Selection, characterization and genetic analysis of laboratory mutants of Botryotinia fuckeliana (Botrytis cinerea) resistant to the fungicide boscalid

Abstract

Resistance to the fungicide boscalid in laboratory mutants of Botryotinia fuckeliana (Botrytis cinerea) was investigated. The baseline sensitivity to boscalid was evaluated in terms of colony growth (EC 50 =0.3-3 µg ml −1 ; MIC=10-30 µg ml −1 ) and conidial germination (EC 50 =0.03-0.1 µg ml −1 ; MIC= 1-3 µg ml −1 ) tests. Mutants were selected in vitro from wild-type strains of the fungus on a fungicide-amended medium containing acetate as a carbon source. Mutants showed two different levels of resistance to boscalid, distinguishable through the conidial germination tests: low (EC 50 ∼0.3 µg ml −1 , ranging from 0.03 to 1 µg ml −1 ; MIC>100 µg ml −1 ) and high (EC 50 > 100 µg ml −1 ) resistance. Analysis of meiotic progeny from crosses between resistant mutants and sensitive reference strains showed that resistant phenotypes were due to mutations in single major gene(s) inherited in a Mendelian fashion, and linked with both the Daf1 and Mbc1 genes, responsible for resistance to dicarboximide and benzimidazole fungicides, respectively. Gene sequence analysis of the four sub-units of the boscalid-target protein, the succinate dehydrogenase enzyme, revealed that single or double point mutations in the highly conserved regions of the iron-sulphur protein (Ip) gene were associated with resistance. Mutations resulted in proline to leucine or phenylalanine replacements at position 225 (P225L or P225F) in high resistant mutants, and in a histidine to tyrosine replacement at position 272 (H272Y) in low resistant mutants. Sequences of the flavoprotein and the two transmembrane sub-units of succinate dehydrogenase were never affected.

A rapid and simple method for determining fungicide resistance in Botrytis

“…With EC 50 values below 0.1 ppm for the most sensitive category of isolates (Table 3), the present test was in the same sensitivity range as previously published conidial germination assays (Stammler & Speakman 2006;De Miccolis Angelini et al 2010) and considerably more sensitive than mycelial growth assays (e.g. Myresiotis et al 2008;De Miccolis Angelini et al 2010).…”

“…Myresiotis et al 2008;De Miccolis Angelini et al 2010). A slightly less sensitive category of isolates (s) displayed resistance factors of 1.7-5.0, similar to those described for MDR strains (Kretschmer et al 2009;Leroux et al 2010).…”

“…The high-resistance category (R) comprised a wide range of EC 50 values from 3.4 to 54.5 (RF = 50-790). A diversity of target mutations in genes encoding subunits of succinate dehydrogenase has been described, and the strains harbouring them may also differ in their general genetic background (De Miccolis Angelini et al 2010;Leroux et al 2010), thus accounting for the wide range of resistance factors recorded with R isolates in the current work. An evaluation of representative inhibition curves (Fig.…”

Detection of Botrytis cinerea field isolates with multiple fungicide resistance from table grape in Sicily

“…On the basis of the literature, pathogen sensitivity to the fungicides was initially related to discriminatory doses as follows: 1 mg mL À1 for carbendazim, iprodione, boscalid and pyrimethanil, and 0.1 mg mL À1 for fenhexamid and fludioxonil (Baroffio et al, 2003;De Miccolis Angelini et al, 2010;Faretra and Pollastro, 1991;Latorre and Torres, 2012;Leroux et al, 1999;Myresiotis et al, 2007;Yourman and Jeffers, 1999;Zhang et al, 2007). Only for boscalid, the authors subsequently considered a Resistance Factor (RF) ¼ 5 (the ratio of the EC 50 value for a boscalid-resistant isolate relative to the EC 50 value for a highly boscalid-sensitive isolate) as distinguishing sensitive from resistant isolates.…”

A real-time PCR assay for detection and quantification of Botrytis cinerea in Pelargonium x hortorum plants, and its use for evaluation of plant resistance

“…The polyphagous nature of B. cinerea and its efficient spread of conidia in the air render it very harmful, especially in greenhouses, where often many susceptible ornamental plants (e.g., cyclamen, gerbera, carnation) are cultivated together and where the humidity and temperature are particularly suitable for disease development. Gray mold is mainly controlled using fungicides, which involves high costs, health risks for greenhouse workers, the possibility of residues in the crops and the environment, and the emergence of fungicide-resistant fungal isolates (Raposo et al 2000;De Miccolis Angelini et al 2010). The use of healthy pelargonium cuttings and the exploitation of cultivar and/or induced resistance are more friendly environmental strategies for controlling this disease.…”