Effect Of Chlorine Dioxide On The Pathogenicity Of Penicillium Digitatum And Its Mechanism

Green mold caused by Penicillium digitatum is the main postharvest disease in citrus fruits.At present,chlorine dioxide（ClO₂）as a green fungicide has been widely used in the preservation of fruits and vegetables.The application of ClO₂in postharvest fruits and vegetables mainly focuses on the preservation and disease resistance of ClO₂-treated fruits.The antimicrobial mechanism of ClO₂mainly focuses on bacteria and viruses.There are few studies on the antifungal effect of ClO₂on postharvest fungal pathogens and its mechanism.In this study,different citrus varieties（kumquats,mandarins,Peru’s oranges,and grapefruits）and the target strain（P.digitatum）were selected.Firstly,the antibacterial effect of ClO₂on P.digitatum on citrus fruits and in vitro was determined.Secondly,the antibacterial mechanism of ClO₂on P.digitatum was explained from the physiological and biochemical level through membrane targeting and analysis of organic acid metabolism.Finally,the antibacterial mechanism of ClO₂on P.digitatum was analyzed from the transcriptological level.The main results are as follows:1.ClO₂ fumigation treatment has a good antifungal effect on P.digitatum.0～250 mg/L of ClO₂could significantly reduce the colony growth of P.digitatum（P<0.05）.0～50 mg/L ClO₂could markedly inhibit the spore germination and germ tube growth of P.digitatum（P<0.05）.With regard to 50 mg/L of ClO₂treatment,no germination of P digitatum was observed during the whole observation period.The results of damage inoculation showed that 200～1800 mg/L of ClO₂significantly inhibited the incidence of green mold on kumquats,mandarins,Peru’s oranges,and grapefruits caused by P.digitatum.ClO₂also actively maintained the physical and chemical qualities,including firmness,mass loss rate,titratable acid,soluble solids,and ascorbic acid,which provided a basis for the application of ClO₂in postharvest storage of citrus.2.ClO₂ can play a membrane-targeted antifungal role by destroying the integrity of the plasma membrane of P.digitatum cells.The results showed that 100and 200 mg/L ClO₂induced early and late apoptosis.The mitochondrial membrane potential decreased by increasing fluorescence intensity（mean 118 to 1225）with 200mg/L ClO₂,the cell viability rate decreased from 96.8%to 6.1%,and the integrity of the plasma membrane was destroyed.Malondialdehyde（MDA）and nucleotide leakage（OD260）were increased,plasma membrane peroxidation and permeability were increased.The morphology of mycelium and mitochondria was changed,such as folds,adhesions,fractures and collapses,and the morphology of plasma membrane was destroyed.3.ClO₂ can reduce the pathogenicity of P.digitatum by inhibiting the synthesis and secretion of citric acid.After treated with ClO₂,the intracellular citric acid content of P.digitatum decreased,and the activities of some enzymes in the tricarboxylic acid cycle,such as succinate dehydrogenase（SDH）,cytoplasmic isocitrate dehydrogenase（ICDHC）and malate dehydrogenase（MDH）decreased.Intracellular ATP content decreased,extracellular ATP content increased,citric acid synthesis and normal energy metabolism were destroyed.At the same time,after inoculation with damaged P.digitatum,the p H of the affected part of citrus fruits decreased by 0.5～1 unit compared with that of healthy fruits,the content of citric acid in fruits increased,the activity of aconitase increased,and the citric acid synthase decreased.However,after ClO₂fumigation,the contents of citric acid in fruits decreased,and the contents of organic acids such as malic acid,fumaric acid and ascorbic acid decreased during storage.The aconitase,citric acid synthase and malate dehydrogenase were not significantly different from those in healthy fruits.These results indicated that ClO₂inhibited the secretion of organic acids by P.digitatum,weakened the effect on the metabolism of organic acids in fruits,and thus reduced the pathogenicity of P.digitatum.4.Transcriptomics analysis revealed significant differences in ClO₂inhibition genes of P.digitatum.ClO₂significantly affected the genes related to cell membrane metabolism and energy metabolism of P.digitatum.The genes of membrane iron osmotic enzyme,exogalacturonase,aldehyde dehydrogenase and chitin synthetase were up-regulated,while the genes of aldehyde dehydrogenase,NADH-cytochrome B5 reductase and phospholipase C PLC-C were down-regulated.Differentiated genes involved in metabolic pathways,cyanoamino acid metabolism,antibiotic biosynthesis,degradation of chloroalkanes and chlorenes,tyrosine metabolism and other 20 major pathways.The expression of differential genes was verified by real-time fluorescence quantitative PCR analysis.The transcriptome data were consistent with the previous results,suggesting that ClO₂could disrupt protein synthesis and enzyme activity by up-regulating or down-regulating the expression of related genes,leading to the blocked metabolic pathway of P.digitatum,thus affecting or inhibiting the growth of P.digitatum.