Studies On Functions Of Biosynthesis Genes DMSs And GART And Antifungal Mechanism Of Calcium Disodium Edetate Against Penicillium Digitatum

Citrus is the most economically important fruit in the south of our country.During pre-and postharvest periods,citrus fruit are susceptible to fungal pathogens due to the unavoidable wounds,which results in enormous economic losses.Green mold caused by Penicillium digitatum is one of the most destructive fungal diseases in citrus during postharvest stage.Nowadays,chemical control is the most popular strategy to prevent citrus postharvest disease.However,the long-time usage of chemical fungicides brings out a series of environmental and health issues,which urges people to explore safer and friendlier control strategies.Hence,this study was conducted from two aspects.First,defected T-DNA insertion mutants were selected,and the functions of mutant genes were characterized to evaluate whether these genes are potential drug targets.Genes included the gene cluster of DHN melanin biosynthesis（DMSs）,and the purine biosynthesis gene Pdgart.Second,the food additive calcium disodium edetate（CDSE）was selected to detect its antifungal activities against P.digitatum,and its biological and molecular mechanisms of antifungal activity were revealed.The candidate target genes toward CDSE were also forecast via transcriptome sequencing.The major findings were displayed as follows:1.The DMSs gene cluster is responsible for the synthesis of the secondary metabolite DHN melanin in P.digitatum,and the melanin is essential for the UV tolerance of pathogen.The T-DNA of the albino mutant mutant N2130 was inserted into the 5’-UTR of polyketide synthase Pks P/Alb1,which is a member of DMSs gene cluster.The 5deletion mutants of DMSs gene cluster were obtained,includingΔPd Pks P,ΔPd Abr1,ΔPd Arp1,ΔPd Arp2 andΔPd Ayg1.The 5 mutants produced albino,brownish,brown,reddish-brown,and yellow conidia,respectively.Compared with wild-type strain N1,deletion mutants showed similarity phenotypes among hyphae growth,conidiation,resistances to high temperature,stress agents and fungicides,as well as conidial hydrophobicity.However,conidia produced by different mutants showed different levels of hypersensitivity under UV-C irradiation treatment,of which the mutantΔPd Pks P was the most sensitive.In addition,the relative expression levels of DMSs gene cluster in the late period（7-d of incubation）of conidia formation is obviously higher than those in the early period（3-d of incubation）,which indicated the cluster is a downstream pathway of growth and development in P.digitatum.2.The purine biosynthesis gene Pdgart is indispensable for the normal energy metabolism of P.digitatum,and it plays a key role in the growth,development and virulence of pathogen.The T-DNA of the defective mutant the coding region of gene Pdgart,T-DNA which is the a member of de novo purine biosynthesis（DNPB）pathway.The knockout and complementation mutants of gene Pdgart have been generated.Deletion mutantΔPdgart displayed similar defects with strain N2535,while the complementation mutants displayed the same as wild-type strain.The phenotypical defects in mutantΔPdgart were thin mycelia,sharply reduced conidia production,and its failed-developed conidiophore.In addition,the sensitivities towards various stress agents and fungicides of strainΔPdgart were significant differences with wild-type strain.The colony morphology defects of strainΔPdgart can be rescued by the addition of exogenous ATP and AMP.During the germination process,most of genes in the DNPB pathway were upregulated,and the conidial ATP levels were sharply accumulated in strain N1,whereas the germination were acutely delayed and the ATP contents were severely deficient ofΔPdgart conidia.Compared with N1 strain,deletion of Pdgart resulted in vague and broken structure of mitochondria,and the expression levels of genes related to energy metabolism in mitochondria were in disorder.Meanwhile,lacking of gene led to attenuated abilities in secretion of organic acids and host cell wall degradation enzymes,which reduced the virulence of P.digitatum and caused weaken and maceration symptom on citrus fruit.3.CDSE displays nice antifungal activity against P.digitatum,and it may chelate the Mn²⁺to inhibit the pyruvate synthesis and the Mn-SOD activity of pathogen,which results in the suppression of fungal growth and development.The minimum inhibitory and fungicidal concentrations（MIC and MFC）of CDSE against P.digitatum P44 strain both were 50 mmol/L.Compared with water treatment（mock）,sweet orange fruit displayed increasingly reduced disease incidences and diameters with increasing concentrations（1/2–5 MIC）of CDSE treatment.In the field storage trial,the storage qualities of“Newhall”navel orange fruit under CDSE treatment,including loss of weight,titratable acid and total soluble solids contents,exhibited only a little difference in consecutive 90 days compared with chemical fungicides（CF）treatment.The rot rates caused by Penicilium spp.and other pathogens also displayed no difference,but the sour rot rates were increasing in CDSE treated group.Treated with CDSE in different concentration,the defense system of sweet orange fruit inoculated with P44 conidia were stimulated in different ways.Low concentration（MIC）of CDSE enhanced the burst of H₂O₂ and GSH,and inhibited the activities of CAT and CHI in citrus fruit.CHI activity was strengthened in fruit under high concentration（5 MIC）of CDSE,but the activities of PG and antioxidant enzymes,as well as H₂O₂ burst.CDSE destroyed the morphology and structure of P.digitatum.The hyphae under CDSE treatment were twisted and shriveled,and could not develop into normal conidiophores.The number septa of hyphae increased,and the septa and cell wall thickened due to the increased chitin.The structures of mitochondria were destroyed as well.But the integrity of cell wall and cell membrane still kept.The transcriptome analyses indicated that differentially expressed genes of CDSE-treated P44 strain were enriched in carbohydrate metabolism pathway.Combined with the q RT-PCR results,CDSE suppressed P44 strain primarily by targeting the synthesis of pyruvate in glycolysis.In addition,exogenous Mn²⁺could cure the mycelial growth and pyruvate synthesis of P44 strain treated with EDTA-Na₂Ca.This study reports the biological functions of the gene cluster of DHN melanin biosynthesis（DMSs）and the purine biosynthesis gene Pdgart in P.digitatum.The results revealed the mechanism of conidial pigmentation in P.digitatum,as well as the mechanism of growth and pathogenicity regulated by Pdgart.It has been confirmed that gene Pdgart is a candidate target to novel antifungal drugs.In addition,current work evaluates the antifungal activities of CDSE against citrus green mold both in vitro/vivo and field,and explored the mechanism of CDSE promoting fruit resistance and suppressing fungi virulence,as well as forecasted the potential target to CDSE,which confirms CDSE is a safer and eco-friendlier preservative agent to citrus fruit.