Resistance Monitoring Of Corynespora Cassiicola From Shandong Province To SDHI Fungicides And Screening Of Effective Fungicides Against Cucumber Corynespora Leaf Spot

As a destructive plant fungal pathogen,Corynespora cassiicola（Berk.&Curt.）Wei seriously affects the quality and quantity of cucumber.Chemical control is one of the most effective measure against Corynespora leaf spot（CLS）.Succinate dehydrogenase inhibitors（SDHI）fungicides is the mainstream fungicides registered for cucumber disease control,including fluopyram and fluxapyroxad for the management of CLS,boscalid for gray mold and isopyrazam for mildew powdery.Gray mold and mildew powdery usually developed alternative or simultaneous with CLS.With the consecutive and frequent usage of SDHI fungicides,the problem of fungicide resistance development become increasingly prominent.In this study,sensitivities of C.cassiicola isolated from different regions in Shandong province to boscalid,fluopyram,fluxapyroxad and isopyrazam were tested.The resistance mechanism in C.cassiicola was analyzed,and molecular docking studies were performed to compare the binding features and energies between chemicals and succinate dehydrogenase（SDH）of different phenotypes.Additionally,several parameters were used to evaluate the fitness costs of resistance mutations.The baseline sensitivity of C.cassiicola to triazole fungicide metconazole and bis-guazatine fungicide iminoctadine tris（albesilate）was established according to the mycelial growth,and the efficacies of metconazole and iminoctadine tris（albesilate）were determined in vivo and in the field.The main results of this study were as followed:1.The EC₅₀ value range of 357 C.cassiicola isolates for boscalid,fluopyram,fluxapyroxad and isopyrazam were 0.11->100,0.19-96.17,0.02-8.77 and 0.11-27.97μg/m L,respectively.The resistance frequency for these four SDHI fungicides were 79.83%,78.43%,83.19%and 49.86%,respectively.The findings of the multiple sequence alignment between amino acid sequence of sdh B,sdh C and sdh D of sensitive and resistant C.cassiicola suggested that totally eight single and three dual amino acid alternations were detected to be linked with SDHI fungicides resistance,including H278Y/L and I280V in sdh B;S73P,N75S and H134R in sdh C;D95E and G109V in sdh D;I280V&S73P,I280V&N75S and S73P&N75S.The sensitivity shifts of different amino acid mutants to these four SDHI fungicides differed.Compared with wild type isolates,no significant fitness costs were observed with resistant phenotype in mycelial growth,spore germination,osmotic sensitivity,oxidative stress sensitivity,sporulation and aggressiveness,except for the H134R phenotype that had fitness superiority in temperature tolerance.The results of molecular docking studies demonstrated that the missense mutation influenced the crystal structure,and then interfered the binding features between chemicals and target protein,associated with the in vitro results.2.The sensitivity of 121 C.cassiicola strains to metconazole was determined based on mycelial growth,and the frequency distribution of EC₅₀ were unimodal,with mean of 0.78±0.41μg/m L.The sensitivity of C.cassiicola was not significantly correlated with that to difenoconazole,tebuconazole,myclobutanil and hexaconazole（P>0.05）.The in vivo assays performed on detached cucumber leaves and potted seedlings suggested that the curative efficacy（87.19%）of metconazole was higher than the protective efficacy（73.62%）.Additionally,the effect exhibited by 150μg/m L difenoconazole was lower than 50μg/m L metconazole.In field trials conducted in 2017 and 2018,the efficacies provided by metconazole at the dosage of 150 g a.i./ha were 77.87%and 73.29%,respectively.Metconazole significantly reduced the disease index of CLS.The findings above suggested that metconazole had a great potential for CLS control.3.The frequency distribution curve for the sensitivity of C.cassiicola to iminoctadine tris（albesilate）tested mycelial growth was unimodal.The range of EC₅₀ values was from 0.12 to1.21μg/m L,with mean of 0.58±0.27μg/m L.Iminoctadine tris（albesilate）affected the morphological development of C.cassiicola with the increase of the mycelial branching.The increased membrane permeability and malondialdehyde（MDA）content after iminoctadine tris（albesilate）treatment suggested a severe cell membrane damage caused by this fungicide.Furthermore,iminoctadine tris（albesilate）had a great potential to reduce the secondary infection source since it could decrease sporulation density of C.cassiicola significantly at the concentration of 0.4μg/m L,from 2.62×10⁴/cm²to 1.50×10⁴/cm².The in vivo protective and curative efficacies of 120μg/m L iminoctadine tris（albesilate）were 98.37%and 81.17%,respectively.In field trials,the control efficacy of iminoctadine tris（albesilate）at the dose of120 g a.i./ha reached 72.92%and 80.92%in 2017 and 2018,respectively.However,the efficacy of the reference fungicide azoxystrobin at the dose of 250 g a.i./ha was only approximately 50%because of the resistance development among C.cassiicola population in the field.Therefore,iminoctadine tris（albesilate）might play an important role in the management of CLS.