Detection of Citrus Psorosis Virus by ELISA, Molecular Hybridization, RT-PCR and Immunosorbent Electron Microscopy and its Association with Citrus Psorosis Disease

Psorosis is a citrus disease of undemonstrated etiology that can be diagnosed by biological indexing on sweet orange seedlings followed by a cross protection test. Its presumed causal agent is Citrus psorosis virus(CPsV), type species of the genus Ophiovirus. We compared detection of CPsV by ELISA, RT-PCR, molecular hybridization and immunosorbent electron microscopy, and examined its association with psorosis disease in 11 biologically characterized isolates and in 47 uncharacterized field sources by observation of field symptoms and by biological indexing including the cross protection test. Detection of CPsV by any of the four procedures always coincided with diagnosis of psorosis by cross protection, but it did not always correlate with observation of symptoms thought to be specific, in field trees or in graft-inoculated indicator plants. Trials to detect CPsV by ELISA, molecular hybridization and RT-PCR in citrus sources from different geographical origins, presumed to be psorosis-infected on the basis of field symptoms or reaction of indicator plants, were sometimes unsuccessful, indicating that psorosis symptoms may be induced by causes other than CPsV.     

Elimination of Citrus psorosis virus by somatic embryogenesis from stigma and style cultures

Somatic embryogenesis was used to eliminate Citrus psorosis virus (CPsV) from three citrus species (common mandarin, sweet orange and Dweet tangor), all of which regenerated somatic embryos with different embryogenic potential from stigma and style explants. CPsV was detected by double antibody sandwich‐indirect‐enzyme‐linked immunosorbent assay (DASI-ELISA) in explants and embryogenic callus, but was not detected in any of the plants obtained from somatic embryos, even 24 months after regeneration. Loss of juvenile characters (disappearance of thorns) was observed in the first year of growth and was retained in plants propagated by grafting from thornless stems. Somatic embryogenesis appears to be a very promising technique for the production of healthy citrus stocks.     

Effect of temperature on RNA silencing of a negative‐stranded RNA plant virus: Citrus psorosis virus

Citrus psorosis virus (CPsV), genus Ophiovirus, causes a bark scaling disease of citrus. CPsV virions are kinked filaments with three negative‐stranded RNA molecules (vRNA) and a 48 kDa coat protein. The effect of temperature on symptom expression, virus accumulation and RNA silencing was examined in sweet orange seedlings (Citrus sinensis) graft‐inoculated with three different CPsV isolates and grown in a glasshouse at 26/18°C or 32/26°C (day/night). Most plants kept in the cooler glasshouse showed a shock reaction in the first flush with shoot necrosis, and then moderate to intense chlorotic flecking and spotting in young leaves, whereas plants incubated at 32/26°C did not exhibit shoot necrosis, and young leaf symptoms were milder. Virus titre estimated by ELISA and by northern and dot blot hybridization paralleled symptom intensity, with significantly higher virus accumulation in plants incubated at 26/18°C. The amount of CPsV‐derived small RNAs (CPsV‐sRNAs) slightly increased at 32/26°C, with the ratio of CPsV‐sRNA/vRNA being higher at 32/26°C than at 26/18°C. These results suggest that (i) CPsV infection induces RNA silencing in citrus plants, (ii) symptom intensity is associated with virus accumulation, and (iii) temperature increase enhances the RNA silencing response of citrus plants and decreases virus accumulation.     

Detection of Citrus psorosis virus in field trees by direct tissue blot immunoassay in comparison with ELISA, symptomatology, biological indexing and cross-protection tests

Serological detection of Citrus psorosis virus (CPsV) by direct tissue blot immunoassay (DTBIA) and by double (DAS) and triple (TAS) antibody sandwich ELISA, was compared in samples from various citrus varieties growing in the glasshouse and in the field. In young shoots and leaves, CPsV was readily detected by the three procedures, whereas DTBIA detection in old leaves was less consistent. DTBIA detection and ELISA readings in nine different citrus varieties were similar, suggesting that CPsV accumulates to equivalent levels in all of them. In infected field trees from Spain or Italy, CPsV was consistently detected by TAS ELISA, even in samples of old leaves in winter, whereas DTBIA detection in the same trees was reliable only when using young shoots. Detection of CPsV by DTBIA and by DAS and TAS ELISA in previously untested field trees correlated perfectly with psorosis diagnostics based on biological indexing, specifically with the capacity of those sources to cross-protect against challenge inoculation with psorosis B. Some trees without bark scaling were shown to be psorosis-infected by biological indexing and to contain CPsV by serological tests; other trees showing psorosis-like bark or leaf symptoms in the field were shown to be psorosis-free by biological indexing and also CPsV-free by serology. This is the first time that the presence of CPsV has been correlated with psorosis infection as diagnosed by biological indexing.     

Association of citrus psorosis B symptoms with a sequence variant of the Citrus psorosis virus RNA 2

Citrus psorosis virus (CPsV), the type species of genus Ophiovirus, is the presumed causal agent of a bark scaling disease in citrus plants. CPsV virions are kinked filaments composed of three negative‐strand RNA molecules and a ～48‐kDa coat protein. The virus induces two different syndromes: psorosis A (PsA), characterized by limited bark scaling lesions in the trunk and main limbs, and a more aggressive form of the disease called psorosis B (PsB) with rampant bark lesions affecting even thin branches and chlorotic blotches in old leaves. In the greenhouse, the PsA and PsB syndromes can be induced by graft inoculating healthy citrus seedlings with non‐lesion or with lesion bark inoculum from PsA‐affected field trees. PsA‐ and PsB‐inducing CPsV sub‐isolates obtained by this procedure from the same tree showed identical single‐strand conformation polymorphism (SSCP) profiles in homologous segments of the RNAs 1 and 3, whereas segments of the RNA 2 enabled discrimination between PsA‐ and PsB‐associated sequence variants. SSCP analysis of the RNA 2 population present in different tissues of psorosis‐infected plants showed that: (i) PsA‐inducing isolates contain PsB‐associated sequence variants at low frequency, (ii) the PsB‐associated sequence variant is predominant in blistered twigs and gummy pustules affecting old leaves, characteristic of PsB isolates, and (iii) the PsB‐associated sequence variant accumulates preferentially in bark lesions of the trunk and limbs. SSCP analysis of the RNA 2 population also enabled monitoring of interference between PsA‐ and PsB‐associated variants in plants co‐inoculated with both psorosis types.     

Improved detection of citrus psorosis virus using polyclonal and monoclonal antibodies

Citrus psorosis is a serious and widespread disease associated with citrus psorosis virus (CPsV), a novel filamentous negative-stranded virus in the genus Ophiovirus . Laborious and costly indexing on test plants has been the only routine diagnostic method available, but recently an antiserum usable in double antibody sandwich (DAS) ELISA has been prepared. Here, major improvements to the DAS-ELISA protocol, a new purification method, and production of two monoclonal antibodies (mabs) to CPsV, an IgG and an IgM are reported. A highly sensitive triple antibody sandwich (TAS) ELISA making use of the mabs is described. In glasshouse citrus the homologous virus was still detectable at a tissue dilution of 1/6250 in DAS and at 1/31250 in TAS-ELISA. Both the DAS and IgG mab-TAS formats detected all CPsV isolates so far tested (from Argentina, Italy, Lebanon, Spain and the USA). A few isolates were not detected by the IgM mab.     

Occurrence of Citrus psorosis virus in Campania, southern Italy

Citrus psorosis virus (CPsV), genus Ophiovirus, is associated with a severe disease of citrus worldwide. Double antibody sandwich (DAS) ELISA using a polyclonal antiserum, and triple antibody sandwich (TAS) ELISAs, employing the IgG monoclonal antibody (mab) 13C5, and the IgM mab 2A3, were used to detect CPsV in orchards of different citrus varieties in Campania, southern Italy. TAS ELISA with 13C5 detected all the infections detected by DAS ELISA. Overall, 14% of trees younger than 15 years were positive, but only 1% of older trees, suggesting that infected propagating material has been increasingly used in recent years, in the absence of certification. Highest infection rates were in younger trees of sweet orange (22.8%) and clementine (18.6%). CPsV could easily be detected at all seasons of the year tested (June–January); these and earlier results indicate that TAS ELISA using 13C5 is a sensitive, broad-spectrum and reliable diagnostic method useful for routine tests and certification programmes. Of 44 field isolates responding strongly to DAS ELISA and 13C5-TAS ELISA, mab 2A3 gave similar results with 29 isolates, but gave low values with the others, thus providing a degree of differentiation among isolates. To confirm that the ELISA tests were indeed detecting CPsV, samples of 42 ELISA-positive plants were analysed by ISEM in a blind test, and in 38 of these, characteristic virus particles were clearly seen. Although CPsV was frequently and consistently detected in the area sampled, bark scaling symptoms were not seen: possible reasons for this are discussed.     

First report of <Emphasis Type="Italic">Citrus psorosis virus</Emphasis> in Japan

Citrus psorosis virus (CPsV) was detected from citrus trees for the first time in Japan. The diagnosis was confirmed by molecular, serological, and biological indexing. RT-PCR detected CPsV from two citrus trees among ca. 200 tested. Both trees were variety Shiranui of [Citrus unshiu Marc. × C. sinensis (L.) Osb.] × C. reticulata Blanco, and neither had the bark scaling symptom typical of CPsV. The CPsV isolate could be genetically related to those from Spain, Italy, Florida, and California.     

Reaction of transgenic Citrus sinensis plants to Citrus tristeza virus infection by Toxoptera citricida

Transgenic Citrus sinensis ‘Hamlin’ and ‘Valencia’ plants containing Citrus tristeza virus (CTV)-derived sequences were propagated and inoculated with CTV. For propagation, selected buds from transgenic and non-transgenic control plants were grafted onto C. aurantium and C. limonia rootstock plants. CTV inoculation was performed via viruliferous aphids (Toxoptera citricida), and viral detection post-inoculation was performed through DASI-ELISA or RT-qPCR. After four inoculations, none of the transgenic lines tested showed complete resistance. However, viral multiplication was undetectable in some of the propagated clones. These resistant clones mainly came from transgenic ‘Valencia’ sweet orange plants grafted onto C. limonia rootstock containing the pCTV-CS gene construct. Although the tested viral inoculation method represents natural field infection conditions, the results did not differ significantly from those previously reported when the same transgenic lines were bud-graft inoculated. This finding indicates that the difficulties in producing CTV-resistant transgenic citrus lines may be unrelated to the inoculation method. Transgene expression level was quantified by RT-qPCR analysis and it was not possible to relate transgene mRNA level with resistance to the pathogen.     

Nucleotide sequence of the coat protein gene of Mirafiori lettuce virus

 The coat protein (CP) gene of Mirafiori lettuce virus (MiLV), a tentative member of the genus Ophiovirus was isolated and sequenced. The established sequence consists of 1514 nucleotides including one open reading frame (ORF) with 1311 nucleotides that encodes 437 amino acids with a relative molecular mass 48 543. When the ORF was expressed in Escherichia coli, the obtained protein was confirmed as CP by Western blotting using an antiserum against MiLV. Database searches showed that the CP gene of MiLV has a sequence similar to that of Citrus psorosis virus (CPsV), the type species of the genus Ophiovirus. The comparison between MiLV and CPsV CP genes revealed that the identities of the nucleotide and amino acid sequences were 46.5% and 30.9%, respectively. Received: July 29, 2002 / Accepted: October 2, 2002     

A combined indexing and budwood propagation method for citrus

Several budwood-transmitted citrus diseases, including citrus tristeza virus, citrus psorosis, citrus impietratura and a range of citrus viroids, were tested both visually and biochemically on a combined indicator (CInd) plant consisting of an Alemow (Citrus macrophylla) rootstock grafted with Etrog citron (C.medica) and Sour orange (C.aurantium) or Sweet orange (C.sinensis) buds. Indexing on CInd plants is economical for limited testing space; an additional advantage is that, by collecting budwood directly from the CInd plants, the risk of diagnostic failure due to uneven pathogen distribution in the budwood source tree is considerably reduced.     

运用一步法RT-PCR检测柑橘鳞皮病毒及其在橘橙不同部位中的全年分布

2005年5月到2006年4月逐月采样,运用一步法RT-PCR检测Citrus psorosis virus(CPV)在Dweet橘橙苗木叶片和枝皮中的分布。保存在控温温室中的Dweet橘橙病株中老叶、老皮、嫩叶和嫩皮全年都可以检测出CPV;保存在网室中的Dweet橘橙病株中老叶、老皮全年均能检测到CPV,而夏梢的嫩叶、嫩皮不能稳定地检测出CPV,春、秋梢的嫩叶、嫩皮均可检测到CPV,表明一步法RT-PCR检测CPV最佳取样部位为老叶和老皮。     

Photosynthetic and antioxidant responses of Mexican lime (Citrus aurantifolia) plants to Citrus tristeza virus infection

The effect of Citrus tristeza virus (CTV) infection on photosynthetic activity and antioxidant metabolism was analysed in plants of the highly susceptible citrus genotype Mexican lime (Citrus aurantifolia). Two virus isolates differing in their virulence (the severe T318 and the mild T385) were used in the experiments. CTV infection caused a reduction in photosynthetic capacity in infected plants. This limitation was mainly due to a reduction in the carboxylative efficiency whereas the limitation of CO₂ diffusion through the stoma had lower impact. The virus did not damage the antennae and did not reduce the efficiency of light harvesting complexes. Oxidative damage occurred in infected plants, as evidenced by the increase in malondialdehyde levels. Indeed, CTV infection caused an increase in ascorbate peroxidase activity in new shoots developed in infected plants during the 2 years of the experiment. Data suggest that the H₂O₂ removal machinery was not damaged as a result of stress but the defence mechanism was overwhelmed with time due to the continuing pressure of biotic stress.     

Evidence for resistance to Citrus tristeza virus in pomelo (Citrus maxima Merr.) grown in Darjeeling and Sikkim hills of India

Darjeeling and Sikkim hills of India are well known for production of mandarin orange (Citrus reticulata). The recent spread of Citrus tristeza virus (CTV) has threatened the citrus cultivation in this region. During a survey in Darjeeling and Sikkim hills, pomelo trees were recorded as CTV free. Since pomelo trees did not show any disease appearance, a study was undertaken to ascertain whether they are resistant to CTV infection or resistant to aphid feeding or both. Toxoptera citricida, the most efficient aphid vector and which is abundantly present in this region, did not feed on pomelo when other Citrus species such as mandarin, kagzi lime and rough lemon were available. Additionally, CTV isolates of Darjeeling and Sikkim hills were not transmissible to pomelo either by T. citricida or grafting. We report for the first time that pomelo is resistant to isolates of CTV present in this region.     

Filamentous flexous particles and serologically related proteins of variable size associated with citrus psorosis and ringspot diseases

Filamentous flexous partic les of unusual morphology, previously associated with several ringspot isolates, were detected also in psorosis A and psorosis B isolates by serologically specific electron microscopy using an antiserum to citrus ringspot. Upon partial purification of six ringspot, six psorosis A, and three psorosis B isolates, a specific protein of 47 kDa was detected in most cases, but two isolates (one psorosis A and one ringspot) had a 46 and a 48 kDa-protein, respectively. These differences in molecular masses were observed when purification was done from different host species or from plants co-inoculated with two isolates differing by their protein size. The three types of protein were serologically related in Western blots. Our results indicate that a common virus with different strains may be involved in psorosis A, psorosis B, and ringspot diseases.     

Fully "Recombinant Enzyme-Linked Immunosorbent Assays" Using Genetically Engineered Single-Chain Antibody Fusion Proteins for Detection of Citrus tristeza virus

ABSTRACT Recombinant single-chain variable fragment antibodies (scFv) that bind specifically to Citrus tristeza virus (CTV), which cause the most detrimental viral disease in the citrus industry worldwide, were obtained from the hybridoma cell lines 3DF1 and 3CA5. These scFv were genetically fused with dimerization domains as well as with alkaline phosphatase, respectively, and diagnostic reagents were produced by expressing these fusion proteins in bacterial cultures. The engineered antibodies were successfully used for CTV diagnosis in plants by tissue print enzyme-linked immunosorbent assay (ELISA) and double antibody sandwich-ELISA. The fully recombinant ELISAs were as specific and sensitive as conventional ELISAs performed with the parental monoclonal antibodies, showing the usefulness of recombinant antibodies for routine detection of a virus in woody plants for the first time.