The Antifungal And Anti-mycotoxin Mechanisms Of Citral And Cinnamaldehyde Against Fusarium Sambucinum

Fusarium sambucinum is an important postharvest fungal pathogen of fruits and vegetables,it could infect potatoes and many other crops.It could not only cause postharvest loss but also produce trichothecenes,inducing potential safety hazard.Citral and cinnamaldehye are common aldehyde constituents of some plant essential oil,and show antifungal activities.However,the antifungal and anti-mycotoxin effects of citral and cinnamaldehyde on F.sambucinum has not yet been elucidated.In this paper,the antifungal activities of citral and cinnamaldehyde against F.sambucinum were studied both in vitro and in vivo.Meanwhile,ultrastructure of spores,accumulation of trichothecenes,conidia cell wall integrity,cell membrane integrity,mitochondrial membrane potential and intracellular reactive oxygen species(ROS)were analyzed.The ergosterol content,malondialdehyde(MDA)content and scavenging enzymes activities were also investigated.The antifungal and anti-mycotoxin mechanism was elucidated with the help of proteomics technology and RT-qPCR method.The results showed as below:1.The minimum inhibitory concentration of citral and cinnamaldehyde against F.sambucinum both were 3 mmol/L,which could obviously inhibit spore germination and colony growth.The percentage of spore germination was reduced by 99.93% and 98.5% after 3 mmol/L treatment,and the colony growth was completely inhibited when the concentration increased to 4 mmol/L.Besides,the lesion diameters of potato slices and tubers also were inhibited,it was reduced by 69.0% and 76.9% after treated with 4 mmol/L citral,and it was reduced by 66.9% and 65.7% after treated with cinnamaldehyde at the same concentration.Meanwhile,the ultrastructural of spores were also changed by citral and cinnamaldehyde,the cell membrane was damaged,the cytoplasm became disorganization and the organelles were destroyed.2.Citral showed inhibiting effect on the accumulation of diacetoxyscirpenol(DAS),the DAS content in vitro and in vivo was reduced by 91.2% and 79.2% when concentration was 3 mmol/L.Further results showed that citral could inhibit the synthesis of DAS,but could not decompose it.On the contrary,cinnamaldehyde could induce the accumulation of DAS.Its content in vitro and in vivo was increased by 3.5 and 1.58 folds after treated with 2 mmol/L cinnamaldehyde.3.Cell membrane was damaged by citral and cinnamaldehyde.The damage percentage of cell membrane was increased by 98.1% and 97.3% when the concentration was 3 mmol/L.The extracellular conductivity was raised by 2.37 and 2.16 folds when treated with 3 mmol/L citral and cinnamaldehyde for 4 h.Meanwhile,the A260 value was also increased by 2.14 and 1.75 folds,the MDA level was increased by 2.3 and 2.0 folds.The ergosterol was also inhibited by citral and cinnamaldehyde,its content was reduced by 63.9% and 67.9% when the concentration was 3 mmol/L.4.The ROS was increased and mitochondrial membrane potential(MMP)was decreased by citral and cinnamaldehyde.The ROS level was increased by 3.3 and 3.1 times when treatment with 3 mmol/L citral and cinnamaldehyde.Besides,the antioxidative enzyme activities were inhibited.Total superoxide dismutase(T-SOD),CuZn superoxide dismutase(CuZn-SOD),catalase(CAT),peroxidase(POD)and glutathione reductase(GR)enzymes activities were decreased by 35.8%,35%,41.3%,38.6% and 35.5% after treated with 3 mmol/L citral.And these enzymes activities were separately decreased by 39.5%,28.7%,48.1%,34.9% and 24.8% when treatment with cinnamaldehyde at the same concentration.5.There were 3512 and 3061 proteins changed after treated with citral and cinnamaldehyde.The KEGG analysis showed that differentially expressed proteins were involved with amino acid,glycan biosynthesis and metabolism,fatty acid biosynthesis,metabolism and degradation,lipid metabolism and energy metabolism.Besides,some differentially expressed proteins were also relevant to carbon metabolism,metabolic pathways,biosynthesis of secondary metabolites,biosynthesis of antibiotics and metabolism of cofactors and vitamins.6.The protein levels of acetyl-CoA C-acetyltransferase,3-hydroxy-3-methyl glutaryl coenzyme A synthase,farnesyl pyrophosphate synthase,squalene synthase,C-14 lanosterol demethylase,C-14 sterol reductase,C-3 sterol dexydrogenase,C-3 ketosterol reductase,C-24 sterol methyltransferase,C-8 sterol isomerase and cytochrome P450 sterol C-22 desaturase in ergosterol pathway were significantly down-regulated in response to citral and cinnamaldehyde treatment,but delta(7)-sterol 5(6)desaturase expression was only inhibited by citral.Meanwhile,SOD1,SOD2,catalase-peroxidase(katG),POD and GR proteins were also down-regulated by citral and cinnamaldehyde,but CAT and glutathione peroxidase(GPx)expression level were only reduced by citral treatment.On the contrary,Cytochrome P450 monooxygenase protein related to DAS synthesis was up-regulated by cinnamaldehyde.RT-qPCR results showed that some differentially expressed genes were not proportionally in transcription and translation level,sometimes even were oppositely.In conclusion,citral and cinnamaldehyde could significantly inhibited the growth and pathogenicity of F.sambucinum,and citral could inhibit the synthesis of DAS at the same time.Besides,citral and cinnamaldehyde could induce the accumulation of ROS,cause lipid peroxidation,inhibit activities of antioxidant enzyme and synthesis of ergosterol,resulting in oxidative stress and cell membrane damage,finally leads to the death of F.sambucinum.And the effects of citral and cinnamaldehyde on fungal growth and DAS synthesis were significantly related with ergosterol pathway,ROS metabolism,and DAS synthesis pathway.