Phytophthora, a genus of oomycetes, contains many devastating plant pathogens, which cause substantial economic losses worldwide. Recently, CRISPR/Cas9-based genome editing tool was introduced into Phytophthora to delineate the functionality of individual genes. The available selection markers for Phytophthora transformation, however, are limited, which can restrain transgenic manipulation in some cases. We hypothesized that PcMuORP1, an endogenous fungicide resistance gene from P. capsici that confers resistance to the fungicide oxathiapiprolin via an altered target site in the ORP1 protein, could be used as an alternative marker. To test this hypothesis, the gene PcMuORP1 was introduced into the CRISPR/Cas9 system and complementation of a deleted gene in P. capsici was achieved using it as a selection marker. All of the oxathiapiprolin-resistant transformants were confirmed to contain the marker gene, indicating that the positive screening rate was 100%. The novel selection marker could also be used in other representative Phytophthora species including P. sojae and P. litchii, also with 100% positive screening rate. Furthermore, comparative studies indicated that use of PcMuORP1 resulted in a much higher efficiency of screening compared to the conventional selection marker NPT II, especially in P. capsici. Successive subculture and asexual reproduction in the absence of selective pressure were found to result in the loss of the selection marker from the transformants, which indicates that the PcMuORP1 gene would have little long term influence on the fitness of transformants and could be reused as the selection marker in subsequent projects. Thus, we have created an alternative selection marker for Phytophthora transformation by using a fungicide resistance gene, which would accelerate functional studies of genes in these species.

Sensitivities of Phytophthora capsici to fluopicolide were investigated in vitro, with results showing that fluopicolide had strong inhibitory activities on each development stage of P. capsici, in particular on the motility of the zoospore. The potential resistance risk for fluopicolide in P. capsici was evaluated. The baseline sensitivities to fluopicolide of 146 isolates obtained from 28 provinces in China were initially determined, and the 50% inhibition of mycelial growth (EC 50) distribution was a unimodal curve with a mean of 0.17 µg/ml. A series of fluopicolide-resistant mutants of P. capsici were obtained by fungicide adaptation, and their biological traits were determined. Most of the resistant mutants showed similar favorable fitness in mycelial growth, sporangium and zoospore production, cystospore germination, and pathogenicity compared with their sensitive parents, with few exceptions. Additionally, the cross-resistance result indicated that the sensitivity of fluopicolide did not correlate with other oomycete fungicides, apart from fluopimomide (LH-2010A). These results suggest a moderate to high resistance risk of P. capsici to fluopicolide in China.

R034-1 is a new member of the piperidinyl thiazole isoxazoline class of fungicides that shows high activity against most plant-pathogenic oomycetes and could effectively inhibit several developmental stages of Phytophthora capsici. Here, the potential resistance risk for R034-1 was evaluated in P. capsici. The baseline sensitivities of 135 isolates to R034-1 showed a unimodal curve, with a mean EC50 value of 0.004 μg/mL. Twelve resistant mutants were generated by fungicide adaptation and displayed lower fitness compared to parental isolates, which suggests that the resistance risk of P. capsici to R034-1 is low. R034-1 and oxathiapiprolin are structurally related, and resistant isolates display cross-resistance to both compounds, suggesting that these fungicides may target the same oxysterol binding protein. Comparison of PcORP1 genes in the resistant mutants and their parental isolates revealed (N767S, N767I, and G700V) amino acid substitutions in the R034-1 resistant mutant. Causality was functionally validated using site-directed mutagenesis of the target gene using the CRISPR/Cas9 system.

SYP-34773 is a pyrimidinamine derivative and a novel fungicide modified from diflumetorim. This study determined the antimicrobial spectrum of SYP-34773, which showed it could strongly inhibit the growth of some important plant pathogens including fungi and oomycetes. In particular, Phytophthora infestans is an oomycete sensitive to SYP-34773, and the mycelium growth stage was found to be the most sensitive stage, with an EC50 value of 0.2030 μg/mL. At a concentration of 200 μg/mL, SYP-34773 displayed an excellent control efficacy of 69.55% and 81.48% against potato and tomato blight disease caused by P. infestans under field conditions, respectively. Mode of action investigations showed that this fungicide could cause severe ultrastructure damage to the mycelia of P. infestans, inhibit its respiration, and increase the cell membrane permeability of this pathogen. The results of this study could provide useful information for the fungicide registration and application of SYP-34773 as a novel fungicide.

The de novo biosynthesis of sterols is critical for the majority of eukaryotes; however, some organisms lack this pathway, including most oomycetes. Phytophthora spp. are sterol auxotrophic but, remarkably, have retained a few genes encoding enzymes in the sterol biosynthesis pathway. Here, we show that PcDHCR7, a gene in Phytophthora capsici predicted to encode Δ7-sterol reductase, displays multiple functions. When expressed in Saccharomyces cerevisiae , PcDHCR7 showed the Δ7-sterol reductase activity. Knocking out PcDHCR7 in P. capsici resulted in loss of the capacity to transform ergosterol into brassicasterol, which means PcDHCR7 has the Δ7-sterol reductase activity in P. capsici itself . This enables P. capsici to transform sterols recruited from the environment for better use. The biological characteristics of ΔPcDHCR7 transformants were compared with those of the wild-type strain and a PcDHCR7 complemented transformant, and the results showed that PcDHCR7 plays a key role in mycelium development and pathogenicity of zoospores. Further analysis of the transcriptome indicated that the expression of many genes changed in the ΔPcDHCR7 transformant, which involve in different biological processes. It is possible that P. capsici compensates for the defects caused by the loss of PcDHCR7 by remodelling its transcriptome.