The Mechanism Of Myrcene Stimulates The Spore Germination Of Penicillium Digitatum On Postharvest Citrus Fruit

Green mold has caused great economic losses to the citrus fruit industry.Studies have shown that the myrcene emitted from the ruptured oil glands in wounded citrus peel tissue can facilitate the infection by promoting the spore germination of Penicillium digitatum.The purpose of this study was to explore the stimulatory effect of myrcene on the spore germination of P.digitatum and its possible mechanism of action,so as to clarify the pathogenicity of myrcene in infection process.This study is significant in exploring the pathogenesis of citrus green mold,promoting the prevention and control of postharvest citrus diseases,and reducing the occurrence of the disease during storage and transportation of citrus fruits.The main results of the study were as follows:(1)The optimal concentration of myrcene that stimulated spore germination of P.digitatum was 0.2 ?L/m L,with the spores germinating at 6 h.The spores of P.digitatum treated with 0.20 ?L/m L myrcene for 6 h and 9 h were collected for transcriptome sequencing.After data quality control,85.47 Gb Clean Data was obtained.Sequencing of the data was conducted for further bioinformatics analysis.A total of 9 447 genes were annotated successfully in the transcriptome of P.digitatum spores,which were annotated into 49 GO classifications in GO analysis,and 26 COG categories were divided in COG analysis.(2)Results of differential gene analysis showed that the glycolysis pathway,citrate cycle,oxidative phosphorylation and amino acid metabolism were significantly affected by myrcene treatment for 6 h and 9 h,and the gene expression was generally up-regulated.By measuring the effects of carbon metabolism and amino acid metabolism related products pyruvate,alanine and taurine on spore germination,the results showed that pyruvate,alanine and taurine could significantly stimulate conidial germination of P.digitatum at the optimum concentrations of 0.2 ?L/m L,0.1g/L and 0.15 g/L,respectively.The effects of respiratory metabolism-related inhibitors iodoacetic acid,malonic acid and sodium phosphate,glutamate transaminase inhibitor chalchalic acid,alanine transaminase and alanine glyoxalate transaminase inhibitor L-cycloserine on spore germination were determined.The results showed that iodoacetic acid,malonic acid and sodium phosphate could inhibit the spore germination of P.digitatum to some extent.However,the spore germination was promoted after treatment with a mixture of inhibitors and myrcene.L-cycloserine could significantly inhibit the spore germination of P.digitatum,but the conidial germination rate was not significantly increased following treatment with a mixture of inhibitors and myrcene,while the conidial germination rate with a mixture of inhibitors and pyruvate was significantly higher than that treated with L-cycloserine.(3)The content of free amino acids in the spore cells of P.digitatum after myrcene treatment was determined by high performance liquid chromatography.The contents of aspartic acid,arginine,alanine,tyrosine,isoleucine,glutamate,threonine and leucine in P.digitatum spores treated with myrcene for 6 h were significantly increased,while the contents of aspartic acid,arginine,alanine,tyrosine,glutamic acid,threonine and methionine in P.digitatum spores treated with myrcene for 9 h were significantly increased.There was a significantly positive correlation between spore germination rate of P.digitatum and pyruvate,arginine,threonine,alanine,and glutamic acid concentrations.Quantitative PCR results showed that myrcene treatment resulted in up-regulation of GPT,gdh A,lta E and gly A genes.By measuring the content of intracellular pyruvate,we found that the content of pyruvate increased significantly after treatment with myrcene,while the content of pyruvate was significantly reduced after treatment with L-cycloserine for 6 h.The results proved that myrcene did not regulate the glycolysis pathway,citrate cycle and pentose phosphate pathway to stimulates the spore germination directly.we speculate that myrcene stimulates the spore germination of P.digitatum related to the production of pyruvate by alanine.Further experiments found that myrcene promoted the spore germination of P.digitatum dependent on the production of pyruvate,and its overall performance was to regulate the up-regulation of GPT,lta E,gly A and gdh A,leading to the production of pyruvate from the alanine,threonine,arginine and glutamic acid,thereby promoting the germination of P.digitatum spores.