| Citrus huanglongbing(HLB)is a devastating systemic disease in citrus production,caused by Candidatus Liberibacter spp.The citrus infected with HLB has symptoms such as yellow shoots,mottled yellow leaves,and"red-nose fruit",and the yield and quality of the citrus are seriously reduced,causing great economic losses.Since there are no effective cures and resistant varieties,early detection is of great significance for the prevention and control of HLB.Metabolomics is a science that studies the overall changes of metabolites in biological systems and their response to internal and external factors.Therefore,it has become an important tool to study the interaction between plants and microorganisms.The researchers analyzed the metabolite differences of HLB tolerant and susceptible citrus varieties through metabolomics methods,and found that the innate immune system and metabolic defense mechanism played a major role in the resistant varieties,but the research on the dynamic response and defense mechanism of citrus during the development of HLB was relatively limited.The objects of this study include“Gannan navel oranges”and“Nanfeng tangerine”,which are national geographical indication products in China.The experimental materials were obtained from artificial grafting in the experimental field and orchards,respectively.The leaves of Newhell navel oranges obtained by artificial grafting include four groups:healthy(Control),incubation(T0),early(T1),and late(T2)periods of HLB-infected.The samples collected from the orchard include three groups:healthy leaves(CK),asymptomatic(T0)and symptomatic(T1)leaves with HLB-infected.The metabolic fingerprints of navel orange leaves were obtained by EESI-MS,combined with multivariate analysis methods such as PCA and PLS-DA to distinguish healthy and different HLB-infected stage leaves.Then,EESI-MS,UPLC-QTOF-MS,and GC-MS were used to analyze the metabolic response and defense reaction process in the development of HLB.Further combined with transcriptome analysis,the transcriptional regulation process and regulation of navel orange in response to HLB were studied.In addition,based on the combined metabolome and transcriptome analysis,the effect of HLB on fruit quality and the metabolic regulation process of pigments that lead to the change of fruit pigment were studied.The main findings are as follows:(1)The Newhell navel orange did not show symptoms during the incubation period(T0)after HLB infection,but the bacterial content detected by q PCR was2.73×10~4 cells/μg DNA.In this study,based on the metabolite changes of navel orange leaves,an EESI-MS technology was established to detect HLB.EESI-MS combined with PCA and PLS-DA achieved early detection of HLB and effective differentiation of the different HLB-infected stage leaves,including Control,T0,T1,and T2 groups.The method is simple in preprocessing and fast in analysis,which is convenient for high-throughput and rapid screening of HLB.Using VIP value>1 and P<0.05 as screening criteria,8 potential markers were screened,among which p-coumaric acid,quinic acid,resveratrol,and isovitexin were enriched in the phenylpropane metabolic branch.The expression of CHI,CHS,C12R,CYP,and HCT in phenylpropane metabolism were up-regulated in T0 and T1 periods,which indicated the phenylpropane metabolic branch was activated in the early stage of HLB development.However,in the late stage of HLB-infected,the expression of the above genes was down-regulated,and the defense response was suppressed.(2)GC-MS was used to detect the changes of volatile components in Newhell navel orange leaves in response to HLB.A total of 27 volatile compounds were identified,including monoterpenes(48.15%),sesquiterpenes(18.52%),alcohols(18.52%),aldehydes(7.41%)and furans(7.41%),20 of them wer common components.The differences in volatile components of the three groups of samples were mainly reflected in the changes in the relative content of volatiles:the content of 3-thujene,D-α-pinene,(+)-3-carene,ψ-limonene andγ-terpinene increased in T0 group;the content of hexanal,trans-2-hexanal,α-phellandrene,β-ocimene decreased,(+)-3-carene,ψ-limonene,and D-limonene and other content increased in T1 and T2 groups.(3)To further analyze the systemic defense response of Newhell navel oranges to HLB,the metabolites in healthy(CK),symptomatic(T0),and symptomatic(T1)leaves with HLB-infected were detected based on UPLC-QTOF-MS.A total of 161metabolites were identified,including 27 categories such as phenolic acids,flavonoids,amino acids,organic acids,and coumarins.PCA and PLS-DA analysis showed that there were significant differences between the three groups.Using VIP value>1 and P<0.05 as criteria,16 and 31 differential metabolites were screened out in T0 and T1groups,respectively.Pathway enrichment found that compared with CK,the arginine metabolic pathway was active in the T1 group,and argininosuccinate and arginine were synthesized in large quantities,which promoted the synthesis of downstream polyamines;however,the response was inhibited in T0 group,salicylic acid content decreased,and downstream defense processes activated by salicylic acid were inhibited.Compared with T0,the metabolic response of T1 group was strong and complex,with more differential metabolites and metabolic pathways,mainly for phenylpropane metabolism,flavonoids,arginine,valine,leucine,and isoleucine biosynthesis pathway.Pathways related to the synthesis of phenolics were involved in the defense response during the development of HLB.The metabolism of arginine and salicylic acid were inhibited in the T0 group,while active in the T1 group,participating in the defense process against pathogens.(4)A total of 701 differentially expressed genes(DEGs)were identified by transcriptome analysis of CK,T0,and T1 navel orange leaves.Compared with CK,70and 631 DEGs(fold change(FC)≥2 or FC≤0.5,and P<0.05)were identified in T0and T1,respectively.The genes related to brassinolide biosynthesis and starch and sucrose metabolism were down-regulated in T0 group.In T1 group,the expression of genes related toα-linolenic acid metabolism,diterpenoids,and zeatin biosynthesis was down-regulated,while the expression of genes related to photosynthesis,porphyrin and chlorophyll metabolism,phenylpropane metabolism,and ubiquinone and other terpenoid quinone biosynthesis were up-regulated.The DEGs involved in phenylpropane metabolism(CYP75B1,GT72E1,4CL1,and 4CL2)and carotenoid biosynthesis(VDE,PSY,Crt Z,and LCYe)were up-regulated during the development of HLB and involved in the defense response of HLB and the formation of mottled yellowing symptoms,respectively.Transcriptome analysis revealed the metabolic response and defense pathways of navel orange to HLB from the transcriptional level,and provided the key DEGs and their involved functional pathways.(5)The content of soluble sugar and soluble solid in HLB-infected fruit decreased significantly,and the content of organic acid increased significantly,the sugar-acid ratio and solid-acid ratio were 4.35 and 5.87,respectively.Ascorbic acid,hesperidin,and quercetin were significantly decreased,and bioactive components were decreased in HLB-infected fruit.After citrus fruit infection with HLB,the nutritional quality and bioactive components of the fruit decreased to varying degrees,and the edible value and commercial value were lost.(6)The determination of bacterial content in the peel of healthy fruit(CK),orange(HLB-O),and green(HLB-G)peels of HLB-infected fruits showed that HLB-O>HLB-G>CK.The content of chlorophyll b in CK and HLB-O was higher than that in chlorophyll a(chlorophyll a/b was 0.56 and 0.60,respectively),and the content of chlorophyll b in HLB-G was lower than that in chlorophyll a(chlorophyll a/b was 1.98);the content of total carotenoids in CK and HLB-O was significantly higher than that in HLB-G.The contents of carotenoids such asα-carotene,violaxanthin,neoxanthin,and lutein in HLB-O were significantly higher than those in HLB-G.Compared with the CK group,the soluble sugar content,SOD,CAT,and POD activities in the diseased peel were significantly increased,and the abscisic acid(ABA)content was significantly decreased.The antioxidant process in the peel of HLB-infected fruit was enhanced,the activities of SOD,POD,and CAT were significantly enhanced,the content of pathogenic bacteria was reduced,the genes related to carotenoid and ABA synthesis were down-regulated,the content of carotenoid was reduced,and the peel was green.Finally,the fruit generally showed the symptoms of"red-nose fruit".Based on the metabolome and transcriptome,this study systematically analyzed the metabolic response and defense response mechanism of Newhell navel orange in different stages of HLB.In the early stage of HLB infection in citrus,the accumulation of phenolic and terpenoid defense substances played a certain antibacterial effect,while the growth process mediated by brassinolide and the immune response mediated by salicylic acid was inhibited.However,the metabolic defense pathways of terpenoids were transformed,and the metabolic activities involving salicylic acid and arginine were enhanced in the later stage of HLB infection in citrus.With the degradation of chlorophyll,changes in photosynthesis and synthesis of carotenoids,leaves and fruits exhibit typical symptoms of uneven coloration.The research results could provide a reference for analyzing the interaction process of citrus and HLB and the dynamic defense mechanism of citrus. |
