Study On The Control Effect And Mechanism Of Peptides O₃TR,C₁₂O₃TR And Jelleine-? On Penicillium Digitatum Of Citrus

Green mold caused by Penicillium digitatum is a serious and common infection of citrus fruit during postharvest storage and transportation,which caused enormous economic losses to the citrus industry.At present,the main way to control postharvest infection of citrus fruits is the use of chemical fungicides.However,the long-term use of chemical fungicides posed a threat to human health and the environment,and increased the resistance of pathogenic bacteria.Therefore,the use of chemical fungicides is subject to more and more restrictions.At present,it has become a hot research topic in postharvest field of fruit and vegetable to find substances or ways that are harmless to human health and the environment to replace or reduce the use of chemical fungicides.In recent years,peptides have attracted more and more attention in the pharmaceutical industry,agriculture,food industry and animal husbandry because of their advantages of high antibacterial activity and resistance to drugs.However,there are few studies on the control of postharvest diseases of citrus fruit with peptide.In this study,three peptides O₃TR,C₁₂O₃TR and Jelleine-I were used as experimental materials to investigate the control effect of three peptides against P.digitatum of citrus in vivo and in vitro.In order to explore the commercial application value of the three peptides,the antifungal stability of three peptides were tested.In addition,in order to explore the mechanism of action of the three peptides,the effect of three peptides on P.digitatum cell membrane integrity was explored by observation of fluorescence microscope and determination of extracellular conductivity and nucleic acid material release.Finally,the body response of P.digitatum mycelia treated by C₁₂O₃TR was analyzed by RNA-Seq technique.The main results wre summarized as follows:?1?Peptides O₃TR,C₁₂O₃TR and Jelleine-I could significantly inhibit the growth of P.digitatum and had a strong lethal effect on its spores and hyphae.The minimum inhibitory concentration?MIC?of peptides O₃TR,C₁₂O₃TR and Jelleine-I were 12.5,6.25 and 6.25?mol L^-1,and the minimum fungicidal concentration?MFC?were 50,12.5 and 12.5?mol L^-1,respectively.When the survival rate of P.digitatum mycelia was less than 50%,the concentration of peptides O₃TR,C₁₂O₃TR and Jelleine-I were50,6.25 and 25?mol L^-1,respectively.O₃TR,C₁₂O₃TR and Jelleine-I could effectively control the occurrence of postharvest green mold in citrus fruit under four inoculation methods?peptides mixed with spore suspension for 0 h or 16 h,inoculated with spore suspension after adding peptide solution,inoculated with spore suspension after adding peptide solution?in vivo.The antifungal activity of peptides O₃TR,C₁₂O₃TR and Jelleine-I against P.digitatum were thermostable and acid-resistant,and were not affected by K⁺.The antifungal activity of O₃TR and Jelleine-I decreased in high concentration of Na⁺,but the antifungal activity of C₁₂O₃TR did not change.The antifungal activity of O₃TR and Jelleine-I was easily affected by protease K,trypsin and a-chymotrypsin,and the original antifungal activity was maintained only in low concentration proteases.However,the antifungal activity of C₁₂O₃TR did not change in protease K and trypsin,and only attenuated under the treatment of high concentration of a-chymotrypsin(1 mg mL^-1).?2?Peptides O₃TR,C₁₂O₃TR and Jelleine-I could increased the cell membrane permeability of P.digitatum mycelium and spore.Then,fluorescent SG and PI combinated with intracellular nucleic acid material,and the part of mycelium and spore presented the green and red fluorescence,respectively.The extracellular conductivity and leakage of intracellular nucleic acid material were increased with increasing peptide concentration and processing time.?3?Using RNA-Seq technique,the differential expression of 2562 and 667 genes was screened from the samples of P.digitatum treated with C₁₂O₃TR for 2 and 12 h,and the reliability of the results of RNA-Seq was verified by fluorescence quantitative PCR test.2 h and 12 h metabolic pathway analysis results showed that the difference in C₁₂O₃TR affect P.digitatum body and energy supplies,cell wall polysaccharides chitin synthesis and metabolism,cell membrane lipid and sterols substances such as anabolic related metabolic pathways,and interference P.digitatum genetic material in the cell's transcription,translation and protein modification,the transfer process.In conclusion,this article evaluated the antifungal effect and antifungal mechanism of peptides O₃TR,C₁₂O₃TR and Jelleine-I against P.digitatum from the several aspects,including the inhibit effect of three peptides against P.digitatum in vitro,the control effect of three peptides on postharvest green mold in citrus fruit,the antifungal stability of three peptides against P.digitatum,the effect of three peptides on P.digitatum cell membranes integrity and the analysis of possible antifungal mechanism of C₁₂O₃TR by using RNA-Seq technique.These comprehensive research provided new ideas and theoretical basis for controlling the green mold of citrus fruit.