| Aaccording to statistics,the past few decades have witnessed a rapid increase in the annual production of citrus fruit,which achieved approximately 158 million tons in 2019,half of which was Citrus sinensis.Furthermore,except for a small part to be consumed freshly,the overwhelming majority of sweet orange are processed for juices,jams,marmalades,etc.Among which,brocade orange,a cultivar belonging to the subgroup Citrus sinensis Osbeck,is extensively cultivated in Sichuan province and Chongqing city of China.It is widely used for producing citrus-based products for its characteristics such as thin skin,rich pulp,moderate sour and sweetness,strong flavor and juicy,and almost no seeds.The production of citrus-based products results in a copious mass of by-products—particularly peels,which account for about 25-40% of raw fruit weight.These generated residues are usually simply discarded directly as waste,representing significant contributors to environmental pollution.However,discarding citrus fruit peels have been identified as a potent,and in some cases even unique,source of a wide variety of beneficial ingredients,especially many flavanones,polymethoxyflavonoids(PMFs),synephrine,etc.,which are currently only or mainly found in citrus plants.Moreover,levels of these bioactive ingredients in citrus peels are equal to or higher than those found in fruit pulp and other tissues.Most promisingly,the biosynthesis of bioactive compounds,derived from fruit primary and secondary metabolism,is easily induced by several biotic and abiotic agents.A widely accepted approach is the application of effective inoculants with beneficial microbial elicitors(MEs)to enhance nutrient bioavailability and uptake,promote accumulation of functional components,and ultimately improve fruit quality.Our previous work demonstrated the capability of Pseudomonas fluorescens ZX to act as an effective biological control agent(BCA)to manage postharvest blue and green molds of citrus fruit via a variety of ways.Among them,the induction of fruit resistance is one of the important mechanisms.Meanwhile,preharvest application of this BCA effectively induced the disease resistance of brocade orange fruit at postharvest,suggesting that P.fluorescens ZX is promising to act as a ME.However,it is unclear whether the preharvest application of this strain can induce and increase the accumulation of active ingredients in brocade orange peels(BOPs).Therefore,in this experiment,the promise of the approach was evaluated by analyzing the profiles and contents of the main bioactive ingredients in BOPs which had been inoculated with P.fluorescens ZX applied under various conditions with respect to the number and timing of preharvest treatments.In particular,the induction effects of phenolic compounds and possible involved mechanisms were expected to provide a theoretical basis for increasing the contents of functional components in BOPs,increasing the added value of agricultural products and strengthening the comprehensive utilization of discarded peels.The main findings are as follows:1.The whole genome sequencing(WGS)of P.fluorescens ZX and its safety assessmentThe fine gene map of P.fluorescens ZX was obtained by WGS.P.fluorescens ZX-WGS was about 6.39 Mb in size,with a GC content of 59.72%.A total of 5865protein-coding genes were predicted,a large number of which were highly related to molecular function and metabolism,indicating that the strain has active metabolism and strong environmental adaptability.Phylogenetic tree and collinearity analysis showed that P.fluorescens ZX is closely related to strain Pseudomonas sp.DSM 29164,and has higher collinearity with P.azotoformans.Homologous gene analysis showed that there are 3204 shared genes and 2470 unique genes,receptively,among P.fluorescens ZX and other 15 selected strains,indicating that P.fluorescens ZX is somewhat special.Meanshile,a great quantity of genes related to composition,synthesis/formation,decomposition,and metabolism of amino acids,small molecular carbohydrates,high-carbon polymers,siderophores,antibiotics,volatile organic compounds,defense enzymes,antioxidants,flagella,cilia,and biofilms were found in P.fluorescens ZX-WGS,suggesting that P.fluorescens ZX is candidate as a ME with multiple survival strategies,tenacious vitality,and excellent colonization ability.In addition,P.fluorescens ZX was not phytopathogenic to plant,nor could it produce hemolysin.Thus,it is safe and can be applied to the field with confidence.2.Effects of preharvest applications of P.fluorescens ZX on the main bioactive components in BOPs and fruit qualityP.fluorescens ZX suspension was preharvest applied during different growth periods of fruit to investigate the response of primary metabolites(PMs)and main active components in BOPs of mature fruit to P.fluorescens ZX,and evaluate the effects of preharvest applications on the fruit quality.The results showed that the preharvest applications with P.fluorescens ZX have significant effects(P < 0.05)on the primary and secondary metabolism of BOPs,which were highly correlated with the applied number.Compared with the control check(CK),low-frequency application of P.fluorescens ZX effectively increased(P < 0.05)the contents of many PMs,including glucose,fructose,inositol,muco-inositol,inositol,fucose,galactose,erythrulose,glycerin,xylose,arabinose,mannose,malic,pyruvate,citric,oxaloacetic,and quinic acids,arginine,glutamate,methionine,phenylalanine,lysine,and valine,while the contents of secondary metabolites(SMs)changed relatively little.On the other hand,high-frequency pre-P.fluorescens ZX-applications,especially when P.fluorescens ZX was applied for 4 times,resulted in obvious decrease(P < 0.05)in contents of most of mentioned PMs,chlorophyll,limonin,and nomilin.While significant increases(P < 0.05)were observed in contents of soluble phenols,insoluble bound phenols(IBPs),flavonoids,synephrine,essential oils,water-soluble pectin,and protopectin,which were increased by about 26.9%,21.6%,39.6%,19.7%,45.4%,33.0%,and 20.7%,respectively,compared with CK.In addition,preharvest applications with P.fluorescens ZX did not impact the fruit quality,and effectively improved the fruit quality in terms of significant increases(P < 0.05)in total soluble solids,soluble sugar,titratable acid,and ascorbic acid in pulp,and limonene,α-pinene,β-pinene,and linalool in BOPs,resulting in effectice improvement in aroma,taste,and nutrients.3.Effect of preharvest applications with P.fluorescens ZX on phenolic compounds in BOPsUltra-high performance liquid chromatography(UHPLC)-quadrupole time of flight mass spectrometry and UHPLC-diode array detector(DAD)were used to qualitatively and quantitatively analyze the phenolic compounds with various fractions in both flavedo and albedo of BOPs in different growth periods.Meanwhile,real-time quantitative polymerase chain reaction was used to determine the expressions of key genes in phenylpropanoid pathway(PPP).The results showed that preharvest applications with P.fluorescens ZX significantly affect(P < 0.05)the compositions and contents of phenolic acids and flavonoids in BOPs,as well as the expressions of key genes in PPP.Precisely,pre-P.fluorescens ZX-applications significantly induced(P < 0.05)the accumulation of a variety of phenolic acids and flavonoids,possibly resulting from up-regulated expressions(P <0.05)of many key genes in PPP.A total of 55 phenolic compounds were identified,including 6 benzoic phenolic acids(HBAs),4 cinnamic phenolic acids(Hydroxycinnamic acids,HCAs),8 flavanones,27 flavonoids(including 20 PMFs),6 flavonols,and 4 other compounds.Then,a total of 23 compounds were quantitatively analyzed,including 4HBAs(benzoic,p-hydroxybenzoic,vanillic,and syringic acids),4 HCAs(caffeic,ferulic,p-coumaric,and sinapic acids),6 flavanones(naringenin,naringin,hesperetin,hesperidin,neohesperidin,and didymin),7 flavonoids(apiin,rhoifolin,vicenin-2,luteolin,sinensetin,tangeretin,and nobiletin),and 2 flavanones(quercetin and rutin).Phenolic acids existed in more forms,most of which were found in free phenols(FPs),esterified BPs,(EBPs),glycosylated BPs(GBPs),and IBPs in both flavedo and albedo,with highest abundance in EBPs.While flavonoids were only detected in FPs,EBPs,and GBPs,among which,flavanones and flavonoids mainly existed in the form of GBPs,hesperidin and PMFs occupied the dominant contents in FPs.In general,flavedo contained more forms and higher levels of HCAs,flavonoids,and PMFs than those of albedo,while the concentrations of flavanones,especially GBPs-flavanones were much higher(P < 0.05)in the albedo.Roughly equal levels of HBAs and flavonols were observed in the two tissues.Moreover,increased preharvest application numbers during fruit development typically elicited stronger stimulus effects.Data highlighted that multiple applications of P.fluorescens ZX dramatically enhancing(P < 0.05)the peels’ biosynthesis and accumulation of beneficial compounds,especially FPs-hesperidin,FPs-sinensetin,FPs-nobiletin,EBPs-ferulic acid,EBPs-p-coumaric acid,and GBPs-hesperetin,the main phenolic compounds in BOPs,and the contents of which in flavedo pre-treated with P.fluorescens ZX for 4 times were about 1.6-,1.3-,1.3-,1.1-,1.2-,and 1.6-fold of those in the CK,respectively.4.Preliminary analysis of the biological mechanisms involved in preharvest applications with P.fluorescens ZX to induce and regulate BOPs metabolism through omics strategyRepresentative flavedo samples of expanding and mature stages were selected to explore the effects of preharvest applications with P.fluorescens ZX on the microbial diversity of fruit using Illumina Miseq high-throughput sequencing.Meanwhile,based on RNA sequencing,series test of cluster(STC)and weighted gene co-expression network analysis(WGCNA)were used to preliminarily analyze the biological mechanism of preharvest applications to regulate the metabolism of BOPs.Preharvest applications of P.fluorescens ZX had no significant effects on the alpha-diversity of bacteria or fungi,however,it significantly changed(P < 0.05)the composition and structure of microbial community,causing the marked increases(P < 0.05)in the abundances of Stenotrophomonas,Burkholderia-Caballeronia-Paraburkholderia,Pseudomonas,paramycosphaerella,and uwebraunia,as well as remarkable reduce(P < 0.05)in the abundances of common disease fungi such as Penicillium and Colletotrichum.Preharvest applications with P.fluorescens ZX did have significant effects(P < 0.05)on the primary and secondary metabolism of flavedo.The pathways significantly enriched in differential expression genes(DEGs)were highly associated with many PMs(such as carbohydrates,lipids,etc.)and SMs(such as glucosinolates,flavonoids,terpenes,etc.)in the expending period,while they were mainly acted on secondary metabolic pathways,especially metabolic pathways and phenylpropanoid biosynthesis in the mature stage.Analysis of the expressions of key genes related to synthesis,metabolism,and signal transduction pathways of signal molecules and plant hormones showed that P.fluorescens ZX may regulate gene expressions in the flavedo mainly depending on the jasmonic acid/ethylene signal transduction pathway.Meanwhile,a variety of plant hormones,especially auxin,brassinolide,and gibberellin,participated in the biological process.Preharvest applications with P.fluorescens ZX significantly up-regulated(P < 0.05)the expression levels of a large number of genes highly related to PPP and flavonoid biosynthesis pathways,and more importantly,the inductive effects offered by pre-treatments were greatly strengthened with increasing number of applications.According to STC,the most significant core pathway affected by preharvest applications with P.fluorescens ZX was the metabolic pathway(ko01100)in the expanding sample,and the most responsive gene might be the Cs＿ont＿5g017430(COMT1).While in the mature stage,21 and 7 hub genes were screened from the orange and pink modules,which were closely related to the selected phenolic compound contents,namely character data,respectively.Among which,Cs＿ont＿1g009820(4CL2)and Cs＿ont＿1g011170(CCR1)might be the key genes in the two modules based on WGCNA,respectively.Taken as a whole,the present work has demonstrated the efficacy of P.fluorescens ZX to be the plant inoculants.Preharvest applications with P.fluorescens ZX significantly stimulated the primary and secondary metabolism of fruit,regulated the microbial community composition,and increased the expressions of related genes,resulting in remarkable improvement in the quality of fruit with higher lecels of nutrients in pulp,and aroma components and bioactive compounds in BOPs.According to both physiological and biochemical reactions and gene expression levels,it is inferred that the inductive effects of increasing the number of P.fluorescens ZX applications can be summarized in two stages: low-frequency applications enhance the accumulation of primary and secondary metabolism,while high-frequency applications strengthen the transformation of primary metabolites towards further biosynthesis of secondary metabolites.Therefore,multiple preharvest applications of P.fluorescens ZX is an effective way to improve fruit quality,regulate the metabolism of BOPs,and induce the accumulation of functional components,especially HCAs,flavanones,PMFs,essential oils,synephrine,and pectin,etc. |
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