A non‐Mendelian inheritance of resistance to strobilurin fungicides in Ustilago maydis

Abstract

Mutants of Ustilago maydis (DC) Corda with high resistance to azoxystrobin (RF 164 to 4714, based on EC50 values), an inhibitor of mitochondrial electron transport at the cytochrome bc1 complex, were isolated in a mutation frequency of 2.3 x 10(-7) after nitrosoguanidine mutagenesis and selection on media containing 1 microgram ml-1 azoxystrobin in addition to 0.5 mM salicylhydroxamate (SHAM), a specific inhibitor of cyanide-resistant (alternative) respiration. Oxygen uptake in whole cells was strongly inhibited in the wild-type strains by azoxystrobin (1.5 micrograms ml-1) in addition to SHAM (1 mM), but not in the mutant isolates. Genetic analysis with nine such mutant isolates resulted in progeny phenotypes which did not follow Mendelian segregation, but satisfied the criteria of non-Mendelian (cytoplasmic) heredity. In crosses between three mutant isolates with the compatible wild-type strains, the sensitivity was inherited by progeny maternally from the wild-type parent strain (criterion of uniparental inheritance). In crosses between wild-type strains and remaining mutant isolates, a continuous distribution of sensitivity in the progeny was found (criterion of vegetative segregation). The third criterion of cytoplasmic resistance (criterion of intracellular selection) was fulfilled by experiments on the stability of resistance phenotypes. With two exceptions, a reduction of resistance was observed in the mutant strains when they were grown on inhibitor-free medium. Recovery of the high resistance level was observed after they were returned to the selection medium. Cross-resistance studies with other fungicides, which also inhibit electron transport through complex III of respiratory chain, showed that mutations for resistance to azoxystrobin were also responsible for reduced sensitivity to kresoxim-methyl (RF 18 to 1199) and to antimycin-A (RF 20 to 305), which act at the Qo and Qi sites of the cytochrome bc1 complex, respectively. Studies of the fitness of azoxystrobin-resistant isolates showed that these mutations appeared to be pleiotropic, having significant adverse effects on growth in liquid culture and pathogenicity on young corn plants.

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).…”

Characterization of Laboratory Mutants of Botrytis cinerea Resistant to QoI Fungicides

“…Similar results have also been reported with laboratory mutants of U. maydis resistant to azoxystrobin (Ziogas et al, 2002), and with field isolates of M. fijiensis (Sierotzki et al, 2000b) and V. inaequalis (Steinfeld et al, 2001). On the contrary, study of the mechanism of resistance to azoxystrobin in S. tritici indicated the presence of an efficient alternative pathway inhibited by SHAM in the mutant isolate (Ziogas et al, 1997).…”

“…Cytochrome b, the target enzyme of the QoIs, is encoded by mitochondrial DNA (Di Rago and Colson, 1989;Zheng and Ko¨ller, 1997) and resistance to QoIs through a target-site modification is expected to be inherited in a non-Mendelian way. Detailed genetic analysis of QoI-resistance in Ustilago maydis provided a satisfaction of the three criteria (uniparental inheritance, vegetative segregation and intracellular selection) of cytoplasmic (non-Mendelian) heredity (Ziogas et al, 2002). Practical failures of QoIs were first recorded in the control of wheat powdery mildew (B. graminis) in northern areas of Germany during 1998 (Sierotzki et al, 2000a) and, more recently in the control of cucumber powdery mildew (Podosphaera fusca) and downy mildew (Pseudoperonospora cubensis) observed in Japan (Ishii et al, 2001) and Plasmopara viticola in Europe (Collina et al, 2004;Sierotzki et al, 2004).…”

In Vitro Influence of Selected Fungicides on Sphacelotheca Reiliana and Ustilago Maydis

“…For wild types, EC 50 in liquid culture was 0.01 µg/ml, and for mutant isolates, it was 1-10 µg/ml (Ziogas et al 2002). A unique relationship was observed for the fungicide azoxystrobin.…”