New cytochrome b haplotypes, harboring L299F or N256S + L299F substitutions, were found in azoxystrobin-resistant Puccinia horiana, the causal agent of chrysanthemum white rust

Rust fungi are generally considered low-risk pathogens in terms of resistance to fungicides. However, Puccinia horiana, which causes white rust in chrysanthemum, has developed resistance to various fungicides via mutations in genes encoding the target proteins of fungicides. We investigated cytochrome b haplotypes of 15 Japanese isolates of P. horiana. Among them, two were wild-type, and the others carried L299F, L275F + L299F, or N256S + L299F amino acid substitutions. To best of our knowledge, L299F and N256S + L299F haplotypes were found for the first time in this study and isolates of each haplotype showed 22- and 222-fold higher azoxystrobin EC50 values than their wild-type counterparts in the basidiospore germination test, respectively. Further tests confirmed cross-resistance among some quinone outside inhibitor (QoI) fungicides in N256S + L299F-harboring isolates whereas L275F-harboring isolates remained sensitive to other QoIs. Our in planta tests revealed that the performance of azoxystrobin against mutant isolates was reduced even at the labeled concentration. The homology model predicted the involvement of L275 and L299 in the interaction between azoxystrobin and cytochrome b, while it was unlikely for N256. In some model organisms such as Saccharomyces cerevisiae, asparagine 256 is known to interact with glycine 137. Considering that G137R substitution confers azoxystrobin resistance in other phytopathogenic fungi, we suggest that N256S might also be responsible for azoxystrobin resistance in P. horiana. This is the first record of a substitution at cytochrome b N256 in azoxystrobin-resistant phytopathogenic fungi.