| Citrus tristeza virus(CTV)efficiently transmitted by the brown citrus aphid Toxoptera citricida(Kirkaldy)could infect Citrus L.and their relatives.CTV occurs in many tropical and subtropical regions around the world,and regarded as a major threat to citrus plants.Their pathogens could enhance the spread to new host plants by affecting vector insects,and previous studies found that the brown citrus aphid was more suitable on CTV-infected plants,which significantly promoted its acquisition of CTV.So far,whether CTV tolerance affects host selection and fitness analysis of the brown citrus aphid between healthy and CTV-infected plants has not been considered when conducting host selection and response to viral infection experiment.Therefore,focusing on the scientific issue of "CTV-mediated feeding selection of the brown citrus aphid",this paper took CTV-susceptible and-tolerant host plants and the brown citrus aphid as the research objects to clarify the adaptability of the brown citrus aphid to different hosts.Furthermore,the mechanism of feeding behavior was studied by Electrical penetration graph(EPG),and the intrinsic physiological and molecular response mechanism of Citrus to CTV infection was revealed by techniques such as chromatography,enzyme activity assay,and transcriptome.The results could provide a theoretical basis for scientific prevention and control of virus epidemics and effective management of vector insects under the new situation.The main results were as follows:1.Comparison of the suitability of brown citrus aphid on different citrus varietiesAphid resistance of eight different citrus varieties(Citrus junos Sieb.ex Tanaka,C.jambhiri Lush.,Fortunella margarita Swingle,C.sinensis(L.)Osbeck,C.reticulata Blanco,C.sinensis,C.limon(L.)Osbeck,and Poncirus trifoliata(L.)Raf)evaluated by EPG technique and population growth experiment.The results showed that the brown citrus aphid feeding EPG parameters varied widely on eight different citrus varieties,with brown citrus aphid spending more time on the path stage and less time on the bast stage on C.limon;the opposite was observed on Citrus junos,Poncirus trifoliata,and C.jambhiri.Aphid population growth experiments showed that aphid population growth was significantly lower on C.limon and Poncirus trifoliata than on other species.The results of the study showed that different host plants significantly influenced the feeding ability of citrus aphids.The brown citrus aphid did not prefer to feed on C.limon compared to the other seven host plants,C.limon showed significant resistance to brown citrus aphid,while Citrus junos and C.jambhiri were susceptible host plants.The results of this study clarified the level of aphid resistance of CTV-susceptible citrus and CTV-tolerant citrus.2.Effect of CTV-infected citrus on the growth and development of brown citrus aphidThe developmental duration,fecundity and feeding behavior of the brown citrus aphid on two different resistant host plants infected by CTV and uninfected were compared.The results showed that the developmental duration of each instar and the pre-adult stage of the brown citrus aphid on healthy or CTV-infected citrus were smaller than those on Poncirus trifoliata plants.The developmental duration of the first instar nymphs on CTV-infected C.junos was 1.83±0.03 d,which was significantly shorter than that in healthy C.junos(1.89±0.03 d).This parameter also showed similar results in Poncirus trifoliata varieties.The development of the brown citrus aphid on CTV-infected C.junos was faster than that in healthy C.junos plants in the pre-adult stage.Compared with healthy C.junos cultivars,there was no significant difference in the pre-adult survival rate of the brown tangerine aphid on CTV-infected C.junos,but there was significantly different on P.trifoliata.The adult lifespan,aphid production period and fecundity of the brown citrus aphid on CTV-infected C.junos were significantly higher than those on healthy plants.The intrinsic growth rate r,the perimeter growth rate λ and the net reproductive rate R0 of the brown citrus aphid on CTV-infected C.junos were significantly higher than those on healthy plants.The average generation time T of CTV-infected C.junos was shorter than that of healthy plants,but the difference was not significant.In words,the different resistant hosts infected by CTV had different effects on the growth and development of the brown citrus aphid,and the growth and development of vector insects are affected by host resistance,and these effects may play a key role in the prevalence of CTV.3.Effect of CTV infection on biochemical indicators in citrus leavesCTV infection of different citrus hosts could significantly affect the amino acid composition of young shoots in citrus hosts.The total sugar content of CTV-infected citrus plants was significantly lower than that of healthy citrus plants.The p H value of the cytosol of the different host plants was significantly different after CTV infection,all of which showed that the p H value of the citrus infected with CTV was significantly lower than that of the uninfected citrus plants.CTV infection had no significant effect on the relative content of chlorophyll in the two citrus hosts,but significantly affected the kinetic parameters of chlorophyll fluorescence.4.Effect of CTV infection and brown citrus aphid feeding on the volatile organic compound profile of different citrus speciesCTV-infected plants significantly attracted more brown citrus aphids compared to non-CTV-infected plants by indoor bioassay.The volatile organic compounds(VOCs)from CTV-infected C.junos/ P.trifoliata,CTV-infected C.junos/ P.trifoliata fed by non-toxic aphids,and uninfected CTV-infected C.junos/ P.trifoliata were collected,identified and analyzed by gas chromatography-mass spectrometry(GC-MS).The results showed that the components of different citrus varieties differed greatly,and the composition and content of volatiles in C.junos were relatively higher than those in P.trifoliata,with the highest relative content of terpene volatiles.Through principal component analysis,the main volatile citrus compounds of 2varieties and 8 treatments showed obvious separation.Both CTV virus infection and brown citrus aphid feeding significantly affected the changes of VOCs in species and composition between the two citrus plants.In C.junos,γ-Terpinene and(-)-β-Elemene decreased significantly after CTV inoculation,while the relative content of β-Pinene increased significantly.The release of β-Pinene,2-Ethyl-1-hexanol and Longifolene increased significantly after aphid feeding.The combined effect of CTV inoculation and aphid feeding resulted in a significant increase in D-Limonene,p-Cymene,β-Ocimene and γ-Terpinene.As well as the combined effect of the two,camphene,β-Phellandrene,Sabinene,β-Ocimene,γ-Elemene,(-)-β-Elemene,Caryophyllene,cis-β-Farnesene,cis-β-Farnesene,andβ-Sesapinene were significantly reduced in P.trifoliata.Humulene andβ-Sesquiphellandrene were significantly lower than those of untreated P.trifoliata.then,the relative contents of 3-Carene in the samples after aphid feeding on P.trifoliata were significantly higher than those of untreated and CTV-infected samples.5.Differences in defense responses caused by CTV infestation and aphid feedingChanges in the activities of important defense enzymes and the content of endogenous jasmonic acid and salicylic acid in citrus were determined after CTV infection and brown citrus aphid feeding in different disease-resistant citrus.The results showed that both CTV infestation and brown citrus aphid feeding significantly affected the activities of superoxide dismutase SOD,catalase CAT,peroxidase POD,lipoxygenase LOX,phenylalanine aminolase PAL and β-1,3-glucanase and endogenous JA and SA in different citrus among different resistance,but there were differences among different resistant citrus varieties.The activities of CAT and POD in P.trifoliata were higher than those of C.junos,and the former was more resistant.CTV-infected C.junos had significantly higher SA levels,and aphid feeding induced more JA.Aphid feeding caused a significant increase in endogenous SA and JA content higher than changes caused by CTV infection.It is speculated that the change of defense substances in the host may be one of the important factors affecting the biological characteristics of the brown citrus aphid in susceptible plants.6.Transcriptome analysis of different resistant citrus plants infected by CTVThe high-throughput sequencing platform was used to sequence the transcriptomes of leaves of two citrus cultivars(C.junos and P.trifoliata)infected and uninfected with CTV.And then high-quality clean reads was spliced using Trinity software,and assembled by de novo,and the assembled unigenes were compared and annotated in the nine major data sets based on Blast software.The results showed that a total of 81,732 unigenes were obtained,of which the N50 length was 2,694 bp.The homology analysis of all genes was carried out with the NR database,indicating that the matching degree of gene similar to C.clementina,C.sinensis and C.unshiu was the highest homology.A total of 1284 differentially expressed genes were identified in C.junos,of which 545 genes were up-regulated and 739 genes were down-regulated,while there were 205 differentially expressed genes in P.trifoliata,of which 186 genes were up-regulated and 19 genes were down-regulated.KEGG enrichment analysis results showed that in C.junos,the most obvious pathway of differential gene enrichment was mainly plant-pathogen interaction,phytohormone signal transduction and MAPK signaling pathway-plant compared CTV infection treatment and healthy control.While in P.trifoliata,differential genes were mainly involved in plant-pathogen interaction,ubiquitin-mediated proteolysis,phytohormone signal transduction.q RT-PCR and the expression profiles obtained by RNA-seq were consistent with each other,thus validating the reliability of the results obtained by RNA-seq.The research results lay a theoretical foundation for in-depth study of the defense mechanism of different citrus varieties. |
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