<0.001. (C) Control experiments demonstrating large differences in 14C-fungicide uptake between living and heat-killed germlings of sensitive (B05.10) and MDR3 (D06.7-33) strains.
Figure 3
Figure 3. B. cinerea MDR strains show constitutive overexpression of efflux transporter genes.
(A) Expression analysis by Northern hybridization of atrB and mfsM2 in B. cinerea germlings. For hybridization with atrB, RNA of the following strains was loaded (from left to right): B05.10, D06.6-15 (sensitive, two lanes each); D06.5-16, D06.7-27 (MDR1, two lanes each); D06.6-5, D06.2-6 (MDR2); D06.7-39, D06.7-33 (MDR3). Below the hybridization signals, the corresponding RNA samples after agarose electrophoresis and ethidium bromide staining are shown as loading controls. ---: no treatment; +: 30 min treatment with 1 mg/l fludioxonil. For hybridization with mfsM2, RNAs of non-treated germlings were loaded in the same order as for atrB. (B) Expression analysis by quantitative RT-PCR of efflux transporter genes in sensitive and MDR strains. Values indicate fold-increases in expression levels, relative to the levels in sensitive strains without fludioxonil treatment (---). Mean values are shown from three strains each with sensitive, MDR1, MDR2, and MDR3 phenotypes. n.d.: Not determined.
Figure 4
Figure 4. The MfsM2 efflux transporter controls MDR2.
(A) Bitertanol (14C-labeled) accumulation after 10 min (shaded) and 60 min (white bars). Significant differences of values (10 min) of the mutants to those of their MDR2 parent strains are indicated: ** p<0.01. (B) Drug sensitivities. Mean values of resistance factors relative to B05.10 are shown, from two MDR2 strains (D06.2-6, D06.6-5; white bars), strains D06.2-6(ΔmfsM2) and D06.6-5(ΔmfsM2) (black bars), and from two transformants of strain B05.Hyg-3(mfsM2ox) (grey bars). Significant differences of corresponding values are indicated between MDR2 (ΔmfsM2) mutants and their parent strains, and between strain B05.Hyg-3(mfsM2ox) and strain B05.Hyg-3: n.s.: Not significant; * p<0.05; ** p<0.01; *** p<0.001. §Due to limited solubility of tolnaftate, no accurate values above 25-fold could be determined. Drugs (abbreviated) are listed in the same order as in Table 1, except for the omission of carbendazim. (C) Drug sensitivity phenotypes on HA plates. 1: D06.2-6(MDR2); 2: D06.2-6(ΔmfsM2); 3: B05.10 (sensitive); 4: B05.Hyg-3(mfsM2ox)-4, 5: D06.6-5(MDR2); 6: D06.6-5(ΔmfsM2); 7: B05.Hyg-3 (sensitive); 8: B05.Hyg-3(mfsM2ox)-11. Strains with mfsM2 mutations showed a slight growth difference to their parent strains. Pictures were taken 3 d.p.i., except for bitertanol (4 d.p.i.). Concentrations of drugs were adjusted to reveal clear differences between the strains which overexpress mfsM2 and those which do not.
Figure 5
Figure 5. MDR1-related mutations in the Mrr1 transcription factor.
Amino acid positions and exchanges found in MDR1 and MDR3 strains, and the observed frequencies of each mutation (in parentheses) are indicated. For a detailed list with Mrr1 sequences of individual strains, see Table S4.
Figure 6
Figure 6. Mrr1 regulates MDR1 phenotypes via modulation of atrB expression.
(A) Expression of ABC transporter genes in strains with different levels of mrr1 expression. Mean values are shown from two sensitive strains (B05.10, B05.-Hyg3), two MDR1 strains (D06.5-16, D06.7-27), two MDR1 mrr1 k.o. transformants (D06.5-16(Δmrr1)-5, -7), and two B05.Hyg-3 transformants expressing mrr1V575M (B05.Hyg-3(+mrr1V575M)-5, -6). (B) Fludioxonil (14C-labeled) accumulation by wild type strains and strains with mutations in atrB and mrr1, after 10 min (shaded) and 60 min (white bars). Significant differences between the values of the mutants and those of their parent strains are indicated separately for 10 min and 60 min values (n.s.: not significant; **: p<0.01; ***: p<0.001). (C) Drug sensitivities of atrB and mrr1 mutants. For each mutant, mean values of two or three transformants were used to calculate resistance factors relative to sensitive strain B05.10 or (in case of B05.Hyg-3(+mrr1V575M) to strain B05.Hyg-3. #MDR1 strain D06.7-27 showed higher resistance to cyprodinil (standard deviation = 12.0) and lower resistance to tolnaftate, compared to other MDR1 strains. Significantly different resistance values of the transformants relative to their parent strains are indicated (O Not significant; * p<0.05; ** p<0.01; *** p<0.001). §Due to limited solubility of tolnaftate, no accurate values above 25-fold could be determined. (D) Fungicide sensitivity phenotypes on agar plates. 1: B05.10 (sensitive); 2: B05.10(ΔatrB)-4; 3: B05.10(ΔatrB)-5; 4: B05.10(Δmrr1)-8; 5: B05.10(Δmrr1)-18; 6: D06.5-16 (MDR1); 7: D06.5-16(ΔatrB)-1; 8: D06.5-16(ΔatrB)-2; 9: D06.5-16(Δmrr1)-5; 10: D06.5-16(Δmrr1)-7; 11: B05.Hyg-3 (sensitive); 12: B05.Hyg-3(+mrr1V575M)-5; 13: B05.Hyg-3(+mrr1V575M)-6; 14: B05.Hyg-3(+mrr1V575M)-10. Top: HA, 2.5 d.p.i.; middle: HA, 0.03 mg/l fludioxonil, 4 d.p.i.; bottom: GB5 (glucose), 0.01 mg/l cyprodinil, 4 d.p.i..
Figure 7
Figure 7. MDR2 strains carry a retroelement-like gene fragment in the mfsM2 promoter.
(A) Structure of the mfsM2 upstream region, and the insertion-deletion rearrangement (in red) in MDR2 and MDR3 strains. The retroelement-like gene fragment encodes truncated reverse transcriptase (RT) and RNase H domains. The deleted region is indicated as hatched bar. (B) The rearrangement leads to activation of the mfsM2 promoter. B. cinerea transformants carrying uidA fusions with mfsM2 upstream fragments from strain B05.10 and MDR2 strain D08.2-12 were stained for ß-glucuronidase activity. Scale bars: 20 µm.
Figure 8
Figure 8. Field competitiveness of an MDR3 strain is increased by fungicide selection.
Recovery rates of B. cinerea isolates from inoculated grapevine plants during grape harvest (autumn), and in the following spring, in two successive years. Fungicide treated (+) and non-treated (−) grapevine plants were inoculated with a 1∶1 mixture of an MDR3 and a sensitive strain. Grey: Introduced MDR3 strain; White: Introduced sensitive strain; Black: Resident strains. Significantly different recovery rates for the introduced strains are shown.
Figure 9
Figure 9. Model for the appearance of MDR phenotypes in B. cinerea vineyard populations.
Regularly alternating treatments with modern fungicides, in particular the anilinopyrimidines pyrimethanil and cyprodinil (since 1990), the phenylpyrrole fludioxonil (since 1995), and the hydroxyanilide fenhexamid (since 2000) are assumed to be responsible for the selection of MDR phenotypes in Champagne vineyards. Repeatedly occurring point mutations in the transcription factor gene mrr1 (blue rose symbol) lead to overexpression of the ABC transporter gene atrB (unlinked to mrr1) and thus to MDR1 phenotype. In contrast, a unique promoter rearrangement in the MFS transporter gene mfsM2 (red asterisk) is responsible for its overexpression and MDR2 phenotype. Strains with MDR3 phenotype, carrying both types of mutations and showing increased MDR, might have originated either by natural MDR1×MDR2 crosses or by secondary mrr1 mutations in MDR2 strains. MDR2 and MDR3 strains, and possibly also MDR1 strains, have migrated within and out of the Champagne, reaching at least the German Wine Road region, 250 km east of the Champagne. Further evidences for the migration are the delayed appearance of MDR2/3 strains in Germany, and the failure until now to detect them in France outside of the Champagne. Gene expression is indicated by arrows, bold arrows indicate overexpression, and the dotted arrows with the ‘+’ sign indicate transcription factor-mediated activation.

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