Resistance Monitoring Of Corynespora Cassiicola To Pyraclostrobin And Molecular Mechanisms Of Resistant To Two New Fungicides

Cucumber target spot,caused by Corynespora cassiicola,has become a global leaf disease,which severely limits the growth and production of cucumbers and causes huge economic losses.Chemical control is one of the most effective methods to control the disease.However,it is diffcult to effectively manage this disease,because C.cassiicola is easy to mutate and has strong destructiveness.In recent year,many reports had showed that the fungicide resistance is a serious problem in C.cassiicola.Therefore,it is imperative to continuously monitor the development of resistance and introduce new fungicides to control cucumber target spot.Florylpicoxamid is a new picolinamide fungicide and could bind the Qi site of the respiratory electron transport chain complex III,and has excellent control effects against the pathogens that display resistant to Qo Is,DMIs or other fungicides.Mefentrifluconazole is a new type of triazole fungicide has and exhibits excellent systemic conductivity and delayed drug resistance because of a unique isopropanol group.At present,the resistance risk and resistance mechanism of two novel fungicides have not yet been reported in C.cassiicola.This study systematically monitored the sensitivity of C.cassiicola from different areas to pyraclostrobin,and systematically assess and analyse the resistance risk and resistant molecular mechanism of C.cassiicola to florylpicoxamid and mefentrifluconazole.The potential of mefentrifluconazole to manage the fungicide resistance in C.cassiicola was also evaluated by determining the activity of absorption conduction and the synergistic effect mixed with other two fungicides.The main results are as follows:1.The sensitivities of 162 C.cassiicola isolates to pyraclostrobin were determined using mycelial growth method.The mean EC₅₀values were 8.21?g/m L,and ranged from 1.67 to97.88?g/m L.The fitness results showed that the growth rate,spores'production,spores germination,and virulence of resistant mutants was similar or better than those of pyraclostrobin-sensitive C.cassiicola isolate.It indicated that Qo Is resistant C.cassiicola isolates were suitable for excellent in survival.On the same dosage,pyraclostrobin showed decreased control effect against resistant isolates compared with sensitive isolates.Pyraclostrobin showed cross-resistance with trifloxystrobin,and no cross-resistance with prochloraz,difenoconazole and fluopyram.Sequencing results showed that all tested C.cassiicola isolates contained a point mutation G143A in Cyt b protein.Molecular docking results showed that G143A mutation in Cyt b changed the binding mode of pyraclostrobin and the target site.The number of surrounding amino acid residues involved in recognition was reduced,and the hydrophobic interaction was weakened,and its ability to bind to the active center was reduced,which ultimately led to drug resistance.A LAMP detection method for the G143A point mutation of C.cassiicola was established.The LAMP method showed excellent specificity and sensitivity(10 times better than traditional PCR),and the reaction time is only 1 h,so it can be used for high-throughput samples detection in the field.2.The sensitivity of 92 of C.cassiicola isolates from different cities to florylpicoxamid was determinanted.The range of EC₅₀values was from 0.001 to 1.37?g/m L and it was selected to establish a baseline sensitive to florylpicoxamid with a mean EC₅₀value of 0.35?g/m L,which can be used for subsequent field resistance monitoring.Seven florylpicoxamid-resistant mutants were obtained by fungicide adaption,and the resistance level was stable after 10 generations culturing on fungicide-free medium.The fitness results showed most of florylpicoxamid-resistant C.cassiicola isolates were suitable for excellent in survival.The control efficiency of florylpicoxamid(30?g/m L)against two sensitive strains were 86.94%and 94.18%,While the control efficacy gagainst the mutants were43.32%-78.90%.There were no cross-resistance between florylpicoxamid and pyraclostrobin,fluopyram,prochloraz and propineb.3.All the florylpicoxamid-resistance mutants have a C?T point mutation at the 110th nucleotide,which resulted in the A37V point mutation in Cyt b protein.Molecular docking results showed that,the pyridine ring part can be combined near Ala37 to form a better shape match before mutation.The molecular docking binding energy is-3.51 kcal/mol.After Ala37is mutated to Val,the volume of the side chain is significantly increased,causing the conformation of the pyridine ring of florylpicoxamid to reverse and change toward Tyr16,which reduces its affinity with the protein,with molecular docking binding energy is-3.28kcal/mol.It confirmed that the A37V mutation in Cyt b could cause florylpicoxamid resistance in C.cassiicola.The molecular detection methods LAMP and AS-PCR for A37V were also established.They could be used to monitor the resistance of florylpicoxamid caused by point mutation A37V in Cyt b protein of C.cassiicola.4.Mefentrifluconazole showed a slightly better activity against germ tube elongation than mycelial growth,but its activity against spore germination is extremely low.Excellent conduction activity of mefentrifluconazole was showed in cucumber leaves and cucumber acropetal the cross-layer control effect was as high as 89.20%.The sensitivity frequency of102 C.cassiicola isolates is distributed into a single peak distribution,The EC₅₀values of which ranged from 0.15 to 14.77?g/m L,with a mean of 4.77?g/m L,thus it could be used as the sensitivity baseline of C.cassiicola to mefentrifluconazole for future field resistance monitoring.5.To analyze the resistance risk and mechanism of mefentrifluconazole,the C.cassiicola mutants were induced by fungicide adaption and UV irradiation.However,only low-resistant mutants were recovered.The resistance level was relatively stable after 10th cultural on fungicide-free medium.The biological characteristics of the mutants obtained by fungicide adaption showed that the fitness of the mutants is not excellent.There was no cross-resistance between mefentrifluconazole and fluconazole,pyraclostrobin,fluopyram,prochloraz,difenconazole and mancozeb.There was no difference in three CYP51 genes'sequence between of all mutants and the corresponding parental isolate.It was found that the expression of Cc CYP51A and Cc CYP51B of mutants was up-regulated and was enhanced significantly after treatment with mefentrifluconazole.While the expression level of Cc CYP51C did not change significantly.The binding ability of CYP51A,CYP51B and CYP51C protein and mefentrifluconazole were analyzed by molecular docking.Mefentrifluconazole bind strong with Cc CYP51A and Cc CYP51B proteins,and binding energy was-9.80 kcal/mol and-9.89 kcal/mol,respectively.Combining the expression analyses results,Cc CYP51A and Cc CYP51B are main targets of mefentrifluconazole.6.In order to delay the development of resistance,the optimal mixture ratio of mefentrifluconazole and isopyrazam or prochloraz were screened at a mixing ratio of 10%to90%.The result showed the optimal ratios were 1:1 and 7:3 for two different combinations respectively.The sensitivity of 10 C.cassiicola isolates to the binary complex were measured,and the synergistic effect was displayed in all experiments.The results of the pot experiment showed that the control effect of the binary compound(Mef:Iso=1:1)was significantly higher than mefentrifluconazole for protective activity,and significantly higher than isopyrazam for the curative activity.The control effect of the binary compound(Mef:Pro=7:3)at 150 and 200?g/m L were significantly higher than that of single mefentrifluconazole or prochloraz.The control effect of the binary compound(Mef:Pro=7:3)at the dosage of 100?g/m L was 77.97%,which showed no significant difference with that of single mefentrifluconazole(150?g/m L)or prochloraz(150?g/m L)treatment.These binary compound,Mef&Iso or Mef&Pro,could be further developed and applied in future to manage the fungicide resistance problem in C.cassiicola.