Synergistic Antifungal Effects Of Thymol And Salicylic Acid On Fusarism Solani And Rhizopus Stolonifer And Their Application In Postharvest Preservation Of Tomato

Natural plants extracts have attracted increasing attention,and become the research direction and hot spot of natural preservation due to their lack of environmental pollution,pesticide residues and drug resistance caused by chemical agents.However,the high price of natural extracts results in high cost of use,especially the agricultural products with low added value are difficult to popularize.Tomatoes are rich in nutrition,but with the characteristics of thin skin and juicy.They are vulnerable to mechanical damage and microbial infection during postharvest storage,resulting in extremely high decay and loss rate.Therefore,how to reduce the use of plants extracts on fresh fruits and vegetables such as tomatoes and reduce their cost is an urgent problem to be solved.In this study,eleven natural agents were collected,and their antifungal capacities toward two common fungi,Fusarium solani?CICC 2603=ATCC 36031?and Rhizopus stolonifer?ATCC12939?that result in the postharvest decay of many fruits and vegetables,especially tomatoes were determined.According to the minimal inhibitory concentration?MIC?values and chessboard method,there was only one group,thymol and salicylic acid(S_TSA)showed synergistic effect on both F.solani and R.stolonifer.Their MIC values decreased significantly from 74.62%to 87.69%?the value of“Combined MIC”from thymol and salicylic acid was divide by the value of“Single MIC”of thymol or salicylic acid for F.solani and R.stolonifer respectively?,reducing cost of application and being more resource friendly.Morphological observation of F.solani and R.stolonifer showed that S_TSASA caused changes to mycelial morphology such as wrinkles and surface protrusions,while decreasing organelles and increasing vacuoles in both fungi.Moreover,S_TSA,thymol and salicylic acid caused different degrees of damage to the structure and function of F.solani and R.stolonifer cell membranes.With the increasing concentrations of S_TSA,thymol and salicylic acid,proportions of F.solani and R.stolonifer staining with propidium iodide?PI?also increased and in dose-dependent manners?r>0.91?.What's more,salicylic acid played a major role in the synergistic effect on the damage of cell membrane integrity of F.solani;thymol and salicylic acid contributed equally to the synergistic effect on the damage of cell membrane integrity of R.stolonifer.Furthermore,the leakage of nucleic acid and protein?at 260 and 280 nm,respectively?increased.There was a significant difference between the action times of each group from 1 h to 24 h?P?27?0.05?,with the highest increase rate reaching 79.52%and 82.65%respectively.Thymol and salicylic acid contributed equally to the synergistic effect of S_TSA with 0.5 and 1 MIC on nucleic acid leakage in F.solani and R.stolonifer cells;thymol played a major role in the synergistic effect of S_TSA with 2 MIC concentration on nucleic acid leakage in F.solani cells;salicylic acid played a major role in the synergistic effect of S_TSA on protein leakage in R.stolonifer cells.In addition,ergosterol content decreased with the increase concentration of fungistat,and showed a concentration dependence?r between-0.8211 and-0.8975?,with equal contributions of thymol and salicylic acid.When S_TSA,thymol and salicylic acid treated with F.solani and R.stolonifer respectively,the mitochondrial membrane potential of both fungi increased significantly and showed concentration-dependent relationship?r between 0.9244 and 0.9972?.Furthermore,the mitochondrial membrane potential?MMP?of F.solani was higher than that of R.stolonifer after treated with the same concentration of fungistat,indicating that the MMP of F.solani was more affected than that of R.stolonifer.The activities of key enzymes in tricarboxylic acid?TCA?cycle,citrate synthase?CS?,isocitrate dehydrogenase?IDH?,alpha-ketoglutarate dehydrogenase??-KGDH?,succinate dehydrogenase?SDH?and malic dehydrogenase?MDH?were inhibited to varying degrees by the fungistat.The decrease rates of enzyme activity were between 71.45%and 98.38%.In addition,the fungistat reduced intracellular citric acid?CA?content by 65.64%?F.solani?and 90.80%?R.stolonifer?respectively;the accumulation of reactive oxygen species?ROS?increased significantly in both fungi,with the maximum growth rates being 86.71%?F.solani?and 97.78%?R.stolonifer?respectively;meanwhile,the fungistat leaded to the mass production of malondialdehyde?MDA?,with the maximum growth rates of73.54%?F.solani?and 80.00%?R.stolonifer?respectively.Therefore,S_TSA caused a lot of leakage of cellular contents?nucleic acids and proteins?,the synthesis of ergosterol was severely blocked,the membrane structure of mitochondria was changed by hyperpolarization of MMP,the decrease of key enzymes activities in TCA cycle affected ATP synthesis and NADH production,destroyed TCA cycle,further caused mitochondrial dysfunction and oxidative damage.Cell peroxidation continueed to affect the activity of key enzymes in mitochondria,aggravated membrane damage and eventually leaded to the death of F.solani and R.stolonifer cells.When S_TSA in vitro antifungal effects against both fungi?F.solani and R.stolonifer?were tested,it was able to completely inhibit mycelial growth of both fungi at concentrations of 0.5,1,and 2 MIC.Moreover,spore germination of both fungi was significantly reduced by S_TSA at all of the tested concentrations of 0.5,1,or 2 MIC.The inhibition of spore germination by both fungi was greater than 96%,and no significant difference was seen?P<0.05?.In vivo antifungal effects were evaluated in terms of protective and therapeutic effects in wound-inoculated or natural tomatoes.The results indicated that S_TSA showed better antifungal effect on F.solani than R.stolonifer.Protective effects of S_TSA in wound-inoculated tomatoes with F.solani were better than therapeutic effects,while there was no significant difference between protective and therapeutic effects of S_TSA in wound-inoculated tomatoes with R.stolonifer.In addition,combined with sensory evaluation,S_TSA with 1 MIC had better preservation effect on good fruit,and S_TSA with 2 MIC was more suitable for tomato fresh-keeping in a rotten condition.The in vitro antifungal effects of S_TSA on F.solani and R.stolonifer showed that the inhibition rates of S_TSA on mycelial growth and spore germination of both spoilage fungi reached 100%and 96%respectively.In vivo tests,S_TSA showed better antifungal effect on F.solani and R.stolonifer.The protective effect of S_TSA on on wound inoculated with F.solani tomatoes is better than that of the therapeutic effect;the protective and therapeutic effects of S_TSA on wound inoculated with R.stolonifer tomatoes were not significantly different.Sensory evaluation showed that S_TSA with 1 MIC concentration was better for the preservation of good fruits,while S_TSA with2 MIC concentration was more suitable for preservation of tomatoes with decay.Furthermore,different concentrations of S_TSA were added to the bleached shellac to form a coating covering the surface of tomatoes.Through the study of the moisture permeability and oxygen permeability of the coatings,it was found that the moisture permeability and oxygen permeability of the S_TSA shellac coating with 10 MIC concentration increased by 6.71%and9.05%respectively,compared with the control group.Compared with the control,tomatoes coated with STSA shellac with 5 MIC concentration had the lowest decay rate and weight loss rate?P<0.05?,the slowest decreasing rate of soluble solids content,as well as the best sensory quality.Different concentrations of S_TSA shellac coatings had no effect on the color of postharvest tomatoes.In addition,1 MIC and 5 MIC S_TSA shellac coatings inhibited the hardness loss of tomatoes,and 10 MIC S_TSA shellac coatings maintained titratable acid content of tomatoes at a high level during storage.1 MIC S_TSA shellac coating delayed the peak of total phenol in postharvest tomatoes,which was conducive to the preservation of tomato flavor.What's more,5 MIC S_TSA shellac coating effectively reduced the accumulation of MDA in tomatoes during storage,effectively preventing fruit senescence and membrane damage.Meanwhile,5 MIC S_TSA shellac coating also induced the activities of two disease resistance related enzymes?polyphenol oxidase and phenylalanine ammonia lyase?in tomatoes to increase in a short time,contributing to improve the immunity of fruit,enhance the ability of postharvest fruit to resist pathogenic fungi infection,and reduce the loss during storage and transportation.The results of this study will expand the application prospects of plant active ingredients,provide basic data and scientific theoretical basis for postharvest tomato preservation,and provide the reference for fruit and vegetable storage and transportation after harvest.