Inhibitory Mechanisms Of Cinnamic Acid On The Growth Of Geotrichum Citri-aurantii

Sour rot is a devastating disease of citrus fruit,and it is also the most difficult to control.Geotrichum citri-aurantii is the main pathogen for sour rot disease of citrus fruit postharvest,which results in its huge economic loss.Chemical control is the important strategy for the control of sour rot disease,but there is a lack of fungicides with better control effects on the market.Thus,there are very necessary for investigating the novel compounds for the control of this fungal and studying their inhibitory mechanisms.Cinnamic acid and cinnamyl alcohol are widely used for the control of pathogenic fungal because of their antiseptic and fungalcidal effects as the main active components of cinnamon.However,the antifungal activity and modes of action of the above two substances against G.citri-aurantii are still unclear.In this study,the antifungal effects of cinnamic acid and cinnamyl alcohol on G.citri-aurantii were comprehensively compared in vitro and in vivo.The damage to the mycelium and spore cells of G.citri-aurantii after cinnamic acid treatment was observed and analyzed by scanning electron microscope（SEM）,transmission electron microscope（TEM）,laser scanning confocal microscope（LSCM）and other techniques;at the same time,its effects on the leakage of cell contents,enzymatic reaction and other physiological levels were determined;Finally,the antifungal molecular mechanism of cinnamic acid on G.citri-aurantii was deeply explored through the combined analysis of transcriptome,proteome and metabolome in order to provide scientific reference for the prevention and control of sour rot disease in citrus fruit postharvest.The main research results are as follow:1.Antifungal effects of cinnamic acid and cinnamyl alcohol on G.citri-aurantiiThe results showed a complete inhibition in the growth of G.citri-aurantii caused by 400 mg L^-1cinnamic acid and 500 mg L^-1cinnamyl alcohol in vitro.The results of antifungal experiment in vivo showed that 4000 mg L^-1cinnamic acid and5000 mg L^-1cinnamyl alcohol had a good inhibitory effect on the growth of G.citri-aurantii and the corresponding incidences of sour rot disease were reduced to38.31%and 30.22%,respectively.In addition,both cinnamic acid and cinnamyl alcohol can significantly inhibit the spore germination and germ tube elongation of G.citri-aurantii.The spore germination rate of G.citri-aurantii was only 2.67%at 4 h after 100 mg L^-1cinnamic acid treatment,while the germ tube length was only 3.33μm at 5 h after 200 mg L^-1cinnamic acid treatment;and when treated with 500 mg L^-1cinnamyl alcohol,the spores did not germinate and the germ tubes did not elongate at4 h.The above results showed that cinnamic acid had the most significant antifungal effect on G.citri-aurantii.2.Effects of cinnamic acid on mycelium morphology,structure and intracellular substances of G.citri-aurantiiSEM and TEM observation results showed that cinnamic acid caused a shrinking and distortion of G.citri-aurantii hyphae and then caused a large amount of leakage and decomposition of intracellular substances.When the concentration of cinnamic acid was 800 mg L^-1,37.72%of G.citri-aurantii spores lost cell membrane integrity.The integrity of the fungus cell membrane was damaged,then the extracellular conductivity was increased,the intracellular osmotic pressure was unbalanced,the permeability of the cell membrane was changed,and substances such as nucleic acids and proteins leaked out of the cell through the damaged cell membrane.Sodium dodecyl sulfate-polycrylamide gel electrophoresis（SDS-PAGE）analysis showed that the number of protein bands were all significantly less,small and macromolecular substances had been leaked out of the cells at 3 h after 200,400 and 800 mg L^-1of cinnamic acid treatments.The results of bicinchoninic acid（BCA）protein detection showed that the content of soluble protein also was decreased significantly.These results all suggested that the cell membrane is the main target of cinnamic acid.3.Effects of cinnamic acid on different enzyme activities in the mycelium of G.citri-aurantiiWhen the concentration of cinnamic acid was 400 mg L^-1,the activity ofβ-1,3-glucanase was decreased continuously,the synthesis and catabolism ofβ-1,3-glucan were unbalanced,and the fungal cell wall was damaged.The activities of SOD and POD were increased significantly,which might cause oxidative stress in the mycelium cells of G.citri-aurantii.For the decreased activity of CAT,the scavenging ability of the H₂O₂also was decreased significantly and then caused the excessive accumulation of H₂O₂.The decreased activity of NAD-MDH and SDH suggested that the TCA pathway of fungal cells were blocked.The activities of Ca²⁺/Mg²⁺-ATP and Na⁺/K⁺-ATPase were decreased suggested that the intracellular ATP content was decreased,while the oxidative phosphorylation pathway was blocked and the cells lost viability.On summary,these results suggested that the cell wall is also one of the targets of cinnamic acid;the enzymatic defense system was damaged and the cells were also damaged by membrane lipid peroxidation;the TCA and oxidative phosphorylation pathways were blocked.4.The effects of cinnamic acid on the related genes,protein expression and metabolites of G.citri-aurantii were detected by multidimensional omicsThe results of RNA-seq analysis showed that the expression levels of chitin synthesis-related genes（TR8159＿c0＿g1 and TR38＿c0＿g1）in the mycelium cells of G.citri-aurantii were inhibited after 200 mg L^-1cinnamic acid treatment,and the expression levels of many genes including TR11432＿c0＿g1,TR13534＿c0＿g1 and TR12600＿c0＿g1 related to cell wall synthesis were down-regulated.Tandem Mass Tag（TMT）labeling combine with LC-MS/MS detected results showed that the glutamine-fructose-6-phosphate amidotransferase protein involved in chitin synthesis and protein glycosylation was down-regulated,and multiple cell wall-related proteins including TR11958＿c0＿g1＿ORF and TR14313＿c0＿g1＿ORF were down-regulated.The expression patterns of genes related to sterol synthesis and energy metabolism were further verified by q RT-PCR assay,and these results were consistent with the results of RNA-seq.UHPLC-Q-TOF/MS results show that decreased metabolic levels of sorbitol,inositol,glutamate and GPL pathways also indicated disruption of cell membrane integrity.The metabolic levels of intracellular pantothenate and succinate were down-regulated,indicating that the TCA pathway was inhibited when fungal cells were stimulated by external substances.It is speculated that cinnamic acid may inhibit the EMP pathway,resulting in the energy supply of amino acids through transamination deamination reactions resulting in the decreased levels of these amino acid metabolites e.g phenylalanine,isoleucine,aspartic acid,leucine and dimethylglycine.On summary,both the cell wall and the cell membrane are the targets of cinnamic acid,and cinnamic acid can lead to the blocking of cellular TCA and EMP pathways.5.Multiple omics combined analysis for the antibacterial molecular mechanism of cinnamic acid on G.citri-aurantiiIn the combined analysis of transcriptome and metabolome,the asparto kinase gene and the phosphoglycerate mutase gene were both up-regulated.The metabolites of aspartic acid,dimethylglycine and tryptophan were all decreased.Significant differences in energy metabolism related to the accumulation of metabolites such asα-D-glucose and 6-phosphate glucose,decreased levels of phenylalanine and succinate metabolites indicated that the EMP pathway of G.citri-aurantii cells was inhibited.In the combined analysis of the proteome and metabolome,the metabolic levels of many urea cycle intermediates were increased,and the metabolic levels of succinate were decreased,indicating that the TCA pathway was inhibited.In the combined analysis of three omics,most genes in the steroid synthesis pathway,such as EGR2,ERG6,SMT1,and FK,were down-regulated,while the LIPA protein was up-regulated,indicating that the steroid synthesis pathway of G.citri-aurantii was blocked after cinnamic acid treatment and cell membranes are destroyed.Additionally,in the oxidative phosphorylation pathway,the MDH-related genes including ND4,ND5 and the cytochrome c-related genes including Cytb and COX1 in mitochondrial complexes I,III and IV were down-regulated,while the differential metabolite succinate in mitochondrial complex II was down-regulated and the ADP metabolite in mitochondrial complex V was up-regulated,suggesting that the oxidative phosphorylation pathway was inhibited.However,through the exclusion of false positives in protein and transcriptomic results from metabolomic results,it is believed that cinnamic acid does not have a substantial impact on the TCA pathway.On summary,after cinnamic acid treatment,the steroid synthesis pathway of G.citri-aurantii was blocked and the cell membrane was damaged;EMP and oxidative phosphorylation were all inhibited.