| Botrytis cinerea causes gray mold of fruits and vegetables leading to not only production loss and quality decline,but also seriously threating their production safety.Owing to lack of disease-resistant variety,gray mold control mainly depends on emergency control using the chemical mothed.However,the pathogen behaves sporulation,more transposons active and variation rate higher leading to easily developing the resistance to all the common used fungicides.At present,gray mold resistance epidemic becomes a limiting factor which restricts fruit and vegetable industrial development and poses a serious challenge for green prevention of gray mold.Fluazinam,a high-efficiency and broad spectrum fungicide,reportedly exhibits unique uncoupling activity of oxidative phosphorylation in animal mitochondria,and excellent field control efficiency against B.cinerea.But the mode of action of fluazinam to filamentous fungi is elusive.Thus,assessing the resistance risk of B.cienrea to fluazinam and research on the toxicology of fluazinam against B.cinerea are important for scientific guidance of fungicide application,exploiting the new-type fungicide,delaying the generation of field resistance and guaranteeing sound development of fruit and vegetable industry.This study established the sensitivity baseline of B.cinerea to fluazinam,assessed the potential resistance risk of B.cinerea to fluazinam;obtained the genes which were related to fluazinam toxicology via screening the Saccharomyces cerevisiae mutant library,and focused on the roles of the four genes,including cystathione beta synthase BcStr2,histone acetyltransferase complex subunit BcElp4,mitochondria GTPase BcMtg2 and mitochondria transfer protein BcMtp1 in regulating the sensitivity of B.cinerea to fluazinam and their biology roles.The results in detail were presented as follow:(1)Establishment of sensitivity baseline of B.cinerea to fluazinam and the phenotypic characteristic of fluazinam-resistant strain.The study established the sensitivity baseline of B.cinerea to fluazinam,and the average EC50 value of fluazinam to B.cinerea was 0.0207±0.0127 ?g/ml.Compared with curative activity,the protection activity of fluazinam was more excellent.All the fluazinam-resistant mutants were obtained by fungicide domestication.The biological characteristics of the mutants showed decreased in biomass,respiratory rate and glycerol synthetic ability,increased in sensitivity to osmotic stress and ATPase activity,and losed ability of pathogenicity and sporulation.There was no cross-resistance between fluazinam and boscalid.The results indicated the resistance risk of B.cinerea to fluazinam is low and the mechanism of B.cinerea resistant to fluazinam is related to energy metabolism.(2)6032 mutants were screened in the S.cerevisiae mutant library and the fluazinam sensitivity-relative genes were obtained.Based on the biology characteristics of the S.cerevisiae parental strain BY4741,the concentration of fluazinam and culture condition for screening were determined.A total of 133 mutants were obtained showing increased sensitivity to fluazinam and the 106 mutant showing decreased sensitivity to fluazinam.Based on protein sequence coded by the deletion gene in S.cerevisiae mutant,the responding genes were identified in nuclear genome database of B.cinerea using the BLASTP method.The function assay result of the 239 genes showed that the genes involve mainly in energy metabolism,amino acid synthesis,nucleic acid transcription and transmembrane transfer pathways.Methionine plays an important role in protein synthesis in organisms.In mammal mitochondria,glutathione can obviously regulate the toxicological effect of fluazinam.In addition,glutathione affects the methionine synthesis of fungi,and y-cystathionine synthase is a significant regulatory enzyme in methionine synthesis pathway which owns the activity for converting cystathionine into homocysteine which is used to synthesize methionine in fungi.Additionally,fluazinam may act on transport protein of mitochondria reducing the proton gradient difference between the matrix and intermembrane interval,uncouple oxidative phosphorylation inhibiting the ATP synthesis,and consequently disturb the energy balance of mitochondria.The screening results of yeast mutant library indicated that Str2,Elp4 Mtg2 and Mtpl may be involved in the toxicological effect of fluazinam.In S.cerevisiae,Str2 coding ?-cystathionine synthase directly involves in the cystathionine synthesis,Elp4 coding a subunit of histone acetyltransferase complex regulating tRNA transport and ATP utilize,Mtg2 coding GTPase regulating ribosome assembly,nucleic acid transcription and energy balance of mitochondria,and Mtpl coding intermembrane transport protein of mitochondria having the activity of transmembrane transport which can transport various biochemical substances.BcStr2?BcElp4?BcMtg2 and BcMtpl in B.cinerea were identified,and the roles of which in the sensitivity to fluazinam and their function in the process of growth and development of B.cinerea were determined.(3)Cystathione beta synthase coding gene BcStr2 regulated the sensitivity of B.cinerea to fluazinam and the vegetative development of B.cinerea.S.cerevisiae Str2 codes a cystathione beta synthase which is involved in methionine biosynthesis process,and the methionine plays important role in protein synthesis and various biochemical pathway in eukaryotes and prokaryotes.The screening results of S.cerevisiae mutant library indicated that the yeast ?Str2 mutant exhibited increased sensitivity to fluazinam,and then we identified the Str2 homologous gene BcStr2 in B.cinerea.Under the fluazinam-treatment condition,the expression level of BcStr2 obviously down-regulated in B.cinerea.The sensitivity of the BcStr2 deletion mutant to fluazinam obviously increased,and the BcStr2 mutant was unable to grow on minimal medium(MM).Supplementation with Met or homocysteine,but not cysteine or glutathione,rescued the defect in mycelial growth of the BcStr2 deletion mutant.The mutant exhibited decreased conidiation and impaired sclerotia development,and the conidia were unable to germinate in water-agar medium.Additionally,the BcStr2 mutant exhibited increased sensitivity to osmotic and oxidative stresses and cell wall-damaging stress agents.The mutant exhibited dramatically decreased pathogenicity on various host plant tissues.All of the defects were restored by genetic complementation of the mutant with wild-type BcStr2,and the sensitivity defect of yeast ?Str2 mutant to fluazinam was restored by genetic complementation of BcStr2.Taken together,the results indicated that BcStr2 involves in regulating the sensitivity of B.cinerea to fluazinam and plays a critical role in the regulation of various cellular processes in B.cinerea.(4)Histone acetyltransferase complex subunit coding gene BcElp4 regulated the sensitivity of B.cinerea to fluazinam of and the phenotypic characteristic of B.cinerea.The S.cerevisiae elongator complex consisting of the six Elp1-Elp6 proteins has been proposed to participate in transcriptional elongation,ATP utilization and formation of modified wobble uridines in tRNA.The screening results of S.cerevisiae mutant library indicated that the yeast ?Elp4 mutant exhibited increased sensitivity to fluazinam,and then we identified the Elp4 homologous gene BcElp4 in B.cinerea.The expression level of BcElp4 significantly decreased in B.cinerea under the fluazinam-treatment condition,and the BcElp4 deletion mutant exhibited increased sensitivity to fluazinam.The mutant was significantly impaired in vegetative growth,sclerotia formation and melanin biosynthesis,while aerial hyphae of the mutant was increased.Additionally,the mutant exhibited decreased sensitivity to osmotic and oxidative stresses as well as cell way-damaging agents and significantly decreased pathogenicity.All these defects of the BcElp4 deletion mutant were restored by genetic complemention of wild-type BcElp4 gene,and the sensitivity defect of yeast ?Elp4 mutant to fluazinam was restored by genetic complementation of BcElp4.The results indicated that BcElp4 was is related to the sensitivity of B.cinerea to fluazinam and involves in regulating vegetative development,environmental adaptation and pathogenicity in B.cinerea.(5)The deletion of mitochondria hydrolase coding gene BcMtg2 affected the sensitivity of B.cinerea to fluazinam and the biological characteristics of B.cinerea.Mtg2 gene encodes the GTP-binding protein(belonging to the Obg protein family),which has an important function in assembling ribosomal subunits,nucleic acid transcription and energy balance.The screening results of S.cerevisae mutant library indicated that the yeast ?Mtg2 mutant exhibited increased sensitivity to fluazinam,and then we identified the Mtg2 homologous gene BcMtg2 in B.cinerea.The expression level of BcMtg2 obviously down-regulated under the fluazinam-treatment condition.The BcMtg2 deletion mutant exhibited increased sensitivity to fluazinam,defected on sporulation,conidial germination and sclerotial formation.Additionally,the mutant exhibited increased sensitivity to various environmental stresses,and decreased pathogenicity on various host plant tissues.All the defects were recovered in the complemented strain ?BcMtg2C.In addition,the sensitivity defect of the yeast ?Mtg2 mutant to fluazinam was restored by genetic complementation of BcMtg2.The results indicated that BcMtg2 involves in the regulating the sensitivity of B.cinerea to fluazinam and plays an important role in mycelial growth,environmental adaptation and pathogenicity in B.cinerea.(6)Mitochondrial transport proteins coding gene BcMtpl involved in the regulation of the sensitivity of B.cinerea to fluazinam and the vegetative development of B.cinerea.In S.cerevisiae,mitochondrial transport proteins(MTP)plays an important role in transmembrane transport.The screening results of S.cerevisiae mutant library indicated that the yeast ?Mtp1 mutant exhibited increased sensitivity to fluazinam,and then we identified the Mtpl homologous gene BcMtpl in B.cinerea.Treated with fluazinam,the expression level of BcMtp1 was obviously down-regulated.The BcMtpl deletion mutant exhibited decreased sensitivity to fluazinam.The mutant defected on vegetative development,conidiation,sclerotia production,respiratory rate,ATP content,sensitivity to osmotic stress,oxidative stress,and cell wall biogenesis inhibitors and pathogenicity.These defective phenotypes were recovered in the complementation strain ?BcMtp1C.In addition,the sensitivity defect of yeast BY4741 ?Mpt1 mutant to fluazinam was restored by genetic complementation of BcMtp1.Together,these data indicated that BcMtpl is critical for mycelial growth,asexual reproduction,stress tolerance and pathogenicity in B.cinerea.In conclusion,fluazinam has the better inhibitory activity for B.cinerea,and the resistance risk of B.cinera to fluazinam is low.Fluazinam may disturb the function of cystathione beta synthase coded by BcStr2,histone acetyltransferase complex subunit coded by BcElp4,mitochondria GTPase coded by BcMtg2 and mitochondria transfer protein coded by BcMtpl of B.cinerea and then affects the vegetative development of B.cinerea.The results may provide thescientific guidance for applying flazinam to control gray mold,the theoretical support for the analysis of fluazinam toxicology and offer the theoretic reserve for exploiting the new-type fungicide. |
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