| Grape is a typical berry fruit,with high tissue moisture content,delicate flesh and exuberant postharvest physiological metabolism.Therefore,the fruit is easily infected by pathogenic fungi and deteriorated during transportation and storage after harvest.The annual rate of decay of the grape after harvest is as high as 50%.The gray mold decay induced by B.cinerea is one of the most common and most damaging diseases in postharvest diseases of grapes.At present,the control of post-harvest invasive diseases of grapes mainly depends on chemical preservatives such as sulfur dioxide and sulfides,and chemical fungicides such as pyrrole and methoxy acrylate,but the problem of pathogen resistance,chemical reagent residues and environmental pollution has become increasingly prominent.Moreover,due to increasing concern about the environmental pollution and food safety recent years,the use of antagonistic microorganisms for biological control has become a research hotspot of fruit and vegetable antisepsis preservation technology.Antagonistic microbes as a biological source elicitor can induce local disease resistance and systemic disease resistance,and enhance the ability of fruits to resist postharvest biotic stress.Therefore,in this research,Pseudofluorococcus ZX(P.fluorescens ZX)obtained by high-throughput screening was used as the test strain,and the fruit of“Kyoho” grape was used as a test material to explore the control efficacy of P.fluorescens ZX on postharvest gray mold decay and the resistance induction of grape fruits.On this basis,the effects of P.fluorescens ZX on the gene expression and substance metabolism in grape fruits were analyzed by means of transcriptomics technology combined with traditional physiological and biochemical analysis methods.The main research contents and results were summarized as follows:1.P.fluorescens ZX could effectively inhibit the growth and reproduction of B.cinerea in vitro,showing extremely high biocontrol potential for gray mold control.When P.fluorescens ZX and B.cinerea were co-cultured in PDB for 16 h,the spore germination rate of B.cinerea spores in the 0.75 MCF bacterial suspension treatment group was only 8.67%.P.fluorescens ZX suspension treatment significantly reduced the incidence of gray mold caused by B.cinerea in postharvest grape berries,and in the concentration range of 0.25 MCF to 1.0 MCF,as the concentration of P.fluorescens ZX increased,the incidence of fruit gray mold gradually decreased.In the storage test,after immersion treatment with 0.5 MCF of P.fluorescens ZX bacterial suspension for 14 days,the natural rot rate of grape fruit was only 23.61%,and there was no significant difference in fruit quality compared with the control treatment,indicating that the bacteria was significantly reduced the natural decay rate of grape berries after harvest can also maintain the quality of the fruits,and has certain commercial prospects.2.In the oral acute toxicity test of P.fluorescens ZX,the LD50 of female and male mice were greater than 10,000 mg / kg body weight.According to the acute toxicity classification standard,it was known that P.fluorescens ZX belongs to safe non-toxic bacteria.P.fluorescens ZX is an important prerequisite for the application of post-harvest biocontrol of fruits and vegetables.This is an important prerequisite for the application of P.fluorescens ZX as a post-harvest biocontrol of fruits and vegetables.3.Combined with in vitro and in vivo tests to analyze and investigate the inhibitory effect and biocontrol mechanism of P.fluorescens ZX on postharvest grape gray mold,the results show that:(1)P.fluorescens ZX bacterial suspension could inhibit the spore germination and elongation of germ tube of B.cinerea in vitro,and it could affect the growth of B.cinerea hypha,which indicated that P.fluorescens ZX can directly inhibit B.cinerea.The volatile antibacterial test results showed that the volatile substances produced by P.fluorescens ZX have a strong antibacterial effect and can significantly inhibit the mycelial growth and colony expansion of B.cinerea;headspace solid phase microextraction and gas chromatography-mass spectrometry were used to determine that P.fluorescens ZX can produce 21 volatile substances.And among them,sulfur-containing compounds were detected,and the relatively high content were organic acids and ketones.Scanning electron microscope observation revealed that when P.fluorescens ZX was co-cultured with B.cinere,P.fluorescens ZX could destroy the morphology of B.cinere and affect the normal growth of B.cinere,indicating that P.fluorescens ZX has a direct inhibitory effect on B.cinere.(2)P.fluorescens ZX suspension treatment significantly reduced the incidence of gray mold caused by B.cinerea in postharvest grape berries,and in the concentration range of 0.25 MCF to 1.0 MCF,as the concentration of P.fluorescens ZX increased,the incidence of fruit gray mold gradually decreased.The culture filtrate and autoclaved culture of P.fluorescens ZX have no obvious inhibitory effect on B.cinere;0.5 MCF of P.fluorescens ZX unwashed cell culture mixture could inhibit the occurrence of grape gray mold,but its antagonistic effect was significantly weaker than the same concentration of washed cell suspension.The inoculation sequence and interval of P.fluorescens ZX relative to B.cinerea spore suspension has a significant effect on the control of P.fluorescens ZX against grape ash disease.The earlier the inoculation of antagonistic bacteria is,the better the control effect is.P.fluorescens ZX could quickly grow and colonize on the surface of grape fruits and wounds of grape fruits stored at 4 ?C and 20 ?C,indicating that the bacteria can adapt to the environment of grape wounds and can compete with pathogens for space and nutrition.This is also one of the main mechanisms by which P.fluorescens ZX inhibits post-harvest rot.(3)During the storage of grape fruits,P.fluorescens ZX suspension treatment could significantly inhibit the occurrence of gray mold,and at the same time enhance the resistance of grape fruit to gray mold,induction of increased activities of CHI,GLU,PAL,POD,CAT and PPO,increased synthesis of total phenols and phytoalexins(resveratrol and resveratrol dehydrodimer).Grape berries treated with P.fluorescens ZX suspension showed a sharp increase in the activity of disease-related enzymes(PAL,PPO,GLU,etc.)and the expression abundance of PRs genes(Vv PPO,Vv POD,Vv CHI,etc.)on the second day of storage.This indicated that the inhibition of P.fluorescens ZX on postharvest grape rot is closely related to the indirect induction of disease resistance.4.The induced disease resistance response of P.fluorescens ZX suspension treatment to postharvest grape fruit was studied from the transcription level.The treatment of P.fluorescens ZX bacteria suspension can effectively induce the rise of key genes of As A-GSH cycle,key genes of reactive oxygen metabolism enzyme system,key genes of phenylpropane metabolism pathway and sucrose metabolism pathway,thereby improving fruit disease resistance.Moreover,it was found that the treatment of P.fluorescens ZX bacteria suspension could induce the expression of PRs genes by regulating different WRKY transcription factors,which provided molecular biological basis for the application of P.fluorescens ZX in the preservation and preservation of postharvest grape fruits. |
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