| Sour rot affected by G.citri-aurantii is a typical citrus postharvest disease second only to blue/green mold.G.citri-aurantii affecting all species and cultivars of citrus in all producing countries,causing serious damage to citrus industry and potential environmental pollution.At present,the most effective fungicide for controlling sour rot in domestic market is iminoctadine tris?the trade name is Bellkute?.However,with the long-term use of chemical fungicides,the sour rot pathogen of G.citri-aurantii in citrus production areas in China have shown different degrees of resistance to Bellkute.Therefore,it is urgent to develop more safe and effective green preservatives or alternative methods.Biological control of postharvest diseases received considerable attention.Using antagonistic yeasts to control postharvest diseases is a very effective biological control method.Most antagonistic yeasts used for postharvest disease control were isolated from the fruit system,which could quickly adapt to the microecological system of the surface of fruit.Yeasts are tolerant to extreme environmental conditions prevailing before and after harvest?low/high temperatures,desiccation,wide range of relative humidity,low oxygen levels,p H fluctuations,UV radiation?.Furthermore,antagonistic yeasts have a relative wide spectrum against pathogens,do not produce allergenic spores or mycotoxins in contrast to filamentous fungi,and they have simple nutritional requirements that enable them to colonize dry surfaces for long periods,which exhibiting great potential for switching applications.M.citriensis,a new species of Metschnikowia.spp,isolated from citrus leaves and identified by our laboratory,was efficient to control postharvest green and blue mold decay caused by Penicillium digitatum and Penicillium italicum on citrus fruit.However,the biocontrol ability of Metschnikowia spp.against G.citri-aurantii remains to be studied.Based on the perspective of biological control,this study investigated the biocontrol efficiency of M.citriensis strain FL01 against G.citri-aurantii,systematically studied its antagonistic action mode,proposed the possible important mechanism and explored its contribution to the biocontrol efficacy.On the basis of these results,the biocontrol effect of M.citriensis was further targeted enhanced,and possible mechanisms involved were also investigated.The main results are as follows:?1?M.citriensis could effectively control the development of sour rot,and significantly inhibit the mycelial growth and spore germination of G.citri-aurantii.Moreover,comparing with the storage condition of 25?,the efficacy of yeast controlling decay incidence was higher at 4?.The cell-free supernatant and VOCs produced by M.citriensis had no inhibitory effect on mycelial growth of G.citri-aurantii.M.citriensis adhered to the hyphae of G.citri-aurantii loosely and sparsely,and the production of lytic enzymes?-1,3-glucanase?GLU?and Chitinase?CHI?could not be induced by G.citri-auranti,indicated the yeast had no direct parasitic effect on the mycelia of G.citri-auranti.M.citriensis also induced resistance of fruit against sour rot.The addition of exogenous Fe Cl3 affected the inhibition of M.citriensis on the growth of G.citri-aurantii and the control efficiency of yeast against sour rot,it is speculated that the competition or depletion of iron by pulcherriminic acid?PA?produced by M.citriensis was the one key mechanism of action.?2?The detailed genome sequence of M.citriensis FL01 was obtained by whole-genome sequencing using combined Illumina PE150 flatform and Pac Bio Sequel platform and de novo assembled.The genome size of M.citriensis FL01 was about 25.74Mb,consisting of 12 contigs,313 t RNA,185 r RNA,52 sn RNA,0.76%scattered repeats accounted and 1.27%tandem repeats.A total of 5189 protein coding genes were predicted,of which 3401 genes were annotated by GO classification,4137 genes were annotated by KEGG,and 1845 genes were matched to functional categories in KOG database.M.citriensis FL01 have rich genes related to intracellular and extracellular signal transduction and metabolism,suggesting that yeast possess the qualities of active metabolism and strong environmental adaptability.Collinearity analysis of nucleic acid showed that M.citriensis FL01 was closer to M.pulcherrima APC 1.2.By genome-wide BLAST of the PUL gene of M.pulcherrima APC 1.2,four PUL genes in M.citriensis FL01 were found,PUL1?1377 bp?,PUL2?1433 bp?,PUL3?960 bp?and PUL4?1638bp?were clustered,and located on Contig4.PUL1 and PUL3 were positively expressed,PUL2 and PUL4 were negatively expressed,and the distribution of PULs genes was consistent with that of M.pulcherrima APC 1.2.?3?Using the CRISPR/Cas9–mediated gene knockout method,the key gene PUL2that responsible for PA in M.citriensis was knocked out and stable mutants without pulcherrimin pigment production were obtained.Compared with wild-type yeast,the mutants of?M.citriensis had no significant change in growth state and spore morphology,but both of them may be lost the ability to produce PA or or the produced PA was too little to be detected.The?M.citriensis mutants lost the inhibition of G.citri-aurantii growth in vitro.While in vivo experiment,the control efficacy of mutant strains against sour rot was significantly lower than that of wild-type M.citriensis,which equivalent to that of wild-type yeast was reduced by about 80%.In addition,there was no significant difference in the biocontrol effect among the mutant strains.These results directly proved that the iron depletion caused by PA production was an important mechanism of M.citriensis in controlling postharvest sour rot of citrus.?4?Based on increasing the yield of PA to improve the biocontrol efficacy of M.citriensis,amino acids that increase the pigment production of M.citriensis were screened from the perspective of physiological regulation.The result showed except Methionine?Met?among the 20 amino acids tested,the exogenous amino acids treatment could significantly induce M.citriensis to produce pigment of pulcherrimin.Serine?Ser?,aspartic amide?Asn?,lysine?Lys?and arginine?Arg?could significantly induce M.citriensis to produce a wider pigment halo compared with other amino acids at the same concentration,and the induction effect of Arg was the most significant one.?5?Arg exhibited the concentration effect on the cell growth,PA production and PUL genes expression of M.citriensis.Low concentration(1 mmol L-1)of Arg had no significant effect on the growth of M.citriensis,while 5 mmol L-1 and 10 mmol L-1 Arg remarkably promoted the growth of M.citriensis.As a secondary metabolite of M.citriensis,PA production by M.citriensis was significantly induced by Arg in the middle and late logarithmic period of culturing.Different concentrations of Arg had different effects on the expression of PUL genes.Within 24 h?72 h of inducting culture,Arg treatment significantly induced the up-regulated expression of PUL genes,while PUL4was inhibited within 12 h of incubation.In general,compared with 1 mmol L-1 of Arg,5mmol L-1 and 10 mmol L-1of Arg could significantly induce the expression of PUL genes thus enhancing the synthesis and secretion more PA of M.citriensis.?6?Arg treatment improved the colonization ability of M.citriensis at fruit wounds,indicating that Arg could improve the tolerance of antagonistic yeast to oxidative stress.Under the oxidative stress induced by H2O2,Arg pretreatment increased the activities of catalase?CAT?,superoxide dismutase?SOD?,glutathione peroxidase?GPX?and trehalose content in M.citriensis cells,and decreased the level of intracellular ROS,thereby inhibited cell apoptosis,plasma membrane damage and intracellular protein oxidation induced by excessive ROS,and then improved the tolerance of M.citriensis to oxidative stress.?7?By comparing the biocontrol effects of Arg-treated yeast which increased PA production and the mutant of?M.c7 which lost PA production capacity,it was found that Arg significantly enhanced the biocontrol efficacy of M.citriensis against sour rot caused by G.citri-aurantii,and the biocontrol efficiency of M.citriensis pretreated with 5 mmol L-1 Arg was exhibited the best.The possible mechanism included:Arg treatment induced the increase of PA production of M.citriensis,increased the colonization ability at the fruit wounds,improved the biofilm formation ability of M.citriensis and enhanced the oxidative stress tolerance of M.citriensis.However,the colonization ability,biofilm formation ability,oxidative stress tolerance,antioxidant capacity and biocontrol efficiency of mutant?M.c7,which lost the ability to produce PA decreased significantly.It was further demonstrated that the PA production ability of M.citriensis was directly related to its biocontrol efficacy,and also affected the intracellular antioxidant reaction of M.citriensis.In addition,PA may act as a quorum sensing molecule to mediate the biofilm formation of M.citriensis. |
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