Effects Of ε-Polylysine On Pathogenicity And Toxin Synthesis Of Alternaria Alternata And Its Mechanism

Alternaria alternata（A.alternata）is the most common strain of Alternaria in postharvest fruits and vegetables,which can infect them and cause their spoilage.At the same time,A.alternata can produce a variety of toxins in the metabolic process,including Altenuene（ALT）,tenuazonic acid（Te A）,tentoxin（Ten）,alternariol（AOH）and alternariol monomethyl ether（AME）,which pose a serious threat to animal and human health.As a natural,safe and efficient fungicide,ε-polylysine（ε-PL）has been widely used in the preservation of various foods.At present,the direct antifungal effect ofε-PL on postharvest pathogens has been widely explored,but there is a lack of comprehensive and systematic research on the control ability of various postharvest diseases of fruits and vegetables caused by A.alternata,and there are few studies on the inhibitory effect ofε-PL on A.alternata and its toxin production.In this thesis,several common important fruits and vegetables,such as crown pear,plateau red apple,winter jujube,tomato,green grape,cherry tomato and red prickly pepper were selected,and A.alternata was used as the target strain to study the antibacterial activity ofε-PL against A.alternata in vitro and on different fruits and vegetables.Secondly,the antibacterial mechanism ofε-PL against A.alternata was explained at the physiological and chemical level by membrane targeting and toxin production analysis of A.alternata.Finally,the antibacterial mechanism ofε-PL against A.alternata was analyzed from the glycomics level.The main results are as follows:1.ε-PL treatment had a good antibacterial effect on A.alternata.50～400 mg/Lε-PL could significantly inhibit the colony growth of A.alternata（P<0.05）,and no signs of colony growth were observed when the concentration ofε-PL was greater than800 mg/L.50～800 mg/Lε-PL significantly decreased spore germination rate and germ tube length（P<0.05）.The results of damage inoculation showed that 800～3200 mg/Lε-PL could effectively control the occurrence of diseases caused by A.alternata in crown pear,plateau red apple,winter jujube,tomato,green grape,cherry tomatoe and red prickly pepper.These results provided the basis for the application ofε-PL in the prevention and control of postharvest Alternaria diseases in various fruits and vegetables.2.ε-PL could exert membrane-targeted antibacterial effect by destroying the cell membrane integrity of A.alternata.50～400 mg/Lε-PL could increase the cell membrane permeability of A.alternata,resulting in the increase of extracellular conductivity,p H value and nucleic acid leakage(OD₂₆₀),inducing membrane lipid peroxidation and more malondialdehyde（MDA）production.200～400 mg/Lε-PL could reduce the mitochondrial membrane potential of A.alternata and induce apoptosis.After treatment with 400 mg/Lε-PL,the mortality rate of A.alternata spores was about73%,and the morphology of mycelium and mitochondria changed,showing rough,broken,shriveled and damaged.3.ε-PL treatment could reduce the pathogenicity of A.alternata by inhibiting the toxin production.50 and 200 mg/Lε-PL promoted the toxin production of A.alternata in vitro,while 400 mg/Lε-PL inhibited the production of Altenuene（ALT）,Tentoxin（Ten）and alternariol（AOH）.Treatment with 3200 mg/Lε-PL could significantly reduce the contents of five major toxins（Altenuene（ALT）,tenuazonic acid（Te A）,tentoxin（Ten）,alternariol（AOH）and alternariol monomethyl ether（AME））in crown pear,tomato,cherry tomato and plateau red apple（P<0.05）.The content of five main toxins in winter jujube and green extract did not change significantly.The relative expression levels of psk J,pks A,omt I,aoh R and ACT1 genes were significantly down-regulated by3200 mg/Lε-PL（P<0.05）,which had a negative effect on the synthesis of alternaria toxin,resulting in a decrease in the pathogenicity of A.alternata and reducing its toxicity production in fruits and vegetables.4.N-glycomic analysis revealed the antibacterial mechanism ofε-PL at the molecular level.The treatment with 400 mg/Lε-PL could change the N-glycan structure and relative content of A.alternata,and the relative content of pentasaccharide core structure（M3）was significantly decreased.The results of N-glycoprotein analysis showed that the N-glycoproteins of the differentially expressed modified peptides were mainly enriched in the extracellular and cell membrane regions.The differentially expressed proteins were mainly enriched in the two important biological processes of lipid biosynthetic process and cellular lipid metabolic process.Most of the differential glycopeptides were located proteins involved in the glycosylphosphatidylinositol（GPI）-anchored biosynthesis pathway,which may had an important effect on fungal adhesion,morphological transformation and cell wall synthesis.These results indicated thatε-p L could negatively affect the N-glycosylation modification process of A.alternata,there by reducing its pathogenicity.