Molecular Mechanism Underlying Difference In Sensitivity Of Fusarium Asiaticum And F. Graminearum To Succinate Dehydrogenase Inhibitors

Fusarium head blight（FHB）caused by Fusarium graminearum species complex（FGSC）is a devastating disease on cereal crops,which occurs in wheat growing areas around the world.The F.asiaticum and F.graminearum are the main pathogens causing wheat FHB in China.Pydiflumetofen,created by Syngenta,is the only succinate dehydrogenase inhibitor（SDHIs）that registered for controlling FHB in wheat.Pydiflumetofen not only has excellent control efficacy on wheat FHB,but also significantly reduce the DON production in wheat grains.Previous study in our lab showed that although the genomes of F.asiaticum and F.graminearum are very similar,the sensitivity of the two fungi to SDHIs is significantly different.The sensitivity of F.graminearum to SDHIs is significantly higher than that of F.asiaticum.Therefore,investigating the molecular mechanism underlying difference in sensitivity of F.asiaticum and F.graminearum to SDHIs is important for the development of anti-resisitance SDHIs,scientific guidance of fungicide application and delaying the development of field resistance.The main results are as follow:（1）Evaluation of the resistance risk of F.asiaticum to pydiflumetofen.In this study,5 pydiflumetofen-resistant mutants were generated by fungicide taming from the wild-type strain F.asiaticum 2021 with a mutation frequency of 0.5%.Sequence results showed that there were three mutation types in the resistant mutants,FaSdhB^H248Y,FaSdhC₁^A64V and FaSdhC₁^R67K.By constructing the replacement mutants,FaSdhB^H248Ywas confirmed caused moderate resistance of F.asiaticum to pydiflumetofen,while FaSdhC₁^A64Vor FaSdhC₁^R67K conferred high resistance of F.asiaticum to this fungicide.Further study showed that the fitness of the resistant mutants exhibited no significant difference with the wild-type strain 2021;the SDH activity and ATP content of the resistant mutants were significantly increased compared with the wild-type strain 2021;FaSdhB^H248Y,FaSdhC₁^A64Vand FaSdhC₁^R67Kmutants exhibited different cross resistance patterns to SDHIs;compared with pydiflumetofen-untreated condition,the relative expression level of FaSdhC₁ of the wild-type strain 2021,FaSdhB^H248Y,FaSdhC₁^A64V and FaSdhC₁^R67K mutants were up-regulated by 967.74,239.30,138.29 and 517.53 folds,respectively,while the relative expression level of FaSdhA,FaSdhB,FaSdhC₂and FaSdhD were changed from 0.78 to 14.04 folds on 5μg/m L pydiflumetofen-untreated condition;Compared with the wild-type strain 2021,the relative expression level of FaSdhC₁ of FaSdhB^H248Y,FaSdhC₁^A64V and FaSdhC₁^R67K mutants were down-regulated by 1.70,2.84 and 2.49 folds on 5μg/m L pydiflumetofen-treated condition.The above results showed that the potential resistance risk of F.asiaticum to pydiflumetofen was estimated at moderate to high level.（2）Investigating the role of FaSdhin regulating the sensitivity of F.asiaticum to SDHIs.In this study,the FaSdhA,FaSdhB,FaSdhC₁,FaSdhC₂and FaSdhD deletion/complementation mutants and GFP/RFP fluorescence labeled mutants were generated.The study showed that FaSdhA,FaSdhB,FaSdhC₁,FaSdhC₂and FaSdhD all localized in mitochondria;the mycelial growth ofΔFaSdhA,ΔFaSdhB andΔFaSdhD was severely impaired,whileΔFaSdhC₁ andΔFaSdhC₂ significantly reduced in mycelial growth,conidiation,DON production,pathogenicity,the SDH activity and ATP content;ΔFaSdhC₁ andΔFaSdhC₂ increased the utilization capacity of non-fermentable carbon sources,while decreased the utilization capacity of fermentable carbon sources;the sensitivity ofΔFaSdhC₁ to oxidative stress was significantly reduced,while the sensitivity ofΔFaSdhC₂to oxidative stress was significantly increased,and the two mutants to osmotic stress was significantly decreased;ΔFaSdhC₁exhibited hypersensitive to SDHIs,but the sensitivity ofΔFaSdhC₂ to boscalid,pydiflumetofen and Y12196 was significantly decreased,and the sensitivity to flubeneteram and thiafluzamide was significantly increased.Compared with pydiflumetofen-untreated condition,the relative expression level of FaSdhA,FaSdhB,FaSdhC₂ and FaSdhD of the wild-type strain 2021were increased 14.00,10.93,14.41 and 6.55 folds,respectively,while the relative expression level of FaSdhC₁ were increased 967.74 folds after treatment 24 h with 5μg/m L pydiflumetofen;Compared with pydiflumetofen-untreated condition,the fluorescence intensity of FaSdhC₁ in conidia,germling and hypha were significantly increased after treatment 24 h with 5μg/m L pydiflumetofen,while the fluorescence intensity of FaSdhA,FaSdhB,FaSdhC₂and FaSdhD exhibited no significant difference at the same conidition.The above results indicated that FaSdhC₁ and FaSdhC₂ have functional differentiation in regulating the sensitivity of F.asiaticum to SDHIs.（3）Evaluation of the resistance risk of F.graminearum to flubeneteram.In this study,4 flubeneteram-resistant mutants were obtained from F.graminearum PH-1 by fungicide domestication with a mutation frequency of 0.4%.Sequencing alignment revealed that there were three mutation types in the resistant mutants:The A to T substitution at the-57nucleotide before the transcription site（ATG）of FgSdhC₁(FgSdhC₁^Pro),FgSdhC₂^T77I and FgSdhC₂^R86C.By constructing the replacement mutants,FgSdhC₁^Pro was confirmed confer high flubeneteram-resistance in F.graminearum,while FgSdhC₂^T77I or FgSdhC₂^R86C confer low flubeneteram-resistance.The fitness of the three genotype replacement mutants was significantly decreased;the SDH activity and ATP production of FgSdhC₁^Pro were not significantly changed,while that of FgSdhC₂^T77I and FgSdhC₂^R86Cwere significantly decreased when compared with the wild-type strain PH-1;FgSdhC₁^Pro,FgSdhC₂^T77Iand FgSdhC₂^R86Cshowed less sensitive to oxidative stress,while FgSdhC₂^T77I and FgSdhC₂^R86C was more sensitive to osmotic stress;FgSdhC₁^Pro,FgSdhC₂^T77I and FgSdhC₂^R86C replacement mutants showed different cross resistance patterns to SDHIs;Under 10μg/m L flubeneteram-treated condition,the relative expression level of FgSdhC₁ of PH-1,FgSdhC₁^Pro,FgSdhC₂^T77I and FgSdhC₂^R86C mutants were up-regulated by 84.28,64.03,52.12 and 107.27 folds,respectively,while the relative expression level of FgSdhA,FgSdhB,FgSdhC₂and FgSdhD were changed from 0.39 to3.78 folds on flubeneteram-untreated condition;On 10μg/m L flubeneteram-treated condition,the relative expression level of FgSdhC₁ of FgSdhC₁^Proand FgSdhC₂^T77Isignificantly decreased 0.74 and 0.23 folds,while the relative expression level of FgSdhC₁ of FgSdhC₂^R86Cwas not significantly changed.The above results suggested that the potential resistance risk of F.graminearum to flubeneteam was estimated at moderate level.（4）Investigating the role of FgSdhin regulating the sensitivity of F.graminearum to SDHIs.In this study,the FgSdhA,FgSdhB,FgSdhC₁,FgSdhC₂and FgSdhD deletion/complementation mutants and GFP/RFP fluorescence labeled mutants were constructed.The results showed that FgSdhA,FgSdhB,FgSdhC₁,FgSdhC₂and FgSdhD all localized in mitochondria;theΔFgSdhC₁ significantly decreased in the mycelial growth,conidiation,DON production,pathogenicity;the SDH activity and ATP production;the mycelial growth ofΔFgSdhA,ΔFgSdhB andΔFgSdhD mutants was severely impaired,while FgSdhC₂ cannot be deleted;the sensitivity ofΔFgSdhC₁ to oxidative stress was significantly decreased,whileΔFgSdhC₁exhibited similar sensitivity to osmotic stress with the wild-type strain PH-1;ΔFgSdhC₁ showed significantly decreased capacity in utilizing non-fermentable sodium citrate and sodium acetate and significantly increased capacity in utilizing fermentable glucose;The sensitivity ofΔFgSdhC₁ to flubeneteram,thiafluzamide and Y12196 was significantly increased,while the sensitivity to pydiflumetofen and boscalid was significantly decreased;Compared with pydiflumetofen-untreated condition,the relative expression level of FgSdhA,FgSdhB,FgSdhC₂ and FgSdhD of PH-1 were increased 15.08,17.05,11.66 and 5.47folds,respectively,while the relative expression level of FgSdhC₁ were increased 659.38folds after treatment 24 h with 5μg/m L pydiflumetofen;Compared with pydiflumetofen-untreated condition,the fluorescence intensity of FgSdhC₁ of conidia,germling and hypha showed no significant difference after treatment 24 h with 5μg/m L pydiflumetofen.The results indicated that FgSdhC₁ play an important role in regulating the mycelial growth,asexual reproduction,pathogenicity and the sensitivity of F.graminearum to SDHIs.（5）Investigating the molecular mechanism underlying differernce in sensitivity of F.asiaticum and F.graminearum to SDHIs.Although the genomes of F.asiaticum and F.graminearum are very similar,there was significant difference in the sensitivity of the two fungi to SDHIs.Amino acid sequence alignment showed that FaSdhA,FaSdhB,FaSdhC₁,FaSdhC₂and FaSdhD had 95.23%,100%,92.30%,98.93%and 98.90%identity with FgSdhA,FgSdhB,FgSdhC₁,FgSdhC₂and FgSdhD.FaSdhA and FgSdhA have 49 different amino acids,and FaSdhA has 15 more amino acids than FgSdhA at N-terminus.FaSdhC₂ had 2 different amino acids with the FgSdhC₂,and FaSdhD had also 2 different amino acids with the FgSdhD.FaSdhC₁ and FgSdhC₁have 26 different amino acids,and FaSdhC₁ has 14 less amino acids than FgSdhC₁ at N-terminus.The 12different amino acids between FaSdhC₁and FgSdhC₁were classified into two categories:there are 4 different amino acids at the N-terminus of between FgSdhC₁ and FaSdhC₁,and 8 different amino acids at the C-terminus of between FgSdhC₁ and FaSdhC₁.The studies showed that FaSdhC₁ and FgSdhC₁ play an important role in regulating the sensitivity of F.asiaticum and F.graminearum to SDHIs,respectively.To investigate whether this difference is caused by the differentiation of SdhC₁ between F.asiaticum and F.graminearum,we constructed the interchanged mutants between FaSdhC₁ and FgSdhC₁（ΔFaSdhC₁::FgSdhC₁,ΔFgSdhC₁::FaSdhC₁）.The results showed that the sentitivities of theΔFaSdhC₁::FgSdhC₁andΔFgSdhC₁::FaSdhC₁to some of the tested SDHIs were only swapped compared with that of their corresponding wild-type strain.To further investigate the roles of the different amino acids between FaSdhC₁ and FgSdhC₁underlying the difference sensitivity of F.asiaticum and F.graminearum to SDHIs,the14 amino acids that FgSdhC₁ more than FaSdhC₁ at the N-terminus,4 different amino acids at the N-terminus and 8 different amino acids at the C-terminusthe were transferred in to F.asiaticum 2021,respectively,and theΔFaSdhC₁::FgSdhC₁/N14aa,ΔFaSdhC₁::FgSdhC₁/N4 andΔFaSdhC₁::FgSdhC₁/C8 were constructed.The results showed the sensitivity ofΔFaSdhC₁::FgSdhC₁/N14aa to SDHIs is significantly lower than that of wild-type strain 2021,while the sensitivity of theΔFaSdhC₁::FgSdhC₁/N4 andΔFaSdhC₁::FgSdhC₁/C8 to pydiflumetofen,boscalid,flubeneteram and Y12196 was significantly increased,but the sensitivity of the two mutants to thifluzamide was significantly reduced when compared with wild-type strain 2021.In conclusion,although FaSdhC₁ and FgSdhC₁ play an important role in regulating the sensitivity of F.asiaticum and F.graminearum to SDHIs,the difference between FaSdhC₁ and FgSdhC₁ is not the main reason that are responsible for the difference in the sensitivity of F.asiaticum and F.graminearum to SDHIs.