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. 2010 Oct;76(19):6615-30.
doi: 10.1128/AEM.00931-10. Epub 2010 Aug 6.

Exploring Mechanisms of Resistance to Respiratory Inhibitors in Field Strains of Botrytis Cinerea, the Causal Agent of Gray Mold

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Exploring Mechanisms of Resistance to Respiratory Inhibitors in Field Strains of Botrytis Cinerea, the Causal Agent of Gray Mold

Pierre Leroux et al. Appl Environ Microbiol. .
Free PMC article

Abstract

Respiratory inhibitors are among the fungicides most widely used for disease control on crops. Most are strobilurins and carboxamides, inhibiting the cytochrome b of mitochondrial complex III and the succinate dehydrogenase of mitochondrial complex II, respectively. A few years after the approval of these inhibitors for use on grapevines, field isolates of Botrytis cinerea, the causal agent of gray mold, resistant to one or both of these classes of fungicide were recovered in France and Germany. However, little was known about the mechanisms underlying this resistance in field populations of this fungus. Such knowledge could facilitate resistance risk assessment. The aim of this study was to investigate the mechanisms of resistance occurring in B. cinerea populations. Highly specific resistance to strobilurins was correlated with a single mutation of the cytb target gene. Changes in its intronic structure may also have occurred due to an evolutionary process controlling selection for resistance. Specific resistance to carboxamides was identified for six phenotypes, with various patterns of resistance levels and cross-resistance. Several mutations specific to B. cinerea were identified within the sdhB and sdhD genes encoding the iron-sulfur protein and an anchor protein of the succinate dehydrogenase complex. Another as-yet-uncharacterized mechanism of resistance was also recorded. In addition to target site resistance mechanisms, multidrug resistance, linked to the overexpression of membrane transporters, was identified in strains with low to moderate resistance to several respiratory inhibitors. This diversity of resistance mechanisms makes resistance management difficult and must be taken into account when developing strategies for Botrytis control.

Figures

FIG. 1.
FIG. 1.
In vitro responses of multidrug-resistant strains from Botrytis group II toward respiratory inhibitors. For given MDR strains, the RF equals the ratio of the EC50s of MDR strains to the mean EC50 of the wild type (see Materials and Methods). Mean RF values were calculated using four strains for each MDR category (Table 1).
FIG. 2.
FIG. 2.
Schematic structure of mitochondrial complexes II and III indicating the main amino acids of the different subunits involved in binding of inhibitors and in resistance. Complex II contains four subunits, whereas complex III consists of 10 to 11 polypeptides. For complex III, only three main subunits (i.e., Cytb, cytochrome b; ISP Rieske, iron sulfur protein with a Rieske-type cluster; cytc1, cytochrome c1) directly involved in the electron transfer are shown. The amino acids involved in the binding of substrates (i.e., ubiquinone or ubiquinol) or of inhibitors were identified in crystallographic studies involving complex II or III from various origins (20, 28, 33, 35, 36, 59, 75). Amino acids are numbered according to B. cinerea sequences.
FIG. 3.
FIG. 3.
Molecular polymorphism of the gene encoding cytochrome b (cytb) in strains of Botrytis group I and group II. (A) Possible structure of cytb according to the presence of the 1,205-bp group I intron and/or the G143A change and possible results observed after CAPS analyses. Presence of the intron changes the length of the PCR fragment, either in Botrytis group I or II. The G143A change was found only in Botrytis group II strains without the 1,205-bp intron. The mutation in cytb is recognized by the SatI restriction enzyme and leads to a two-band pattern after digestion of the PCR fragment. (B) Ethidium bromide-stained gel of PCR fragments subjected to electrophoresis after Qo13ext/Qo14ext amplification of the cytochrome b gene in Botrytis group I and group II (first row) and after SatI digestion of these fragments (second row) (CAPS test). Strains 1 and 3, Botrytis group I with and without intron, respectively; strains 8, 21, 24, and 25, Botrytis group II. Strain 25 is strobilurin resistant (Table 1). Strain 8 exhibits an intron after codon 143. Vv, Vitis vinifera (grapevine); PviS1 and PviS3, Plasmopara viticola (downy mildew; PViS3 is strobilurin resistant); En, Erysiphe necator (powdery mildew); Sc, Saccharomyces cerevisiae (yeast).

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