Biological Control Of Volatile Organic Compounds Produced By Pseudomonas Fluorescens ZX Against Gray Mold On Postharvest Grapes And Its Inhibition Mechanism

Grapes are susceptible to be infected by various plant pathogens due to its own characteristics,which caused huge economic losses to postharvest grapes industry.Therein,gray mold caused by Botrytis cinerea is one of the major diseases of postharvest grapes.Currently,primary strategy against postharvest fungal diseases mainly depends on various chemical fungicides,which are low cost,acted quickly,strong effect.However,abuse of fungicides is ubiquitous to bring a larger negative influence on human health and natural environment.Therefore,in recent years,how to reduce the use of chemical fungicides and select alternative options become a urgent conundrum.Biological control has some significant advantage such as green,safety,and effective,which has attracted many researchers.Of these,biological control agent(BCAs)are mostly some microorganisms that have strong biocontrol efficacy against plant pathogens in postharvest fruits and vegetables.They are not able to infect fruits and vegetables,and can inhibit the growth and reproduction of pathogens through multiple pathways to lighten the severity of postharvest fungal diseases of fruits and vegetables.Multiple studies have now demonstrated that some BCAs can produce various volatile organic compounds(VOCs)during growth,which have certain broadspectrum antibacterial activity to strongly inhibit many plant pathogens.Using the biofumigation of VOCs can avoid direct contact with fruits and vegetables,and therefore BCAs-VOCs are considered to be a promising potential biocontrol strategy for postharvest diseases.Pseudomonas fluorescens exists widely in nature,mainly in the soil and rhizosphere.Available studies suggest that P.fluorescens is the most potential BCAs,which can control the growth of many pathogens.P.fluorescens ZX was obtained from China-Hungary cooperation project,and prior test demonstrated that P.fluorescens ZX has excellent biocontrol capability for pathogens via multiple pathways,including competition for nutrients and space,production of antibacterial metabolites,induced resistance,and direct parasitism.Meanwhile,P.fluorescens ZX can also produce diverse VOCs,and however,researches regarding the biocontrol efficacy of these VOCs have not been fully explored.Thus,the goal of current study is to explore the antifungal activity of P.fluorescens ZX-VOCs on B.cinerea in vitro,control ability on gray mold of grapes,the antifungal mechanisms of P.fluorescens ZX-VOCs combining with transcriptomics,and the effects of VOCs on grapes quality.These results will provide a theoretical support for the actual application of P.fluorescens ZX-VOCs in postharvest fruits and vegetables industry.Our study revealed the following:1、The “two-plates” method was used to explore the antifungal activity of P.fluorescens ZX-VOCs against B.cinerea in vitro test.The results showed that compared to the control,the diameter growth of mycelium and spore suspensions were inhibited by 55.8% and 40.9% respectively,after B.cinerea fumigated by VOCs.In parallel,spore germination rate and sporulation showed a significant decline.In vivo test showed that the incidence and disease index of grapes fumigated by VOCs from direct fumigation,prior fumigation,and P.fluorescens ZX colonized grapes were a significant decrease.Of which,the biocontrol efficacy of direct and prior fumigation was better.The pure substances test in vitro showed that the effective composition of P.fluorescens ZX-VOCs were acetic acid,dimethyl disulfide,geranyl formate,butyric acid,dimethyl butyric acid,isobutyric acid,isovaleric acid,dimethyl trisulfide.These substances could inhibit the incidence and disease index of grapes in vivo test,which illustrated again that them were the effective composition of VOCs.2、The “two-plates” method was used to assay some indicators of B.cinerea fumigated by VOCs to evaluate the antifungal mechanisms of P.fluorescens ZX-VOCs against B.cinerea.The results demonstrated that the mechanisms of P.fluorescens ZXVOCs were mainly in bringing a obvious damage for cell membrane.Specific manifestation was as follows: increasing the permeability of B.cinerea cell membrane,resulting in leakage of intracellular material,significantly inhibiting the ergosterol biosynthesis in cell membrane,disrupting the integrity of cell membranes,inducing a significant increase in malondialdehyde levels,and causing membrane lipid peroxidation.On the other hand,VOCs fumigation could also damage the morphological structure of B.cinerea mycelium,resulting in multiple fractures.In addition,VOCs fumigation significantly reduced the activities of ATPase,malate dehydrogenase,and succinate dehydrogenase in mycelium,which significantly interfered with the respiration and energy metabolism of B.cinerea.3 、 Based on transcriptomic techniques,the analysis of B.cinerea samples fumigated with P.fluorescens ZX-VOCs,acetic acid and isovaleric acid was performed to reveal the antifungal mechanisms of VOCs against B.cinerea from a molecular biology perspective and to verify the accuracy of the preliminary inhibition test.The results indicated that after three different treatments,B.cinerea exhibited significant changes in gene expression,generating a significant number of differentially expressed genes(DEGs),with the largest number in the VOCs group.GO enrichment assays revealed that these DEGs were mainly enriched in the GO term related to membrane function,which again demonstrated the effect of VOCs on B.cinerea cell membranes.The results of KEGG enrichment revealed that multiple metabolic pathways of B.cinerea produced significant differences after the three treatments,with the most amino acid-related pathways,affecting mainly the expression of enzymes in the pathway.The expression of genes related to the inhibitory mechanisms showed that most of the DEGs related to ergosterol biosynthesis,ATPase,mitochondrial respiratory chain,malate dehydrogenase,and cell membrane were down-regulated,suggesting some disturbance of these physiological functions.This was in general agreement with the preliminary determination of physicochemical indicators.4、To determine the effect of VOCs on postharvest grape quality,we compared grapes fumigated by P.fluorescens ZX-VOCs with fresh grapes.The results showed that VOCs did not have a significant negative impact on the sensory quality,storage quality,textural characteristics,color and odor of the grapes.It can even inhibit microorganisms on the surface of the fruit,and delay natural decay,and have a certain ability to preserve freshness and increase the shelf life of the fruit.In conclusion,the experimental results above indicate that P.fluorescens ZXVOCs exhibit significant antifungal activity against B.cinerea,significantly inhibiting its growth and controlling postharvest gray mold in grapes.The antifungal mechanisms of VOCs are mainly to increase the permeability of cell membranes,disrupt its integrity,disrupt mycelial morphological structures,and interfere with the respiration of Pathogens and the expression of enzymes in various metabolic pathways.In addition,VOC fumigation does not reduce the quality of the fruit and has a certain effect on freshness,which has good potential for biological control and broad application prospects.