Molecular Basis Of Selective Activity Of Succinate Dehydrogenase Inhibitor Fungicides Against Colletotrichum Spp.

Succinate dehydrogenase inhibitor fungicides(SDHIs),the third fungicide worldwide,have been received extensive attentions owing to its novel structure,high efficient and broad-spectrum.In the early development stage of pesticides,SDHIs were mainly used to control the disease caused by basidiomycetes,but after boscalid development,the SDHIs had broad spectrum activity.In our previous study,the novel SDHIs showed selective activity against different pathogenic fungi and the mechanism of this phenomenon was not clear.The interaction mode between pesticide structure and targetome structure will point out the direction of broad spectral and high effective fungicide development.To determine the selective activity of SDHIs,we selected benzovindiflupyr,isopyrazam,fluxapyroxad,pydiflumetofen,fluopyram and boscalid as the tested fungicides to evaluate their application potential and fungicidal activity.We evaluated the function of SDHA/B/C/D in selective activity of SDHIs and the differential binding affinity between SDHIs and SDHB/C/D was also determined.Additionally,the membrane permeability of the six fungicides was also determined.In this study,we systematically expounded the selective activity of SDHIs in target and non-target mechanism.The main results are as follows:1.The difference in antifungal spectrum for SDHIsTo determine the antifungal spectrum of SDHIs,the inhibitory activity of SDHIs against19 pathogenic fungi was measured.There were no significant differential sensitivity of Colletotrichum spp.to SDHIs.Compared with other fungicides,benzovindiflupyr showed high activity against the mycelial growth,spore germination and germ tube elongation of the eight Colletotrichum spp.with EC₅₀<1.27 mg/L.The inhibitory activity of isopyrazam and fluxapyroxad against Colletotrichum spp.was lower than benzovindiflupyr with EC₅₀values of 3.56-13.54 and 7.09-20.71,>500 and>500,>500 and>500 mg/L.Colletotrichum spp.were inherently resistant to pydiflumetofen,fluopyram and boscalid with EC₅₀>500 mg/L.The in vivo and field experiments also showed benzovindiflupyr and isopyrazam at 180 g a.i./ha can effectively control pepper anthracnose with efficacy higher than 63.37%,which demonstrated that benzovindiflupyr and isopyrazam had high potential for anthracnose disease control.Fluopyram and boscalid showed unsatisfactory activity against Magnaporthe oryzae,Botryosphaeria berengriana,Fusarium graminearum,F.pseudograminis and F.oxysporum,which was similar with Colletotrichum spp..F.graminearum was more sensitive to SDHIs than other Fusarium spp..The six tested SDHIs showed favorable activity against the different growth stages of Botrytis cinerea,Sclerotinia Sclerotiorum,Corynespora cassiicola,Cladosporium fulvum,Monilinia fructicola,and Alternaria Kikuchiana with EC₅₀<7.79 mg/L.The antifungal activity of pyrazole amide was higher than picolinamide and pyridine ethyl benzoamide.2.The positive correlation between antifungal activity and mitochondrial function affected by SDHIsBenzovindiflupyr showed the strongest effect on respiration rate,mitochondrial activity,ATP content and mitochondrial membrane potential.All the treatments,including the controls,exhibited first increased and then decreased respiration over time.The respiration rate of C.gloeosporioides was severely affected by benzovindiflupyr.With elongated treated time,the respiration inhibition rate increased.The similar tendency for respiration was observed in isopyrazam and fluxapyroxad treatments.However,pydiflumetofen,fluopyram and boscalid had little effect on respiration of C.gloeosporioides.Along with the increase of the fungicide treatment time,the mitochondrial activity was decreased.After 12 h treatments,all fungicides decreased the activity of mitochondria relative to the untreated control with reduction rate of 49.95%-57.84%,55.58%-61.53%,48.07%-60.91%,38.41%-8.38%,50.76%-52.47%and 49.15%-57.71%for benzovindiflupyr,isopyrazam,fluxapyroxad,pydiflumetofen,fluopyram and boscalid,respectively.However,no correlation was observed between fungicides treated time and ATP content.Benzovindiflupyr can also significantly decrease the ATP production in C.gloeosporioides.After 6 h fungicide treatments,the mitochondrial membrane potential was determined by flow cytometry as shown in apoptotic cell(Q3).The subpopulation rates of Q3 treated with benzovindiflupyr,isopyrazam,fluxapyroxad,pydiflumetofen,fluopyram and boscalid were88.5%,70.7%,60.6%,51.7%,38.5%and 20.3%,respectively.In addition,the mitochondrial function of B.cinerea was significantly affected by the six tested SDHIs.3.The significant difference in enzyme activity reduced by SDHIsColletotrichum spp.exhibited the conserved domains of SDHB/C/D and placed in a clade with a homolog from Fusarium spp.and M.oryzae,then some conserved amino acid included SDHB-113Q/G,185S,254T,256A;SDHC-69E/G,94F,162W,148Y;SDHD-89D,99I,127T/M,137V,142Y,170S were observed.Meanwhile,the genetic distance for oomycetes and plants or nematode and mammal was close.Compared with other subunits,SDHB was conservative that has 9 different motif models.The expression characteristics of SDHA/B/C/D is an indicator for evaluating the activity of target affected by SDHIs.Results indicated that the expression level of SDHA was not significantly changed after fungicide treatments.The SDHB/C/D genes were abundantly expressed in C.gloeosporioides and B.cinerea after treated with benzovindiflupyr,isopyrazam and fluxapyroxad.Pydiflumetofen,fluopyram and boscalid can lead to the high expression of SDHB/C/D genes in B.cinerea,while their expression level in C.gloeosporioides was not changed dramatically.These results demonstrated that the different expression level of SDHB/C/D was related to the various protein-ligands binding affinity and the penetration ability of fungicides.The in vivo enzyme activity revealed different tendency with the in vitro enzyme activity affected SDHIs,which was consistent with their antifungal activity.The IC₅₀values of the in vitro enzyme activity for benzovindiflupyr,isopyrazam,fluxapyroxad,pydiflumetofen,fluopyram and boscalid were 0.16,0.49,1.21,31.31,22.45,and 16.35?M.The high inhibitory activity of fluxapyroxad for SDH enzyme activity suggesting that the other mechanism was involved in the selective activity of SDHIs except for target pathway.4.The heterologous expression system for Cg SDHA/B/C/DTo evaluate the binding affinity between SDHIs and target protein,we using C.gloeosporioides c DNA as template,the Cg SDHB/C/D genes were successfully cloned,and the recombinant plasmids p ET28A-SDHA,p ET28A-SDHB,p ET28A-SDHC and p ET28A-SDHD were constructed.Then the recombinant plasmids were transformed into Escherichia coli ROSETTA2,and fusion proteins for Cg SDHA/B/D were successfully induced.The Cg SDHC protein was not expressed in ROSETTA2,BL21 and C43 because of its transmembrane structure.Therefore,Cg SDHA/B/C/D genes were constructed in the same plasmid to induce the expression of Cg SDHC and then target strip was not observed.We tried to express the three transmembrane domains using the plasmid with the label of MBP,respectively and the combinatorial protein of 2+3 domains was obtained.These proteins were purified by Ni⁺ion affinity chromatography,but the proteins were not conserved steadily after solvent exchange in microscale thermophoresis(MST)assay.Therefore,MST assay is not suitable for determination the binding affinity between Cg SDHA/B/C/D and SDHIs.In the future,we will try other protein expression systems or DTNB-SH to measure the binding capacity between SDHIs and protein.5.Differential binding affinity between SDHIs and Cg SDHBDeletion of SDHB in C.gloeosporioides lead to decreased sensitivity to benzovindiflupyr(31.99 mg/L),with the exception of isopyrazam(5.61 mg/L),fluxapyroxad(8.06 mg/L),pydiflumetofen(>500 mg/L),fluopyram(>500 mg/L)and boscalid(>500mg/L).No significant difference in EC₅₀values between?Cg SDHB-C and wild type isolates.These results demonstrated that the differential binding affinity between fungicides and SDHB may be the main mechanism for the selective activity of SDHIs.No correct Cg SDHC/D gene knockout transformants was obtained by verifying more than 300transformants,which may be attributed to the transmembrane domains and Cg SDHC/D were regarded as the lethal genes.The mycelial growth rate for?Cg SDHB mutants was decreased relative to wild type strain.The offshoot of hyphal tip for?Cg SDHB mutants increased and the diaphragm of hyphae was not fully developed.Meanwhile,the conidia production and germination in?Cg SDHB mutants were obviously decreased.Furthermore,the SDHB deletion mutant also exhibited satisfactory resistance to osmotic,oxidative stress and cell-wall perturbing agents.6.The different binding mode between SDHIs and Cg SDHB/C/DMolecular dynamics study revealed the various binding affinity between the six tested SDHIs and Cg SDHB/C/D.Among them,benzovindiflupyr associated more tightly with Cg SDHB/C/D than isopyrazam,fluopyroxad,pydiflumetofen,fluopyram and boscalid with binding energy of-18.22,-16.80,-14.40,-10.07,-6.32,and-5.61 kcal/mol,respectively.The main forces promoted the selective bindings of SDHIs to Cg SDHB/C/D from the difference in the electrostatic energy,van der Waals interactions and nonpolar interactions of SDHIs with protein,while polar interactions provided adverse effect.Molecular dynamic model assay indicated that the conformation for Cg SDHB/C/D-benzovindiflupyr was more stable,it can generate hydrogen bonds and hydrophobic interactions with active site in binding pocket.The residue-based free energy decomposition method was used to analysis the selective inhibitory activity of the six tested SDHIs against Colletotrichum spp..The results showed that the binding difference of SDHIs to the residue(B-200P?B-204W?C-76S?C-77I?D-135Y)drive the selectivity of inhibitors toward Colletotrichum spp..In the future,we will use the method of site-directed mutation of amino acid to explore key sites.7.The favorable membrane permeability for benzovindiflupyrThe membrane permeability of pesticides play an important role in their antifungal activity.The six tested SDHIs showed various tendency in penetration ratio for C.gloeosporioides and B.cinerea.The penetration ratio in C.gloeosporioides was benzovindiflupyr>isopyrazam>pydiflumetofen>boscalid>fluopyram>fluxapyroxad.The penetration ratio for all tested fungicides in low concentration was higher than in high concentration.Except for isopyrazam,the penetration ratio of other tested fungicides was increased with the prolonged treated time.The penetration ratio for B.cinerea was benzovindiflupyr>isopyrazam>pydiflumetofen>fluxapyroxad>boscalid>fluopyram.According to dynamic simulation,the penetration rate decreased with the extension of fungicide treatment times in C.gloeosporioides and B.cinerea.