Characterization of Laboratory Mutants of Botrytis cinerea Resistant to QoI Fungicides

Abstract

Mutants of Botrytis cinerea with moderate and high resistance to pyraclostrobin, a Qo inhibitor of mitochondrial electron transport at the cytochrome bc 1 complex, were isolated at a high mutation frequency, after nitrosoguanidine mutagenesis and selection on medium containing pyraclostrobin and salicylhydroxamate (SHAM), a specific inhibitor of cyanide-resistant (alternative) respiration. Oxygen uptake in whole cells was strongly inhibited in the wild-type strain by pyraclostrobin and SHAM, but not in the mutant isolates. Cross-resistance studies with other Qo and Qi inhibitors (QoIs and QiIs) of cytochrome bc 1 complex of mitochondrial respiration showed that the mutation(s) for resistance to pyraclostrobin also reduced the sensitivity of mutant strains to other QoIs as azoxystrobin, fluoxastrobin, trifloxystrobin and picoxystrobin, but not to famoxadone and to the QiIs cyazofamid and antimycin-A. An increased sensitivity of pyraclostrobin-resistant strains to the carboxamide boscalid, an inhibitor of complex II, and to the anilinopyrimidine cyprodinil, a methionine biosynthesis inhibitor, was observed. Moreover, no effect of pyraclostrobin resistance mutation(s) on fungitoxicity of the hydroxyanilide fenhexamid, the phenylpyrrole fludioxonil, the benzimidazole benomyl, and to the phenylpyridinamine fluazinam, which affect other cellular pathways, was observed. Study of fitness parameters in the wild-type and pyraclostrobin-resistant mutants of B. cinerea showed that most mutants had a significant reduction in the sporulation, conidial germination and sclerotia production. Experiments on the stability of the pyraclostrobin-resistant phenotype showed a reduction of resistance, mainly in moderate resistant strains, when the mutants were grown on inhibitor-free medium. However, a rapid recovery of the resistance level was observed after the mutants were returned to a selective medium. Studies on the competitive ability of mutant isolates against the wild-type parent strain, by applications of a mixed conidial population, showed that, in vitro, all mutants were less competitive than the wild-type strain. However, the competitive ability of high resistant mutants was higher than the moderate ones. Pathogenicity tests on cucumber seedlings showed that all mutant strains tested exhibited an infection ability similar with the wild-type parent strain. Preventive applications of the commercial product of F-500 25EC (pyraclostrobin) were effective against lesion development on cotyledons by the wild-type, but ineffective, even at high concentrations, against disease caused by the pyraclostrobin-resistant isolates. Boscalid (F-510 50WG) was found equally effective against the disease caused by the wild-type or pyraclostrobin-resistant mutants. This is the first report indicating the appearance of B. cinerea strains resistant to QoI fungicides by the biochemical mechanism of site modification and the risk for field resistance.

In vitro toxicity of alternative oxidase inhibitors salicylhydroxamic acid and propyl gallate on Fusicladium effusum

“…Other evidence suggests that the fungal AOX system is active during infections in planta, but can be inhibited by plant secondary metabolites (Wood and Hollomon 2003). In contrast to our results with F. effusum, no toxic effects of SHAM or PG were reported in previous in vitro studies of several other fungi, including Alternaria solani ( Jones et al 2007;Malandrakis et al 2006;Markoglou et al 2006;Pasche et al 2004;Ziogas et al 2002). However, there are very few examples of such studies that included any biochemical evidence of the suppression of AOX, e.g., measurements of oxygen uptake.…”

“…Due to the inhibition of electron flow through complex III by QoI fungicides, an alternative respiration system located on the mitochondrial membrane can be activated to avoid the fatal effects of the fungicide (Kameko and Ishii 2009;Wood and Hollomon 2003;Ypema and Gold 1999). Therefore, it is a common practice to include alternative oxidase (AOX) inhibitors, e.g., salicylhydroxamic acid (SHAM), in the medium to suppress alternative respiration (Avila-Adame et al 2003b;Avila-Adame and Köller 2003a;Kameko and Ishii 2009;Markoglou et al 2006;Malandrakis et al 2006;Jones et al 2007;Ishii et al 2009;Kim et al 2003;Köller et al 2004;Pasche et al 2004;Steinfeld et al 2001;Walker et al 2009;Wood and Hollomon 2003;Ziogas et al 2002;Ziogas et al 1997).…”

A rapid and simple method for determining fungicide resistance in Botrytis

“…Four isolates with high sensitivity to trifloxystrobin were also sensitive to pyraclostrobin and azoxystrobin, whereby the EC 50 values for the former two compounds were comparable whilst those for azoxystrobin were 4-10 times higher (Table 3). This observation is in line with published sensitivity differences in Botrytis (Markoglou et al 2006) and other pathogens (Wise et al 2008). The most sensitive values (ss) obtained in the present work (Table 3) were very similar to those reported by other workers for baseline strains of Botrytis (Myresiotis et al 2008;Banno et al 2009;Leroux et al 2010).…”

Biological and molecular characterization of laboratory mutants of Cercospora beticola resistant to Qo inhibitors

“…The absence of cross-resistance between Qo and Qi inhibitors in pyraclostrobin-resistant mutants of C. beticola would suggest changes only in the configuration at the Qo-site of cytochrome b, which do not transmit a structural change to the Qi-site. A positive cross-resistance, with high resistance factors, among QoI has also been observed in many other phytopathogenic fungi (Heaney et al, 2000;Sierotzki et al, 2000a, b;Chin et al, 2001a, b;Ishii et al, 2001;Steinfeld et al, 2001;Vincelli and Dixon, 2002;Ziogas et al, 2002;Pasche et al, 2005;Markoglou et al, 2006).…”