| Geotrichum citri-aurantii is a kind of pathogenic fungus that infects fruit and vegetable.It mainly infects fruits with large moisture like grapes and citrus causing acid rot,which lead to fruits epidermal rot,producing white colonies and wounds.It has characteristics of strong infectivity and wide spread,which seriously affects post-harvest quality and commercial value.In this paper,L-menthol which the Peo main functional component was used to inhibit G.citri-aurantii growth,and further more explored molecular mechanism of L-menthol on controlling G.citri-aurantii.The present results provided new insights for exploring fungi inhibition mechanism,and provided theoretical basis for development and application Peo in food safety field.The main results are as follows:1.The inhibitory effect of different concentrations Peo on G.citri-aurantii was investigated by in vitro plate antibacterial experiment.The experimental results showed that Peo could inhibit hypha growth and spore germination of G.citri-aurantii,and the inhibitory effect was positively connect to Peo concentration.The inhibitory rate was reached to 100%in 2.0 L/mL Peo treatment groups.The pathogenicity effect in grape fruit of G.citri-aurantii was investigated,and results showed that Peo could effectively protected grapes suffer from acid rot.while physical evaluation indicated that Peo could significantly enhanced fruit hardness.GC-MS analysis was used to obtain Peo composition list,and four compounds were screened with high proportion and potential antibacterial effect.(L-menthol,caryophyllene,cineole and n-hexane).2.The components inhibitory effects of mycelial growth and spore germination were shown that from strong to weak was L-menthol>caryophyllene>eucalyptol>n-hexane.At the same time,L-menthol had stronger inhibitory effect than Peo.Meanwhile,Peo and L-menthol treatment groups could destroy cell membrane,resulting in sugars content and cell membrane alkaline phosphatase AKP content changes.The spore membrane structure was broken and precipitated particle on surface after L-menthol treatment.At the same time,spores morphology became glued and difficultly disperse.3.The antibacterial mechanism of Peo and L-menthol was further studied.L-menthol could destroy spore membrane,and membrane conductivity was significantly increased by PI fluorescent probe assay.The content of ROS were sharply increased in spores,and antioxidant enzymes activities and genes expression level of synthesize these enzymes were dramatically changed.The genetic material was differently degree degraded in spore by AO staining.The SPLOM model was used to screen correlation induced by L-menthol treatment,and results showed that spore membrane permeability change may lead to spore membrane protein degradation,reactive oxygen species and genetic material contents changed,while spore membrane protein may induce POD enzyme activity changed.4.The results of transcriptome sequencing showed that gene expression level was differently between L-menthol treatment and control groups,and a total of 1704 DEGs were screened.GO functional analysis showed that DEGs were annotated into 2125 functional classes,involving 1436 biological processes,182 cell composition,507 molecular functions.KEGG results showed that DEGs were mainly distributed in two primary pathways of bioinformatics(70 DEGs)and metabolism(356 DEGs),and main secondary pathway was MAPK signaling pathway,tyrosine metabolism and pyruvate metabolism.In DEGs interaction network diagram,77 genes were found with correlation coefficient more than 0.95,and 10 hub genes with the most nodes were obtained which were CIT 1,CDC 21,RAD 27,TOP 2,MLS 1,YDJ 1,CYB 2,RNR 2,POL 30,and SMC 3,respectively.These genes were enriched in glyoxylic acid and dicarboxylic acid metabolism.Results of fluorescence quantitative analysis of hub genes showed that POL 30 was the most obvious changed gene.Molecular docking simulation results showed that L-menthol and POL30 were linked by hydrogen bond at 44 ARG residue,which bond energy 3.67 and reaction range 3.2.The flow cytometry results showed that L-menthol treatment caused cell cycle S phase stagnate and lead to partial cell apoptosis. |
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