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
The CRISPR/Cas9 system has shown great promise in engineering targeted mutations in a genome.The efficiency of Cas9-mediated genome editing is temperature sensitive.A high-temperature regime can increase the mutation ...The CRISPR/Cas9 system has shown great promise in engineering targeted mutations in a genome.The efficiency of Cas9-mediated genome editing is temperature sensitive.A high-temperature regime can increase the mutation efficiency induced by the CRISPR/Cas9 system in many plant species.However,a heat stress treatment has not been applied during the tissue culture process in citrus.To develop an efficient heat stress regime to improve the efficiency of CRISPR/Cas9-mediated targeted mutagenesis,three and five cycles of heat stress treatments were used during callus induction in citrus.The results showed that the heat stress treatment with three cycles of 24 h at 37℃,followed by 24 h at 26℃,increased the mutation efficiency by 11.6%compared with no heat stress treatment,and that five cycles of heat stress treatment were optimal,from which 50%mutants had a 100%mutation rate.The mutation profiles of Cas9 at 28℃ for 10 d and 37℃ for three or five cycles were similar,indicating that heat stress treatment did not affect the non-homologous end joining repair pathway.No detectable off-target mutation was detected in the potential off-target sites with four nucleotide mismatches compared with the designed on-target site.This study demonstrated that five cycles of heat stress treatment during callus induction could efficiently increase the mutation efficiency mediated by the CRISPR/Cas9 system without observable negative effects,and provided an efficient Cas9-mediated citrus genome editing system to produce mutants with a high mutation rate.展开更多
The genus Actinidia is primarily functionally dioecious,and early sex identification plays a crucial role in improving breeding efficiency and reducing production costs.In this study,the accuracy of three sex-linked m...The genus Actinidia is primarily functionally dioecious,and early sex identification plays a crucial role in improving breeding efficiency and reducing production costs.In this study,the accuracy of three sex-linked molecular markers(SyGI[Shy Girl],FrBy[Friendly Boy],and SmY1)in sex identification was evaluated in various Actinidia species.The selected marker products were subsequently cloned and sequenced in six wild Actinidia species.Ninety-six wild A.chinensis chinensis accessions and 74 A.chinensis deliciosa accessions,most of which were wild,with only one cultivated,were used for comprehensive primer validation.Thirty-three juvenile A.chinensis chinensis hybrid seedlings were used for practical application tests.The results showed that the marker SyGI accurately identified the sex of 20 samples from six Actinidia species and 96 A.chinensis chinensis accessions with 100%reliability.For Actinidia chinensis deliciosa,the identification accuracy reached 98.65%.Sequence analysis revealed that SyGI shared the highest similarity with the male-specific genomic region.Furthermore,SyGI achieved 100%accuracy in identifying the sex of 33 juvenile A.chinensis chinensis individuals.The findings confirm that the SyGI marker possesses high accuracy,strong specificity,and broad applicability,making it a valuable tool for kiwifruit breeding programs.The cloned sequences from wild Actinidia species also provide important references for future research on the mechanisms of sexual evolution and determination.展开更多
Simple sequence repeats(SSRs)were investigated in the unigene sequences from expressed sequence tags(EST)of sweet orange(Citrus sinensis osbeck),trifoliate orange(Poncirus trifoliata Raf.)and other citrus species and ...Simple sequence repeats(SSRs)were investigated in the unigene sequences from expressed sequence tags(EST)of sweet orange(Citrus sinensis osbeck),trifoliate orange(Poncirus trifoliata Raf.)and other citrus species and cultivars.A total of 37802 citrus unigene sequences corresponding to 23.29 Mb were searched,resulting in the identification of 8218 SSRs.Among them there were 4913(59.8%)mono-,1419(17.3%)di-,1709(20.8%)tri-,114(1.39%)tetra-,23(0.28%)penta-and 40(0.49%)hexa-nucleotide SSRs.The estimated frequency of SSRs was approximately 1/2.8 kb,which could be extrapolated to 1 SSR-containing unigene in 4.6 unigenes.The maximum length of the SSR ranged from 40 to 105 bp depending on the repeating numbers of the motif in the SSR.The overall average length of SSRs was 20.9 bp.The frequencies of different SSR types(di-,tri-,tetra-,and penta-nucleotide repeats)were very similar between sweet orange and trifoliate orange.The mononucelotide repeats appeared to be the most abundant SSRs within sweet orange and trifoliate orange,followed by trimeric repeats.The adenine rich repeats such as A/T,AG,AT,AAG,AAAT,AAAG,AAAT,AAAAG,AAAAT etc.were predominant in each type of SSRs(mono-,di-,tri-,tetra-,and penta-),whereas the C/G,CG,CCG repeats were less abundant.Twenty-five primer pairs flanking EST-SSR loci were designed to detect the possible polymorphism of six citrus cultivars including sweet orange and trifoliate orange.The PCR result with all these 25 primer pairs revealed the existence of polymorphism within six citrus cultivars confirming that citrus EST database could be efficiently exploited for the development of gene-derived SSR markers.展开更多
Single nucleotide polymorphisms （SNPs） are the most abundant sequence variations found in plant genomes and are widely used as molecular genetic markers in cultivar identification and genetic diversity studies. The ...Single nucleotide polymorphisms （SNPs） are the most abundant sequence variations found in plant genomes and are widely used as molecular genetic markers in cultivar identification and genetic diversity studies. The objective of this study was to identify SNP markers useful for discrimination of citrus cultivars, since large numbers of expressed sequence tags （ESTs） of sweet orange are available from the National Center for Biotechnology Information （NCBI）. We now have the opportunity to discover SNP markers suitable for determining the haplotypes with which to distinguish very closely related cultivars and to assess genetic diversity within or between related species of citrus. SNPs and small insertions/deletions （Indels） from ESTs of sweet orange and satsuma were identified by the in silico SNP discovery strategy. 55 296 EST sequences of sweet orange and 2 575 of satsuma retrieved from the NCBI repository were mined for potential SNPs. Cleaved amplified polymorphic sequences （CAPS） and sequencing approaches were used to validate putative SNPs in a sample of 30 citrus accessions. A total of 3 348 putative SNPs were identified based on the abundance of sequences and haplotype cosegregation. Of these 3 348 SNPs, the transitions, transversions and Indels ratios were 47.9, 36.1 and 16.0%, respectively. The SNPs occurred on average at a frequency of 1 per 164 bp in the coding region of citrus. 14 SNPs were randomly selected and genotyped according to 30 citrus accessions including 23 accessions of sweet orange; 11 SNPs displayed polymorphism with an average polymorphism information content （PIC） of 0.20 among 30 citrus accessions. The genetic diversity present in sweet orange was low, so the 14 SNP markers failed to discriminate different cultivars of sweet orange, but they did succeed in distinguishing accessions of inter-species of citrus. In this study, SNPs were mined from EST sequences of sweet orange and satsuma, which displayed potential capability as molecular markers to discriminate inter-species accessions of citrus. It is anticipated that these putative SNPs could be applied in citrus genetics research and breeding.展开更多
The total phenolic and flavonoid contents in the fruit tissues （peels, pulp residues, seeds, and juices） of 19 citrus genotypes belonged to Citrus reticulata Blanco were evaluated and their antioxidant capacity was ...The total phenolic and flavonoid contents in the fruit tissues （peels, pulp residues, seeds, and juices） of 19 citrus genotypes belonged to Citrus reticulata Blanco were evaluated and their antioxidant capacity was tested by 2,2-diphenyl-l-picrylhydra- zyl radicals （DPPH） method and 2,2＇-azino-bis（3-ethylbenzthiozoline-6）-sulphonic acid （ABTS） method. The total phenolic and flavonoid contents, and their antioxidant capacity varied in different citrus fruit tissues. Generally, the peel had both the highest average of total phenolics （27.18 mg gallic acid equivalent （GAE） g^-1 DW） and total flavonoids （38.97 mg rutin equivalent （RE） g^-1 DW）. The highest antioxidant capacity was also the average of DPPH value （21.92 mg vitamin C equiv- alent antioxidant capacity （VCEAC） g^-1 DW） and average of ABTS value （78.70 mg VCEAC g-1 DW） in peel. The correlation coefficient between the total phenolics and their antioxidant capacity of different citrus fruits tissues ranged from 0.079 to 0.792, and from -0.150 to 0.664 for the total flavonoids. The antioxidant capacity of fruit tissues were correlated with the total phenoilc content and flavonoid content except in case of the peel. In addition, the total phenolic content and antioxidant capacity varied in different citrus genotypes. Manju and Karamandarin were better genotypes with higher antioxidation and the phenolic content, however Shagan was the poorest genotype with lower antioxidation and the phenolic content.展开更多
Citrus tristeza virus(CTV)exists in citrus as a large number of distinct strains differing in biological characters.The control strategies such as mild strains cross protection(MSCP)require a clear understanding of th...Citrus tristeza virus(CTV)exists in citrus as a large number of distinct strains differing in biological characters.The control strategies such as mild strains cross protection(MSCP)require a clear understanding of the characterization of CTV.For better understanding of the structure of CTV population and the relationship between molecular and biological characterization,72 CTV samples collected from five provinces in China were studied,using biological indexing,p25/Hinf I restriction fragment length polymorphism(RFLP),multiple molecular markers,and bidirectional RT-PCR assay.The mixture of severe stem pitting isolates was found to be dominant in the field.CTV isolates with p25/HinfⅠRFLP group 3 and p23/BD-PCR groupⅠ,Ⅲwere the main cause of epidemics,and most CTV isolates were found to be the mixture of T30 and VT genotypes.More accurate identification of strain mixtures in the field and better understanding of the biological traits of the isolates may be achieved by applying the three molecular detection methods simultaneously.展开更多
Scarcity of water and nutrients in orchards are the major causes of low productivity and decline of citrus in tropics. With a hypothesis that the optimal amount of water and nutrients application through drip irrigati...Scarcity of water and nutrients in orchards are the major causes of low productivity and decline of citrus in tropics. With a hypothesis that the optimal amount of water and nutrients application through drip irrigation （DI） could save substantial irrigation and fertilizers comparative to that under traditional basin irrigation （BI） with band placement of fertilizer （BPF）, this study was conducted in citrus in vertisol of central India. Three DI regimes： I1, I2 and I3 scheduled at 50% class-A pan evaporation rate （Ep）, 75% Ep, and 100% Ep atong with three fertilizer doses： F1, F2 and F3 at 50% recommended dose of fertilizer （RDF, N：P2Os：K20=600 g：200 g：100 g）, 75% of RDF and 100% of RDF were applied to mandai＇in plants respectively. BI with band placement of fertilizer （BPF） at 100% RDF was taken for comparison. All the drip-fertigation treatments （except I1F1） produced higher plant growth and fruit yield, with better quality fruits compared with BI＋BPF. The highest fruit yield （16.39t ha^-1）, water use efficiency （3.9 kg m^-3） and fertilizer use efficiency （87.3 kg kg^-1） were observed in I2F2. The concentrations of N, K and Fe in leaves were significantly higher in I2F2 compared to other treatments. The leaf photosynthesis, stomatal conductance （G） and transpiration were the highest in I3F3. However, leaf water use efficiency （LWUE, photosynthesis/transpiration） was the highest in I2F2. The overall results of this study demonstrated that the application of optimum quantity of water and fertilizers （I2F2） through DI could be a productive and water efficient option in citrus production in vertisol.展开更多
The Arabidopsis ICEI （inducer of CBF expression 1） gene was cloned through RT-PCR of Arabidopsis cDNAs and introduced into the lemon （Citrus Limon （L.） Burm. F. cv. Eureka） genome using Agrobacterium-mediated tr...The Arabidopsis ICEI （inducer of CBF expression 1） gene was cloned through RT-PCR of Arabidopsis cDNAs and introduced into the lemon （Citrus Limon （L.） Burm. F. cv. Eureka） genome using Agrobacterium-mediated transformation method. Epicotyl segments from in vitro grown lemon seedlings were co-cultivated with A. tumefaciens strain EHA 105 carrying the binary plasmid pMVICE1, whose T-DNA region contain ICEI gene driven by 35S CaMV promoter. Among 320 epicotyl segments inoculated, 71 explants responded and regenerated 51 elongated shoots. These shoots were subjected to an extra month of kanamycin exposure. In this way, the number of escapes reduced. Thirteen of 31 survived shoots formed roots and 7 were tested positive using PCR technique. Southern blot analyses confirmed PCR results and demonstrated that more than two copies of the ICE1 gene were integrated into the lemon genome.展开更多
Citrus tristeza virus （CTV） causes economically important losses to the citrus industry worldwide. Mild strain cross protection （MSCP） against tristeza has hardly been practised due to mixed infection of different...Citrus tristeza virus （CTV） causes economically important losses to the citrus industry worldwide. Mild strain cross protection （MSCP） against tristeza has hardly been practised due to mixed infection of different CTV-strains and little background of its molecular biology in China. For better cognition on CTV, 192 sweet orange samples collected from eight provinces （Chongqing, Sichuan, Fujian, Hunan, Guangxi, Yunnan, Guangdong and Jiangxi） were tested by direct tissue blot immuno-assay （DTBIA）, and 158 of them were tested positively, which therefore were subjected to coat protein gene （CPG）/Hinf Ⅰ restriction fragment length polymorphism （RFLP） analysis. Sample bulks were compared between Chongqing and Fujian by some statistical data, including ratios of single infection and mixed infection to local samples, proportions of CTV isolates with single RFLP groups, and rates of each RFLP group. The simplified analysis of samples from the other six provinces were then conducted. This study suggests that CTV isolates with CPG/Hinf Ⅰ RFLP groups Ⅲ and Ⅰ are the main epidemic ones in China, and mixed infection of CTV in fields are popular. Based on observation of severity of stem-pitting symptom in field trees, CTV isolates with CPG/Hinf Ⅰ RFLP groups Ⅲ and Ⅰ caused severe stem-pittings in sweet oranges in China.展开更多
Citrus tristeza virus （CTV） is one of the most economically important citrus viruses and harms the citrus industry worldwide. To develop reliable and effective serological detection assays of CTV, the major capsid p...Citrus tristeza virus （CTV） is one of the most economically important citrus viruses and harms the citrus industry worldwide. To develop reliable and effective serological detection assays of CTV, the major capsid protein （CP） gene of CTV was expressed in Escherichia coli BL21 （DE3） using the expression vector pET-28a and purified through Ni＊-NTA affinity chromatography. The recombinant protein was used to immunize BALB/c mice. Four hybridoma cell lines （14B10, 14Hll, 20D5, and 20G12） secreting monoclonal antibodies （MAbs） against CTV were obtained through conventional hybridoma technology. The titers of MAb-containing ascitic fluids secreted by the four hybridoma lines ranged from 10-6 to 10.7 in indirect enzyme-linked immunosorbent assay （ELISA）. Western blots showed that all four MAbs could specifically react with CTV CP. Using the prepared MAbs, dot-ELISA, Tissue print-ELISA, and triple antibody sandwich （TAS）-ELISA were developed to detect CTV in tree nurseries and epidemiological studies. The developed dot-ELISA and TAS-ELISA methods could detect CTV in crude extracts of infected citrus leaves with dilutions of 1：2560 and 1：10, 240 （w/v, g/mL）, respectively. Tissue print-ELISA was particularly useful for large-scale field sample detection, mainly owing to its simplicity and lack of sample preparation requirements. The field survey revealed that CTV is prevalent on citrus trees in the Chongqing Municipality, Jiangxi Province, and Zhejiang Province of China. The coincidence rate of serological and RT-PCR test results reached more than 99.5%. The prepared MAbs against CTV and established sensitive and specific serological assays have a significant role in the detection and prevention and control of CTV in our country.展开更多
Huanglongbing （HLB, or citrus greening） is the most destructive disease of citrus, which is associated with Candidatus Liberibacter asiaticus （Las）. Few management options are available, aside from preventive meas...Huanglongbing （HLB, or citrus greening） is the most destructive disease of citrus, which is associated with Candidatus Liberibacter asiaticus （Las）. Few management options are available, aside from preventive measures such as removing infected plants, planting disease-free seedlings, and managing the insect vector. In this study, we assessed the efficacy of thermotherapy against HLB under controlled greenhouse conditions. A total of 60 two-year-old, graft-infected Citrus reticulata Blanco plants were used. The plants were randomly divided into three groups （45℃, 48℃, and untreated control）, with five plants/replicate （rep） and four reps/treatment. The treated plants were placed in phytotrons for a 4-h treatment session, repeated once per week for three consecutive weeks. Disease remission was observed eight weeks post-treatment. Real-time PCR assays revealed that Las titers in HLB-affected seedlings were significantly reduced in both 45 and 48℃ treatments four weeks after treatment, with the exception of eight plants. In contrast, Las titers in the untreated control plants increased significantly during the same period, with a maximum increase of 28-fold. Except for seven plants, Las titers in the new flushes of treated plants decreased more than 90% eight weeks after treatment. Las titers in mature leaves of treated plants decreased 56 and 60% in average at 45 and 48℃, respectively, eight weeks after treatment. The HLB symptoms and Las titer of seedings were markedly alleviated eight weeks after treatment in both 45 and 48℃ treatments. Our results laid a good foundation for the further development of citrus free-disease seedling cultivation and Huanglongbing control in the field. The whole plants were replaced for scion or branch in previous as the research object in this study, and the expression of Huanglongbing symptoms combined with real-time polymerase chain reaction （PCR） were used to evaluate the effect of heat treatment in the greenhouse.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
Two scab diseases are currently recognized on citrus：citrus scab, caused by Elsino？ fawcettii, and sweet orange scab, caused by E. australis. Although these pathogens are economically important, there is no molecula...Two scab diseases are currently recognized on citrus：citrus scab, caused by Elsino？ fawcettii, and sweet orange scab, caused by E. australis. Although these pathogens are economically important, there is no molecular data on these species in China. Here we use internal transcribed spacer sequence data to report on host-speciifcity and genetic relationships among 46 isolates collected from the main citrus varieties grown across China. All strains isolated were E. fawcettii. Based on pathogenicity testing on 9 different citrus species, isolates were divided into 11 pathotypes （SM, FBHR, SJCR, SPOJCR, SR, SOJG, SPOJC, SRGC, Lemon and two unnamed pathotypes）. SM is a new pathotype, and two isolates did not ift into any of the known pathotypes of E. fawcettii. Inter-simple sequence repeat （ISSR-PCR） assays separated the E. fawcettii isolates into 10 subgroups;the groupings basically corresponded to the pathogenicity test.展开更多
Pathological hypertrophy(cell enlargement)plays an important role in the development of citrus canker,but its regulators are largely unknown.Although WRKY22 is known to be involved in pathogen-triggered immunity and p...Pathological hypertrophy(cell enlargement)plays an important role in the development of citrus canker,but its regulators are largely unknown.Although WRKY22 is known to be involved in pathogen-triggered immunity and positively regulates resistance to bacterial pathogens in Arabidopsis,rice and pepper,the CRISPR/Cas9-mediated partial knockout of CsWRKY22 improves resistance to Xanthomonas citri subsp.citri(Xcc)in Wanjincheng orange(Citrus sinensis Osbeck).Here,we demonstrate that CsWRKY22 is a nucleus-localized transcriptional activator.CsWRKY22-overexpressing plants exhibited dwarf phenotypes that had wrinkled and thickened leaves and were more sensitive to Xcc,whereas CsWRKY22-silenced plants showed no visible phenotype changes and were more resistant to Xcc.Microscopic observations revealed that the overexpression of CsWRKY22 increased cell size in the spongy mesophyll.Transcriptome analysis showed that cell growth-related pathways,such as the auxin and brassinosteroid hormonal signaling and cell wall organization and biogenesis pathways,were significantly upregulated upon CsWRKY22 overexpression.Interestingly,CsWRKY22 activated the expression of CsLOB1,which is a key gene regulating susceptibility to citrus canker.We further confirmed that CsWRKY22 bound directly to the W-boxes just upstream of the transcription start site of CsLOB1 in vivo and in vitro.We conclude that CsWRKY22 enhances susceptibility to citrus canker by promoting host hypertrophy and CsLOB1 expression.Thus,our study provides new insights into the mechanism regulating pathological hypertrophy and the function of WRKY22 in citrus.展开更多
Citrus yellow vein clearing virus(CYVCV)is the causal agent of yellow vein clearing disease,a significant and devastating disease of most citrus species including lemon and sour orange.CYVCV,a single-strand positive-s...Citrus yellow vein clearing virus(CYVCV)is the causal agent of yellow vein clearing disease,a significant and devastating disease of most citrus species including lemon and sour orange.CYVCV,a single-strand positive-sense RNA virus containing six ORFs(Open Reading Frames),represents a new species in the genus Mandarivirus of the Alphaflexiviridae family.The virus can cause particularly serious damage,resulting in reduced tree vigor,lower yields,and decreased marketability of fruit production,and it has been found in India,Turkey,Pakistan,China and Iran.Here we described the geographical distribution of the virus,its transmission mode by grafting,mechanical inoculation,and insects,as well as currently available techniques for detection.In addition,we also discussed practical measures aimed at controlling the disease and provided theoretical guidance to prevent the acquisition and spread of the disease that is a significant step toward ensuring the health of the citrus industry.展开更多
Citrus is one of the most important commercial fruit crops worldwide.With the vast genomic data currently available for citrus fruit,genetic relationships,and molecular markers can be assessed for the development of m...Citrus is one of the most important commercial fruit crops worldwide.With the vast genomic data currently available for citrus fruit,genetic relationships,and molecular markers can be assessed for the development of molecular breeding and genomic selection strategies.In this study,to permit the ease of access to these data,a web-based database,the citrus genomic variation database(CitGVD,http://citgvd.cric.cn/home)was developed as the first citrusspecific comprehensive database dedicated to genome-wide variations including single nucleotide polymorphisms(SNPs)and insertions/deletions(INDELs).The current version(V1.0.0)of CitGVD is an open-access resource centered on 1,493,258,964 high-quality genomic variations and 84 phenotypes of 346 organisms curated from in-house projects and public resources.CitGVD integrates closely related information on genomic variation annotations,related gene annotations,and details regarding the organisms,incorporating a variety of built-in tools for data accession and analysis.As an example,CitGWAS can be used for genome-wide association studies(GWASs)with SNPs and phenotypic data,while CitEVOL can be used for genetic structure analysis.These features make CitGVD a comprehensive web portal and bioinformatics platform for citrus-related studies.It also provides a model for analyzing genome-wide variations for a wide range of crop varieties.展开更多
Citrus viroids are the small but economically important RNA pathogens. For investigating their occurrence and distribution in China, 65 viroid samples collected from 8 major citrus cultivated regions were evaluated us...Citrus viroids are the small but economically important RNA pathogens. For investigating their occurrence and distribution in China, 65 viroid samples collected from 8 major citrus cultivated regions were evaluated using one-step or multiplex onestep RT-PCR and biological indexing for specifically detection of Citrus exocortis viroid （CEVd）, Citrus bent leaf viroid （CBLVd）, Hop stunt viroid （HSVd）, Citrus viroid-Ⅲ （CVd-Ⅲ） and Citrus viroid-Ⅳ （CVd-Ⅳ）. The results showed that there were at least 4 kinds of citrus viroids （CEVd, CBLVd, HSVd, and CVd-Ⅲ） on citrus trees in China. Most of the infected citrus plants harbored more than one viroid species, and two plants were infected with up to 4 citrus viroids. Sweet orange was more frequently infected by viroids than other citrus varieties. It is the preliminary report on the species and distribution of citrus viroids in China.展开更多
Citrus bacterial canker(CBC)is a disease resulting from Xanthomonas citri subsp.citri(Xcc)infection and poses a grave threat to citrus production worldwide.Wall-associated receptor-like kinases(WAKLs)are proteins with...Citrus bacterial canker(CBC)is a disease resulting from Xanthomonas citri subsp.citri(Xcc)infection and poses a grave threat to citrus production worldwide.Wall-associated receptor-like kinases(WAKLs)are proteins with a central role in resisting a range of fungal and bacterial diseases.The roles of WAKLs in the context of CBC resistance,however,remain unclear.Here,we explored the role of CsWAKL08,which confers resistance to CBC,and we additionally analyzed the molecular mechanisms of CsWAKL08-mediated CBC resistance.Based on systematic annotation and induced expression analysis of the CsWAKL family in Citrus sinensis,CsWAKL08 was identified as a candidate that can be upregulated by Xcc infection in the CBC-resistant variety.CsWAKL08 can also be induced by the phytohormones salicylic acid(SA)and methyl jasmonic acid(MeJA)and spans the plasma membrane.Overexpression of CsWAKL08 resulted in strong CBC resistance in transgenic sweet oranges,whereas silencing of CsWAKL08 resulted in susceptibility to CBC.The peroxidase(POD)and superoxide dismutase(SOD)activities were significantly enhanced in the CsWAKL08-overexpressing plants compared to the control plants,thereby mediating reactive oxygen species(ROS)homeostasis in the transgenic plants.Moreover,the JA levels and the expression of JA biosynthesis and JA responsive genes were substantially elevated in the CsWAKL08 overexpression plants relative to the controls upon Xcc infection.Based on these findings,we conclude that the wall-associated receptor-like kinase CsWAKL08 positively regulates CBC resistance through a mechanism involving ROS control and JA signaling.These results further highlight the importance of this kinase family in plant pathogen resistance.展开更多
This study aimed to provide a theoretical basis for adopting suitable cultivation measures to tackle calcium （Ca） deficiency in citrus leaves. The Newhall navel orange （Citrus sinensis Osbeck） canopy was sprayed w...This study aimed to provide a theoretical basis for adopting suitable cultivation measures to tackle calcium （Ca） deficiency in citrus leaves. The Newhall navel orange （Citrus sinensis Osbeck） canopy was sprayed with 20.0 mmol L-1 of Ca（NO3）2 during physiological fruit drop period, fruit expanding period, and fruit maturing period on 30, 90, and 210 days after full bloom （DAFB）, respectively, and its effects on leaf gas exchange parameters and leaf mineral nutrition and fruit quality were analyzed. The results showed that： （1） The photosynthetic rate （ACO2） at 9：00 a.m. and 16：00 p.m. of fruit expanding period with 30 and 90 DAFB Ca（NO3）2treatments slightly or significantly improved mainly by decreasing stomatal limitation and nonstomatal limitation, respectively. （2） Compared with control （CK）, the Ca concentration in leaves with 30, 90, and 240 DAFB Ca（NO3）2 treatments increased by 127.16; 97.53, and 33.33%, respectively, and the leaf magnesium concentra- tion also increased by more than 32.26%. However, Ca（NO3）2 canopy spraying on 30 DAFB significantly reduced the leaf potassium concentration, by 22.14% compared with CK. （3） Ca（NO3）2 canopy spraying on 30 DAFB decreased the second fruit drop rate by 30.55% and increased the weight per fruit by 25.04%, thus resulting in a significant increase in citrus yield. （4） Spraying Ca（NO3） on 30 DAFB mainly affected the metabolism of titratable acid （TA） to improve the maturity of citrus fruits. Whilst it improved the external quality and the coloring of citrus fruit significantly. Therefore, Ca（NO3）2 canopy spraying during physiological fruit drop period has a better influence on the tree character and fruit quality of Newhall navel orange （Citrus sinensis Osbeck）.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 Key Research and Development Program of China(Grant No.2022YFD1201600)Earmarked Fund for China Agriculture Research System(Grant No.CARS-26)+1 种基金Chongqing Natural Science Foundation Project(Grant No.CSTB2023NSCQ-MSX1085)Cultivar Improvement of Nanfeng Orange.
文摘The CRISPR/Cas9 system has shown great promise in engineering targeted mutations in a genome.The efficiency of Cas9-mediated genome editing is temperature sensitive.A high-temperature regime can increase the mutation efficiency induced by the CRISPR/Cas9 system in many plant species.However,a heat stress treatment has not been applied during the tissue culture process in citrus.To develop an efficient heat stress regime to improve the efficiency of CRISPR/Cas9-mediated targeted mutagenesis,three and five cycles of heat stress treatments were used during callus induction in citrus.The results showed that the heat stress treatment with three cycles of 24 h at 37℃,followed by 24 h at 26℃,increased the mutation efficiency by 11.6%compared with no heat stress treatment,and that five cycles of heat stress treatment were optimal,from which 50%mutants had a 100%mutation rate.The mutation profiles of Cas9 at 28℃ for 10 d and 37℃ for three or five cycles were similar,indicating that heat stress treatment did not affect the non-homologous end joining repair pathway.No detectable off-target mutation was detected in the potential off-target sites with four nucleotide mismatches compared with the designed on-target site.This study demonstrated that five cycles of heat stress treatment during callus induction could efficiently increase the mutation efficiency mediated by the CRISPR/Cas9 system without observable negative effects,and provided an efficient Cas9-mediated citrus genome editing system to produce mutants with a high mutation rate.
基金funded by Sichuan Science and Technology Program,grant numbers 2021YFYZ0010,2023YFH0006,2025YFHZ0295The Basic Research Program of Sichuan Provincial Research Institutes,grant numbers 2024JDKY0001 and 2023JDKY0001.
文摘The genus Actinidia is primarily functionally dioecious,and early sex identification plays a crucial role in improving breeding efficiency and reducing production costs.In this study,the accuracy of three sex-linked molecular markers(SyGI[Shy Girl],FrBy[Friendly Boy],and SmY1)in sex identification was evaluated in various Actinidia species.The selected marker products were subsequently cloned and sequenced in six wild Actinidia species.Ninety-six wild A.chinensis chinensis accessions and 74 A.chinensis deliciosa accessions,most of which were wild,with only one cultivated,were used for comprehensive primer validation.Thirty-three juvenile A.chinensis chinensis hybrid seedlings were used for practical application tests.The results showed that the marker SyGI accurately identified the sex of 20 samples from six Actinidia species and 96 A.chinensis chinensis accessions with 100%reliability.For Actinidia chinensis deliciosa,the identification accuracy reached 98.65%.Sequence analysis revealed that SyGI shared the highest similarity with the male-specific genomic region.Furthermore,SyGI achieved 100%accuracy in identifying the sex of 33 juvenile A.chinensis chinensis individuals.The findings confirm that the SyGI marker possesses high accuracy,strong specificity,and broad applicability,making it a valuable tool for kiwifruit breeding programs.The cloned sequences from wild Actinidia species also provide important references for future research on the mechanisms of sexual evolution and determination.
基金This work was supported by the Chinese National Programs for Science and Technology Development(No.2004-06)and the Natu-ral Science Foundation of Chongqing.
文摘Simple sequence repeats(SSRs)were investigated in the unigene sequences from expressed sequence tags(EST)of sweet orange(Citrus sinensis osbeck),trifoliate orange(Poncirus trifoliata Raf.)and other citrus species and cultivars.A total of 37802 citrus unigene sequences corresponding to 23.29 Mb were searched,resulting in the identification of 8218 SSRs.Among them there were 4913(59.8%)mono-,1419(17.3%)di-,1709(20.8%)tri-,114(1.39%)tetra-,23(0.28%)penta-and 40(0.49%)hexa-nucleotide SSRs.The estimated frequency of SSRs was approximately 1/2.8 kb,which could be extrapolated to 1 SSR-containing unigene in 4.6 unigenes.The maximum length of the SSR ranged from 40 to 105 bp depending on the repeating numbers of the motif in the SSR.The overall average length of SSRs was 20.9 bp.The frequencies of different SSR types(di-,tri-,tetra-,and penta-nucleotide repeats)were very similar between sweet orange and trifoliate orange.The mononucelotide repeats appeared to be the most abundant SSRs within sweet orange and trifoliate orange,followed by trimeric repeats.The adenine rich repeats such as A/T,AG,AT,AAG,AAAT,AAAG,AAAT,AAAAG,AAAAT etc.were predominant in each type of SSRs(mono-,di-,tri-,tetra-,and penta-),whereas the C/G,CG,CCG repeats were less abundant.Twenty-five primer pairs flanking EST-SSR loci were designed to detect the possible polymorphism of six citrus cultivars including sweet orange and trifoliate orange.The PCR result with all these 25 primer pairs revealed the existence of polymorphism within six citrus cultivars confirming that citrus EST database could be efficiently exploited for the development of gene-derived SSR markers.
基金supported by the National Natural Science Foundation of China(2006BAD13B06-1-4)the Natural Science Foundation of Chongqing,China (CSTC,2007BB1343)
文摘Single nucleotide polymorphisms （SNPs） are the most abundant sequence variations found in plant genomes and are widely used as molecular genetic markers in cultivar identification and genetic diversity studies. The objective of this study was to identify SNP markers useful for discrimination of citrus cultivars, since large numbers of expressed sequence tags （ESTs） of sweet orange are available from the National Center for Biotechnology Information （NCBI）. We now have the opportunity to discover SNP markers suitable for determining the haplotypes with which to distinguish very closely related cultivars and to assess genetic diversity within or between related species of citrus. SNPs and small insertions/deletions （Indels） from ESTs of sweet orange and satsuma were identified by the in silico SNP discovery strategy. 55 296 EST sequences of sweet orange and 2 575 of satsuma retrieved from the NCBI repository were mined for potential SNPs. Cleaved amplified polymorphic sequences （CAPS） and sequencing approaches were used to validate putative SNPs in a sample of 30 citrus accessions. A total of 3 348 putative SNPs were identified based on the abundance of sequences and haplotype cosegregation. Of these 3 348 SNPs, the transitions, transversions and Indels ratios were 47.9, 36.1 and 16.0%, respectively. The SNPs occurred on average at a frequency of 1 per 164 bp in the coding region of citrus. 14 SNPs were randomly selected and genotyped according to 30 citrus accessions including 23 accessions of sweet orange; 11 SNPs displayed polymorphism with an average polymorphism information content （PIC） of 0.20 among 30 citrus accessions. The genetic diversity present in sweet orange was low, so the 14 SNP markers failed to discriminate different cultivars of sweet orange, but they did succeed in distinguishing accessions of inter-species of citrus. In this study, SNPs were mined from EST sequences of sweet orange and satsuma, which displayed potential capability as molecular markers to discriminate inter-species accessions of citrus. It is anticipated that these putative SNPs could be applied in citrus genetics research and breeding.
基金supported by the Identification of the Common Nutrients of Edible Agricultural Products and the Character Nutrients of Special Agricultural Products and Their Key Control Points of Quality,China (GJFP201701501)the Chongqing Program for Production of Late Maturing Citrus Fruits,China (20174-4)+2 种基金the Program for Talent Introduction of Chongqing Three Gorges University,China (14RC05)the Program for Chongqing Municipal Education Commission,China (KJ1501015)the Program for Chongqing Science & Technology Commission,China (cstc2016jcyj A0555)
文摘The total phenolic and flavonoid contents in the fruit tissues （peels, pulp residues, seeds, and juices） of 19 citrus genotypes belonged to Citrus reticulata Blanco were evaluated and their antioxidant capacity was tested by 2,2-diphenyl-l-picrylhydra- zyl radicals （DPPH） method and 2,2＇-azino-bis（3-ethylbenzthiozoline-6）-sulphonic acid （ABTS） method. The total phenolic and flavonoid contents, and their antioxidant capacity varied in different citrus fruit tissues. Generally, the peel had both the highest average of total phenolics （27.18 mg gallic acid equivalent （GAE） g^-1 DW） and total flavonoids （38.97 mg rutin equivalent （RE） g^-1 DW）. The highest antioxidant capacity was also the average of DPPH value （21.92 mg vitamin C equiv- alent antioxidant capacity （VCEAC） g^-1 DW） and average of ABTS value （78.70 mg VCEAC g-1 DW） in peel. The correlation coefficient between the total phenolics and their antioxidant capacity of different citrus fruits tissues ranged from 0.079 to 0.792, and from -0.150 to 0.664 for the total flavonoids. The antioxidant capacity of fruit tissues were correlated with the total phenoilc content and flavonoid content except in case of the peel. In addition, the total phenolic content and antioxidant capacity varied in different citrus genotypes. Manju and Karamandarin were better genotypes with higher antioxidation and the phenolic content, however Shagan was the poorest genotype with lower antioxidation and the phenolic content.
基金supported by National Natural Science Foundation of China(30471205).
文摘Citrus tristeza virus(CTV)exists in citrus as a large number of distinct strains differing in biological characters.The control strategies such as mild strains cross protection(MSCP)require a clear understanding of the characterization of CTV.For better understanding of the structure of CTV population and the relationship between molecular and biological characterization,72 CTV samples collected from five provinces in China were studied,using biological indexing,p25/Hinf I restriction fragment length polymorphism(RFLP),multiple molecular markers,and bidirectional RT-PCR assay.The mixture of severe stem pitting isolates was found to be dominant in the field.CTV isolates with p25/HinfⅠRFLP group 3 and p23/BD-PCR groupⅠ,Ⅲwere the main cause of epidemics,and most CTV isolates were found to be the mixture of T30 and VT genotypes.More accurate identification of strain mixtures in the field and better understanding of the biological traits of the isolates may be achieved by applying the three molecular detection methods simultaneously.
文摘Scarcity of water and nutrients in orchards are the major causes of low productivity and decline of citrus in tropics. With a hypothesis that the optimal amount of water and nutrients application through drip irrigation （DI） could save substantial irrigation and fertilizers comparative to that under traditional basin irrigation （BI） with band placement of fertilizer （BPF）, this study was conducted in citrus in vertisol of central India. Three DI regimes： I1, I2 and I3 scheduled at 50% class-A pan evaporation rate （Ep）, 75% Ep, and 100% Ep atong with three fertilizer doses： F1, F2 and F3 at 50% recommended dose of fertilizer （RDF, N：P2Os：K20=600 g：200 g：100 g）, 75% of RDF and 100% of RDF were applied to mandai＇in plants respectively. BI with band placement of fertilizer （BPF） at 100% RDF was taken for comparison. All the drip-fertigation treatments （except I1F1） produced higher plant growth and fruit yield, with better quality fruits compared with BI＋BPF. The highest fruit yield （16.39t ha^-1）, water use efficiency （3.9 kg m^-3） and fertilizer use efficiency （87.3 kg kg^-1） were observed in I2F2. The concentrations of N, K and Fe in leaves were significantly higher in I2F2 compared to other treatments. The leaf photosynthesis, stomatal conductance （G） and transpiration were the highest in I3F3. However, leaf water use efficiency （LWUE, photosynthesis/transpiration） was the highest in I2F2. The overall results of this study demonstrated that the application of optimum quantity of water and fertilizers （I2F2） through DI could be a productive and water efficient option in citrus production in vertisol.
基金the National Natural Science Foundation of China(30070528)
文摘The Arabidopsis ICEI （inducer of CBF expression 1） gene was cloned through RT-PCR of Arabidopsis cDNAs and introduced into the lemon （Citrus Limon （L.） Burm. F. cv. Eureka） genome using Agrobacterium-mediated transformation method. Epicotyl segments from in vitro grown lemon seedlings were co-cultivated with A. tumefaciens strain EHA 105 carrying the binary plasmid pMVICE1, whose T-DNA region contain ICEI gene driven by 35S CaMV promoter. Among 320 epicotyl segments inoculated, 71 explants responded and regenerated 51 elongated shoots. These shoots were subjected to an extra month of kanamycin exposure. In this way, the number of escapes reduced. Thirteen of 31 survived shoots formed roots and 7 were tested positive using PCR technique. Southern blot analyses confirmed PCR results and demonstrated that more than two copies of the ICE1 gene were integrated into the lemon genome.
基金supported by the National Natural Science Foundation of China(30471205)under the program"Comparison Research on Strains of Citrus tristeza virus."
文摘Citrus tristeza virus （CTV） causes economically important losses to the citrus industry worldwide. Mild strain cross protection （MSCP） against tristeza has hardly been practised due to mixed infection of different CTV-strains and little background of its molecular biology in China. For better cognition on CTV, 192 sweet orange samples collected from eight provinces （Chongqing, Sichuan, Fujian, Hunan, Guangxi, Yunnan, Guangdong and Jiangxi） were tested by direct tissue blot immuno-assay （DTBIA）, and 158 of them were tested positively, which therefore were subjected to coat protein gene （CPG）/Hinf Ⅰ restriction fragment length polymorphism （RFLP） analysis. Sample bulks were compared between Chongqing and Fujian by some statistical data, including ratios of single infection and mixed infection to local samples, proportions of CTV isolates with single RFLP groups, and rates of each RFLP group. The simplified analysis of samples from the other six provinces were then conducted. This study suggests that CTV isolates with CPG/Hinf Ⅰ RFLP groups Ⅲ and Ⅰ are the main epidemic ones in China, and mixed infection of CTV in fields are popular. Based on observation of severity of stem-pitting symptom in field trees, CTV isolates with CPG/Hinf Ⅰ RFLP groups Ⅲ and Ⅰ caused severe stem-pittings in sweet oranges in China.
基金supported by Public Science and Technology Research Funds Projects of Agriculture (20120307605)
文摘Citrus tristeza virus （CTV） is one of the most economically important citrus viruses and harms the citrus industry worldwide. To develop reliable and effective serological detection assays of CTV, the major capsid protein （CP） gene of CTV was expressed in Escherichia coli BL21 （DE3） using the expression vector pET-28a and purified through Ni＊-NTA affinity chromatography. The recombinant protein was used to immunize BALB/c mice. Four hybridoma cell lines （14B10, 14Hll, 20D5, and 20G12） secreting monoclonal antibodies （MAbs） against CTV were obtained through conventional hybridoma technology. The titers of MAb-containing ascitic fluids secreted by the four hybridoma lines ranged from 10-6 to 10.7 in indirect enzyme-linked immunosorbent assay （ELISA）. Western blots showed that all four MAbs could specifically react with CTV CP. Using the prepared MAbs, dot-ELISA, Tissue print-ELISA, and triple antibody sandwich （TAS）-ELISA were developed to detect CTV in tree nurseries and epidemiological studies. The developed dot-ELISA and TAS-ELISA methods could detect CTV in crude extracts of infected citrus leaves with dilutions of 1：2560 and 1：10, 240 （w/v, g/mL）, respectively. Tissue print-ELISA was particularly useful for large-scale field sample detection, mainly owing to its simplicity and lack of sample preparation requirements. The field survey revealed that CTV is prevalent on citrus trees in the Chongqing Municipality, Jiangxi Province, and Zhejiang Province of China. The coincidence rate of serological and RT-PCR test results reached more than 99.5%. The prepared MAbs against CTV and established sensitive and specific serological assays have a significant role in the detection and prevention and control of CTV in our country.
基金funded by the United States Department of Agriculture (USDA)-APHIS-PPQ-CPHST and North Carolina State University joint project (2012-0195-01)the Special Fund for Agro-Scientific Research in the Public Interest, China (201003067-05)
文摘Huanglongbing （HLB, or citrus greening） is the most destructive disease of citrus, which is associated with Candidatus Liberibacter asiaticus （Las）. Few management options are available, aside from preventive measures such as removing infected plants, planting disease-free seedlings, and managing the insect vector. In this study, we assessed the efficacy of thermotherapy against HLB under controlled greenhouse conditions. A total of 60 two-year-old, graft-infected Citrus reticulata Blanco plants were used. The plants were randomly divided into three groups （45℃, 48℃, and untreated control）, with five plants/replicate （rep） and four reps/treatment. The treated plants were placed in phytotrons for a 4-h treatment session, repeated once per week for three consecutive weeks. Disease remission was observed eight weeks post-treatment. Real-time PCR assays revealed that Las titers in HLB-affected seedlings were significantly reduced in both 45 and 48℃ treatments four weeks after treatment, with the exception of eight plants. In contrast, Las titers in the untreated control plants increased significantly during the same period, with a maximum increase of 28-fold. Except for seven plants, Las titers in the new flushes of treated plants decreased more than 90% eight weeks after treatment. Las titers in mature leaves of treated plants decreased 56 and 60% in average at 45 and 48℃, respectively, eight weeks after treatment. The HLB symptoms and Las titer of seedings were markedly alleviated eight weeks after treatment in both 45 and 48℃ treatments. Our results laid a good foundation for the further development of citrus free-disease seedling cultivation and Huanglongbing control in the field. The whole plants were replaced for scion or branch in previous as the research object in this study, and the expression of Huanglongbing symptoms combined with real-time polymerase chain reaction （PCR） were used to evaluate the effect of heat treatment in the greenhouse.
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 China Agriculture Research System (CARS-27)the Technology Application Research Program for Public Interest of Zhejiang Province,China
文摘Two scab diseases are currently recognized on citrus：citrus scab, caused by Elsino？ fawcettii, and sweet orange scab, caused by E. australis. Although these pathogens are economically important, there is no molecular data on these species in China. Here we use internal transcribed spacer sequence data to report on host-speciifcity and genetic relationships among 46 isolates collected from the main citrus varieties grown across China. All strains isolated were E. fawcettii. Based on pathogenicity testing on 9 different citrus species, isolates were divided into 11 pathotypes （SM, FBHR, SJCR, SPOJCR, SR, SOJG, SPOJC, SRGC, Lemon and two unnamed pathotypes）. SM is a new pathotype, and two isolates did not ift into any of the known pathotypes of E. fawcettii. Inter-simple sequence repeat （ISSR-PCR） assays separated the E. fawcettii isolates into 10 subgroups;the groupings basically corresponded to the pathogenicity test.
基金the National Key Research and Development Program of China(2018YFD1000300,to S.C.)the National Natural Sciences Foundation of China(31972393,to X.Z.)+2 种基金the Natural Science Foundation Project of Chongqing City(cstc2019jcyj-bshX0024 to Q.L.)the Fundamental Research Funds for the Central Universities(XDJK2019C027,to Q.L.)the Earmarked Fund for China Agriculture Research System(CARS-26,to S.C.).
文摘Pathological hypertrophy(cell enlargement)plays an important role in the development of citrus canker,but its regulators are largely unknown.Although WRKY22 is known to be involved in pathogen-triggered immunity and positively regulates resistance to bacterial pathogens in Arabidopsis,rice and pepper,the CRISPR/Cas9-mediated partial knockout of CsWRKY22 improves resistance to Xanthomonas citri subsp.citri(Xcc)in Wanjincheng orange(Citrus sinensis Osbeck).Here,we demonstrate that CsWRKY22 is a nucleus-localized transcriptional activator.CsWRKY22-overexpressing plants exhibited dwarf phenotypes that had wrinkled and thickened leaves and were more sensitive to Xcc,whereas CsWRKY22-silenced plants showed no visible phenotype changes and were more resistant to Xcc.Microscopic observations revealed that the overexpression of CsWRKY22 increased cell size in the spongy mesophyll.Transcriptome analysis showed that cell growth-related pathways,such as the auxin and brassinosteroid hormonal signaling and cell wall organization and biogenesis pathways,were significantly upregulated upon CsWRKY22 overexpression.Interestingly,CsWRKY22 activated the expression of CsLOB1,which is a key gene regulating susceptibility to citrus canker.We further confirmed that CsWRKY22 bound directly to the W-boxes just upstream of the transcription start site of CsLOB1 in vivo and in vitro.We conclude that CsWRKY22 enhances susceptibility to citrus canker by promoting host hypertrophy and CsLOB1 expression.Thus,our study provides new insights into the mechanism regulating pathological hypertrophy and the function of WRKY22 in citrus.
基金This research was supported by Fundamental Research Funds for the CentralUniversities,China(Grant No.XDJK2015D026).Cuihua Liu was the recipient of a scholarship from the China Scholarship Council.Hai Liu,John Hurst,and Michael P.Timko were supported in part by a grant(Award No.GI14732)from 22nd Century Limited,LLC.
文摘Citrus yellow vein clearing virus(CYVCV)is the causal agent of yellow vein clearing disease,a significant and devastating disease of most citrus species including lemon and sour orange.CYVCV,a single-strand positive-sense RNA virus containing six ORFs(Open Reading Frames),represents a new species in the genus Mandarivirus of the Alphaflexiviridae family.The virus can cause particularly serious damage,resulting in reduced tree vigor,lower yields,and decreased marketability of fruit production,and it has been found in India,Turkey,Pakistan,China and Iran.Here we described the geographical distribution of the virus,its transmission mode by grafting,mechanical inoculation,and insects,as well as currently available techniques for detection.In addition,we also discussed practical measures aimed at controlling the disease and provided theoretical guidance to prevent the acquisition and spread of the disease that is a significant step toward ensuring the health of the citrus industry.
基金funded by the National Key Research and Development Program of China(2018YFD1000306)Earmarked Funds for the China Agriculture Research System(CARS-26)the Guangxi Science and Technology Key Project(GuiKeAA18118046-6).
文摘Citrus is one of the most important commercial fruit crops worldwide.With the vast genomic data currently available for citrus fruit,genetic relationships,and molecular markers can be assessed for the development of molecular breeding and genomic selection strategies.In this study,to permit the ease of access to these data,a web-based database,the citrus genomic variation database(CitGVD,http://citgvd.cric.cn/home)was developed as the first citrusspecific comprehensive database dedicated to genome-wide variations including single nucleotide polymorphisms(SNPs)and insertions/deletions(INDELs).The current version(V1.0.0)of CitGVD is an open-access resource centered on 1,493,258,964 high-quality genomic variations and 84 phenotypes of 346 organisms curated from in-house projects and public resources.CitGVD integrates closely related information on genomic variation annotations,related gene annotations,and details regarding the organisms,incorporating a variety of built-in tools for data accession and analysis.As an example,CitGWAS can be used for genome-wide association studies(GWASs)with SNPs and phenotypic data,while CitEVOL can be used for genetic structure analysis.These features make CitGVD a comprehensive web portal and bioinformatics platform for citrus-related studies.It also provides a model for analyzing genome-wide variations for a wide range of crop varieties.
基金the National Key Technology R&D Program of China (2007BAD61B04)the National Natural Science Foundation of CQ CSTC, China (2006BB1351)
文摘Citrus viroids are the small but economically important RNA pathogens. For investigating their occurrence and distribution in China, 65 viroid samples collected from 8 major citrus cultivated regions were evaluated using one-step or multiplex onestep RT-PCR and biological indexing for specifically detection of Citrus exocortis viroid （CEVd）, Citrus bent leaf viroid （CBLVd）, Hop stunt viroid （HSVd）, Citrus viroid-Ⅲ （CVd-Ⅲ） and Citrus viroid-Ⅳ （CVd-Ⅳ）. The results showed that there were at least 4 kinds of citrus viroids （CEVd, CBLVd, HSVd, and CVd-Ⅲ） on citrus trees in China. Most of the infected citrus plants harbored more than one viroid species, and two plants were infected with up to 4 citrus viroids. Sweet orange was more frequently infected by viroids than other citrus varieties. It is the preliminary report on the species and distribution of citrus viroids in China.
基金funded by the National Key Research and Development Program of China(2018YFD1000306)the Fundamental Research Funds for the Central Universities(SWU115025)+1 种基金the Guangxi Science and Technology Key Project(GuiKeAA18118046-6)the Earmarked Funds for the China Agriculture Research System(CARS-26).
文摘Citrus bacterial canker(CBC)is a disease resulting from Xanthomonas citri subsp.citri(Xcc)infection and poses a grave threat to citrus production worldwide.Wall-associated receptor-like kinases(WAKLs)are proteins with a central role in resisting a range of fungal and bacterial diseases.The roles of WAKLs in the context of CBC resistance,however,remain unclear.Here,we explored the role of CsWAKL08,which confers resistance to CBC,and we additionally analyzed the molecular mechanisms of CsWAKL08-mediated CBC resistance.Based on systematic annotation and induced expression analysis of the CsWAKL family in Citrus sinensis,CsWAKL08 was identified as a candidate that can be upregulated by Xcc infection in the CBC-resistant variety.CsWAKL08 can also be induced by the phytohormones salicylic acid(SA)and methyl jasmonic acid(MeJA)and spans the plasma membrane.Overexpression of CsWAKL08 resulted in strong CBC resistance in transgenic sweet oranges,whereas silencing of CsWAKL08 resulted in susceptibility to CBC.The peroxidase(POD)and superoxide dismutase(SOD)activities were significantly enhanced in the CsWAKL08-overexpressing plants compared to the control plants,thereby mediating reactive oxygen species(ROS)homeostasis in the transgenic plants.Moreover,the JA levels and the expression of JA biosynthesis and JA responsive genes were substantially elevated in the CsWAKL08 overexpression plants relative to the controls upon Xcc infection.Based on these findings,we conclude that the wall-associated receptor-like kinase CsWAKL08 positively regulates CBC resistance through a mechanism involving ROS control and JA signaling.These results further highlight the importance of this kinase family in plant pathogen resistance.
基金supported by a grant from the National Natural Science Foundation of China(NSFC,31470408)the Science and Technology Support Project of Chongqing,China(cstc2014fazktjcsf 80031)+1 种基金the Fundamental Research Funds for the Central Universities,China(XDJK2016 A012XDJK2013 A002)
文摘This study aimed to provide a theoretical basis for adopting suitable cultivation measures to tackle calcium （Ca） deficiency in citrus leaves. The Newhall navel orange （Citrus sinensis Osbeck） canopy was sprayed with 20.0 mmol L-1 of Ca（NO3）2 during physiological fruit drop period, fruit expanding period, and fruit maturing period on 30, 90, and 210 days after full bloom （DAFB）, respectively, and its effects on leaf gas exchange parameters and leaf mineral nutrition and fruit quality were analyzed. The results showed that： （1） The photosynthetic rate （ACO2） at 9：00 a.m. and 16：00 p.m. of fruit expanding period with 30 and 90 DAFB Ca（NO3）2treatments slightly or significantly improved mainly by decreasing stomatal limitation and nonstomatal limitation, respectively. （2） Compared with control （CK）, the Ca concentration in leaves with 30, 90, and 240 DAFB Ca（NO3）2 treatments increased by 127.16; 97.53, and 33.33%, respectively, and the leaf magnesium concentra- tion also increased by more than 32.26%. However, Ca（NO3）2 canopy spraying on 30 DAFB significantly reduced the leaf potassium concentration, by 22.14% compared with CK. （3） Ca（NO3）2 canopy spraying on 30 DAFB decreased the second fruit drop rate by 30.55% and increased the weight per fruit by 25.04%, thus resulting in a significant increase in citrus yield. （4） Spraying Ca（NO3） on 30 DAFB mainly affected the metabolism of titratable acid （TA） to improve the maturity of citrus fruits. Whilst it improved the external quality and the coloring of citrus fruit significantly. Therefore, Ca（NO3）2 canopy spraying during physiological fruit drop period has a better influence on the tree character and fruit quality of Newhall navel orange （Citrus sinensis Osbeck）.
