Production of Polyclonal Antibodies to the Recombinant Coat Protein of Citrus tristeza virus and their Effectiveness for Virus Detection

Citrus tristeza virus (CTV) is distributed worldwide and causes the most economically important virus diseases of citrus. Enzyme‐linked immunosorbent assay (ELISA) and/or immunoprinting have become an indispensable tools for large‐scale diagnosis of CTV worldwide. Several CTV detection kits are commercially available, based on either polyclonal or monoclonal antibodies developed against purified virus preparations. We have developed polyclonal antibodies to recombinant p25 CTV coat proteins (rCP) and determined their effectiveness for both trapping and as the intermediate antibody in double‐antibody sandwich indirect (DASI) ELISA. The p25 coat protein gene of three CTV isolates was amplified by RT‐PCR and further cloned and expressed in Escherichia coli cells. The rCP was injected into rabbits and goats for antibody production. Western blotting assays with the rCP CTV‐specific antibodies reacted positively with the homologous and heterologous rCP of the three CTV isolates and with the corresponding native coat protein present in crude sap extracts of CTV‐infected citrus tissue, but not with extracts from healthy tissue. The rCP antibodies from goat and rabbit reacted as both plate trapping and intermediate antibodies in DASI‐ELISA, discriminating healthy and CTV‐infected citrus, with optical density (OD₄₀₅) values in the range of 0.151–2.415 for CTV‐infected samples and less than 0.100 for healthy tissue. Commercially available anti‐CTV antibodies were used as a reference. Previous reports indicate that antibodies developed to recombinant antigens, including those of CTV, may not be functional for trapping the target antigens under non‐denaturing conditions. Our results showed the feasibility of CTV antibodies developed to the rCP for use as both trapping and intermediate antibodies in DASI‐ELISA, when the recombinant antigen was fractioned with polyacrylamide electrophoresis gel and further extensively dialysed against phosphate buffer saline prior to its use as immunogen.     

The resistance of sour orange to Citrus tristeza virus is mediated by both the salicylic acid and RNA silencing defence pathways

Citrus tristeza virus (CTV) induces in the field the decline and death of citrus varieties grafted on sour orange (SO) rootstock, which has forced the use of alternative decline‐tolerant rootstocks in affected countries, despite the highly desirable agronomic features of the SO rootstock. Declining citrus plants display phloem necrosis below the bud union. In addition, SO is minimally susceptible to CTV compared with other citrus varieties, suggesting partial resistance of SO to CTV. Here, by silencing different citrus genes with a Citrus leaf blotch virus‐based vector, we have examined the implication of the RNA silencing and salicylic acid (SA) defence pathways in the resistance of SO to CTV. Silencing of the genes RDR1, NPR1 and DCL2/DCL4, associated with these defence pathways, enhanced virus spread and accumulation in SO plants in comparison with non‐silenced controls, whereas silencing of the genes NPR3/NPR4, associated with the hypersensitive response, produced a slight decrease in CTV accumulation and reduced stunting of SO grafted on CTV‐infected rough lemon plants. We also found that the CTV RNA silencing suppressors p20 and p23 also suppress the SA signalling defence, with the suppressor activity being higher in the most virulent isolates.     

The movement and distribution of citrus tristeza virus and citrus exocortis viroid in citrus seedlings1

The systemic movement of citrus tristeza virus (CTV) in sour orange (Citrus aurantium) seedlings and of citrus exocortis viroid (CEVd) in Etrog citron (C. medica) seedlings was studied. The movement of the two pathogens was analysed by detection in sections of roots and stems at different time intervals. Both pathogens were detected initially in the basal parts and the roots and subsequently spread to the shoot. CTV and CEVd moved in young citrus seedlings at similar rates. The findings are consistent with long distance phloem transport of the virus and the viroid. The practical implications of the pattern of systemic movement for diagnosis of infected trees are discussed.     

Pathological categorization of the stem‐pitting and mild strains Citrus tristeza virus in Taiwan and their genomic analysis

Citrus tristeza virus (CTV), the causal agent of tristeza disease, causes the devastating diseases worldwide. In Taiwan, complex cultivars and long‐term infection by CTV result in more than 90% of infected citrus trees, but local strain identification and classification are still incomplete. Here, six CTV strains were categorized by grafting onto eight citrus cultivars and the pathological characteristics of the stem‐pitting mild strains were identified. After 6 months of inoculation, the pummelo stem‐pitting severe strain (CTV‐Pum/SP/T1) only caused severe symptoms in Wentan pummelo (WP) and the mild strain (CTV‐Pum/M/T5) was symptomless in every cultivar; the sweet orange (SO) stem‐pitting severe strain (CTV‐SwO/SP/T7) affected SO, WP and Ponkan mandarin (PM), and the mild strain (CTV‐SwO/M/T51) caused no symptoms in SO except for WP; the mandarin stem‐pitting severe strain (CTV‐Man/SP/T46) caused severe impacts in PM, WP and Eureka lemon, whereas the mild strain (CTV‐Man/M/T2) only caused severe stem‐pitting in WP. The full‐length sequencing of both pummelo stem‐pitting strains and phylogenetic analysis revealed that CTV‐Pum/SP/T1 and CTV‐Pum/M/T5 were related to the HA18‐9 and HA16‐5 strains from Hawaii, respectively. Moreover, recombination analysis revealed that TCT repeat sequences existed at open reading frame 1a in both the CTV‐Pum/SP/T1 and the T36 strains from the United States, indicating that the possible evolution relationship between two regions. Furthermore, improved universal and specific primer pairs were designed for more specific, sensitive detection to meet the needs for quarantine and early prevention. The understanding of strain pathogenicity and genomic analysis provided further characterization of each strain and enabled practical challenge inoculation against CTV disease.     

Citrus tristeza virus replicates and forms infectious virions in protoplasts of resistant citrus relatives

Citrus tristeza virus (CTV) is the most economically important viral disease of citrus worldwide. Cultivars with improved CTV tolerance or resistance are needed to manage CTV-induced diseases. The citrus relatives Poncirus trifoliata (L.) Raf., Swinglea glutinosa (Blanco) Merr., and Severinia buxifolia (Poir) Ten. are potential sources of CTV resistance, but their resistance mechanisms are poorly characterized. As a first step to examine the mechanisms of resistance to CTV in these citrus relatives and selected Citrus × Poncirus hybrids, it was necessary to develop methods for protoplast isolation and viral inoculation to allow examination of CTV multiplication in this range of citrus varieties and relatives. Leaf and/or cultured cell protoplasts were isolated and inoculated with four biologically distinct CTV isolates. Northern-blot hybridization analyses for progeny RNAs and immuno-electron microscopy assays for newly produced virions showed that CTV replicated and produced infectious particles in protoplasts from all of the resistant plants tested. These results suggest that resistance to CTV observed at the plant level results from a lack of virus movement and/or some induced resistance response, rather than lack of viral multiplication at the cellular level.     

Detection and Inoculation of Peanut Witches' Broom Phytoplasma (16SrII‐A) and Periwinkle Leaf Yellowing Phytoplasma (16SrI‐B) in Citrus Cultivars in Taiwan

To clarify the phytoplasma associated with Huanglongbing (HLB), a detection survey of phytoplasma in field citrus trees was performed using the standardized nested PCR assay with primer set P1/16S‐Sr and R16F2n/R16R2. The HLB‐diseased citrus trees with typical HLB symptoms showed a high detection of 89.7% (322/359) of HLB‐Las, while a low detection of phytoplasma at 1.1% (4/359) was examined in an HLB‐affected Wentan pummelo (Citrus grandis) tree (1/63) and Tahiti lime (C. latifolia) trees (3/53) that were co‐infected with HLB‐Las. The phytoplasma alone was also detected in a healthy Wentan pummelo tree (1/60) at a low incidence total of 0.3% (1/347). Healthy citrus plants were inoculated with the citrus phytoplasma (WP‐DL) by graft inoculation with phytoplasma‐infected pummelo scions. Positive detections of phytoplasma were monitored only in the Wentan pummelo plant 4 months and 3.5 years after inoculation, and no symptoms developed. The citrus phytoplasma infected and persistently survived in a low titre and at a very uneven distribution in citrus plants. Peanut witches' broom (PnWB) phytoplasma (16SrII‐A) and periwinkle leaf yellowing (PLY) phytoplasma belonging to the aster yellows group (16SrI‐B) maintained in periwinkle plants were inoculated into healthy citrus plants by dodder transmission. The PnWB phytoplasma showed infection through positive detection of the nested PCR assay in citrus plants and persistently survived without symptom expression up to 4 years after inoculation. Positive detections of the phytoplasma were found in a low titre and several incidences in the other inoculated citrus plants including Ponkan mandarin, Liucheng sweet orange, Eureka lemon and Hirami lemon. None of the phytoplasma‐infected citrus plants developed symptoms. Furthermore, artificial inoculation of PLY phytoplasma (16SrI‐B) into the healthy citrus plants demonstrated no infection. The citrus symptomless phytoplasma was identified to belong to the PnWB phytoplasma group (16SrII‐A).     

Expanded Strain‐Specific RT‐PCR Assay for Differential Detection of Currently Known Citrus Tristeza Virus Strains: a Useful Screening Tool

Genotypic characterization of Citrus tristeza virus (CTV) strains has progressed significantly, but their phenotypic expression is poorly established as CTV naturally occurs as mixed‐strain populations. A screening system for the analysis of mixed‐strain populations is required for population studies and the correlation with symptom expression. In this study, a published CTV strain‐specific detection assay was expanded and improved to facilitate detection of currently known CTV strains. Supplementary RT‐PCR assays were developed for two variant groups of the RB strain and the HA16‐5 strain, and assays for the T36 strain and generic CTV detection were improved. The value of the strain‐specific assays was shown by the ability to identify the strain components of two CTV cross‐protecting sources, GFMS35 and LMS6, used in the South African budwood certification scheme and to demonstrate the segregation of strains in budwood source trees.     

Spatial and temporal spread of Citrus tristeza virus and its aphid vectors in the North western area of Morocco

First report of Citrus tristeza virus (CTV,Closterovirus) in Morocco datesback to 1961 in collections of citrus varieties. An exhaustive survey of citrus in the north of the country in 2009 revealed that CTV was spread all over the citrus production area. We attempted to evaluate the relative contribution of different aphid species in the spread of CTV disease in a Citrus reticulata orchard at the Loukkous region during 2 years (2012 and 2013). The overall CTV incidence estimated in the experimental site increased from 17.8% in 2012 to 31.15% in 2013. The most abundant aphid species colonising clementine trees was Aphis spiraecola and A. gossypii. Both aphid species reached their maximum peaks during the spring season. The rate of viruliferous aphids, estimated by real‐time RT‐PCR of single aphid, revealed that 35.4% of winged A. gossypii and 28.8% of winged A. spiraecola were viruliferous, confirming a high inoculum pressure in the area surrounding the experimental site. The aphid species Toxoptera citricida, which is able to transmit the aggressive isolates of CTV, was not found in the Loukkous region. The study of the spatial distribution of the CTV showed that in general, the disease was randomly distributed in the field. Overall, the results seem to indicate that A. spiraecola may be considered as the major aphid species contributing to CTV spread in our experimental conditions. The prevalence of mild strains in the region and the high level of aphid flight activity could explain the rapid evolution of CTV incidence in the experimental area.     

Transgenic expression in citrus of single-chain antibody fragments specific to <Emphasis Type="Italic">Citrus tristeza virus</Emphasis> confers virus resistance

Citrus tristeza virus (CTV) causes one of the most destructive viral diseases of citrus worldwide. Generation of resistant citrus genotypes through genetic engineering could be a good alternative to control CTV. To study whether production of single-chain variable fragment (scFv) antibodies in citrus could interfere and immunomodulate CTV infection, transgenic Mexican lime plants expressing two different scFv constructs, separately and simultaneously, were generated. These constructs derived from the well-referenced monoclonal antibodies 3DF1 and 3CA5, specific against CTV p25 major coat protein, whose mixture is able to detect all CTV isolates characterized so far. ScFv accumulation levels were low and could be readily detected just in four transgenic lines. Twelve homogeneous and vigorous lines were propagated and CTV-challenged by graft inoculation with an aggressive CTV strain. A clear protective effect was observed in most transgenic lines, which showed resistance in up to 40–60% of propagations. Besides, both a delay in symptom appearance and attenuation of symptom intensity were observed in infected transgenic plants compared with control plants. This effect was more evident in lines carrying the 3DF1scFv transgene, being probably related to the biological functions of the epitope recognized by this antibody. This is the first report describing successful protection against a pathogen in woody transgenic plants by ectopic expression of scFv recombinant antibodies.     

Citrus tristeza virus (CTV) resistance in transgenic citrus based on virus challenge of protoplasts

Summary A strategy for the sereening of candidate virus-derived sequences to provide RNA-mediated citrus tristeza virus (CTV) resistance and early selection of virus-resistant citrus is presented. The system is based on the polyethylene glycol-(PEG) mediated cotransformation of protoplasts using virus-derived sequences and green fluorescent protein as a single selectable marker, followed by an in vitro assay of virus inoculation into transgenic protoplasts to determine the level of citrus tristeza virus replication. A cotransformation rate higher than 20% allowed selection of several clones carrying the desired transgenes. Efficient in vitro inoculation of virus in transgenic protoplasts was performed. Tobacco mosaic virus virions were used as a control in order to check eitrus protoplast viability. Different CTV replication levels were detected in transgenic clones. Only one clone showed no replication of CTV. Considerations regarding selection of candidate virusderived sequences and virus challenge of transgenic cells are presented.     

Monoclonal antibody-based serological methods for detecting <Emphasis Type="Italic">Citrus tristeza virus</Emphasis> in citrus groves

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, 14H11, 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.

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Symptoms induced by transgenic expression of p23 from Citrus tristeza virus in phloem‐associated cells of Mexican lime mimic virus infection without the aberrations accompanying constitutive expression

Citrus tristeza virus (CTV) is phloem restricted in natural citrus hosts. The 23‐kDa protein (p23) encoded by the virus is an RNA silencing suppressor and a pathogenicity determinant. The expression of p23, or its N‐terminal 157‐amino‐acid fragment comprising the zinc finger and flanking basic motifs, driven by the constitutive 35S promoter of cauliflower mosaic virus, induces CTV‐like symptoms and other aberrations in transgenic citrus. To better define the role of p23 in CTV pathogenesis, we compared the phenotypes of Mexican lime transformed with p23‐derived transgenes from the severe T36 and mild T317 CTV isolates under the control of the phloem‐specific promoter from Commelina yellow mottle virus (CoYMV) or the 35S promoter. Expression of the constructs restricted to the phloem induced a phenotype resembling CTV‐specific symptoms (vein clearing and necrosis, and stem pitting), but not the non‐specific aberrations (such as mature leaf epinasty and yellow pinpoints, growth cessation and apical necrosis) observed when p23 was ectopically expressed. Furthermore, vein necrosis and stem pitting in Mexican lime appeared to be specifically associated with p23 from T36. Phloem‐specific accumulation of the p23Δ158–209(T36) fragment was sufficient to induce the same anomalies, indicating that the region comprising the N‐terminal 157 amino acids of p23 is responsible (at least in part) for the vein clearing, stem pitting and, possibly, vein corking in this host.     

Detection of Australian Field Isolates of Citrus Tristeza Virus by Double-Stranded RNA Analysis

Double-stranded RNA analysis indicated that widespread latent infection by citrus tristeza virus (CTV) occurs naturally in a citrus variety collection at Merbein, Australia. Detection was based on the presence of the putative replicative form (RF) of the virus (ca 19. 5 kilobase pairs) and the presence or absence of three previously known putative subgenomic dsRNAs with molecular sizes of ca 3. 1,1.7 and 0.8 kilobase pairs. With the exception of some citrus relatives normally used as rootstocks, the dsRNAs were readily detectable in all the trees tested. Variability was noticed among the dsRNAs profiles of individual seedling trees of Microcitrus australasica var. sanguinea. By contrast, dsRNA patterns of trees of open-pollinated Ellendale tangor were indistinguishable. A number of dsRNA bands found in West Indian lime infected with CTV were not detected when this isolate was graft-inoculated to healthy sweet orange seedlings. A simple method for enhancing the sensitivity of detection of the CTV dsRNA bands by silver staining is described which involves incubating the gels in RNase A prior to staining; this allowed detection of the putative CTV RF in only 40 mg of green hark material.     

Enhanced proliferation and efficient transmission of Candidatus Liberibacter asiaticus by adult Diaphorina citri after acquisition feeding in the nymphal stage

We carried out a quantitative detection of Candidatus Liberibacter asiaticus, the bacterium associated with the disease of huanglongbing, in the vector psyllid Diaphorina citri by using a TaqMan real‐time PCR assay. The concentration of the bacterium was monitored at 5‐day intervals for a period of 20 days after psyllids were exposed as fifth instar nymphs or adults to a Ca. L. asiaticus‐infected plant for an acquisition access period of 24 h. When adults fed on Ca. L. asiaticus‐infected plant, the concentration of the bacterium did not increase significantly and the pathogen was not transmitted to any citrus seedlings. In contrast, when psyllids fed on infected plant as nymphs, the concentration of the pathogen significantly increased by 25‐, 360‐ and 130‐fold from the initial acquisition day to 10, 15 and 20 days, respectively. Additionally, the pathogen was successfully transmitted to 67% of citrus seedlings by emerging adults. Our data suggested that multiplication of the bacterium into the psyllids is essential for an efficient transmission and show that it is difficult for adults to transmit the pathogen unless they acquire it as nymphs.     

A TaqMan PCR Method for Routine Diagnosis of the Quarantine Fungus Guignardia citricarpa on Citrus Fruit

With respect to disease risk for the quarantine fungus Guignardia citricarpa on citrus fruit an accurate diagnosis for routine analysis is required. Also, when inspections have to be performed on imported citrus fruits, a fast detection method is urgently needed. A fast automated DNA extraction method based on magnetic beads combined with a real‐time PCR assay was optimized to improve and advance the routine diagnosis of citrus black spot disease. Real‐time PCR was used for detection of the pathogen G. citricarpa in planta. A specific primer/TaqMan probe combination that discriminates between G. citricarpa and the harmless citrus endophyte Guignardia mangiferae, was designed based on the internal transcribed spacer region of the multi‐copy rDNA gene. Co‐amplification of target DNA along with an internal competitor DNA fragment made the diagnostic assay more reliable to check for false negatives. The real‐time PCR was specific, since no cross reaction was observed with a series of citrus pathogens and related species. The diagnostic assay was performed on lesions dissected from imported diseased oranges. Comparison between the conventional PCR and the real‐time PCR methods showed that the TaqMan method was more sensitive.     

Efficient search for new resistant genotypes to the citrus tristeza closterovirus in the orange subfamily Aurantioideae

 Virulent isolates of the citrus tristeza virus (CTV) are continuously arising and their spread threatens the world citrus industry. Methods for effective utilization of material conserved in germplasm banks are needed in plant improvement. Two objectives are pursued in the present paper: a search for new CTV-resistant genotypes and tests of two strategies for this search. One of these tests is based on a study of genetic relationships among genera and species of the orange subfamily and the other on scores of molecular markers known to be linked to the CTV-resistant locus. Sampled plants were graft-inoculated with a mild CTV isolate (T-346) and two virulent ones (T-388 and T-305). Susceptible plants were those where CTV multiplication was detected beyond 4 months after inoculation. All cultivars of Poncirus trifoliata tested, as well as Severinia buxifolia and Atalantia ceylanica, were resistant to the three CTV isolates; Fortunella crassifolia (Meiwa kumquat) resists two of them. The finding of CTV resistance in this species, closely related to cultivated Citrus species, opens a new arena for CTV-resistance improvement of oranges and mandarines by sexual hybridization. The searching strategy based on phylogenetic data has been successful, whereas the other one may be worthwhile only when the search is restricted to the species where linkage analysis is available. A good documentation system that allows quick sampling of accessions to build up core collections and where the location of new and useful genes could be easily worked out, is suggested to enhance germplasm utilization. Received: 27 April 1997 / Accepted: 5 June 1997