Resistance to the DMI fungicide mefentrifluconazole in Monilinia fructicola: risk assessment and resistance basis analysis.

BACKGROUND: Brown rot disease, caused by Monilinia fructicola, poses a significant challenge to peach production in China. The efficacy of mefentrifluconazole, a new triazole fungicide, in controlling brown rot in peaches has been remarkable. However, the resistance risk and mechanism associated with this fungicide remain unclear. This study was designed to assess the resistance risk of M. fructicola to mefentrifluconazole and reveal the potential resistance mechanism. RESULTS: The mean median effective concentration (EC50 ) of 101 M. fructicola isolates to mefentrifluconazole was 0.003 µg mL-1 , and the sensitivity exhibited a unimodal distribution. Seven mefentrifluconazole-resistant mutants were generated from three parental isolates in the laboratory through fungicide adaption. The biological characteristics of the resistant mutants revealed that three of them exhibited enhanced survival fitness compared to the parental isolates, whereas the remaining four mutants displayed reduced survival fitness. Mefentrifluconazole showed strong positive cross-resistance with fenbuconazole, whereas no cross-resistance was observed with pyrimethanil, procymidone or pydiflumetofen. No overexpression of MfCYP51 gene was detected in the resistant mutants. Multiple sequence alignment revealed that three resistant mutants (MXSB2-2, Mf12-1 and Mf12-2) had a point mutation (G461S) in MfCYP51 protein. Molecular docking techniques confirmed the contribution of this point mutation to mefentrifluconazole resistance. CONCLUSION: The risk of M. fructicola developing resistance to mefentrifluconazole is relatively low-to-medium and point mutation G461S in MfCYP51 could confer mefentrifluconazole resistance in M. fructicola. This study provided essential data for monitoring the emergence of resistance and developing resistance management strategies for mefentrifluconazole. © 2023 Society of Chemical Industry.

Resistance Risk Assessment for the New OSBP Inhibitor Y18501 in Pseudoperonospora cubensis and Point Mutations (G705V, L798W, and I812F) in PscORP1 that Confer Resistance.

Y18501 is a new oxysterol-binding protein inhibitor (OSBPI) that shows strong inhibitory activity against Pseudoperonospora cubensis. In this study, the sensitivities of 159 Ps. cubensis isolates to Y18501 were determined, with EC50 values ranging from 0.001 to 11.785 µg/mL, indicating that a Y18501-resistant subpopulation has appeared in the field. Ten Y18501-resistant mutants were obtained by fungicide adaptation and displayed fitness equal to or stronger than their parental isolates, which suggests that the resistance risk of Ps. cubensis to Y18501 is high. The consecutive applications of Y18501 in the field resulted in the rapid resistance of Ps. cubensis and decreased control efficacy of cucumber downy mildew (CDM), which could be alleviated by compounding with mancozeb. A positive cross-resistance was detected between Y18501 and oxathiapiprolin. The amino acid substitutions G705V, L798W, and I812F in PscORP1 conferred resistance to Y18501 in Ps. cubensis, which was validated by molecular docking and molecular dynamics simulations.

Resistance to pydiflumetofen in Botrytis cinerea: risk assessment and detection of point mutations in sdh genes that confer resistance.

BACKGROUND: Gray mold caused by Botrytis cinerea Pers. is one of the most significant airborne diseases. It can infest a wide range of crops, causing significant losses in yield and quality worldwide. Pydiflumetofen, a new generation succinate dehydrogenase inhibitor (SDHI), is currently being registered in China to control gray mold in a variety of crops. The baseline sensitivity, resistance risk, and resistance mechanism of Botrytis cinerea to pydiflumetofen were assessed in this study. RESULTS: A total of 138 strains of B. cinerea from 10 different regions were tested for their sensitivity to pydiflumetofen, and the mean EC50 value was 0.0056 µg mL-1 . Eight mutants were obtained by fungicide adaption from five sensitive parental isolates, and the resistance factor (RF) ranged from 51 to 135. The mutants exhibited strong adaptive traits in conidial production, conidial germination, and pathogenicity. Positive cross-resistance was only observed between other SDHIs (i.e. boscalid, fluopyram, and isopyrazam). Two different types of pydiflumetofen-resistant mutants were identified: point mutation P225L in sdhB and double mutation G85A and I93V in sdhC. The in vivo control efficacy of pydiflumetofen on the resistant mutants carrying P225L in sdhB as well as G85A and I93V in sdhC was significantly decreased to 52.62% and 32.27%, respectively. CONCLUSION: The fitness was significantly higher for all pydiflumetofen-resistant mutants than the corresponding parental. Two types of point mutations, sdhB-P225L and sdhC-G85A and I93V, might confer resistance to pydiflumetofen in B. cinerea. A precautionary resistance management strategy should be implemented. © 2021 Society of Chemical Industry.

Resistance assessment of pyraoxystrobin in Magnaporthe oryzae and the detection of a point mutation in cyt b that confers resistance.

Pyraoxystrobin is a new QoI fungicide developed in China. The present study was aimed at determining the baseline sensitivity of M. oryzae to pyraoxystrobin and investigating the potential resistance risk and resistance mechanism of pyraoxystrobin in M. oryzae. The results showed that the mean EC50 of 109 M. oryzae isolates to pyraoxystrobin was 0.0094 µg/mL and the sensitivity exhibited a unimodal distribution. The established baseline sensitivity could provide critical data for monitoring sensitivity changes of M. oryzae to pyraoxystrobin in rice fields. The potential resistance risk was assessed by investigating the biological characteristics of the resistant mutants obtained by fungicide adaptation. The results indicated that the resistance risk of pyraoxystrobin in M. oryzae was medium to high with positive cross-resistance between pyraoxystrobin and azoxystrobin, but without cross resistance between pyraoxystrobin and carbendazim, isoprothiolane, and prochloraz. Further investigation revealed that the pyraoxystrobin-resistant mutants had a G143S mutation in the cyt b protein. Molecular docking confirmed that the G143S substitution conferred high resistance to pyraoxystrobin in M. oryzae. Collectively, the results of this study provided essential data for monitoring the emergence of resistance and developing resistance management strategies for pyraoxystrobin.

Survival Cost and Diverse Molecular Mechanisms of Magnaporthe oryzae Isolate Resistance to Epoxiconazole.

Rice blast caused by Magnaporthe oryzae is one of the most destructive diseases on rice worldwide. Epoxiconazole is a 14α-demethylation inhibitor (DMI) with excellent control on rice blast; to date, no resistant isolates have been observed in the field. Four mutants resistant to epoxiconazole were generated from three parental isolates via fungicide adaptation. Resistance was stable after 10 weekly consecutive transfers on fungicide-free medium. Three parameters, including growth rate, sporulation in vitro, and aggressiveness, were significantly lower for mutants compared with their parental isolates, with the exception of the low-resistance isolate. Sporulation and aggressiveness were negatively correlated with effective concentration values for 50% inhibition of mycelial growth for parental isolates and mutants (P < 0.05). Cross-resistance was found between epoxiconazole and prochloraz (ρ = 0.863, P = 0.000) or difenoconazole (ρ = 0.861, P = 0.000). The resistance factor for mutants was positively correlated with the relative expression of MoCYP51A in epoxiconazole treatment (r = 0.977, P = 0.02). In addition, two putative amino acid substitutions in MoCYP51A were found in two resistant mutants: Y126F in the high-resistance mutant and I125L in the low-resistance mutant. Mutation Y126F reduced the affinity of MoCYP51A with epoxiconazole, whereas I125L was not in the binding pocket of epoxiconazole. No amino acid change or overexpression in MoCYP51B was found in any of the mutants studied. To our knowledge, this is the first study to report DMI resistance observed in M. oryzae. The survival cost of M. oryzae resistance to epoxiconazole might be the reason why DMI resistance has not yet emerged in field populations worldwide.

Activity and Resistance Assessment of a New OSBP Inhibitor, R034-1, in Phytophthora capsici and the Detection of Point Mutations in PcORP1 that Confer Resistance.

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.

Sensitivity of Different Developmental Stages and Resistance Risk Assessment of Phytophthora capsici to Fluopicolide in China.

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 (EC50) 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.

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

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.