Study On Control Effect Of Sour Rot On Postharvest Citrus Fruit And Mechanisms Of Peptides Mastoparan-S,thanatin And Ponericin W1

Sour rot,caused by Geotrichum citri-aurantii,is a serious infectious postharvest disease.Citrus fruit are susceptible to the disease when the storage environment is wet,which can lead to significant economic losses.Sour rot can be controlled in some degree by some commercial fungicides which are the primary methods for controlling postharvest diseases of citrus fruit.However,the long-term using of fungicides has lead to some problems,such as tolerance,fungicide residues,environmental pollution and human health.Therefore,there is a need to develop safe and effective alternative methods to control citrus sour rot.Antimicrobial peptides?AMPs?is a kind of small molecule peptide with broad-spectrum antibacterial activity.It has been reported that some antibacterial peptides can effectively control peach brown rot,strawberry gray mold and apple blue mold.AMPs are considered as potential alternatives to fungicides due to their strong antimicrobial activity,lack of pollution,low level of drug resistant.Thus,the antimicrobial peptides have a good prospect in controlling postharvest diseases of citrus fruit.This study investigated the antifungal activity against G.citri-aurantii in vitro and the control effect on sour rot in vivo?i.e.,thanatin,ponericins W1 and mastoparan-S?.In order to understand the effect of thanatin,ponericins W1 and mastoparan-S on the cell membrane integrity and DNA binding ability,the extracellular conductivity and the release of cytoplasmic constituents were determined.It was also studied by the scanning electron microscopy assay,fluorescence microscopy assay,gel retardation assay,UV spectrum analysis and fluorescence spectrum analysis.The main results were summarized as follows:?1?The fungicidal activity of the three peptides against G.citri-aurantii in vitro.Ponericin W1,thanatin and mastoparan-S could dramatically inhibit the growth of G.citri-aurantii and kill conidia in vitro in a dose-dependent manner.The minimum inhibitory concentrations?MIC?of mastoparan-S,thanatin and ponericin W1 were 8?mol L^-1,8?mol L^-1 and 2?mol L^-1,respectively.The minimum fungicidal concentration?MFC?of mastoparan-S,thanatin and ponericin W1 were 8?mol L^-1,8?mol L^-1 and 4?mol L^-1,respectively.?2?Effects of the three peptides on sour rot development in citrus fruit.When the peptides and pathogens were inoculated in the same wounds,ponericin W1,thanatin and mastoparan-S could effectively control citrus fruit decay in vivo,which was dependent on concentration and treatment methods.The peptides were mixed with the pathogen and were incubated for 0 h?or 2 h?prior to inoculation.The other treatments were that the peptides were applied before?or after?the pathogen was inoculated.The control effect of the former was better than that of the latter.When the peptides were mixed with the pathogen and were incubated for 0 h?or 2 h?prior to inoculation,mastoparan-S,thanatin and ponericin W1 could cause 16.67%?21.43%?,28.57%?28.57%?,22.62%?22.62%?incidence reduction,respectively,after storage for 15 d.When the peptides were applied into wounds after the pathogen had been incubated,the control effect of mastoparan-S improved with increasing concentrations.However,thanatin and ponericin W1 exhibited a good effect at 100?mol L^-1,which had no significant?P<0.05?difference among the different concentrations.All the three peptides could not induce disease resistance in citrus fruits when the peptides and the pathogen were inoculated in the different wounds.?3?Effects of the three peptides on the membrane integrity of G.citri-aurantiiThe morphology of the hyphae and conidia of G.citri-aurantii was altered to varying degrees after treatment with ponericin W1,thanatin and mastoparan-S.The hyphae were collapsed and folded and the conidia were concaved.Ponericin W1 showed a strongest destructive effect The membrane integrity disruption after treatment with peptides was determined by extracellular conductivity,the release of cytoplasmic constituents and fluorescence microscopy assay.These results indicated that small molecular substances,and ions leaked out of the cells,so they lost their membrane integrity.In addition,the effect of ponericin W1 on the cell membrane was the strongest among the three peptides.Ponericin W1 showed a strong destructive effect on the G.citri-aurantii cell membrane,which was much stronger than that of thanatin and mastoparan-S.Thanatin and mastoparan-S had a low degree of damage to cell membrane integrity at high concentrations.In addition,the laser confocal microscopy was used to observe the localization of these three peptides.It was founded that ponericin W1,thanatin and mastoparan-S could also cross the cell membrane,indicating that there may be an interacellular mechanism of action.?4?The effects of the three peptides on the G.citri-aurantii DNA.Gel retardation,UV spectra and fluorescence spectra analysis indicated that mastatopara-S,thanatin,and ponericin W1 could bind to the G.citri-aurantii genomic DNA through electrostatic interaction firstly,and intercalate into the base pairs in a helix of DNA,then wreck the double-helix structure.In addition,mastatopara-S,thanatin,and ponericin W1 can also inhibit the synthesis of DNA and RNA of G.citri-aurantii.In summary,mastatopara-S,thanatin,and ponericin W1 have strong antifungal activity against G.citri-aurantii in vitro and the control effect on sour rot in vivo.The peptides could disrupt membrane integrity and bind to the genomic DNA of G.citri-aurantii crossing the membrane.These comprehensive research provided new ideas and theoretical basis for controlling the sour rot of citrus fruit.