Abstract
The novel agricultural fungicide 3-[5-(4-chlorophenyl)-2,3-dimethyl-3-isoxazolidinyl] pyridine (SYP-Z048) developed by China Shenyang Research Institute of Chemical Industry has been confirmed to be an ergosterol biosynthesis inhibitor (EBI). Previous studies have shown that EBIs target the proteins from a range of genes, including CYP51, ERG2 and/or ERG24, and ERG27, which are involved in the ergosterol biosynthesis pathway. In the current study the ERG2, ERG24, and ERG27 genes were cloned from wild type and resistant mutants of Monilinia fructicola in an attempt to clarify the target site of SYP-Z048. Comparative analysis of the deduced aa sequence of these genes, as well as CYP51, revealed several point mutations that resulted in amino acid variation among the sensitive and resistant isolates. However, sensitivity assays indicated that only one, the substitution of phenylalanine (F) for the tyrosine (Y) at 136 in CYP51, was correlated with reduced sensitivity to SYP-Z048. Heterologous expression of MfCYP51-136Y (MfCYP136Y) and MfCYP51-136F (MfCYP136F) in Pichia pastoris revealed that MfCYP136F significantly reduced sensitivity to SYP-Z048, increasing the average EC50 of the transformants 11-fold relative to those carrying MfCYP136Y. However, neither the additional copy of MfCYP136Y nor multiple copies of MfCYP136F were found to reduce sensitivity relative to the empty vector control or single copy transformants, respectively. Molecular docking experiments using SYP-Z048 with HsCYP145Y and the mutated version HsCYP145F as substitutes for MfCYP136Y and MfCYP136F, respectively, indicated that the reduced affinity of HsCYP145F for SYP-Z048 resulted from the loss of a hydrogen bond between the fungicide and the active site. Taken together these results indicate that MfCYP51 is the major target site of SYP-Z048 in M. fructicola, which has important implications for the resistance management of this fungicide in the field.
Highlights
Fungal diseases are one of the most important limiting factors in crop production, reducing yields and quality, but in some cases tainting the produce with toxins that are harmful to both livestock and human consumers
The results of a BLAST analysis showed that the deduced aa sequences of ERG2, ERG24 and ERG27 from M. fructicola exhibited a high degree of similarity to the corresponding sequences from closely related species
In our previous study several highly resistant mutants of M. fructicola were produced by UV-irradiation of spores (Chen et al, 2012), where only the MfCYP51 gene in sensitive and resistant isolates was analyzed
Summary
Fungal diseases are one of the most important limiting factors in crop production, reducing yields and quality, but in some cases tainting the produce with toxins that are harmful to both livestock and human consumers. The development of plant cultivars exhibiting disease resistance is the method of choice for reducing the damage caused by pathogenic fungi, in practice many diseases require the application of agrochemicals for effective control, which has become a critical element in plant disease management programs. Winter) Honey, a ubiquitous pathogen that is the primary causal agent of brown rot in stone fruit (EPPO/CABI1). The EC50 value for SYP-Z048 in baseline populations of M. fructicola is 0.017 μg/ml (Chen et al, 2012), which is similar to the values for propiconazole (0.03 μg/ml) (Zehr et al, 1999) and tebuconazole (0.016 μg/ml) (Yoshimura et al, 2004), and field trials have indicated that SYP-Z048 effectively controls brown rot in peach orchards (Chen et al, 2014)
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