Evaluation Of Yeast Isolates From Kimchi With Antagonistic Activity Against Green Mold In Citrus And Elucidating The Action Mechanisms Of Three Yeasts

The production of citrus fruit is saddled with post-harvest decay of the fruits.This phenomenon is alarming because it increases losses significantly,reduces the quality of the citrus fruits thereby affecting their economic value.The losses also affect the profitability of farmers,the citrus supply chain as well as the consumer.The challenges in the citrus production chain are associated with poor control of pathogens during pre-harvest,mechanical damage during harvest,transportation,and insufficient storage.The most dangerous post-harvest disease of citrus fruit is green mold produced by P.digitatum.Postharvest deterioration of citrus caused by the disease has been largely controlled by the use of fungicides whose active ingredients are hazardous to human health and the environment.Useful organisms,genes,and metabolites from them are used in biological control to decrease the actions of one or more plant diseases while promoting positive plant responses.To find more innovative antagonistic yeasts to control post-harvest infections in fruits,researchers must investigate a variety of microbial habitats,including fermented dishes.In this study,kimchi,a fermented dish was screened for antagonistic yeast isolates,and after that,further investigations were conducted into the possible mechanism of action of the most effective yeast species.Results of the evaluation are presented as follows:（1）90 yeast strains were isolated from kimchi,and in vitro screening experiments revealed that ten of the ninety isolates inhibited the growth of the pathogen by forming an inhibition zone.The antagonistic yeast isolates were identified using molecular methods,PCR amplification,and sequencing of the internal transcribed spacer regions.80 of the entire yeast isolates（88.9%）showed no apparent inhibitory halo,which could be due to inadequate or no antifungal substance synthesis.Pichia sp.,K.marxianus,Y.lipolytica,and I.orientalis were among the 10 effective isolates.3 isolates examined in vivo in the orange were able to lower the incidence of disease from 18%to 57%.P.kudriavzevii S₂C reduced%Disease incidence in orange by 57%,with a mean lesion diameter（LD）of 45 mm on the 9^th day.On the 9^th day,K.marxianus S₃A₂ and Y.lipolytica S₄A reduced the%Disease incidence by 28%and 18%,respectively,with the control giving a%Disease incidence of 99%.Three antagonistic yeasts were studied further to learn more about their mechanisms of action.（2）The approach utilized by a beneficial microbe against a disease-causing pathogen in plants is referred to as the mechanism of action of BCAs.Knowledge of biological control agents’mechanisms of action is critical for selecting an exceptional strain of the biocontrol agent,as well as creating appropriate marketable formulations and application methods to optimize its usage.Antibiosis,the development of lytic enzymes,parasitism,the promotion of host resistance,the production of volatile metabolites,and competition for limited nutrients and space are all common biocontrol mechanisms.Following that,the mode of actions of the three isolates was investigated by ascertaining the yeast isolates’biofilm development ability,screening the isolates for lytic enzyme activity,assessing the effect of yeast isolates’VOCs on P.digitatum,examining the ability of the microorganisms to compete for nutrients,and inducing of resistance against P.digitatum in oranges,evaluating the population dynamics of the yeast organisms on the lesion sites of oranges,and so on.By forming biofilms,competing for nutrients,and releasing volatile organic substances,P.kudriavzevii was the most potent isolate that reduced the incidence of green mold much better than the two other isolates.By inducing resistance and colonizing the lesion spots,K.marxianus demonstrated antifungal efficacy.P.digitatum was suppressed by Y.lipolytica adherence to P.digitatum mycelia and the secretion of lytic enzymes.（3）The use of VOCs to substitute fungicides in combating plant infections appears to be a promising and long-term strategy.The interaction between the antagonist and the pathogen is such that,direct contact between them is not feasible.The volatile organic compounds（VOCs）of 3 yeast organisms,Pichia kudriavzevii S₂C,Kluyveromyces marxianus S₃A₂,and Yarrowia lipolytica S₄A,that had an antagonistic effect against green mold in citrus in previous research by our team,were profiled with the help of GC-IMS equipment.The identified VOCs were distinct and quantified with Y.lipolytica S₄A producing 16（42.11%）,K.marxianus S₃A_2,13（34.21%）,and P.kudriavzevii S₂C synthesizing 9（23.68%）of the total VOCs produced.With a tolerable deviation of less than5%,38 distinct volatile organic compounds were detected using the GC-IMS based on the normalized drift time of standard substances in the user-built image database and retention time index.The 38 volatile compounds identified are 2-butylaldehyde,benzaldehyde,isoamyl acetate,1-butanol,2-phenyl ethyl alcohol,1-pentanol,butyl acetate,butyl propanoate,ethyl propanoate acetophenone,cyclohexene-2-one,3-methyl heptane-2-one,2,3-pentanedione,3-methyl-2-isobutyl pyrazine,hexanoic acid,3-hydroxybutanone,2-methyl butyraldehyde benzaldehyde,4-methyl-1-pentanol,isoamyl acetate,propyl butanoate,butyl propanoate,propanal,γ-decalactone,2,4-decadienal,heptanoic acid,ethyl butyrate,triethylamine,2,4-nonadienal,benzene acetaldehyde,2-phenyl ethyl alcohol,pentyl propanoate,2-octanone,isobutyric acid,2-octanal,2-nonenal,isoamyl butanoate,2-butanone.The research includes information on the compound’s retention index retention time,migration time,molecular formula,molecular weight,and odour attribute.These compounds were classified under the functional groups of aldehydes（26.31%）,alcohol（13.10%）,esters（26.31%）,ketones（26.31%）,and others（13.10%）.3-methyl-2-isobutyl pyrazine was the compound with the highest concentration of（414.55±10.36 a.u）detected in the isolate Y.lipolytica S₄A and had2-Phenyl ethyl alcohol as the VOC with the lowest concentration of（31.81±10.36 a.u）.The compound with the highest concentration in K.marxianus was 2,4-Decadienal（559.50±14.76 a.u）and Butyl propanoate had the lowest concentration of（37.21±14.76 a.u）.P.kudriavzevii S₂C also had Isobutyric acid as the highest concentration of（572.69±17.78 a.u）and the compound with the lowest concentration of（103.13±17.78 a.u）was Isoamyl butanoate.9 synthetic compounds（1-Butanol,Benzaldehyde,2-Phenyl ethyl alcohol,Isobutyric acid,Butyl propanoate,Isoamyl acetate,3-Methyl-2-isobutylpyrazine,2,4-Decadienal,?-Decalactone）derived from the VOCs identified were selected and tested in vitro to confirm the results obtained previously.Out of the nine compounds tested,four（2-phenyl alcohol PA,isobutyric acid IS,2,4-Decadienal DD,and?-Decalactone DL）were able to prevent P.digitatum from growing.For the 6 days of storage,the selected compounds inhibited P.digitatum at a rate ranging from（29.70±0.32%）to（83.67±0.28%）.They had varying degrees of impact on P.digitatum mycelia development,the pathogen’s spore and mycelia morphology,membrane integrity,membrane permeability,and cellular constituent leakage.The concentration of the four most effective compounds（2,4-Decadienal DD,?-Decalactone DL,2-Phenyl ethyl alcohol PA,Isobutyric acid IS）altered the spore morphology causing the spore walls to appear rough,depressed with shrunk surfaces,whereas the spores of the control had a regular morphology,normal size,shape,and the intracellular content appearing green in colour.The compound,2,4-Decadienal DD was the most effective antifungal compound having a MIC of 0.2μl m L^-1and an MFC of 0.4μl m L^-1 and having the highest inhibitory mycelia growth rate of 83.67%after 6 hours at 25 ^oC.For the first time,the compounds isobutyric acid IS,?-Decalactone DL,and 2,4-Decadienal DD have been identified as having the capacity to suppress growth.（4）The transcriptional components and changes in gene expression patterns in orange peel treated with K.marxianus were investigated,as well as the impact on important defence-related enzyme activities and metabolites.This study was carried out by the method of RNA sequencing,and the use of HPLC-MS and GC-MS equipment.A total of 3785 genes were transcribed between the yeast-treated and control fruits,nevertheless,based on the threshold,1287 genes were expressed significantly in the K.marxianus-treated and control fruit after 48h of incubation.682 genes were up-regulated representing 52.99%of the total significant expressed genes.605 genes were also found to be down-regulated representing 47.00%of the total significant expressed genes.The sequencing data revealed that the induction of K.marxianus in the orange peel activated the expression of genes in 90 biological pathways.Pathways such as ribosome and phenylpropanoid biosynthesis,carbohydrate metabolism,amino acid biosynthesis,secondary metabolites,etc.were up-regulated and those of cutin,suberin,and wax synthesis,linoleic and linolenic acid metabolism,fatty acid elongation,etc.were down-regulated.Gene expression changes in some of the pathways in the treated orange peel increased the production of some defence-related enzymes,as well as the synthesis of secondary metabolites like flavonoids,phenolic compounds,and also,an increase in the production of lignin.The accumulation of these compounds contributed to the improvement of the antifungal activity and defence responses in the orange peel.After 72 hours of storage,caffeic acid,syringic acid,sinapic acid,rutin,kaempferol,p-coumaric acid,ferulic acid,and chlorogenic acids were detected in various quantities in the samples.The sugars evaluated in the study were glucose,maltose,fructose,ribofuranose,xylose,mannose,psicose,inositol,sucrose,arabinose,fucose,gentibiose,and the organic acids determined also consisted of citric acid,malic acid,palmitic acid,stearic acid,and succinic acid with different concentrations.Alanine,serine,glutamate,threonine,leucine,glycine aspartate,cysteine,valine,tyrosine,phenylalanine,isoleucine,lysine,histidine proline,arginine,and methionine were among the estimated amino acids with varying amounts.This study’s findings have added to the knowledge of the potential of certain yeasts isolated from a fermented dish called kimchi in reducing green mold post-harvest decays in citrus fruits.The RNA sequencing data also contributes to a better understanding of Kluyveromyces marxianus’induced resistance mechanism in citrus.In general,these studies serve as a springboard for enhancing citrus fruit quality and identifying long-term alternatives to chemical fungicides in reducing postharvest losses.