Inhibitory Effects Of Typical Components In Plant Essential Oils Aganist The Two Postharvest Pathogens Of Citrus Fruit

Invasive fungal infection is the main cause of postharvest citrus fruit decay,resulting in huge economic loss of citrus industry every year.Green mold and acid rot,caused by Penicillium digitatum and Geotrichunm citri-aurantii,respectively,are the most serious postharvest diseases.Synthetic fungicides can effectively control these diseases,but chemical fungicides have the disadvantages of high residual,strong toxic and inducing resistance of pathogens.Previous studies showed that plant essential oils exhibited strong antifungal activities against some postharvest pathogens of vegetables and fruits,with the advantages of safety,high efficiency and no residue,which are promising fungicides.Previously,we have observed that several components of essential oils,such as citral(CI)and cinnamaldehyde(CN),significantly inhibited the growth of P.digitatum and G citri-aurantii,and had good effects on controlling green mold and acid rot,which are promising substances that can be used in biological control of postharvest diseases in citrus.However,the underlying molecular mechanism is barely understood.Therefore,this study aims to analyze the antifungal mechanism of CI and CN against P.digitatum and G.citri-aurantii,respectively,and screen out the essential oils combinations which could apply in postharvest diseases of citrus fruit,in an effort to provide theoretical and technical data for the storage of citrus.The main results were as follows:(1)The antifungal mechanism of CI against P.digitatum myceliaResults showed that CI rapidly entered P.digitatum by its lipotropy,which caused the decrease of lipid and ergosterol contents and the accumulation of MDA and H2O2,resulting in the membrane lipid peroxidation and the disruption of cell membrane integrity.RNA-seq results showed that 1/2 minimum inhibitory concentration(1/2MIC)of CI mainly affected ABC transporters,glycolysis/gluconeogenesis and steroid biosynthesis.PCR results showed that 1/2MIC of CI down-regulated the expression levels of four ergosterol biosynthetic genes(ERG11,ERG6,ERG3,and ERGS).In addition,the expression of ERG11 was always lower than that of the control group,which was observed to the potential target gene in response to CI.iTRAQ results showed that 1/2MIC of CI mainly affected oxidative phosphorylation associating with the production of ROS and ATP.And the activities of mitochondrial complex ? and complex ? were apparently suppressed,whereas mitochondrial complex ?,complex ? and complex V were significantly induced.These changes caused a large accumulation of ROS,the increase of antioxidant enzymes activities,and the decrease of glutathione and intracellular ATP contents and mitochondrial membrane potential.Moreover,exogenous addition of cysteine could delay the accumulation of ROS and membrane damage.These results indicated that CI inhibited the growth of P.digitatum by inhibiting the ergosterol biosynthesis,affecting the oxidative phosphorylation,and inducing the accumulation of ROS.(2)Effects of different components of essential oils on ergosterol metabolism of P.digitatumResults showed that 1/2MIC of citronellal(CT),eugenol(EG)and a-terpineol(TE)could destroy the cell membrane integrity and significantly change the contents of lanosterol and ergosterol,the intermediate metabolites of ergosterol biosynthesis,and the ergosterol-related gene expressions of P.digitatum imazalil-sensitive and resistant strains.Exogenous addition of appropriate ergosterol could decreased the antifungal activities of CT,EG,and TE against P.digitatum,repair the cell membrane damage,restore the ergosterol content,change the intermediate metabolites of ergosterol biosynthesis,and reduce the difference of the expression levels of test genes between the treatment groups and the control group.After 1/2MIC of CT treatment,the lanosterol contents of P.digitatum were increased,while the ergosterol contents were decreased.In addition,the expression of pathway genes(except for ERG6)in sensitive strain and genes ERG5 and ERG26 in resistant strain were inhibited.Among them,ERG11 and ERG26 were the potential key genes of CT affecting ergosterol synthesis of sensitive and resistant strains,respectively.After treated with 1/2MIC of EG,lanosterol content in sensitive strain was increased while that in resistant strain was decreased,and ergosterol contents in P.digitatum were increased.PCR results showed that EG could affect ergosterol biosynthesis by suppressing the expression of the main chain gene ERG4 of P.digitatum,but it induced compensating for the loss by opening the side chain and stimulated the increase of ergosterol contents.After treated with 1/2MIC of TE,the ergosterol contents were both decreased,but the lanosterol content of sensitive strain was increased and the content of resistant strain was decreased.In addition,the expressions of ERG27 in sensitive strain and ERG9 in resistant strain were the key down-regulated genes in reponse to TE.(3)The antifungal mechanism of CN against G.citri-aurantii myceliaResults showed that the extracellular alkaline phosphatase activity of G.citri-aurantii was significantly increased and the fluorescence of cell wall became weaker after 30 min treatment with 1/2MIC of CN,indicating that the permeability and integrity of the cell wall were destroyed.The cell membrane was damaged at 120 min treatment,but the lipid and ergosterol contents were not decreased.In addition,the content of H2O2 did not increased during treatment period,indicating that CN did not cause oxidative stress.RNA-seq results showed that MAPK signaling pathway,starch and sucrose metabolism,amino sugar and nucleotide sugar metabolism were significantly affected after 1/2MIC of CN treatment.PCR results showed that the expression levels of chitin synthesis-related genes(CHS2,chsA,chsB,chsG,and UAP1)and glucan hydrolysis-associated genes(glul and glu2)were inhibited,and the expression level of CHI1 was induced.These results were consistent with the decrease of chitin content.These results showed that CN may exhibit its antifungal activity against G.citri-aurantii by blocking chitin synthesis,accelerating hydrolysis of chitin,and destroying the cell wall integrity.(4)Controlling effects of CN combinate with CI on green mold and acid rotBy comparison,In vitro assays results showed that the combination of CA(CN+CI,v/v:1:4)and CO(CN+CT,v/v:5:16)significantly inhibited the mycelial growth of P.digitatum with MFC of 0.80 and 0.40 ?L/mL,respectively,which could synergistically inhibit the germination of spores,disrupt the formation of cell wall and destroy the cell membrane integrity.Meanwhile,the combination of CA(CN+CI,v/v:1:2)and CO(CN+CT,v/v:5:16)had strong antifungal activities on G.citri-aurantii with MFC of 0.80 and 1.60 ?L/mL,respectively,and these combinations could synergistically inhibit the spore's germination and cell wall formation of G citri-aurantii.In vivo test results showed that wax supplemented with 0.40 ?L/mL and 16.00 ?L/mL of CO had the best preservative effects on Miyagawa fruits inoculated with P.digitatum and G citri-aurantii,respectively,and when the rotting rate of the control reached 100%,the rotting rates of the above treatments were only 38%(5 d)and 53%(8 d),respectively.In addition,these treatments had no negative effects on fruit qualities,such as weight loss rate,coloring index,hardness,pH,total soluble solid,solid acid ratio,and Vc content.This study revealed the mechanism of CI and CN aganist P.digitatum and G.citri-aurantii,respectively,and obtained the essential oils combinations that could significantly reduce the decay of green mold and acid rot,which can provide theoretical data for the study of antifugal meachanisms of essential oils and the development of antifungal agents.