Investigation Of ε-poly-l-lysine Inhibition On Postharvest Blue Mold In Apples And Its Potential Mechanisms

Apples are the most common fruit in daily life.But,they are highly vulnerable to pathogenic fungal infections during storage and transportation.Blue mold caused by Penicillium expansum is one of the most common postharvest diseases of apples and produces severe decay and losses.The inhibition of postharvest blue mold in apples mainly relies on chemical fungicides.Although the control efficacy is good,fungicides have some problems,such as food safety,generation of drug-resistant strains,and pollution of environment.Therefore,developing disease control methods with higher safety and better control effect is of high application value.Epsilon-poly-L-lysine（ε-PL）is a safe,non-toxic,natural food preservative that can be decomposed into essential lysine after entering the human body.ε-PL has been applied in controlling the postharvest diseases of citrus,jujube,tomato,etc.,but is relatively rare in preventing postharvest diseases in apples.In order to explore a safe and efficient control method for postharvest blue mold in apples,this thesis studied the effect ofε-PL on inhibition of postharvest blue mold in apples,revealed the possible mechanism ofε-PL against P.expansum in vitro,and also exposed the physiological and molecular inhibitory mechanism ofε-PL on inhibition of postharvest blue mold in apples through physiology,biochemistry omics approaches and gene function verification.The results provide a new idea and theoretical reference for preventing postharvest blue mold in apples,which has good scientific significance and practical application value.The research outcomes are as follows:（1）Theε-PL treatment on apples effectively controlled the natural decay,damaged inoculation-induced decay and had no adverse effect on the storage quality.The inhibitory effect ofε-PL against P.expansum was positively correlated with its concentration.Above 200 mg/L ofε-PL significantly inhibited spore germination,germ tube elongation,and mycelium growth of P.expansum.Applyingε-PL（600 mg/L）after 24 h of pathogen inoculation had the best control effect on postharvest blue mold in apples.Theε-PL control effect was negatively correlated with the concentration of inoculated P.expansum spores in apples.600 mg/L ofε-PL could significantly inhibit blue mold in apples caused by a low concentration of spores but the inhibitory effect was weak on a high concentration of spores.（2）The inhibitory effect ofε-PL on postharvest blue mold could result from the combined action ofε-PL on the integrity of P.expansum spores and induced activities of apple-resistant enzymes or resistant substances.ε-PL treatment of P.expansum in vitro led to spore reactive oxygen species（ROS）eruption.Excessive ROS can oxidase and damage the spore cell membrane,resulting in the leakage of a number of intracellular substances,thus inhibiting spore germination and mycelial growth.ε-PL treatment also stimulated the systemic resistance reaction of apple tissue.600 mg/Lε-PL induced and improved the activities of polyphenol oxidase（PPO）,peroxidase（POD）,phenylalanine ammonia-lyase（PAL）and chitinase（CHI）enzymes and up-regulated the expression of corresponding genes.ε-PL treatment also induced the secretion of total phenols,flavonoids,lignin and other disease resistance-related substances against blue mold in apples.（3）ε-PL treatment enhanced the apple resistance against blue mold by regulating the metabolisms of respiration,ROS and membrane lipids.The critical enzyme activities of respiratory metabolism were enhanced in apples treated withε-PL,including the rate-limiting enzymes in EMP and TCA pathways and respiratory chain cytochrome oxidase（CCO）,which accelerated the EMP,TCA and respiratory chain processes.Thus,the ATP and ADP content and energy charge（EC）levels of apples were increased,leading to enhanced apple resistance against blue mold.ε-PL treatment increased the antioxidant enzyme activities related to ROS metabolism,scavenged excessive superoxide anions（O₂ˉ^·）,H₂O₂,and reduced oxidative damage to cell membrane caused by ROS.In addition,ε-PL treatment effectively inhibited the activities of phospholipase D（PLD）,lipoxygenase（LOX）,and esterase,which promoted membrane lipid degradation,reduced the degree of phospholipase hydrolysis and protected the membrane integrity.（4）ε-PL treatment could improve the apple resistance against blue mold by regulating the expression of resistance-related genes in the apple metabolic pathway.The RNA sequencing（RNA-Seq）results showed thatε-PL induced ROS signaling,Ca²⁺signaling,PAMP triggered immunity（PTI）,and effector proteins triggered immunity（ETI）,and up-regulated the expression of genes related to ROS synthesis pathway.Thus,ε-PL could promote ROS outbreak and generate a local hypersensitive response（HR）,causing cell consolidation and stomatal closure to improve apple resistance against exogenous stress.ε-PL also up-regulated the expressions of genes related to resistant substances synthesis and encoding antioxidant enzymes POD,PPO and Glutathione S-transferase（GST）in apples,leading to enhanced apple resistance against blue mold decay.In addition,ε-PL could regulate the expression of genes encoding cell strength-related enzymes to improve the strength of apple cell wall and cuticle.Without P.expansum inoculation,ε-PL treatment down-regulated the genes encoding pectin hydrolase and cellulolytic enzymes and up-regulated the genes related to lignin and keratin wax synthesis in apples.When inoculated with P.expansum,ε-PL treatment down-regulated the genes encoding pectin esterase and cellulolytic enzymes of apples and up-regulated the genes associated with pectin synthetase and keratin wax synthesis genes.（5）ε-PL treatment could induce systemic acquired resistance（SAR）in apples.The SAR-induced resistance depends on the salicylic acid（SA）pathway and is closely related to MAPK cascade signal transduction.RNA-seq results showed thatε-PL directly up-regulated expression of defense-related genes such as NPR1,TGA10,and PR1 in the SA pathway and activated SAR without P.expansum inoculation.However,in the case of P.expansum inoculation,ε-PL induced the up-regulation of PAL1 gene and accumulated SA.The SA accumulation stimulated the up-regulation of TGA9,TGA4,and PR1,promoting the expression of pathogenesis-related proteins（PRs）in the whole fruit range and improving apple resistance against blue mold.In addition,MAPK cascades play a pivotal role in the signal transduction of apple resistance induced byε-PL.ε-PL can activate the MAPK cascade pathway of apple cells in response to external stress and the infection of P.expansum.ε-PL treatment induced the transcription activation of resistance genes,phytoalexins（PA）synthesis,cell wall strengthening,HR,ROS burst,stomatal closure,and plant hormone signal transduction in apples.（6）In P.expansum infecting apples,ε-PL inhibited the expressions of antioxidant genes,pathogenicity genes,energy metabolism-related genes,and cell function and cell integrity-related genes.It also inhibited the expression of antioxidant enzymes encoding genes of P.expansum,such as SOD2,CAT,tpc F,hx A,and dao1,which could decrease the ROS scavenging ability of cells.ε-PL also inhibited the expressions of genes pme A,ply A,and bgl G that encode cell wall degrading enzymes（CWDEs）of P.expansum and reduced the ability of P.expansum for degradation cell wall.It also down-regulated the expression of rate-limiting enzymes encoding genes of energy metabolic pathways such as EMP,TCA cycle and oxidative phosphorylation,resulting in insufficient ATP synthesis and weakening the resilience of P.expansum to adversity.In addition,ε-PL inhibited the expression of P.expansum cell membrane lipid synthetase,ergosterol synthetase,and multidrug transporter genes,which hindered the differentiation of conidia and damaged the cell membrane integrity.（7）Based on the homologous recombination technique of Agrobacterium tumefaciens mediated transformation（ATMT）,the hx A knockout mutant（Δhx A）and the complementation strain（Δhx A-C）were successfully constructed.The hx A was identified as the key gene of P.expansum infection in apples through functional verification experiments.This study further explained that the blue mold inhibitory mechanism ofε-PL in postharvest apples is related to the inhibition of hx A expression.Using Cytoscape software,the possible interactions of genes with hx A of P.expansum were analyzed,and a total of 702 genes have a high correlation with hx A（|Cor|>0.8,P<0.05）.Among them,19 genes were highly correlated with hx A and down-regulated expression.These genes were also associated with the antioxidant capacity,infection capacity,ATP synthesis capacity,cell function and integrity of P.expansum.