Rhizoctonia solani is a widely distributed soilborne plant pathogen, and can cause significant economic losses to crop production. In chemical controls, SYP-14288 is highly effective against plant pathogens, including R. solani. To examine the sensitivity to SYP-14288, 112 R. solani isolates were collected from infected rice plants. An established baseline sensitivity showed that values of effective concentration for 50% growth inhibition (EC50) ranged from 0.0003 to 0.0138 μg/ml, with an average of 0.0055 ± 0.0030 μg/ml. The frequency distribution of the EC50 was unimodal and the range of variation factor (the ratio of maximal over minimal EC50) was 46.03, indicating that all wild-type strains were sensitive to SYP-14288. To examine the risk of fungicide resistance, 20 SYP-14288-resistant mutants were generated on agar plates amended with SYP-14288. Eighteen mutants remained resistant after 10 transfers, and their fitness was significantly different from the parental strain. All of the mutants grew more slowly but showed high virulence to rice plants, though lower than the parental strain. A cross-resistance assay demonstrated that there was a positive correlation between SYP-14288 and fungicides having or not having the same mode of action with SYP-14288, including fluazinam, fentin chloride, fludioxonil, difenoconazole, cyazofamid, chlorothalonil, and 2,4-dinitrophen. This result showed a multidrug resistance induced by SYP-14288, which could be a concern in increasing the spectrum of resistance in R. solani to commonly used fungicides.
BACKGROUND: Fungicide resistance has become a serious problem for different mode of action groups except for uncouplers, which makes their resistance mechanism a hot topic, which until now, has not been well clarified. SYP-14288, a newly developed diarylamine fungicide modeled on fluazinam, has shown good toxicity to both oomycete and fungus by the action of uncoupling. In this research, the resistance of Phytophthora capsici to SYP-14288 was studied to clarify the resistance mechanism of uncouplers.RESULTS: The toxicity tests of resistant strains against SYP-14288 showed multidrug resistance. The high-performance liquid chromatography (HPLC) results showed that resistant strains could efflux the fungicide, and this ability could be inhibited by the efflux pump inhibitor amitriptyline. The target protein of amitriptyline is P-glycoprotein (P-gp), which was overexpressed in resistant strains. Three products of nitrate reduction of SYP-14288 were detected and determined by HPLC-Q-TOF. Eight cytochrome P450 monooxygenase (P450) proteins were differentially involved in the reduction reaction.CONCLUSION: Both fungicide efflux and detoxification metabolism were involved in the resistance mechanisms of P. capsici to SYP-14288.
The probenazole fungicide is used for controlling rice blast (Magnaporthe grisea) primarily by inducing disease resistance of the plant. To investigate the mechanism of induced plant defense, rice seedlings were treated with probenazole at 15 days post emergence, and non-treated plants were used for the control. The plants were infected with M. grisea 5 days after chemical treatment and incubated in a greenhouse. After 7 days, rice seedlings were sampled. The metabolome of rice seedlings was chemically extracted and analyzed using gas chromatography and mass spectrum (GC-MS). The GC-MS data were processed using analysis of variance (ANOVA), principal component analysis (PCA) and metabolic pathway elucidation. Results showed that probenazole application significantly affected the metabolic profile of rice seedlings, and the effect was proportionally leveraged with the increase of probenazole concentration. Probenazole resulted in a change of 54 metabolites. Salicylic acid, γ-aminobutyrate, shikimate and several other primary metabolites related to plant resistance were significantly up-regulated and some metabolites such as phenylalanine, valine and proline were down-regulated in probenazole-treated seedlings. These results revealed a metabolic pathway of rice seedlings induced by probenazole treatment regarding the resistance to M. grisea infection.
SYP-14288 is a fungicide as an uncoupler of oxidative phosphorylation, which is effective in controlling fungal pathogens like Rhizoctonia solani. To determine whether R. solani can develop SYP-14288 resistance and possibly multi-drug resistance (MDR), an SYP-14288-resistant mutant of R. solani X19-7 was generated from wild-type strain X19, and the mechanism of resistance was studied through metabolic and genetic assays. From metabolites of R. solani treated with SYP-14288, three compounds including M1, M2, and M3 were identified according to UPLC-MS/MS analysis, and M1 accumulated faster than M2 and M3 in X19-7. When X19-7 was treated by glutathione-S-transferase (GST) inhibitor diethyl maleate (DEM) and SYP-14288 together, or by DEM plus one of tested fungicides that have different modes of action, a synergistic activity of resistance occurred, implying that GSTs promoted metabolic resistance against SYP-14288 and therefore led to MDR. By comparing RNA sequences between X19-7 and X19, six cytochrome P450s (P450s) and two GST genes were selected as a target, which showed a higher expression in X19-7 than X19 both before and after the exposure to SYP-14288. Furthermore, heterologous expression of P450 and GST genes in yeast was conducted to confirm genes involved in metabolic resistance. In results, the P450 gene AG1IA_05136 and GST gene AG1IA_07383 were related to fungal resistance to multiple fungicides including SYP-14288, fluazinam, chlorothalonil, and difenoconazole. It was the first report that metabolic resistance of R. solani to uncouplers was associated with P450 and GST genes.
Ethaboxam is a β-tubulin inhibitor registered for the control of oomycete pathogens. The current study was established to determine the ethaboxam sensitivity of the plant pathogen Phytophthora sojae and investigate the potential for the emergence of fungicide resistance. The effective concentration for 50% inhibition (EC50) of 112 Phytophthora sojae isolates exhibited a unimodal distribution with a mean EC50 for ethaboxam of 0.033 µg/ml. Establishing this baseline sensitivity provided critical data for monitoring changes in ethaboxam-sensitivity in field populations. The potential for fungicide resistance was investigated using adaptation on ethaboxam-amended V8 agar, which resulted in the isolation of 20 resistant mutants. An assessment of the biological characteristics of the mutants including mycelial growth, sporulation, germination rate and pathogenicity indicated that the resistance risk in Phytophthora sojae was low to medium with no cross-resistance between ethaboxam and cymoxanil, metalaxyl, flumorph, and oxathiapiprolin being detected. However, positive cross-resistance was found between ethaboxam and zoxamide for Q8L and I258V but negative cross-resistance for C165Y. Further investigation revealed that the ethaboxam-resistant mutants had point mutations at amino acids Q8L, C165Y, or I258V of their β-tubulin protein sequences. CRISPR/Cas9-mediated transformation experiments confirmed that the Q8L, C165Y, or I258V mutations could confer ethaboxam resistance in Phytophthora sojae and that the C165Y mutation induces high levels of resistance. Taken together, the results of the study provide essential data for monitoring the emergence of resistance and resistance management strategies for ethaboxam, as well as for improving the design of novel β-tubulin inhibitors for future development.
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