| Xiangxi ponkan is a characteristic agricultural product of Hunan Province’s "targeted poverty alleviation".Due to the relatively concentrated maturity period of ponkan,it is easily infected by fungi during storage,causing fruit rot and causing huge economic losses to the ponkan industry.Chemical fungicides can effectively control the occurrence of postharvest diseases of ponkan,but there are problems such as large residues,environmental pollution,and easy induction of pathogenic bacteria to develop resistance.The previous research of this research group found that sodium dehydroacetate in food preservatives has a good control effect on postharvest green mildew and acid rot of citrus,and has potential application prospects in the storage and preservation of citrus.The purpose of this study was to isolate pathogenic fungi from naturally rotten and pesticide-treated Xiangxi ponkan,identify the pathogenic bacteria species by morphological and molecular biology methods,and screen out the prochloraz with drug resistance and strong pathogenicity.On this basis,the effect and potential antibacterial mechanism of sodium dehydroacetate on Penicillium italicum were investigated.The main findings are as follows:(1)Isolation and identification of postharvest pathogenic bacteria in ponkan mandarin and isolation of drug-resistant strainsA total of 38 strains of pathogenic fungi were obtained by tissue separation method,and the strains were preliminarily identified according to Koch’s rule.The results showed that 12,10 and 6 pathogenic fungi were screened in Xixi town,Tanxi town and Wuxi town of Luxi county,respectively.Traditional morphological observation combined with r DNA-ITS molecular identification showed that 28 of them were Penicillium italicum.The virulence test results showed that P.italicum isolated from Wuxi town had the fastest disease incidence.When stored for 3 d,the incidence rate of ponkan fruits inoculated with 6 strains of P.italicum reached 100 %.Followed by Xixi town,the incidence rate of ponkan fruits inoculated with 12 strains of P.italicum reached 100% when stored for 5 d.The incidence of P.italicum isolated from Tanxi town was the slowest.On 6 d,the incidence rate of ponkan fruits inoculated with 10 strains of P.italicum reached 100 %.Prochloraz resistance experiments showed that P.italicum numbered RXX2 and RXX4-1 both grew at a concentration of 10 μg/m L and belonged to the prochloraz-resistant strains,while the remaining 26 strains did not grow at a concentration of 1 μg/m L that belonged to the prochloraz sensitive strain.(2)Inhibitory effect of sodium dehydroacetate on Penicillium italicumThe minimum inhibitory concentration(MIC)and minimum antifungal concentration(MFC)of sodium dehydroacetate on the mycelium growth of 28 strains of P.italicum were determined by agar dilution method.Sodium dehydroacetate inhibited28 strains of P.italicum to varying degrees and showed better inhibitory effect on drug-resistant strains,with MIC and MFC of 0.2 mg/m L and 0.4 mg/m L for RXX2,RXX3 and XX1 strains.It can be seen that RXX2 and RXX4-1 are prochloraz-resistant strains,and the incidence of RXX2 is higher.Since the incidence of XX1 is in 6 strains in Xixi town(isolated from naturally rotten ponkan fruits)Prochloraz sensitive strains were the highest,and the incidence rate of RXX3 was the lowest among the four prochloraz sensitive strains in Xixi town(isolated from pesticide-treated ponkan fruits).As well as the differences between different pathogenic strains,combined with the results of pathogenicity,drug resistance and the effect of sodium dehydroacetate on the growth of P.italicum mycelium,RXX2,RXX3 and XX1 were identified as follow-up experimental strains.Further in vivo experiments showed that sodium dehydroacetate could effectively reduce the incidence of penicillosis in ponkan fruits inoculated with RXX2,RXX3 and XX1 strains.The control group needed 4 d,6 d and 4 d respectively from inoculation to whole fruit rot.At this time,8×MFC(3.2 mg/m L)treatment groups’ incidence rates of were 10.18 %、75.00 % and 40.00 %,respectively,and the diameter of the lesions was significantly lower than that of the control group.With the increase of the concentration,the bacteriostatic effect was more obvious.The effects of sodium dehydroacetate on the physiological and biochemical properties of prochloraz sensitive(XX1 and RXX3)and resistant strains(RXX2)were further explored.Scanning electron microscopy(SEM)results showed that the hyphae appeared shriveled,folded,twisted,and even broken after sodium dehydroacetate treatment.The results of CFW(Calcofluor White)fluorescence staining showed that within 120 min of treatment with different concentrations of sodium dehydroacetate,there was no significant difference in the fluorescence intensity between the treatment group and the control group,indicating that the integrity of the cell wall was not damaged.The results of propidium iodide(PI)staining showed that the integrity and permeability of RXX2,RXX3 and XXI cell membranes changed after sodium dehydroacetate treatment.The results of intracellular reactive oxygen species(ROS)content and mitochondrial membrane potential(MMP)experiments showed that no significant changes were found in the ROS content and MMP of RXX2,RXX3 and XXI,indicating that sodium dehydroacetate did not cause damage to the normal function of mitochondria.(3)The effect of sodium dehydroacetate on gene expression of Penicillium italicumThe overall gene expression of RXX2,RXX3 and XX1 treated with 1/2 MIC(0.1mg/m L)sodium dehydroacetate was analyzed by transcriptome sequencing(RNA-seq)technology.The results showed that 320,1246 and 2939 differentially expressed genes(DEGs)were generated after treatment of RXX2,RXX3 and XX1 with sodium dehydroacetate,respectively.The results of GO analysis showed that the DEGs of the prochloraz-resistant strain RXX2 were mainly related to the functions of organic acid metabolism,oxyacid metabolism and cellular amino acid metabolism;the DEGs of the prochloraz-sensitive strains RXX3 and XX1 were mainly related to ribosome synthesis,RNA metabolism and RNA processing functions.KEGG enrichment results showed that RXX2 differentially expressed genes were mainly involved in ribosome biosynthesis,amino acid and alanine,aspartate and glutamate metabolism and other metabolic pathways;RXX3 differentially expressed genes were mainly involved in amino acid biosynthesis and antibiotic biosynthesis,etc.Metabolic pathways;XX1 differentially expressed genes are mainly related to metabolic pathways such as ribosome and proteasome.What is particularly interesting is that sodium dehydroacetate has a significant effect on RXX2,RXX3 and XX1 cell membrane glycerol phosphate metabolic pathways,and phospholipase D is down-regulated and phospholipid synthesis is blocked.The phospholipid metabolism pathway destroys the integrity of the cell membrane,thereby achieving the inhibition of the growth of the strain. |
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