In this review,the advantages and advances in applying high-throughput sequencing(HTS)in the management of viral diseases in citrus,along with some challenges,are discussed to provide perspectives on future prospects....In this review,the advantages and advances in applying high-throughput sequencing(HTS)in the management of viral diseases in citrus,along with some challenges,are discussed to provide perspectives on future prospects.Since the initial implementation of HTS in citrus virology,a substantial number of citrus viruses have been identified,with a notable increase in the last 7 years.The acquisition of viral genomes and various HTS-based omics analyses serve as crucial pillars for advancing research in the etiology,epidemiology,pathology,evolution,ecology,and biotechnology of citrus viruses.HTS has notably contributed to disease diagnosis,such as the diagnoses of concave gum and impietratura,as well as to the surveillance of new virus risks and the preparation of virus-free materials.However,certain inherent defects in HTS and coupled bioinformatics analysis,such as challenges with sequence assembly and the detection of viral dark matter,require improvement to enhance practical efficiency.In addition,the utilization of HTS for the systematic management of citrus viral diseases remains limited,and drawing insights from other virus-plant pathosystems while integrating emerging compatible techniques and ideas may broaden its specific applications.展开更多
Citrus tristeza virus （CTV） is one of the most important causal agents of citrus diseases and exists as numerous strains. CTV is replicated in phloem cells of plants within the family Rutaceae and is transmitted by ...Citrus tristeza virus （CTV） is one of the most important causal agents of citrus diseases and exists as numerous strains. CTV is replicated in phloem cells of plants within the family Rutaceae and is transmitted by a few of aphid species. CTV epidemics have caused death of millions of citrus trees in many regions all over the world, where the sour orange （Citrus aurantium） was used as rootstock. Also the production of grapefruit （C. paradisi） and sweet orange （C. sinensis） has been affected by CTV strains. CTV gives uplift to three prominent syndromes, namely quick-decline （tristeza）, stempitting and seedling-yellows. The disease is graft-transmissible in nature but not seed-transmitted. However, the tristeza disease in most citrus groves was a man-made problem created by the desire of horticulturists to introduce cultivars from other citrus growing areas. The utmost importance of the disease called for review articles in numbers of plant protection, epidemiology books, citriculture and proceedings. This review collects the information with respects to disease history, distribution host range, virus isolates association, identification and detection, transmission and management; especially on the current status of CTV prevailing and controlling in Pakistan. It provides valuable information for CTV disease and its controlling approaches.展开更多
Citrus bacterial canker(CBC) is resulted from Xanthomonas citri subsp. citri(Xcc) infection and poses a significant threat to citrus production.Glutathione S-transferases(GSTs) are critical in maintaining redox homeos...Citrus bacterial canker(CBC) is resulted from Xanthomonas citri subsp. citri(Xcc) infection and poses a significant threat to citrus production.Glutathione S-transferases(GSTs) are critical in maintaining redox homeostasis in plants, especially in relation to abiotic and biotic stress responses. However, the function of GSTs in resisting CBC remains unclear. Here, citrus glutathione S-transferases were investigated applying a genome-wide approach. In total, 69 CsGSTs belonging to seven classes were identified, and the phylogeny, chromosomal distribution, gene structures and conserved motifs were analyzed. Several CsGSTs responded to Xcc infection, as observed in the upregulation of CsGSTF1 and CsGSTU18 in the CBC-sensitive ‘Wanjincheng' variety but not in the resistant ‘Kumquat' variety. CsGSTF1 and CsGSTU18 were localized at the cytoplasm. Transient overexpression of CsGSTF1 and CsGSTU18 mediated reactive oxygen species(ROS) scavenging, whereas the virus-induced gene silencing(VIGS) of CsGSTF1 and CsGSTU18 caused strong CBC resistance and ROS burst. The present study investigated the characterization of citrus GST gene family, and discovered that CsGSTF1 and CsGSTU18 negatively contributed to CBC through modulating ROS homeostasis. These findings emphasize the significance of GSTs in infection resistance in plants.展开更多
One of the main diseases that adversely impacts the global citrus industry is citrus bacterial canker(CBC),caused by the bacteria Xanthomonas citri subsp.citri(Xcc).Response to CBC is a complex process,with both prote...One of the main diseases that adversely impacts the global citrus industry is citrus bacterial canker(CBC),caused by the bacteria Xanthomonas citri subsp.citri(Xcc).Response to CBC is a complex process,with both proteinDNA as well as protein–protein interactions for the regulatory network.To detect such interactions in CBC resistant regulation,a citrus high-throughput screening system with 203 CBC-inducible transcription factors(TFs),were developed.Screening the upstream regulators of target by yeast-one hybrid(Y1H)methods was also performed.A regulatory module of CBC resistance was identified based on this system.One TF(CsDOF5.8)was explored due to its interactions with the 1-kb promoter fragment of CsPrx25,a resistant gene of CBC involved in reactive oxygen species(ROS)homeostasis regulation.Electrophoretic mobility shift assay(EMSA),dual-LUC assays,as well as transient overexpression of CsDOF5.8,further validated the interactions and transcriptional regulation.The CsDOF5.8–CsPrx25 promoter interaction revealed a complex pathway that governs the regulation of CBC resistance via H2O2homeostasis.The high-throughput Y1H/Y2H screening system could be an efficient tool for studying regulatory pathways or network of CBC resistance regulation.In addition,it could highlight the potential of these candidate genes as targets for efforts to breed CBC-resistant citrus varieties.展开更多
Citrus yellow vein clearing virus(CYVCV)is a new citrus virus that has become an important factor restricting the development of China’s citrus industry,and the CYVCV coat protein(CP)is associated with viral pathogen...Citrus yellow vein clearing virus(CYVCV)is a new citrus virus that has become an important factor restricting the development of China’s citrus industry,and the CYVCV coat protein(CP)is associated with viral pathogenicity.In this study,the Eureka lemon zinc finger protein(ZFP)ClDOF3.4 was shown to interact with CYVCV CP in vivo and in vitro.Transient expression of ClDOF3.4 in Eureka lemon induced the expression of salicylic acid(SA)-related and hypersensitive response marker genes,and triggered a reactive oxygen species burst,ion leakage necrosis,and the accumulation of free SA.Furthermore,the CYVCV titer in ClDOF3.4 transgenic Eureka lemon plants was approximately 69.4%that in control plants 6 mon after inoculation,with only mild leaf chlorotic spots observed in those transgenic plants.Taken together,the results indicate that ClDOF3.4 not only interacts with CP but also induces an immune response in Eureka lemon by inducing the SA pathways.This is the first report that ZFP is involved in the immune response of a citrus viral disease,which provides a basis for further study of the molecular mechanism of CYVCV infection.展开更多
Citrus leaf blotch virus （CLBV） is a member of the genus Citrivirus, in the family Betaflexiviridae. It has been reported CLBV could infect kiwi, citrus and sweet cherry in China. Of 289 citrus samples from six regi...Citrus leaf blotch virus （CLBV） is a member of the genus Citrivirus, in the family Betaflexiviridae. It has been reported CLBV could infect kiwi, citrus and sweet cherry in China. Of 289 citrus samples from six regions of China, 15 were detected to be infected with CLBV in this study. The complete genome of four isolates of CLBV was obtained from Reikou in Sichuan （CLBV-LH）, Yura Wase in Zhejiang （CLBV-YL）, Bingtangcheng in Hunan （CLBV-BT）, Fengjie 72-1 in Chongqing （CLBV- F J）, respectively. While they all represented 8 747 nucleotides in monopartite size, excluding the poly（A） tail, each of the isolates coded three open reading frames （ORFs）. Identity of the four isolates ranged from 98.9 to 99.8% to each other and from 96.8 to 98.1% to the citrus references in GenBank by multiple alignment of genomes. A phylogenetic tree based on the genome sequences of available CLBV isolates indicated that the four isolates were clustered together, suggesting that CLBV isolates from citrus in China did not have obvious variation. This is the first report of the complete nucleotide sequences of CLBV isolates infecting citrus in China.展开更多
The most damaging citrus diseases are Huanglongbing(HLB)and citrus canker,which are caused by Candidatus Liberibacter asiaticus(CaLas)and Xanthomonas citri pv.citri(Xcc),respectively.Endolysins from bacteriophages are...The most damaging citrus diseases are Huanglongbing(HLB)and citrus canker,which are caused by Candidatus Liberibacter asiaticus(CaLas)and Xanthomonas citri pv.citri(Xcc),respectively.Endolysins from bacteriophages are a possible option for disease resistance in plant breeding.Here,we report improvement of citrus resistance to HLB and citrus canker using the LasLYS1 and LasLYS2 endolysins from CaLas.LasLYS2 demonstrated bactericidal efficacy against several Rhizobiaceae bacteria and Xcc,according to inhibition zone analyses.The two genes,driven by a strong promoter from Cauliflower mosaic virus,35S,were integrated into Carrizo citrange via Agrobacterium-mediated transformation.More than 2 years of greenhouse testing indicated that LasLYS2 provided substantial and long-lasting resistance to HLB,allowing transgenic plants to retain low CaLas titers and no obvious symptoms while also clearing CaLas from infected plants in the long term.LasLYS2 transgenic plants with improved HLB resistance also showed resistance to Xcc,indicating that LasLYS2 had dual resistance to HLB and citrus canker.A microbiome study of transgenic plants revealed that the endolysins repressed Xanthomonadaceae and Rhizobiaceae populations in roots while increasing Burkholderiaceae and Rhodanobacteraceae populations,which might boost the citrus defense response,according to transcriptome analysis.We also found that Lyz domain 2 is the key bactericidal motif of LasLYS1 and LasLYS2.Four endolysins with potential resistance to HLB and citrus canker were found based on the structures of LasLYS1 and LasLYS2.Overall,the work shed light on the mechanisms of resistance of CaLas-derived endolysins,providing insights for designing endolysins to develop broad-spectrum disease resistance in citrus.展开更多
Huanglongbing (HLB) is the most destructive disease of citrus and is associated with ‘Candidatus Liberibacter asiaticus’(CLas), a member of the α-proteobacteria. Citrus tristeza virus (CTV) is another pathogen of c...Huanglongbing (HLB) is the most destructive disease of citrus and is associated with ‘Candidatus Liberibacter asiaticus’(CLas), a member of the α-proteobacteria. Citrus tristeza virus (CTV) is another pathogen of citrus with very great historic as well as current importance. Both CLas and CTV are phloem-restricted pathogens. A severe CTV isolate, CTV-B6, and CLas-B232 induce a group of symptoms of phloem dysfunction that overlap, but the mild isolate CTV-B2 does not cause any loss to commercial trees. Prior inoculation and establishment of CLas-B232 did not affect subsequent establishment of either CTV-B2 or CTV-B6, while super infection by CLas-B232 was reduced by prior establishment of CTV-B2 and to a lesser extent by prior infection with CTV-B6. Trees co-infected with CTV-B6 and CLas-B232 developed more severe symptoms, typical of CTV-B6, than either of the two pathogens co-infected with CTV-B2. In this study, we confirmed that CLas established in the rootlets earlier and with higher concentration than in leaves. The distribution of CLas in the plant infected by CLas-B438 alone and with CTV-B2 fits a previously proposed model but CLas was more sporadically distributed in a plant co-infected by CLas and CTV-B2 than in a plant infected by CLas alone. These biological phenomena are aligned with previously analyzed transcriptome data and the study provides a novel idea that mild CTV strains may provide some protection against CLas by limiting its multiplication and spread. The protective effect may be due to opposite regulation of key host defense pathways in response to CTV-B2 and CLas-B438.展开更多
Traumatic brain injury(TBI)is closely related to neuro-inflammatory response and causes a complex pathological process and serious consequence.In this study,we explored whether the formatted citrus peel extract gold l...Traumatic brain injury(TBI)is closely related to neuro-inflammatory response and causes a complex pathological process and serious consequence.In this study,we explored whether the formatted citrus peel extract gold lotion(GL)could have a therapeutic effect on rats suffered TBI.TBI rat model was prepared by the electric Cortical Contusion Impactor(eCCI)device.Prevention against TBI by GL was assessed by the behavioral,organizational of rats and the molecular markers in the brain injury area.The results showed that GL could reduce nerve damage and neurological dysfunction.The mechanisms probably due to that GL could not only reduce the associated damage markers including GFAP,OX-42,TNF-α,COX-2,NF-κB and TLR4 but also promote the expression of VEGF which is responsible for protecting neuronal cells in the injury area.These findings revealed that GL could be a promising preparation for TBI prevention.展开更多
As the bacterial etiologic agent causing citrus bacterial canker(CBC),Xanthomonas citri subsp.citri(Xcc)seriously impacts citrus plantation and fruit production globally.In an earlier study,we demonstrated that CsBZIP...As the bacterial etiologic agent causing citrus bacterial canker(CBC),Xanthomonas citri subsp.citri(Xcc)seriously impacts citrus plantation and fruit production globally.In an earlier study,we demonstrated that CsBZIP40 can positively impact CBC resistance in the sweet orange(Citrus sinensis).However,the mechanistic basis for the protective benefits conferred by CsBZIP40 is yet to be delineated.Here,we show that CsBZIP40 positively regulates CBC resistance and reactive oxygen species(ROS)homeostasis in transgenic sweet orange overexpressing CsBZIP40.CsBZIP40 directly binds to the TGA-box of the CsWRKY43 promoter to repress its transcriptional activity.CsWRKY43 overexpression induces CBC susceptibility in transgenic sweet oranges.In contrast,its inhibition produces strong resistance to CBC.CsWRKY43 directly binds to the W-boxes of the CsPrx53 and CsSOD13 promoters to positively regulate the activities of these antioxidant enzymes,resulting in the negative regulation of ROS homeostasis and CBC resistance in sweet orange plants.CsPrx53/CsSOD13 knockdown enhances ROS accumulation and CBC resistance.Overall,our results outline a regulatory pathway through which CsBZIP40 transcriptionally represses CsWRKY43-CsPrx53/CsSOD13 cascade-mediated ROS scavenging in a manner conducive to CBC resistance.These mechanisms underscore the potential importance of CsBZIP40,CsWRKY43,CsPrx53,and CsSOD13,providing promising strategies for the prevention of CBC.展开更多
As one of the most widely distributed and highly conserved transcription factors in eukaryotes,basic leucine zipper proteins(bZIPs)are involved in a variety of biological processes in plants,but they are largely unkno...As one of the most widely distributed and highly conserved transcription factors in eukaryotes,basic leucine zipper proteins(bZIPs)are involved in a variety of biological processes in plants,but they are largely unknown in citrus.In this study,56 bZIP family members were identified genome-wide from an important citrus rootstock,namely trifoliate orange(Poncirus trifoliata L.Raf.),and these putative bZIPs were named PtbZIP1—PtbZIP56.All PtbZIPs were classified into 13 subgroups by phylogenetic comparison with Arabidopsis thaliana bZIPs(AtbZIPs),and they were randomly distributed on nine known(50 genes)chromosomes and one unknown(6 genes)chromosome.Sequence analysis revealed the detailed characteristics of PtPZIPs,including their amino acid length,isoelectric point(pI),molecular weight(MW),predicted subcellular localization,gene structure,and conserved motifs.Prediction of promoter elements suggested the presence of drought,low-temperature,wound,and defense and stress responsive elements,as well as multiple hormone-responsive cis-acting elements.Spatiotemporal expression analysis showed the transcriptional patterns of PtbZIPs in different tissues and under dehydration,high salt,ABA,and IAA treatments.In addition,21 PtbZIPs were predicted to have direct or indirect protein—protein interactions.Among these,PtbZIP49 was experimentally proven to interact with PtbZIP1 or PtbZIP11 by using a yeast two-hybrid assay and bimolecular fluorescence complementation(BiFC).Subcellular localization analysis further revealed that PtbZIP1,PtbZIP11,and PtbZIP49 were localized in the nucleus.Moreover,PtbZIP49 was functionally identified as having an important role in salt stress via ectopic expression in A.thaliana and silenced in trifoliate orange using virus-induced gene silencing(VIGS).This study provided comprehensive information on PtbZIP transcription factors in citrus and highlights their potential functions in abiotic stress.展开更多
The publication of several high-quality genomes has contributed greatly to clarifying the evolution of citrus.However,due to their complex genetic backgrounds,the origins and evolution of many citrus species remain un...The publication of several high-quality genomes has contributed greatly to clarifying the evolution of citrus.However,due to their complex genetic backgrounds,the origins and evolution of many citrus species remain unclear.We assembled de novo the 294-Mbp chromosome-level genome of a more than200-year-old primitive papeda(DYC002).Comparison between the two sets of homologous chromosomes of the haplotype-resolved genome revealed 1.2%intragenomic variations,including 1.75 million SNPs,149,471 insertions and 154,215 deletions.Using this genome as a reference,we resequenced and performed population and phylogenetic analyses of378 representative citrus accessions.Our study confirmed that the primary origin center of core Citrus species is in South China,particularly in the Himalaya-Hengduan Mountains.Papeda species are an ancient Citrus type compared with C.ichangensis.We found that the evolution of the Citrus genus followed two radiations through two routes(to East China and Southeast Asia)along river systems.Evidence for the origin and evolution of some individual citrus species was provided.Papeda probably played an important role in the origins of Australian finger lime,citrons,Honghe papeda and pummelos;Ichang papeda originated from Yuanjiang city of Yunnan Province,China,and C.mangshanensis has a close relationship with kumquat and Ichang papeda.Moreover,the Hunan and Guangdong Provinces of China are predicted to be the origin center of mandarin,sweet orange and sour orange.Additionally,our study revealed that fruit bitterness was significantly selected against during citrus domestication.Taken together,this study provides new insight into the origin and evolution of citrus species and may serve as a valuable genomic resource for citrus breeding and improvement.展开更多
The Asian citrus psyllid,Diaphorina citri,is the primary vector of the HLB pathogen,Candidatus Liberibacter asiaticus(CLas).The acquisition of CLas shortens the developmental period of nymphs,accelerating the emergenc...The Asian citrus psyllid,Diaphorina citri,is the primary vector of the HLB pathogen,Candidatus Liberibacter asiaticus(CLas).The acquisition of CLas shortens the developmental period of nymphs,accelerating the emergence into adulthood and thereby facilitating the spread of CLas.Cuticular proteins(CPs)are involved in insect emergence.In this study,we investigated the molecular mechanisms underlying CLas-promoted emergence in D.citri via CP mediation.Here,a total of 159 CP genes were first identified in the D.citri genome.Chromosomal location analysis revealed an uneven distribution of these CP genes across the 13 D.citri chromosomes.Proteomic analysis identified 54 differentially expressed CPs during D.citri emergence,with 14 CPs exhibiting significant differential expression after CLas acquisition.Five key genes,Dc18aa-1,Dc18aa-2,DcCPR-24,DcCPR-38 and DcCPR-58,were screened from the proteome and CLas acquisition.The silencing of these 5 genes through a modified feeding method significantly reduced the emergence rate and caused various abnormal phenotypes,indicating the crucial role that these genes play in D.citri emergence.This study provides a comprehensive overview of the role of CPs in D.citri and reveals that CLas can influence the emergence process of D.citri by regulating the expression of CPs.These key CPs may serve as potential targets for future research on controlling huanglongbing(HLB)transmission.展开更多
‘Candidatus Liberibacter asiaticus(CLas)’,which causes citrus Huanglongbing(HLB)disease,has not been successfully cultured in vitro to date.Here,a rapid multiplication system for CLas was established through in vitr...‘Candidatus Liberibacter asiaticus(CLas)’,which causes citrus Huanglongbing(HLB)disease,has not been successfully cultured in vitro to date.Here,a rapid multiplication system for CLas was established through in vitro regeneration of axillary buds from CLas-infected‘Changyecheng’sweet orange(Citrus sinensis Osbeck).Stem segments with a single axillary bud were cultured in vitro to allow CLas to multiply in the regenerating axillary buds.A high CLas titer was detected in the regenerated shoots on an optimized medium at 30 days after germination(DAG).This titer was 28.2-fold higher than in the midribs from CLas-infected trees growing in the greenhouse.To minimize contamination during in vitro regeneration,CLas-infected axillary buds were micrografted onto seedlings of‘Changyecheng’sweet orange and cultured in a liquid medium.In this culture,the titers of CLas in regenerated shoots rapidly increased from 7.5×10^(4)to 1.4×10^(8)cellsμg^(-1)of citrus DNA during the first 40 DAG.The percentages of shoots with>1×10^(8)CLas cellsμg^(-1)DNA were 30 and 40%at 30 and 40 DAG,respectively.Direct tissue blot immunoassay(DTBIA)indicated that the distribution of CLas was much more uniform in regenerated plantlets than in CLas-infected trees growing in the greenhouse.The disease symptoms in the plantlets were die-back,stunted growth,leaf necrosis/yellowing,and defoliation.The death rate of the plantlets was 82.0%at 60 DAG.Our results show that CLas can effectively multiply in citrus plantlests in vitro.This method will be useful for studying plant-HLB interactions and for rapid screening of therapeutic compounds against CLas in citrus.展开更多
Selection of rootstock is very important for citrus production. Besides its major role on resistance, the rootstock also can affect fruit production and quality. Currently, the main concerns on selection of rootstock ...Selection of rootstock is very important for citrus production. Besides its major role on resistance, the rootstock also can affect fruit production and quality. Currently, the main concerns on selection of rootstock for citrus production are compatibility and resistance, due to less information on the impacts of rootstock to the performance of scion varieties. This study aims to provide information on performances of navel orange varieties on different rootstocks. Three late-ripening navel orange varieties(Citrus sinensis var. Powell, Chislett and Banfield) grafted on seven rootstocks(Swingle citrumelo(C. paradisi× P. trifoliata), Carrizo citrange(C. sinensis×P. trifoliata), X639(C. reticulata×P. trifoliata), MXT(C. sinensis×P. trifoliata), Hongju(C. reticulata), Ziyang Xiangcheng(C. junos) and trifoliate orange(P. trifoliata)) were used as plant materials for comprehensive comparison of the performances on tree growth, fruit yield and quality in 21 scion–stock combinations. Investigation was carried out in these combinations in field nine years after planting. Vigorous growth of all the three late-ripening navel orange varieties was observed on Carrizo citrange with the largest canopy volume at 33.34 m3 and the highest yield at 29.43 kg per tree, but a low yield efficiency at 2.87 kg m–3. On the contrary, those on trifoliate orange had the smallest canopy volume at 10.79 m3 and the lowest fruit yield at 12.51 kg per tree, but the highest yield efficiency at 3.95 kg m–3. Rootstocks did not show significant effects on fruit size, fruit shape index, peel thickness and the edible rate of the fruits, but fruit quality was significantly affected by the rootstocks. Fruits from the trees grafted on trifoliate orange presented the best quality with significantly higher total soluble solids(TSS) content than those on Ziyang Xiangcheng and Hongju, and also the highest ratio of TSS/titratable acidity(TA). The TA content was observed from the fruits on X639 at 0.59 g 100 mL–1. Vitamin C(Vc) content of fruits on Hongju was the highest at 49.25 mg 100 mL–1. Growth vigor of the trees was positively correlated with fruit yield at an extremely significant level. The canopy volume was negatively correlated with yield efficiency, but positively correlated with compatibility index. Results of this study indicated that the rootstock has great impacts on the growth vigor of the tree, yield efficiency and quality of the fruit. In order to achieve good quality and yield efficiency for navel orange production, less growth vigor rootstock such as trifoliate orange is highly recommended.展开更多
Drought is the most common form of abiotic stress that reduces plant growth and productivity.It causes plant injuries through elevated production of reactive oxygen species(ROS).Potassium(K)is a vital plant nutrient t...Drought is the most common form of abiotic stress that reduces plant growth and productivity.It causes plant injuries through elevated production of reactive oxygen species(ROS).Potassium(K)is a vital plant nutrient that notably ameliorates the detrimental effect of drought stress in the plant.A pot experiment was conducted at the Laboratory of Plant Stress Responses,Faculty of Agriculture,Kagawa University,Japan,under controlled environment of green house to explore the role of K in mitigating drought severity in wheat(Triticum asevitum L.)seedlings.Three days after germination,seedlings were exposed to three water regimes viz.,100,50,and 20%field capacity(FC)for 21 days.Potassium was adjusted in Hoagland nutrient solution at 0,6 and 12 mM concentration and applied to pot instead of normal water.Results show that,water deficit stress notably reduced plant growth,biomass accumulation,leaf relative water content(RWC)along with reduced photosynthetic pigments.Increased amount of biochemical stress markers viz.,malondialdehyde(MDA),hydrogen peroxide(H_(2)O_(2)),methylglyoxal(MG),proline(Pro)as well as an impaired antioxidant defense system were observed in drought affected wheat plants.On the contrary,K supplementation resulted in improvement of biochemical and physiological parameters that worked behind in improving growth and development of the wheat plants.In addition,enzymes of ascorbateglutathione(AsA-GSH)cycle were also enhanced by supplemented K that accelerated the ROS detoxification process in plant.Although glyoxalse system did not performed well till MG was detoxified might following another short stepped pathways.Our results revealed that drought stressed plants showed better performances in terms of biochemical and physiological attributes,antioxidant defense and glyoxalase system,as well as ROS detoxification due to K supplementation with better performance at 12 mM K added in 50%FC growing condition.展开更多
Genetic transformation with mature material as the explants could shorten the transgenic period and avoid seed dependence compared with genetic transformation using the epicotyl seedling stem segments as the receptor....Genetic transformation with mature material as the explants could shorten the transgenic period and avoid seed dependence compared with genetic transformation using the epicotyl seedling stem segments as the receptor. Here, we constructed an Agrobacterium tumefaciensmediated transformation for generation of marker-free transgenic plants from navel orange(Citrus sinensis Osbeck) mature stems using a CreloxP recombination system. To efficiently recover the regenerated buds from mature tissues, five recovery methods were compared: in vitro micrografting of 0.1-0.5(1-2 weeks), > 0.5 cm(3-4 weeks) and > 1 cm long lignified bud and in vitro micrografting of explants with a bud and rooting regenerated bud. The data showed that in vitro micrografting of > 1 cm long regenerated bud with expanded leaves after one month of continuous culture for lignification was the optimal solution for plant recovery from mature tissues. Transgenic plants without selectable marker genes were created from navel orange(Citrus sinensis Osbeck) tissue using a transformation vector PLI-35SPR1aCB containing a Cre/loxP system recombination together with genes encoding the selectable marker isopentenyl transferase(IPT) and an anti-bacterial peptide(PR1aCB).Using IPT positive selection, the transformation efficiency determined by PCR was 0.9%, and in total, 20 transgenic plants were obtained.Southern blotting confirmed further their transgenicity. PCR and sequencing analysis demonstrated that both the Cre and IPT genes had been successfully removed from the transgenic plants(deletion efficiency 100%). Over all, using Cre/loxP system recombination together with the IPT positive selection, marker-free transgenic plants can be recovered efficiently from mature tissues of navel orange(Citrus sinensis Osbeck), which provides a potential method for production of transgenic plants from citrus mature tissue.展开更多
Citrus Huanglongbing（HLB, yellow shoot disease） is one of the most serious citrus diseases worldwide. To better improve the detection sensitivity, a droplet digital PCR（ddPCR） assay was developed for the rapid det...Citrus Huanglongbing（HLB, yellow shoot disease） is one of the most serious citrus diseases worldwide. To better improve the detection sensitivity, a droplet digital PCR（ddPCR） assay was developed for the rapid detection of ‘Candidatus Liberibacter asiaticus＇（Las）, the putative causal agent of HLB. The detection of sensitivity comparison using positive plasmid indicated that dd PCR was superior to quantitative PCR（qPCR） for detecting and quantifying Las at low concentrations. The Las detection of 40 field samples also showed that six of 13 asymptomatic samples（46.15%） with high Ct value（〉35） were positive by dd PCR. This methodology showed great potential for early HLB infection diagnosis.展开更多
Two miniature inverted-repeat transposable elements（MITEs）, MCLas-A and MCLas-B, were recently identified from ‘Candidatus Liberibacter asiaticus＇ known to be associated with citrus Huanglongbing（HLB, yellow shoo...Two miniature inverted-repeat transposable elements（MITEs）, MCLas-A and MCLas-B, were recently identified from ‘Candidatus Liberibacter asiaticus＇ known to be associated with citrus Huanglongbing（HLB, yellow shoot disease）. MCLas-A was suggested as an active MITE because of its mobility. The immediate upstream gene of the two MITEs was predicted to be a putative transposase. The goal of this study is to analyze the sequence variation in the upstream putative transposase of MITEs and explore the possible correlation between sequence variation of transposase gene and MITE activity. PCR and sequence analysis showed that 12 sequence types were found in six major amplicon types from 43 representative ‘Ca. L. asiaticus＇ isolates from China, the United States and Brazil. Out of the 12 sequence types, three（T4, T5-2, T6） were reported for the first time. Recombination events were found in the two unique sequence types（T5-2 and T6） which were detected in all Brazilian isolates. Notably, no sequence variation or recombination events were detected in the upstream putative transposase gene of MCLas-A, suggesting the conservation of the transposase gene might be closely related with the MITE activity. Phylogenetic analysis demonstrated two well supported clades including five subclades were identified, clearly reflecting the geographical origins of isolates, especially that of Ruili isolates, S？o Paulo isolates and a few Florida isolates.展开更多
基金supported by National Natural Science Foundation of China(Grant Nos.32370005,32072389)Chongqing Science Funds for Distinguished Young Scientists(Grant No.CSTB2022NSCQ-JQX0027)+3 种基金Innovation Research 2035 Pilot Plan of Southwest University(Grant Nos.SWU-XDPY22002,SWUXDZD22002)Special Fund for Youth Team of Southwest University(Grant No.SWU-XJLJ202310)Chongqing Talents of Exceptional Young Talents Project(Grant No.cstc2022ycjh-bgzxm0143)Chongqing Municipal Training Program of Innovation and Entrepreneurship for Undergraduates(Grant No.S202310635160)。
文摘In this review,the advantages and advances in applying high-throughput sequencing(HTS)in the management of viral diseases in citrus,along with some challenges,are discussed to provide perspectives on future prospects.Since the initial implementation of HTS in citrus virology,a substantial number of citrus viruses have been identified,with a notable increase in the last 7 years.The acquisition of viral genomes and various HTS-based omics analyses serve as crucial pillars for advancing research in the etiology,epidemiology,pathology,evolution,ecology,and biotechnology of citrus viruses.HTS has notably contributed to disease diagnosis,such as the diagnoses of concave gum and impietratura,as well as to the surveillance of new virus risks and the preparation of virus-free materials.However,certain inherent defects in HTS and coupled bioinformatics analysis,such as challenges with sequence assembly and the detection of viral dark matter,require improvement to enhance practical efficiency.In addition,the utilization of HTS for the systematic management of citrus viral diseases remains limited,and drawing insights from other virus-plant pathosystems while integrating emerging compatible techniques and ideas may broaden its specific applications.
基金supported by the National Natural Science Foundation of China(30471205)the Special Fund for Agroscientific Research in the Public Interest,China(200903004-06)+1 种基金the Program for Changjiang Scholars and Innovative Research Team in University,China(PCSIRT,IRT0976)the Chinese Scholarship Council,China(CSC)
文摘Citrus tristeza virus （CTV） is one of the most important causal agents of citrus diseases and exists as numerous strains. CTV is replicated in phloem cells of plants within the family Rutaceae and is transmitted by a few of aphid species. CTV epidemics have caused death of millions of citrus trees in many regions all over the world, where the sour orange （Citrus aurantium） was used as rootstock. Also the production of grapefruit （C. paradisi） and sweet orange （C. sinensis） has been affected by CTV strains. CTV gives uplift to three prominent syndromes, namely quick-decline （tristeza）, stempitting and seedling-yellows. The disease is graft-transmissible in nature but not seed-transmitted. However, the tristeza disease in most citrus groves was a man-made problem created by the desire of horticulturists to introduce cultivars from other citrus growing areas. The utmost importance of the disease called for review articles in numbers of plant protection, epidemiology books, citriculture and proceedings. This review collects the information with respects to disease history, distribution host range, virus isolates association, identification and detection, transmission and management; especially on the current status of CTV prevailing and controlling in Pakistan. It provides valuable information for CTV disease and its controlling approaches.
基金funded by the National Key Research and Development Program of China (Grant No.2022YFD1201600)Natural Science Foundation of Chongqing (Grant No.cstc2020jcyj-msxmX1064)+1 种基金Earmarked Funds for the China Agriculture Research System (Grant No.CARS-26)Three-year Action Plan of Xi'an University (Grant No.2021XDJH41)。
文摘Citrus bacterial canker(CBC) is resulted from Xanthomonas citri subsp. citri(Xcc) infection and poses a significant threat to citrus production.Glutathione S-transferases(GSTs) are critical in maintaining redox homeostasis in plants, especially in relation to abiotic and biotic stress responses. However, the function of GSTs in resisting CBC remains unclear. Here, citrus glutathione S-transferases were investigated applying a genome-wide approach. In total, 69 CsGSTs belonging to seven classes were identified, and the phylogeny, chromosomal distribution, gene structures and conserved motifs were analyzed. Several CsGSTs responded to Xcc infection, as observed in the upregulation of CsGSTF1 and CsGSTU18 in the CBC-sensitive ‘Wanjincheng' variety but not in the resistant ‘Kumquat' variety. CsGSTF1 and CsGSTU18 were localized at the cytoplasm. Transient overexpression of CsGSTF1 and CsGSTU18 mediated reactive oxygen species(ROS) scavenging, whereas the virus-induced gene silencing(VIGS) of CsGSTF1 and CsGSTU18 caused strong CBC resistance and ROS burst. The present study investigated the characterization of citrus GST gene family, and discovered that CsGSTF1 and CsGSTU18 negatively contributed to CBC through modulating ROS homeostasis. These findings emphasize the significance of GSTs in infection resistance in plants.
基金funded by the National Key Research and Development Program of China(2022YFD1201600)the earmarked fund for the China Agriculture Research System(CARS-26)+1 种基金the Fundamental Research Funds for the Central Universities,China(SWU-XDJH202308)the Science and Technology Research Program of Chongqing Municipal Education Commission,China(KJQN202001418)。
文摘One of the main diseases that adversely impacts the global citrus industry is citrus bacterial canker(CBC),caused by the bacteria Xanthomonas citri subsp.citri(Xcc).Response to CBC is a complex process,with both proteinDNA as well as protein–protein interactions for the regulatory network.To detect such interactions in CBC resistant regulation,a citrus high-throughput screening system with 203 CBC-inducible transcription factors(TFs),were developed.Screening the upstream regulators of target by yeast-one hybrid(Y1H)methods was also performed.A regulatory module of CBC resistance was identified based on this system.One TF(CsDOF5.8)was explored due to its interactions with the 1-kb promoter fragment of CsPrx25,a resistant gene of CBC involved in reactive oxygen species(ROS)homeostasis regulation.Electrophoretic mobility shift assay(EMSA),dual-LUC assays,as well as transient overexpression of CsDOF5.8,further validated the interactions and transcriptional regulation.The CsDOF5.8–CsPrx25 promoter interaction revealed a complex pathway that governs the regulation of CBC resistance via H2O2homeostasis.The high-throughput Y1H/Y2H screening system could be an efficient tool for studying regulatory pathways or network of CBC resistance regulation.In addition,it could highlight the potential of these candidate genes as targets for efforts to breed CBC-resistant citrus varieties.
基金supported by the China Agriculture Research System of MOF and MARA(CARS26-05B)the Innovation Research 2035 Pilot Plan of Southwest University,China(SWU-XDPY22002)+1 种基金the Guangxi Science and Technology Planed Project,China(Gui Ke AD23026090)the Guangxi Natural Science Foundation,China(2023GXNSFBA026285).
文摘Citrus yellow vein clearing virus(CYVCV)is a new citrus virus that has become an important factor restricting the development of China’s citrus industry,and the CYVCV coat protein(CP)is associated with viral pathogenicity.In this study,the Eureka lemon zinc finger protein(ZFP)ClDOF3.4 was shown to interact with CYVCV CP in vivo and in vitro.Transient expression of ClDOF3.4 in Eureka lemon induced the expression of salicylic acid(SA)-related and hypersensitive response marker genes,and triggered a reactive oxygen species burst,ion leakage necrosis,and the accumulation of free SA.Furthermore,the CYVCV titer in ClDOF3.4 transgenic Eureka lemon plants was approximately 69.4%that in control plants 6 mon after inoculation,with only mild leaf chlorotic spots observed in those transgenic plants.Taken together,the results indicate that ClDOF3.4 not only interacts with CP but also induces an immune response in Eureka lemon by inducing the SA pathways.This is the first report that ZFP is involved in the immune response of a citrus viral disease,which provides a basis for further study of the molecular mechanism of CYVCV infection.
基金supported by the National Natural Science Foundation of China (31501611)the Chongqing Research Program of Basic Research and Frontier Technology, China (cstc2017jcyjB X0016)+2 种基金the Chongqing Science and Technology Commission Project, China (cstc2016shmsztzx80003)the Fundamental Research Funds for the Central Universities, China (XDJK2016B21, SWU116012)the Special Fund for Agro-scientific Research in the Public Interest, China (201203076-01)
文摘Citrus leaf blotch virus （CLBV） is a member of the genus Citrivirus, in the family Betaflexiviridae. It has been reported CLBV could infect kiwi, citrus and sweet cherry in China. Of 289 citrus samples from six regions of China, 15 were detected to be infected with CLBV in this study. The complete genome of four isolates of CLBV was obtained from Reikou in Sichuan （CLBV-LH）, Yura Wase in Zhejiang （CLBV-YL）, Bingtangcheng in Hunan （CLBV-BT）, Fengjie 72-1 in Chongqing （CLBV- F J）, respectively. While they all represented 8 747 nucleotides in monopartite size, excluding the poly（A） tail, each of the isolates coded three open reading frames （ORFs）. Identity of the four isolates ranged from 98.9 to 99.8% to each other and from 96.8 to 98.1% to the citrus references in GenBank by multiple alignment of genomes. A phylogenetic tree based on the genome sequences of available CLBV isolates indicated that the four isolates were clustered together, suggesting that CLBV isolates from citrus in China did not have obvious variation. This is the first report of the complete nucleotide sequences of CLBV isolates infecting citrus in China.
基金supported by the National Key Research and Development Program of China(2021YFD1400800,2022YFD1400200)the National Natural Sciences Foundation of China(31972393)+1 种基金the Fundamental Research Funds for the Central Universities(SWU-XDJH202308,XDJK2018B016)the Earmarked Fund for China Agriculture Research System(CARS-27).
文摘The most damaging citrus diseases are Huanglongbing(HLB)and citrus canker,which are caused by Candidatus Liberibacter asiaticus(CaLas)and Xanthomonas citri pv.citri(Xcc),respectively.Endolysins from bacteriophages are a possible option for disease resistance in plant breeding.Here,we report improvement of citrus resistance to HLB and citrus canker using the LasLYS1 and LasLYS2 endolysins from CaLas.LasLYS2 demonstrated bactericidal efficacy against several Rhizobiaceae bacteria and Xcc,according to inhibition zone analyses.The two genes,driven by a strong promoter from Cauliflower mosaic virus,35S,were integrated into Carrizo citrange via Agrobacterium-mediated transformation.More than 2 years of greenhouse testing indicated that LasLYS2 provided substantial and long-lasting resistance to HLB,allowing transgenic plants to retain low CaLas titers and no obvious symptoms while also clearing CaLas from infected plants in the long term.LasLYS2 transgenic plants with improved HLB resistance also showed resistance to Xcc,indicating that LasLYS2 had dual resistance to HLB and citrus canker.A microbiome study of transgenic plants revealed that the endolysins repressed Xanthomonadaceae and Rhizobiaceae populations in roots while increasing Burkholderiaceae and Rhodanobacteraceae populations,which might boost the citrus defense response,according to transcriptome analysis.We also found that Lyz domain 2 is the key bactericidal motif of LasLYS1 and LasLYS2.Four endolysins with potential resistance to HLB and citrus canker were found based on the structures of LasLYS1 and LasLYS2.Overall,the work shed light on the mechanisms of resistance of CaLas-derived endolysins,providing insights for designing endolysins to develop broad-spectrum disease resistance in citrus.
基金funded by the National Natural Science Foundation of China (31671992)the National Key R&D Program of China (2018YFD0201500)+2 种基金the Fundamental Research Funds for the Central Universities of China (XDJK2018C032)the China Postdoctoral Fund (2018M633307)supported by the Agricultural Research Serves, United States Department of Agriculture (USDA ARS)
文摘Huanglongbing (HLB) is the most destructive disease of citrus and is associated with ‘Candidatus Liberibacter asiaticus’(CLas), a member of the α-proteobacteria. Citrus tristeza virus (CTV) is another pathogen of citrus with very great historic as well as current importance. Both CLas and CTV are phloem-restricted pathogens. A severe CTV isolate, CTV-B6, and CLas-B232 induce a group of symptoms of phloem dysfunction that overlap, but the mild isolate CTV-B2 does not cause any loss to commercial trees. Prior inoculation and establishment of CLas-B232 did not affect subsequent establishment of either CTV-B2 or CTV-B6, while super infection by CLas-B232 was reduced by prior establishment of CTV-B2 and to a lesser extent by prior infection with CTV-B6. Trees co-infected with CTV-B6 and CLas-B232 developed more severe symptoms, typical of CTV-B6, than either of the two pathogens co-infected with CTV-B2. In this study, we confirmed that CLas established in the rootlets earlier and with higher concentration than in leaves. The distribution of CLas in the plant infected by CLas-B438 alone and with CTV-B2 fits a previously proposed model but CLas was more sporadically distributed in a plant co-infected by CLas and CTV-B2 than in a plant infected by CLas alone. These biological phenomena are aligned with previously analyzed transcriptome data and the study provides a novel idea that mild CTV strains may provide some protection against CLas by limiting its multiplication and spread. The protective effect may be due to opposite regulation of key host defense pathways in response to CTV-B2 and CLas-B438.
文摘Traumatic brain injury(TBI)is closely related to neuro-inflammatory response and causes a complex pathological process and serious consequence.In this study,we explored whether the formatted citrus peel extract gold lotion(GL)could have a therapeutic effect on rats suffered TBI.TBI rat model was prepared by the electric Cortical Contusion Impactor(eCCI)device.Prevention against TBI by GL was assessed by the behavioral,organizational of rats and the molecular markers in the brain injury area.The results showed that GL could reduce nerve damage and neurological dysfunction.The mechanisms probably due to that GL could not only reduce the associated damage markers including GFAP,OX-42,TNF-α,COX-2,NF-κB and TLR4 but also promote the expression of VEGF which is responsible for protecting neuronal cells in the injury area.These findings revealed that GL could be a promising preparation for TBI prevention.
基金This study was funded by the National Key Research and Development Program of China(2022YFD1201600,2021YFD1600800)National Natural Sciences Foundation of China(32202425)Earmarked Funds for the China Agriculture Research System(CARS-26).
文摘As the bacterial etiologic agent causing citrus bacterial canker(CBC),Xanthomonas citri subsp.citri(Xcc)seriously impacts citrus plantation and fruit production globally.In an earlier study,we demonstrated that CsBZIP40 can positively impact CBC resistance in the sweet orange(Citrus sinensis).However,the mechanistic basis for the protective benefits conferred by CsBZIP40 is yet to be delineated.Here,we show that CsBZIP40 positively regulates CBC resistance and reactive oxygen species(ROS)homeostasis in transgenic sweet orange overexpressing CsBZIP40.CsBZIP40 directly binds to the TGA-box of the CsWRKY43 promoter to repress its transcriptional activity.CsWRKY43 overexpression induces CBC susceptibility in transgenic sweet oranges.In contrast,its inhibition produces strong resistance to CBC.CsWRKY43 directly binds to the W-boxes of the CsPrx53 and CsSOD13 promoters to positively regulate the activities of these antioxidant enzymes,resulting in the negative regulation of ROS homeostasis and CBC resistance in sweet orange plants.CsPrx53/CsSOD13 knockdown enhances ROS accumulation and CBC resistance.Overall,our results outline a regulatory pathway through which CsBZIP40 transcriptionally represses CsWRKY43-CsPrx53/CsSOD13 cascade-mediated ROS scavenging in a manner conducive to CBC resistance.These mechanisms underscore the potential importance of CsBZIP40,CsWRKY43,CsPrx53,and CsSOD13,providing promising strategies for the prevention of CBC.
基金supported by grants from the National Natural Science Foundation of China(Grant Nos.32072550,31902084)the National Key Research and Development Program of China(Grant No.2018YFD1000300)。
文摘As one of the most widely distributed and highly conserved transcription factors in eukaryotes,basic leucine zipper proteins(bZIPs)are involved in a variety of biological processes in plants,but they are largely unknown in citrus.In this study,56 bZIP family members were identified genome-wide from an important citrus rootstock,namely trifoliate orange(Poncirus trifoliata L.Raf.),and these putative bZIPs were named PtbZIP1—PtbZIP56.All PtbZIPs were classified into 13 subgroups by phylogenetic comparison with Arabidopsis thaliana bZIPs(AtbZIPs),and they were randomly distributed on nine known(50 genes)chromosomes and one unknown(6 genes)chromosome.Sequence analysis revealed the detailed characteristics of PtPZIPs,including their amino acid length,isoelectric point(pI),molecular weight(MW),predicted subcellular localization,gene structure,and conserved motifs.Prediction of promoter elements suggested the presence of drought,low-temperature,wound,and defense and stress responsive elements,as well as multiple hormone-responsive cis-acting elements.Spatiotemporal expression analysis showed the transcriptional patterns of PtbZIPs in different tissues and under dehydration,high salt,ABA,and IAA treatments.In addition,21 PtbZIPs were predicted to have direct or indirect protein—protein interactions.Among these,PtbZIP49 was experimentally proven to interact with PtbZIP1 or PtbZIP11 by using a yeast two-hybrid assay and bimolecular fluorescence complementation(BiFC).Subcellular localization analysis further revealed that PtbZIP1,PtbZIP11,and PtbZIP49 were localized in the nucleus.Moreover,PtbZIP49 was functionally identified as having an important role in salt stress via ectopic expression in A.thaliana and silenced in trifoliate orange using virus-induced gene silencing(VIGS).This study provided comprehensive information on PtbZIP transcription factors in citrus and highlights their potential functions in abiotic stress.
基金supported by the China Agriculture Research System(CARS-Citrus),Fundamental Research Funds for the Central Universities(Grant No.SWU120021)the National Natural Science Foundation of China(Nos.32060664,31901995)funds from Fujian Agriculture and Forestry University,and Fundamental Research Funds for the Central Universities(SWU-XDJH202308)。
文摘The publication of several high-quality genomes has contributed greatly to clarifying the evolution of citrus.However,due to their complex genetic backgrounds,the origins and evolution of many citrus species remain unclear.We assembled de novo the 294-Mbp chromosome-level genome of a more than200-year-old primitive papeda(DYC002).Comparison between the two sets of homologous chromosomes of the haplotype-resolved genome revealed 1.2%intragenomic variations,including 1.75 million SNPs,149,471 insertions and 154,215 deletions.Using this genome as a reference,we resequenced and performed population and phylogenetic analyses of378 representative citrus accessions.Our study confirmed that the primary origin center of core Citrus species is in South China,particularly in the Himalaya-Hengduan Mountains.Papeda species are an ancient Citrus type compared with C.ichangensis.We found that the evolution of the Citrus genus followed two radiations through two routes(to East China and Southeast Asia)along river systems.Evidence for the origin and evolution of some individual citrus species was provided.Papeda probably played an important role in the origins of Australian finger lime,citrons,Honghe papeda and pummelos;Ichang papeda originated from Yuanjiang city of Yunnan Province,China,and C.mangshanensis has a close relationship with kumquat and Ichang papeda.Moreover,the Hunan and Guangdong Provinces of China are predicted to be the origin center of mandarin,sweet orange and sour orange.Additionally,our study revealed that fruit bitterness was significantly selected against during citrus domestication.Taken together,this study provides new insight into the origin and evolution of citrus species and may serve as a valuable genomic resource for citrus breeding and improvement.
基金supported by the National Key R&D Program of China(2021YFD1400805)the National Natural Science Foundation of China(32160625)the Science and Technology Projects in Jiangxi Province(20225BCJ22005)and the China Agriculture Research System:CARS-26.
文摘The Asian citrus psyllid,Diaphorina citri,is the primary vector of the HLB pathogen,Candidatus Liberibacter asiaticus(CLas).The acquisition of CLas shortens the developmental period of nymphs,accelerating the emergence into adulthood and thereby facilitating the spread of CLas.Cuticular proteins(CPs)are involved in insect emergence.In this study,we investigated the molecular mechanisms underlying CLas-promoted emergence in D.citri via CP mediation.Here,a total of 159 CP genes were first identified in the D.citri genome.Chromosomal location analysis revealed an uneven distribution of these CP genes across the 13 D.citri chromosomes.Proteomic analysis identified 54 differentially expressed CPs during D.citri emergence,with 14 CPs exhibiting significant differential expression after CLas acquisition.Five key genes,Dc18aa-1,Dc18aa-2,DcCPR-24,DcCPR-38 and DcCPR-58,were screened from the proteome and CLas acquisition.The silencing of these 5 genes through a modified feeding method significantly reduced the emergence rate and caused various abnormal phenotypes,indicating the crucial role that these genes play in D.citri emergence.This study provides a comprehensive overview of the role of CPs in D.citri and reveals that CLas can influence the emergence process of D.citri by regulating the expression of CPs.These key CPs may serve as potential targets for future research on controlling huanglongbing(HLB)transmission.
基金supported by the National Key R&D Program of China (2018YFD0201500 and 2018YFD1000300)the National Natural Science Foundation of China (31972393)the China Agriculture Research System of MOF and MARA (CARS-26)
文摘‘Candidatus Liberibacter asiaticus(CLas)’,which causes citrus Huanglongbing(HLB)disease,has not been successfully cultured in vitro to date.Here,a rapid multiplication system for CLas was established through in vitro regeneration of axillary buds from CLas-infected‘Changyecheng’sweet orange(Citrus sinensis Osbeck).Stem segments with a single axillary bud were cultured in vitro to allow CLas to multiply in the regenerating axillary buds.A high CLas titer was detected in the regenerated shoots on an optimized medium at 30 days after germination(DAG).This titer was 28.2-fold higher than in the midribs from CLas-infected trees growing in the greenhouse.To minimize contamination during in vitro regeneration,CLas-infected axillary buds were micrografted onto seedlings of‘Changyecheng’sweet orange and cultured in a liquid medium.In this culture,the titers of CLas in regenerated shoots rapidly increased from 7.5×10^(4)to 1.4×10^(8)cellsμg^(-1)of citrus DNA during the first 40 DAG.The percentages of shoots with>1×10^(8)CLas cellsμg^(-1)DNA were 30 and 40%at 30 and 40 DAG,respectively.Direct tissue blot immunoassay(DTBIA)indicated that the distribution of CLas was much more uniform in regenerated plantlets than in CLas-infected trees growing in the greenhouse.The disease symptoms in the plantlets were die-back,stunted growth,leaf necrosis/yellowing,and defoliation.The death rate of the plantlets was 82.0%at 60 DAG.Our results show that CLas can effectively multiply in citrus plantlests in vitro.This method will be useful for studying plant-HLB interactions and for rapid screening of therapeutic compounds against CLas in citrus.
基金financially supported by the National Key R&D Program of China (2018YFD1000101 and2018YFD0201503)the earmarked fund for China Agriculture Research System (CARS-26)+3 种基金the Fundamental Research Funds for the Central Universities,China(XDJK2016B024)the National Citrus Engineering Research Center,China (NCERC)the earmarked fund for Chongqing Special&Economic Agriculture Research System on Late Maturation Citrus,Chinathe Basic Research and Frontier Exploration Projects in Chongqing,China(cstc2018jcyjAX0400)。
文摘Selection of rootstock is very important for citrus production. Besides its major role on resistance, the rootstock also can affect fruit production and quality. Currently, the main concerns on selection of rootstock for citrus production are compatibility and resistance, due to less information on the impacts of rootstock to the performance of scion varieties. This study aims to provide information on performances of navel orange varieties on different rootstocks. Three late-ripening navel orange varieties(Citrus sinensis var. Powell, Chislett and Banfield) grafted on seven rootstocks(Swingle citrumelo(C. paradisi× P. trifoliata), Carrizo citrange(C. sinensis×P. trifoliata), X639(C. reticulata×P. trifoliata), MXT(C. sinensis×P. trifoliata), Hongju(C. reticulata), Ziyang Xiangcheng(C. junos) and trifoliate orange(P. trifoliata)) were used as plant materials for comprehensive comparison of the performances on tree growth, fruit yield and quality in 21 scion–stock combinations. Investigation was carried out in these combinations in field nine years after planting. Vigorous growth of all the three late-ripening navel orange varieties was observed on Carrizo citrange with the largest canopy volume at 33.34 m3 and the highest yield at 29.43 kg per tree, but a low yield efficiency at 2.87 kg m–3. On the contrary, those on trifoliate orange had the smallest canopy volume at 10.79 m3 and the lowest fruit yield at 12.51 kg per tree, but the highest yield efficiency at 3.95 kg m–3. Rootstocks did not show significant effects on fruit size, fruit shape index, peel thickness and the edible rate of the fruits, but fruit quality was significantly affected by the rootstocks. Fruits from the trees grafted on trifoliate orange presented the best quality with significantly higher total soluble solids(TSS) content than those on Ziyang Xiangcheng and Hongju, and also the highest ratio of TSS/titratable acidity(TA). The TA content was observed from the fruits on X639 at 0.59 g 100 mL–1. Vitamin C(Vc) content of fruits on Hongju was the highest at 49.25 mg 100 mL–1. Growth vigor of the trees was positively correlated with fruit yield at an extremely significant level. The canopy volume was negatively correlated with yield efficiency, but positively correlated with compatibility index. Results of this study indicated that the rootstock has great impacts on the growth vigor of the tree, yield efficiency and quality of the fruit. In order to achieve good quality and yield efficiency for navel orange production, less growth vigor rootstock such as trifoliate orange is highly recommended.
文摘Drought is the most common form of abiotic stress that reduces plant growth and productivity.It causes plant injuries through elevated production of reactive oxygen species(ROS).Potassium(K)is a vital plant nutrient that notably ameliorates the detrimental effect of drought stress in the plant.A pot experiment was conducted at the Laboratory of Plant Stress Responses,Faculty of Agriculture,Kagawa University,Japan,under controlled environment of green house to explore the role of K in mitigating drought severity in wheat(Triticum asevitum L.)seedlings.Three days after germination,seedlings were exposed to three water regimes viz.,100,50,and 20%field capacity(FC)for 21 days.Potassium was adjusted in Hoagland nutrient solution at 0,6 and 12 mM concentration and applied to pot instead of normal water.Results show that,water deficit stress notably reduced plant growth,biomass accumulation,leaf relative water content(RWC)along with reduced photosynthetic pigments.Increased amount of biochemical stress markers viz.,malondialdehyde(MDA),hydrogen peroxide(H_(2)O_(2)),methylglyoxal(MG),proline(Pro)as well as an impaired antioxidant defense system were observed in drought affected wheat plants.On the contrary,K supplementation resulted in improvement of biochemical and physiological parameters that worked behind in improving growth and development of the wheat plants.In addition,enzymes of ascorbateglutathione(AsA-GSH)cycle were also enhanced by supplemented K that accelerated the ROS detoxification process in plant.Although glyoxalse system did not performed well till MG was detoxified might following another short stepped pathways.Our results revealed that drought stressed plants showed better performances in terms of biochemical and physiological attributes,antioxidant defense and glyoxalase system,as well as ROS detoxification due to K supplementation with better performance at 12 mM K added in 50%FC growing condition.
基金supported by the Fundamental Research Funds for the Central Universities (Grant No. XDJK 2018B016)the National Natural Sciences Foundation of China (Grant No. 31972393)+1 种基金he earmarked fund for China Agriculture Research System (Grant No. CARS-26)the Natural Science Foundation of Chongqing (Grant No. cstc2020jcyj-msxmX1064)。
文摘Genetic transformation with mature material as the explants could shorten the transgenic period and avoid seed dependence compared with genetic transformation using the epicotyl seedling stem segments as the receptor. Here, we constructed an Agrobacterium tumefaciensmediated transformation for generation of marker-free transgenic plants from navel orange(Citrus sinensis Osbeck) mature stems using a CreloxP recombination system. To efficiently recover the regenerated buds from mature tissues, five recovery methods were compared: in vitro micrografting of 0.1-0.5(1-2 weeks), > 0.5 cm(3-4 weeks) and > 1 cm long lignified bud and in vitro micrografting of explants with a bud and rooting regenerated bud. The data showed that in vitro micrografting of > 1 cm long regenerated bud with expanded leaves after one month of continuous culture for lignification was the optimal solution for plant recovery from mature tissues. Transgenic plants without selectable marker genes were created from navel orange(Citrus sinensis Osbeck) tissue using a transformation vector PLI-35SPR1aCB containing a Cre/loxP system recombination together with genes encoding the selectable marker isopentenyl transferase(IPT) and an anti-bacterial peptide(PR1aCB).Using IPT positive selection, the transformation efficiency determined by PCR was 0.9%, and in total, 20 transgenic plants were obtained.Southern blotting confirmed further their transgenicity. PCR and sequencing analysis demonstrated that both the Cre and IPT genes had been successfully removed from the transgenic plants(deletion efficiency 100%). Over all, using Cre/loxP system recombination together with the IPT positive selection, marker-free transgenic plants can be recovered efficiently from mature tissues of navel orange(Citrus sinensis Osbeck), which provides a potential method for production of transgenic plants from citrus mature tissue.
基金funded by the National Natural Sciences Foundation of China (31671992, 31301635)the Chongqing Science and Technology Commission Project, China (cstc2016shms-ztzx80003)the Guangxi Key Laboratory of Citrus Biology, Guangxi Academy of Specialty Crops, China (SYS2015K004)
文摘Citrus Huanglongbing（HLB, yellow shoot disease） is one of the most serious citrus diseases worldwide. To better improve the detection sensitivity, a droplet digital PCR（ddPCR） assay was developed for the rapid detection of ‘Candidatus Liberibacter asiaticus＇（Las）, the putative causal agent of HLB. The detection of sensitivity comparison using positive plasmid indicated that dd PCR was superior to quantitative PCR（qPCR） for detecting and quantifying Las at low concentrations. The Las detection of 40 field samples also showed that six of 13 asymptomatic samples（46.15%） with high Ct value（〉35） were positive by dd PCR. This methodology showed great potential for early HLB infection diagnosis.
基金provided by the Special Fund for Agro-Scientific Research in the Public Interest, China (201003067-02)the Natural Science Foundation Project of CQ CSTC (cstc2012jj A80025)the Fundamental Research Funds for the Central Universities, China (XDJK2014A001, XDJK2014D004)
文摘Two miniature inverted-repeat transposable elements（MITEs）, MCLas-A and MCLas-B, were recently identified from ‘Candidatus Liberibacter asiaticus＇ known to be associated with citrus Huanglongbing（HLB, yellow shoot disease）. MCLas-A was suggested as an active MITE because of its mobility. The immediate upstream gene of the two MITEs was predicted to be a putative transposase. The goal of this study is to analyze the sequence variation in the upstream putative transposase of MITEs and explore the possible correlation between sequence variation of transposase gene and MITE activity. PCR and sequence analysis showed that 12 sequence types were found in six major amplicon types from 43 representative ‘Ca. L. asiaticus＇ isolates from China, the United States and Brazil. Out of the 12 sequence types, three（T4, T5-2, T6） were reported for the first time. Recombination events were found in the two unique sequence types（T5-2 and T6） which were detected in all Brazilian isolates. Notably, no sequence variation or recombination events were detected in the upstream putative transposase gene of MCLas-A, suggesting the conservation of the transposase gene might be closely related with the MITE activity. Phylogenetic analysis demonstrated two well supported clades including five subclades were identified, clearly reflecting the geographical origins of isolates, especially that of Ruili isolates, S？o Paulo isolates and a few Florida isolates.
