| Citrus,belonging to Rutaceae,are widely planted in tropical,subtropical and intermediate climatic regions of the world.According to statistics,the annual output of citrus fruits has reached 120 million tons and ranked first among all fruits,surpassing apples,grapes and bananas.However,citrus are typical non-climacteric fruits,which are close to physiological maturity with week disease resistance when picking.Therefore,citrus fruits are susceptible to a wide variety of postharvest fungal diseases,e.g.,Penicillium decay.Of these diseases,green(Penicillium digitatum)and blue(Penicillium italium)mold are the most devastating,causing heavy economic and resource losses around the world.The application of chemical fungicides,such as thiabendazole,carbendazim and imazalil is currently the main method of controlling postharvest decay.However,continuous use of large volumes of the same synthetic fungicides facilitates the development of resistance in fungi.Furthermore,public concern has been growing towards the effect of fungicides on food safety,chemical residues,and environmental pollution.Thus,there is an increasing demand for research on alternative non-chemical fungicidal methods to inhibit postharvest diseases.Recently,interest in microbial biocontrol agent development has grown.In comparison to chemical fungicides,biological control is an approach to inhibiting pathogen growth and spread that is considered both safe and environmentally friendly.Pseudomonas fluorescens is widely distributed in nature,and one of the most valuable biocontrol.To the best of our knowledge,little is known about P.fluorescens biocontrol of postharvest disease of citrus fruits.Thus,P.fluorescens ZX obtained by high throughput screening was used as the test strain,and "Beibei 447" Jincheng fruits were used as the test materials.First of all,the viability,antimicrobial activity and biocontrol effect of P.fluorescens ZX in postharvest Jincheng fruits were evaluated.The mechanisms of P.fluorescens ZX were analyzed both through in vitro and in vivo tests,which may provide a theoretical reference for further application of P.fluorescens ZX.The main results are as follows:1.The properties of P.fluorescens ZX were studied by microbiological techniques to evaluate its survivability and stress resistance.The results showed that optimum growth conditions of P.fluorescens ZX are 30oC,neutral environment,and 20 g/L Na Cl.P.fluorescens ZX,with certain heat resistance,higher ultraviolet resistance and wider antibiotic resistance,is sensitive to strong acid and strong alkali.P.fluorescens ZX began to enter the logarithmic growth stage after incubating for 4 h,showing strong vitality and environmental adaptability.2.The antimicrobial activity of P.fluorescens ZX and its biocontrol efficacy against blue and green mold decay in postharvest Jincheng fruits were evaluated via both in vitro and in vivo tests.Whether in vitro or in vivo,P.italicum and P.digitatum were significantly inhibited by bacterial fluid(P.fluorescens ZX in nutrient broth liquid medium)and bacterial suspension(P.fluorescens ZX in sterile distilled water)with living cells.P.fluorescens ZX could completely inhibit the growth of P.italicum and P.digitatum when incubated with spore suspension in agar plate.The results in vivo showed that the inhibitory of P.fluorescens ZX suspension was related to the concentration,the higher concentration,the lower disease incidence and the smaller lesion diameter.In addition,P.fluorescens ZX was effective when applied preventatively but not when applied curatively.Cell-free autoclaved cultures or culture filtrate had limited capacity to suppress P.italicum and P.digitatum,whose biocontrol efficacy was much lower than that of the bacterial fluid and bacterial suspension.3.The biological control mechanisms of P.fluorescens ZX against blue and green mold of postharvest Jincheng orange were analyzed,the results were as follows:(1)Pathogen hyphae after different treatments were smooth,healthy,and bore numerous conidia on their phialides.The adsorption,distortion,malformation,cavity and protoplast leakage were observed to be normal,indicating the P.fluorescens ZX isolate could not colonize the hyphae or attack the conidia of P.italium and P.digitatum.(2)Nutrition and space competition was the most important method for P.fluorescens ZX to inhibit P.italicum and P.digitatum.P.fluorescens ZX could colonize in fruit wounds effectively.The population of P.fluorescens ZX increased 28-and 34-fold more,being incubated at 20oC for 8 days and 4oC for 16 days,respectively.Competition tests showed P.fluorescens ZX could use fructose,sucrose,glucose and free amino acid better than P.italicum and P.digitatum.P.fluorescens ZX could also compete for limited iron in wounds by secreting siderophores.Furthermore,an effective biofilm that peaked after a 24-h incubation at 30oC was formed by the P.fluorescens ZX isolate,creating a mechanical barrier that prevents pathogens from coming in contact with wounds and acquiring nutrients needed for germination and growth.(3)When molecular analyses were performed on the P.fluorescens ZX isolate,the genes encoding phenazine-1-carboxylic acid,pyrrolnitrin,pyoluteorin,2,4-diacetylphloroglucinol and hydrogen cyanide production were determined to be absent.P.fluorescens ZX isolate displayed protease,but not chitinase,glucanase,or cellulose activity,and produced volatile organic compounds with antifungal abilities.After P.fluorescens ZX fumigation treatment,the disease incidence of blue and green mold decay and the lesion diameter were significantly reduced.(4)P.fluorescens ZX could significantly enhance the disease resistance of Jincheng fruits within a specific induction time range,especially after a 48-h induction.Further analysis showed that P.fluorescens ZX was able to induce resistance on citrus peel by increasing the chitinase,?-1,3-glucanase,peroxidase and phenylalanine ammonia lyase activitives and advancing the activities peaks of ?-1,3-glucanase and peroxidase.Foremore,P.fluorescens ZX treatment could effectively improve the internal reduction potential of fruits,and induce the accumulation of nitric oxide,hydrogen peroxide,glutathione,reductive coenzyme II,total phenols and scoparone in fruits.In addition,P.fluorescens ZX treatment could effectively inhibit or delay membrane lipid peroxidation by significantly inhibiting the increase of lipid oxygenase activity and malondialdehyde content in fruits.3.The fruits were soaked with P.fluorescens ZX suspension and the changes of quality indexs of the fruits before and after storage were monitored.The results showed that P.fluorescens ZX could effectively prevent the invasion of pathogens and inhibit the disease of fruits,prolonging the storage time significantly.Moreover,there was little difference on storage quality,sensory quality and aroma components between the treatment group with P.fluorescens ZX and the control group.In conclusion,the results of this study showed that the application of P.fluorescens ZX was observed to be effective in controlling bllu and green mold in citrus fruits.The possible modes of action may include inhibiting spore germination and mycelium growth,competition for nutrient substance and space sites,and inducing disease resistance.Therefore,the P.fluorescens ZX islate can potentially be used as a biocontrol agent against Penicillium italicum and P.digitatum in postharvest citrus. |
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