Resistance Assessment for Oxathiapiprolin in Phytophthora capsici and the Detection of a Point Mutation (G769W) in PcORP1 that Confers Resistance

Abstract

The potential for oxathiapiprolin resistance in Phytophthora capsici was evaluated. The baseline sensitivities of 175 isolates to oxathiapiprolin were initially determinated and found to conform to a unimodal curve with a mean EC50 value of 5.61 × 10-4 μg/ml. Twelve stable oxathiapiprolin-resistant mutants were generated by fungicide adaptation in two sensitive isolates, LP3 and HNJZ10. The fitness of the LP3-mutants was found to be similar to or better than that of the parental isolate LP3, while the HNJZ10-mutants were found to have lost the capacity to produce zoospores. Taken together these results suggest that the risk of P. capsici developing resistance to oxathiapiprolin is moderate. Comparison of the PcORP1 genes in the LP3-mutants and wild-type parental isolate, which encode the target protein of oxathiapiprolin, revealed that a heterozygous mutation caused the amino acid substitution G769W. Transformation and expression of the mutated PcORP1-769W allele in the sensitive wild-type isolate BYA5 confirmed that the mutation in PcORP1 was responsible for the observed oxathiapiprolin resistance. Finally diagnostic tests including As-PCR and CAPs were developed to detect the oxathiapiprolin resistance resulting from the G769W point mutation in field populations of P. capsici.

Oxysterol‐binding protein‐related protein 2 is not essential for Phytophthora sojae based on CRISPR/Cas9 deletions

“…Only two ORPs were found, which we named PsORP1 (Protein ID: 558498) and PsORP2 (Protein ID: 470921) respectively. According to previous reports, ORP1 is the target protein of oxathiapiprolin in Phytophthora capsici and Phytophthora infestans (Andreassi et al ., ; Miao et al ., ). Furthermore, mutations in PsORP1 protect P. sojae against oxathiapiprolin (Miao et al ., submitted).…”

“…To examine PsORP2 transcription throughout the asexual life cycle and during infection, we measured PsORP2 transcript levels in P . sojae using quantitative real‐time polymerase chain reaction (qRT‐PCR) using the primers shown in Supporting Information Table S1 and the methods described by Miao and colleagues (). PsORP2 showed similar transcript levels in mycelia, during sporulation, in zoospores, in cysts and during early infection (Fig.…”

“… Oxathiapiprolin sensitivity of PsORP2 knockout transformants and wild‐type isolates. Mycelial growth was measured after 4 days' growth on 10% V8 agar medium amended with the indicated concentrations of oxathiapiprolin, as described by Miao and colleagues (). All the plates were incubated in darkness at 25°C.…”

“…In this study, Phytophthora sojae was selected as a target pathogen. The P. sojae genome database at the JGI (https://genome.jgi.doe.gov/Physo3/Physo3.home.html) was searched by BlastP using PcORP1 (Miao et al, 2016b) as a query. Only two ORPs were found, which we named PsORP1 (Protein ID: 558498) and PsORP2 (Protein ID: 470921) respectively.…”

“…According to previous studies, the EC 50 and EC 90 values of oxathiapiprolin to Phytophthora species were as low as 10 24 and 10 23 mg ml 21 (Miao et al, 2016a). It is the most effective oomycete inhibitor and has no crossresistance with any other commercial fungicides (Miao et al, 2016b). Until now, the biological functions of ORPs in oomycetes have been unclear.…”