Identification And Analysis Of Patulinproducing Fungi From Kiwifruit And The Study Of Their Control Mechanism

Kiwifruit,with abundant of nutritional compounds,is beneficial to human health.However,kiwifruit,a kind of climacteric fruit,is prone to soft rot and susceptible to the aggressiveness of many kinds of pathogens,causing decrease in fruit quality and economic losses during storage.Researchers have reported that kiwifruit is favorable for P.expansum growth and patulin production,showing potential risks of patulin contamination in kiwifruit.However,the fungal flora distribution and the occurrence of patulin-producing fungi from orchards and processing plant of kiwifruit are lack of investigation.Recently,chemical fungicide is still widely used to control P.expansum,but the extensive use of chemical fungicides have induced the occurrence of resistant pathogen,and resulted in fungicides residue,which is harmful to environment and human health,so the bio-control agents and plant derived natural products are promising alternatives against P.expansum.The present work takes the fungal flora distribution,identification,contamination level,growth and toxin-production features of patulin-producing fungi from kiwifruit orchards and plant as basis.Then this study focuses on the inhibition effect of antagonists and plant derived natural compounds on the isolated fungi—P.expansum.After that,this project investigate the inhibitory mechanism of cinnamaldehyde and citral combination on P.expansum according to biochemistry,molecular biology and transcriptomics methods.Lastly,the effects of cinnamaldehyde and citral fumigation on the induction of antimicrobial and antioxidant ability of kiwifruit are evaluated.The main contents and results of this study were as the follows:?1?Fungal flora and patulin-producing fungi were analyzed from samples of kiwifruit orchards and processing plant,the results indicated that dropped fruits showed more fungal population than that on fresh fruits.Fungal population of sections in processing plant conducted outdoor was higher than indoor by 92.8%,NaClO treatment reduced the fungal population of kiwi slices by 83.18%.Fungal flora comprised of nine genera,Penicillium,Aspergillus,Fusarium,Alternaria,Cladosporium,Cochliobolus,Mucor,Rhizopus,Trichoderma.Aspergillus,with Penicillium and Aspergillius as the predominant ones.Eight patulin-producing fungi were all isolated from Zhouzhi County,therein,five isolates were P.expansum from dropped fruits,two isolates were P.griseofulvum from dropped fruits,and one was P.paneum from soil.All patulin-producing fungi contained two key genes of patulin biosynthesis pathway,6-MSAS and IDH genes.The occurrence of patulin-producing fungi showed the potential risks of patulin contamination in kiwifruits.?2?The growth and patulin-production of three patulin-producing fungi from kiwifruit were evaluated in different media and the results showed that their colony diameter increased approximately linearly,and their patulin continued to increase in KPA and PDA medium,P.paneum grown fastest,followed by P.expansum and P.griseofulvum.The biomass and patulin content of three strains in KJ and PDB medium increased first and then decreased.The growth of the three strains was well fitted using modified Gompertz model and Baranyi and Roberts model?R²?0.95?.Patulin accumulation was better fitted using Luedeking-Piret model in solid medium than liquid medium.The colony diameter and PAT content in KPA medium of P.expansum and P.griseofulvum revealed a significant positive correlation based on Pearson correlation coefficient,and their patulin showed significant migration in KPA medium.After ten days of inoculation of P.expansum in eight different cultivars of fresh kiwi juice at 25°C,P.expansum grown and produced patulin in all juices with the highest as 268.88?g/mL and the lowest as 45.10?g/mL.?3?The antifungal activity of three selected antagonists against P.expansum were evaluated in vitro and in vivo.The results showed that B.subtilis CICC 10034,R.sphaeroides CGMCC 1.2182 and A.tumefaciens CGMCC 1.2554 both inhibited the growth and patulin accumulation of P.expansum,with B.subtilis showed the best efficacy.The growth of P.expansum can be inhibited by antagonists when their cells contacted directly,indicating that the adhesion of antagonists to mycelia,or the nutrition and space competition may be the main reason of antagonism.Simultaneous inoculation of P.expansum and antagonists in apples resulted in a significant decrease in P.expansum quantity,and the survive of antagonists in rotten apple tissues containing patulin suggested that three antagonists have tolerance to patulin.Additionally,the living cells and autoclaved inactivated cells of antagonists could remove patulin directly.?4?The plant derived natural products were evaluated for their inhibitory ability against P.expanusm,and the results showed that protocatechuic acid,coumarin,caffeic acid,hyperin and cinnamon acid showed better inhibitory effect among phenolic compounds,their minimum inhibitory concentration?MIC?and minimum fungicidal concentration?MFC?were more than200 mg/L.Cinnamaldehyde,citral,garlic oil,eugenol and thymol showed better antifungal effect than other three essential oils.The MIC and MFC of cinnamaldehyde,citral,garlic oil were 90,100 mg/L,140,150 mg/L and 60,100 mg/L,respectively.While the MIC and MFC of eugenol and thymol were also?200 mg/L.Then the antifungal effects of dual combinations among cinnamaldehyde,citral and garlic oil were assessed based on Fractional inhibitory concentration index?FICI?,and the result indicated that cinnamaldehyde and citral showed additive effect?FICI=1?.Cin/Cit could inhibit the growth and patulin production simultaneously when P.expanusm was treated at early stage or without large number of mycelial biomass and patulin accumulation.When mycelial biomass and patulin of P.expanusm accumulated a lot,Cin/Cit could reduced the biomass to a certain extent but have little effect on patulin production.?5?The inhibitory effect of Cin/Cit on P.expanusm was evaluated based on cell structure,composition and metabolism etc.,and the result showed that Cin/Cit could inhibit mycelial growth and spore germination of P.expanusm effectively which was related to the dosage and treated time.TEM and SEM revealed that Cin/Cit caused hollow and wrinkle of spore surface,degradation of cellular content,separation of cell membrane and cell wall.FT-IR spectra reflected that Cin/Cit mainly influenced the peptide,protein,phospholipids,nucleic acid,fatty acid etc.Cin/Cit did not influenced the content of glucan,chitin and protein of cell wall significantly.However,Cin/Cit remarkably caused cell membrane damage and cellular proteins leakage;inhibited the ergosterol synthesis,induced unsaturated fatty acid biosynthesis;reduced mitochondrial membrane potential and caused mitochondrial dysfunction.Additionally,Cin/Cit induced oxidative stress on P.expanusm cell,which resulted in that ROS,MDA,and H₂O₂content increased,SOD activity increased and CAT activity declined.Lastly,the volatile metabolites profile of P.expanusm changed significantly after Cin/Cit treatment,and cinnamaldehyde and citral could be metabolized into cinnamic alcohol,geraniol,citronellol and other products through detoxification by P.expanusm under this unfavorable condition.?6?P.expanusm F-WY-12-02 was further subjected to RNA-seq to explain the antifungal mechanism in a molecular scale.11468 genes were obtained from RNA-Seq,11022 genes belonged to known cluster and 445 genes were novel genes.Besides,approximately 79%of the total genes expressed?FPKM>1?,and more than 2400 genes were highly expressed?FPKM>60?.There were 1713 differently expressed genes?DEGs?were identified from control and treatment group,920 DEGs were up-regulated,793 DEGs were down-regulated.GO analysis showed that the up-regulated DEGs were mainly enriched in oxidation-reduction process and oxidoreductase activity,etc.,and the down-regulated genes were mainly enriched in transmembrane transporter activity and ATPase activity coupled to movement of substances.KEGG analysis demonstrated that the up-regulated DEGs were mainly involved in oxidation-reduction process and fatty acid metabolism pathways,etc.,and the down-regulated genes were mainly involved in amino acid metabolism pathway.The DEGs were further subjected to functional analysis and the results showed that considerable alterations in genes expression levels related to stress response,cell integrity,cellular ABC transport system,oxidation-reduction process,patulin biosynthesis were influenced by Cin/Cit.Genes related to translational activity,energy metabolism of cells were down-regulated,genes related to heat shock protein and Inositol phosphate metabolism were up-regulated.Genes involved in chitin synthase,chitinase,glycoside hydrolase were mostly repressed.Genes related to biosynthesis of unsaturated fatty acids,and fatty acid degradation were up-regulated,but the key gene of the ergosterol biosynthesis pathway?ERG6?was repressed.Besides,genes related to ABC transmembrane transporter,amino acid transporter were significantly repressed,and the genes involved in multidrug resistance and multi antimicrobial extrusion protein were down-regulated.Considering the cellular oxidation-reduction process,genes related to SOD and peroxidase were induced to be up-regulated to release the oxidative stress caused by Cin/Cit in P.expansum.The RNA-seq data and the biochemical experimental results basically kept consistent.The whole 15 genes involved in patulin biosynthesis pathway were found in P.expanusm F-WY-12-02,RNA-seq results indicated that 12 genes were repressed after Cin/Cit treatment,changes in expression of other three genes?PatB,PatG,PatL?were not significant.After that,the biochemical test of patulin production of P.expanusm F-WY-12-02 treated by Cin/Cit showed remarkable reduction in patulin content,and the alterations in the 15 genes expression through qRT-PCR analysis kept the similar trend as RNA-seq results.?7?The effect of cinnamaldehyde and citral fumigation on the antimicrobial and antioxidant capability,and other physiological characteristics of stored kiwifruit were evaluated.The result showed that the two compounds could effectively inhibited the microbial growth on the surface of kiwifruit on a certain period,and promoted the accumulation of more phenolics and flavonoids,delayed the loss of vitamin C,reduced the level of MDA,H₂O₂ and·O₂^–,which enhanced the antioxidant activity of kiwifruit.Additionally,cinnamaldehyde and citral decreased the content of reduced sugar,increased the level of soluble protein and hydrolyzed amino acids,delayed the softness of kiwifruit.In general,cinnamaldehyde and citral treatment contributed to delay the senescence of kiwifruit and improve the resistant ability to external stress.