| Cherry tomato is especially popular world wide due to its pleasant taste and its rich in vitamin C,vitamin E,?-carotene,lycopene and flavonoids.It is one of the most consumed agricultural products in the world.As a kind of climacteric fruit,the cherry tomato is thin and juicy,and the fruit is prone to infect fungal diseases during storage and transportation,which cause quality loss and greatly limit the development of the cherry tomato industry.Although chemical synthetic fungicides are the most commonly used for controlling postharvest disease of fruit,they could cause the emergence of drug-resistant strains,environmental pollution and fruit safety issues.Antibacterial lipopeptides are secondary metabolites produced by Bacillus spp..They induce defense responses in plants,and can effectively prevent a variety of postharvest diseases of fruits and vegetables.Our team obtained a strain of Bacillus subtilis fmbJ which has a good inhibitory effect on Rhizopus stolonifer.The main inhibitory ingredient was found to be Bacilllomycin D.Therefore,this paper used Bacilllomycin D as an antifungal substance to explore its antibacterial mechanism on Rhizopus stolonifer and its effect on postharvest diseases of cherry tomatoes.The contents and results are as follows:1.The antifungal substances with antifungal activity against R.stolonifer was separated and purified from the fermentation broth of Bacillus subtilis fmbj,acid precipitation,Sephadex LH-20,organic extraction and preparative high performance liquid chromatography were used.We obtained 3 purified peaks with antifungal activity against Rhizopus stolonifer.LC-MS and NMR showed that its structure was n-C14 Bacillomycin D,iso-C15 Bacillomycin D and iso-C16 Bacillomycin D.2.The mechanismof Bacilllomycin D antimicrobial activity on R.stolonifer examined using scanning electron microscopy(SEM)and measuring cell membrane integrity.The results showed that the MICs of Bacilllomycin D-C14,Bacilllomycin D-C15 and Bacilllomycin D-C16 to R.stolonifer were 64 ?g/mL,64 ?g/mL and 16 ?g/mL,respectively.Bacilllomycin D with longer fatty acid chain(Bacillomyicn D-C16)could more effectively inhibit R.stolonifer mycelium growth and spore germination.Scanning electron microscopy observations showed that Bacillumycin D-C16 may cause mycelial deformation and collapse.Bacillomycin D-C16 disruption of membrane integrity,of R.slolonifer mycelial,resulting in nucleic acids and proteins releave.Meanwhile,Bacillomycin D-C16 could induce mycelia to produce more ROS.The accumulation of ROS can cause membrane lipid peroxidation,which leads to impaired cell function and loss of vitality.3.The effect of Bacillomycin D-C 16 on soft rot of cherry tomato was determined by artificial inoculation with pathogenic fungi.Results indicated that Bacillomycin D-C 16 had a significant direct inhibitory effect on soft rot of cherry tomato.Meanwhile,after the induction time of 36 h,Bacillomycin D-C 16treatment could significantly enhance the resistance of cherry tomato fruits to soft rot,and effectively reduce the incidence of soft rot disease and its losses.Bacillomycin D-C 16 could induce resistance in cherry tomato by activating the expression of crucial defense-related genes(NPR1,PR1,CHI,GLU,PR5,PR10 and WRKY53)and the activities of defense-related enzymes(CHI,GLU,PAL and POD),thus activating comprehensive defense reaction against pathogen invasion.4.Base on RNA-Seq,differentially expressed genes were screened from Bacillomycin D-C 16-treated samples.The results from gene ontology(GO)classification showed that these differentially expressed genes(DEGs)were mainly involved in metabolic processes,cellular process,single-organism process,response to stimulus and so on.The results from Kyoto Encyclopedia of Genes and Genomes(KEGG)pathway classification showed that these DEGs were mainly involved in phenylpropanoid biosynthesis,plant hormone signal transduction and plant-pathogen interaction pathway.Activation of phenylpropanoids biosynthesis pathway was correlated with the increasing production of caffeic acid,ferulic acid,chlorogenic acid and lignin,indicating antifungal metabolites indeed contributed to Bacillomycin D-C 16 enhanced cherry tomato protection.5.The effect of Bacillomycin D combination with chitosan on growth of Rhizopus stolomifer and Botrytis cinerea and on cherry tomato preservation was determined by immersion method.The results indicated that the combination of Bacillomycin D and chitosan showed an additive inhibition on the growth of R.stohmifer and B.cinerea as compared to that of its individualcompound.In addition.the combination of Bacilloinycin D and chitosan reduced the incidence of soft rot and gray mold in cherry tomato,caused by R.siolonifer and B.cinei-ea,respectively.Meanwhile,the combination of Bacillomycin D and chitosan treatment exhibited lower weight loss and higher firmness and higher contents of total soluble solids,titratable acidity and ascorbic acid,compared to those treated with sterile water(control).The kinetics models demonstrated that the shelf life of cherry tomato treated with the combination of Bacillomycin D and chitosan could be extended about 15 days longer than the control. |
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