| China has the largest planting area and the associated fruit yield of citrus fruits in the whole world.The citrus fruits are mainly eaten fresh but losses due to postharvest decay are huge.The most serious postharvest disease of citrus fruits is the green mold caused by Penicillium digitatum.The disease is mainly prevented and controlled by the use of chemical fungicides but this is associated with a number of problems including environmental pollution,human health hazards and emergence of drug-resistant strains,which have restricted the development of the citrus fruit industry.It is therefore urgent to find green fungicides that can replace traditional chemical fungicides.Sodium dehydroacetate(SD)is a highly effective and broad-spectrum food additive.Previous studies have shown that SD has a good control effect on citrus green mold and blue mold,but its potential mechanism of action is still unclear.In this paper,the inhibition mechanism of SD on Penicillium digitatum was systematically studied at the physiological and molecular levels,and the effects of SD combined with 2,4-D sodium on the storage and manintaining of the fruits fresh were investigated.The main results are as follows:(1)Inhibition of SD on imazalil sensitive strain(Pds01)and imazalil resistant strain(Pdw03)of Penicillium digitatumResults of in vitro experiments showed that different concentrations of SD could effectively inhibit the growth of Pdw03 mycelium,with the minimum inhibitory concentration(MIC)and minimum bactericidal concentration(MFC)being 0.1mg/m L and 0.2 mg/m L,respectively.The results of in vivo experiment showed that SD treatment could significantly reduce the incidence of fruit rot caused by green mold.When Pds01 and Pdw03 were inoculated on Miyagawa and Kumquat respectively,the rot rates of Miyagawa treated with 8×MFC SD were 60.0%and51.7%respectively,and the diameter of plaque was significantly lower than that of control when the rot rates of control were 100%;The decay rates of kumquat treated with 16×MFC SD were 45.8%and 64.6%,respectively,and the effect became more obvious with the increase in inhibitory concentration.The surface morphology of Pds01 and Pdw03 mycelia gradually changed from smooth and plump to twisted,wrinkled and shriveled with increase in time treatment time.Results of cell wall fluorescence staining and extracellular alkaline phosphatase(AKP)activity showed that there was no significant difference in fluorescence intensity between the treatment group and the control group,indicating that the integrity of cell wall was not destroyed.Results of propidium iodide(PI)staining showed that the cell membrane permeability of both Pds01 and Pdw03 mycelia were seriously damaged by 1/2 MIC and MIC SD treatments for 60 min.Analysis of lipid content showed that the total lipid content was 396.60±19.63 mg/g DW and 366.81±59.11 mg/g DW after 60 min of SD treatment,which was significantly lower than that of the control group 524.63±33.40 mg/g DW(P<0.05);The change of lipid content in Pdw03 was similar to that in Pds01,indicating that the integrity of cell membrane was destroyed again.The results of HPLC analysis showed that the content of intracellular SD of Penicillium digitatum increased gradually with the extension of treatment time,and then decreased slowly.When the mycelia were treated with 1/2MIC SD for 60 min,the content of intracellular SD reached the highest value of1.40×10-3mg/m L;When the mycelia were treated with MIC SD for 30 min,the intracellular SD content reached the peak value of 2.20×10-3mg/m L,which was significantly higher than that of the control group(P<0.05),while the extracellular SD content remained unchanged.In addition,SD treatment significantly decreased the mitochondrial membrane potential(MMP)and activated Na+/K+-ATPase in mycelia of Pds01 and Pdw03,suggesting that SD treatment may alter the intracellular energy level of Penicillium digitatum.According to the above results,it can be inferred that the antifungal activity of SD on Penicillium digitatum may be related to the damage of mycelial cell membrane structure and mitochondrial function.(2)Molecular mechanism of SD inhibiting the mycelial growth of Pds01 and Pdw03The effects of SD treatment on the mycelial growth of Pds01 and Pdw03 were studied by RNA-seq analysis.After Pds01 and Pdw03 were treated with 1/2 MIC SD for 30 min,3107 and 2194 differentially expressed genes(DEGs)were obtained,respectively(|log2FC|?1,Pvalue<0.05).The results of go enrichment analysis showed that 1515 DEGs co-existed in the two strains,and these DEGs were mainly related to"ribosome","translation"and"RNA metabolism".The results of KEGG enrichment analysis showed that the differentially expressed genes were mainly related to the metabolic pathways of ribosome,amino acid metabolism and membrane lipid metabolism.The most influential ribosomal anabolic pathway was selected for quantitative PCR validation.The Results showed that SD treatment significantly decreased the expression of ribosome synthesis genes RPSA,RPS9,RPL7a and RPL5in Pds01 and Pdw03 mycelia,indicating that SD treatment interfered with the genetic material transcription,translation and protein synthesis pathways of Pds01 and Pdw03by affecting ribosome biosynthesis.So as to achieve the purpose of inhibiting its growth.(3)Effect of SD and 2,4-D Sodium Compound Treatment on Storage and Fresh-keeping of Orange2,4-D Sodium is the most effective chemical used to maintain the freshness of citrus fruits in production.The effects of SD and 2,4-D Sodium on the storage and fresh-keeping of citrus fruits were studied.After preliminary screening,8×MFC SD and 0.05 mg/m L sodium 2,4-D were selected to treat citrus fruits.The results showed that there was no significant difference in the occurrence of disease between the compound treatment group and the SD treatment group within 0?30 d of storage,and the decay rate of the compound treatment group was 7.08±0.27%,which was significantly lower than that of the water control group(9.35±0.09%)(P<0.05).In addition,the combined treatment could maintain higher Vc content and effectively improve the phenomenon of fruit aging and blackening,but had no significant effect on other fruit qualities(color index,hardness,p H,solid acid ratio,soluble solids).This situation may be related to the low concentration of 2,4-D sodium,or may be related to the proportion of compound.This study revealed the mechanism of SD inhibiting the mycelium growth of Penicillium digitatum,and preliminarily discussed the effects of SD combined with2,4-D sodium on the storage and preservation of citrus fruits,which could provide theoretical and technical reference for the preservation and preservation of citrus. |
PDF Full Text Request