Inhibition Of The Antimicrobial Peptide Thanatin On The Citrus Pathogen Penicillium Digitatum And Its Mechanism

Penicillium digitatum（P.digitatum）is the most serious infectious fungus in citrus fruit.The green mold disease infected by P.digitatum accounts for more than 90%of all post-harvest losses,causing huge economic losses.At present,traditional chemical fungicides are mainly used to control the P.digitatum on citrus,but the abuse of chemical reagents can easily lead to the formation of drug-resistant strains.Also,chemical pollution may damage the environment and harm human health.As people pay more and more attention to food safety and agricultural production safety,it is urgent to find a new and effective method to control P.digitatum.Antimicrobial peptides（AMPs）are a type of peptides with a wide range of sources and broad-spectrum antimicrobial properties.AMPs have been identified as a potential new class of antibiotics to combat bacterial resistance to conventional drugs.And AMPs are relatively friendly to the human body and the environment.Thanatin is the first insect-derived AMP found with broad-spectrum antimicrobial activity at physiological concentrations.Thanatin can inhibit a variety of pathogens such as gram-positive bacteria,gram-negative bacteria,and filamentous fungi.These excellent features make thanatin become a prospective candidate for traditional chemical fungicides.Some studies have shown that AMPs have application prospects in the field of fruit and vegetable disease control.However,there are still few studies on the application of thanatin to the control of fruit or vegetable diseases,and the antimicrobial mechanism still needs to be explored deeply.In our study,the tests（in vitro and in vivo）were carried out to identify the effect of thanatin on P.digitatum.The gene expression level in P.digitatum after thanatin treatment were analyzed by transcriptomics technology（RNA-seq）.Based on the RNA-seq results,the mechanisms were explored including three aspects including the membrane integrity,the gene information process,and the oxidative stress response of P.digitatum.（1）Thanatin could inhibit P.digitatum in vitro effectively.The longer incubation time and higher concentrations could cause a better antimicrobial effect.Thanatin could significantly inhibit the growth of P.digitatum spores with a MIC value of 2μmol L^-1.The survival rate of spores could effectively reduce with thanatin treatment on MFC value of 4μmol L^-1.The mycelial viability and the ability of mycelium to produce spore were inhibited with thanatin treatment.Moreover,the injury incubation test indicated that the disease rate and lesion diameter of green mold on citrus were controlled effectively with thanatin treatment.And thanatin could not induce citrus fruits to develop resistance to green mold disease.（2）RNA analysis identified 938 differentially expressed genes（DEGs）in P.digitatum,out of which 556 DEGs were upregulated,and 382 DEGs were downregulated.These genes were involved in multiple metabolic pathways and cellular processes.The relative expression levels of 9 DEGs were verified by q RT-PCR,and the result indicated it was consistent with the result of RNA-seq.（3）Thanatin could affect the integrity of the cell membrane of P.digitatum.RNA-seq analysis showed that thanatin could increase the expression level of DEGs involved in the cell wall and cell membrane composition（such as chitin,dextran,sterol,phospholipid,etc.）.The cell membrane permeability of P.digitatum was increased under thanatin treatment observed by a fluorescence microscope.Moreover,thanatin caused cellular organelles leakage and cytoplasm disorganization of P.digitatum by transmission electron microscopy analysis.The extracellular conductivity and leakage of intracellular nucleic acid and protein material were increased when exposed to thanatin treatment.（4）Thanatin could affect the genetic information process of P.digitatum.RNA-seq analysis showed that thanatin inhibited DEGs expression involved in multiple processes such as RNA polymerase,ribosomal biosynthesis,and amino acid metabolism.Moreover,thanatin could reduce the DNA,RNA,and protein content of P.digitatum.We also carried out gel blocking,ultraviolet spectroscopy,and DNA thermal denaturation tests,the results showed that thanatin could directly bind to DNA and RNA,destroy the double helix structure of DNA,and decrease the melting point of DNA.（5）Thanatin could induce oxidative stress in P.digitatum.RNA-seq results showed that the transcription level of DEGs associated with oxidative stress balance processes（peroxisomes,glutathione metabolism,oxidoreductase,and sulfur redox protein）in cells was upregulated under thanatin treatment.And the ROS content of P.digitatum was increased when exposed to thanatin using the DCFH-DA probe method.In summary,this study explored the effect of thanatin on P.digitatum in vitro and in vivo.The RNA-seq technology was used to analyze the effect of thanatin on P.digitatum gene expression.And then,the tests associated with P.digitatum cell membrane integrity,genetic information,and oxidative stress were carried out to explore the antimicrobial mechanism of thanatin on P.digitatum,respectively.This work provided a theoretical reference for thanatin to control citrus green mold disease,and offered a new opportunity to develop strategies of other AMPs in controlling pathogenic fungi during postharvest.