Mutations in ORP1 Conferring Oxathiapiprolin Resistance Confirmed by Genome Editing using CRISPR/Cas9 in Phytophthora capsici and P. sojae.

Oxathiapiprolin is a novel fungicide that was recently registered in a number of countries to control plant-pathogenic oomycetes such as Phytophthora capsici. In our previous study, point mutations G770V and G839W in oxysterol binding protein-related protein 1 (ORP1) were detected in oxathiapiprolin-resistant P. capsici isolates (PcORP1). Here, we used the CRISPR/Cas9 system to verify the effects of these two point mutations on P. capsici phenotypes. Transformants containing heterozygous G770V and G839W mutations in PcORP1 showed high levels of oxathiapiprolin resistance. The G770V transformants showed otherwise similar phenotypes compared with the wild-type isolate BYA5, including sporangia and zoospore production, cyst germination, and pathogenicity. However, two independent transformants with heterozygous G839W mutations in PcORP1 could not produce sporangia. Three transformants with an unexpected point mutation in PcORP1 (ΔN837) showed high oxathiapiprolin resistance, and either similar or significantly reduced fitness compared with BYA5. The same deletion (ΔN837) was confirmed to confer oxathiapiprolin resistance in P. sojae by using CRISPR/Cas9. These homozygous P. sojae mutants also showed either similar or strongly reduced fitness compared with the wild-type parent isolate P6497. These results improve our understanding of oxathiapiprolin resistance in Phytophthora spp., and will be useful for the development of novel oxysterol-binding protein homolog inhibitor fungicides.

Pseudoperonospora cubensis in China: Its sensitivity to and control by oxathiapiprolin.

The novel fungicide oxathiapiprolin has potential for the control of downy mildew of cucumber, which is caused by Pseudoperonospora cubensis. In this study, an in vitro bioassay with detached leaves was used to determine the baseline sensitivity to oxathiapiprolin for 77 Ps. cubensis isolates from 11 provinces in China. The baseline sensitivity curve was continuous, and the average EC50 value was 2.23 × 10-4 µg ml-1. In field trials, the control of downy mildew of cucumber was greater with oxathiapiprolin at 20 or 30 g a.i. ha-1 than with dimethomorph at 262.5 g a.i. ha-1. Oxathiapiprolin was taken up by cucumber roots and transported upwards to stems and leaves. The full length of PscORP1, the gene that encodes the target protein of oxathiapiprolin in Ps. cubensis, was sequenced for the first time. Our results suggested that oxathiapiprolin will be an excellent alternative fungicide for control of cucumber downy mildew. However, as Ps. cubensis is a high-risk pathogen, resistance development to oxathiapiprolin should be monitored and managed.