Citrus tristeza closterovirus

Abstract

EPPO BulletinVolume 34, Issue 2 p. 239-246 Diagnostic protocols for regulated pests†Free Access Citrus tristeza closterovirus First published: 10 September 2004 https://doi.org/10.1111/j.1365-2338.2004.00725.xCitations: 14 European and Mediterranean Plant Protection Organization PM 7/31(1) Organisation Européenne et Méditerranéenne pour la Protection des Plantes AboutSectionsPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinked InRedditWechat Specific scope This standard describes a diagnostic protocol for Citrus tristeza closterovirus. Specific approval and amendment This Standard was developed under the EU DIAGPRO Project (SMT 4-CT98-2252) by partnership of contractor laboratories and intercomparison laboratories in European countries. Approved as an EPPO Standard in 2003-09. Introduction The Closterovirus Citrus tristeza virus (CTV) causes one of the most damaging diseases of citrus (Bar-Joseph & Lee, 1989) and is the most economically important pathogen of this crop (Lee & Bar-Joseph, 2000). CTV probably originated in Asia and has been disseminated to almost all citrus-growing countries by movement of infected plant material. Subsequent spread by aphid vectors has created major epidemics. Epidemics of tree losses on sour orange rootstock were first reported from South Africa in the early part of the 20th century, and in Argentina and Brazil in the 1930s following the importation of CTV-infected plants and the efficient aphid vector Toxoptera citricida. More than 80 million trees grafted on sour orange (Citrus aurantium) rootstock have been killed or rendered unproductive by CTV-induced decline. The losses caused in Argentina (more than 10 million trees), Brazil (more than 6 million trees) and USA (more than 3 million trees) have been reported by Bar-Joseph et al. (1989). In Spain alone, more than 40 million trees, mainly sweet orange (Citrus sinensis) and mandarin (Citrus reticulata) grafted on sour orange, have declined progressively (Cambra et al., 2000a). In addition, CTV may cause stem pitting in some citrus cultivars regardless of the rootstock used, leading to significant losses in fruit quality and yield. As a member of the genus Closterovirus (Karasev et al., 1995), CTV has virions which are flexuous (2000 × 11 nm in size) and contain a non-segmented, positive-sense, single-stranded RNA genome. The sequence of the CTV genome contains 12 open reading frames (ORFs), potentially encoding at least 17 proteins. ORFs 7 and 8 encode proteins with estimated molecular weights of 27.4 (P27) and 24.9 kDa that have been identified as the capsid proteins. The complete sequence of several CTV isolates has been reported (Pappu et al., 1994; Karasev et al., 1995; Mawassi et al., 1996; Vives et al., 1999; Yang et al., 1999; Albiach-Marti et al., 2000; Suastika et al., 2001), including the sequence of a typical mild Spanish CTV isolate (Vives et al., 1999). Transmission CTV is readily transmitted by graft and, in a semipersistent manner, by the main aphid species visiting citrus: T. citricida, Aphis gossypii, Aphis spiraecola and Toxoptera aurantii. T. citricida (not yet present in continental Europe or in the Mediterranean Basin) is a much more efficient vector than A. gossypii, but epidemic spread has occurred in Spain when A. gossypii was the predominant aphid species (Cambra et al., 2000a). A. spiraecola is not an efficient vector but, since its populations can become so high, it may be a significant factor in CTV spread in some areas. T. aurantii apparently transmits only certain CTV isolates (Lee & Bar-Joseph, 2000). Eight aphid species (T. citricida not included) have been assayed as vectors of different Mediterranean CTV isolates (Hermoso de Mendoza et al., 1984, 1988). A. gossypii was always the most efficient vector and transmission efficiencies were up to 78%, whereas A. spiraecola and T. aurantii had very low efficiencies (0–6%). The spatial and temporal spread of tristeza disease has been studied in European citrus orchards (Cambra et al., 1988, 1990a; Gottwald et al., 1996, 1997; Cambra et al., 2000a). A long time may elapse between the introduction of a primary source of inoculum and the development of a disease epidemic (Garnsey & Lee, 1988). Isolate differentiation Field CTV isolates may vary in pathogenicity and may contain multiple genomic virus variants that can be separated by aphids or graft transmission to different citrus host species. The subisolates segregated in this way can be differentiated by pathogenicity tests in different hosts, by dsRNA patterns (Moreno et al., 1993) or serologically using specific monoclonal antibodies (Cambra et al., 1993). The monoclonal antibody MCA13 (Permar et al., 1990) was described in Florida (US) as specific for severe and CTV decline-inducing isolates. Reaction with MCA13 is not necessarily correlated with the decline of trees in the Mediterranean Basin, but constitutes a good indication of potential aggressiveness of an isolate. There are no molecular methods allowing reliable typing of CTV isolates according to their aggressiveness. It has been demonstrated that the haplotype distribution of two CTV genes can be altered after host change or aphid transmission (Ayllón et al., 1999). Molecular hybridization (Albiach et al., 1995) and single-strand conformation polymorphisms analysis of the coat protein gene (Rubio et al., 1996) have been used to differentiate Mediterranean CTV isolates. Principal host plants Most species of Citrus and some species in other genera of the family Rutaceae (Aegle marmelos, Aeglopsis chevalieri, Afraegle paniculata, Citropsis gilletiana, Microcitrus australis and Pamburus missionis) have been reported as hosts for CTV. Most trifoliate orange clones and many of their hybrids are resistant to infection. Protoplasts of Nicotiana benthamiana have been experimentally infected by CTV. Identity Name: Citrus tristeza virus Acronym: CTV Taxonomic position: Viruses, Closteroviridae, Closterovirus Bayer computer code: CTV000 Phytosanitary categorization: EPPO A2 list n°93, EU Annex II/AII for European isolates EU Annex II/AI for non European isolates Detection Symptoms Symptom expression in citrus hosts is highly variable and affected by environmental conditions, host species and the aggressiveness of the CTV isolate. Some CTV isolates are mild and produce no noticeable effect on most commercial citrus species. In general, mandarins are especially tolerant to CTV infection. Sweet orange, sour orange, rough lemon (C. jambhiri) and Rangpur lime (C. limonia) are usually symptomless but may react to some aggressive isolates. Reactive hosts include lime, grapefruit (C. paradisi), some cultivars of pummelo (C. grandis), alemow (C. macrophylla), some cultivars of sweet orange, some citrus hybrids and some citrus relatives above mentioned. Stunting, leaf cupping, vein clearing and chlorosis, stem pitting, and reduced fruit size are common symptoms of susceptible hosts. One of the most economically significant symptoms of tristeza disease is the decline of trees grafted on sour orange. Sweet orange, mandarin and grapefruit on sour orange rootstock become stunted, chlorotic and often die after a period of several months or years (slow decline), or some days after the first symptoms (quick decline). The decline results from the effects of the virus on the phloem of the sour orange rootstock just below the bud union. Trees that decline slowly generally have a bulge above the bud union, and inverse pinhole pitting (honey combing) on the inner face of the sour orange bark. Some isolates of the virus do not induce decline symptoms, even in trees on sour orange, for many years. Aggressive CTV isolates can severely affect trees, inducing stem pitting on the trunk and branches of lime, grapefruit and sweet orange. Stem pitting may sometimes cause a bumpy or ropy appearance of the trucks and limbs of adult trees. Deep pits in the wood are present under depressed areas of the bark. Fruit quality and yield are greatly reduced in trees with severe stem pitting. Nevertheless, most CTV isolates are able to cause stem pitting in C. macrophylla rootstocks and reduce tree vigour. Symptoms caused by CTV are presented in Web Fig. 1. Identification The classic identification procedure for CTV is to graft-inoculate indicator seedlings of Mexican lime (Wallace & Drake, 1951) and observe them for vein clearing, leaf cupping, and stem pitting. Electron and light microscopy can be used to identify CTV particles and inclusions, but DAS-ELISA (Bar-Joseph et al., 1979; Cambra et al., 1979) has revolutionized diagnosis, making it feasible to test many samples during surveys of large citrus areas, for CTV control in nurseries and for epidemiological studies. Polyclonal antibodies from antisera were used from 1978 to 1983 for routine ELISA tests. The production of monoclonal antibodies specific to CTV (Vela et al., 1986; Permar et al., 1990) and others reported by Nikolaeva et al. (1996) solved the problems of specificity and increased sensitivity of ELISA tests. A mixture of two monoclonal antibodies (3DF1 and 3CA5) or their recombinant versions (Terrada et al., 2000) recognizes all CTV isolates tested from different international collections (Cambra et al., 1990b). A detailed description and characterization of these monoclonal antibodies has been summarized (Cambra et al., 2000a). The development of Tissue print-ELISA (Garnsey et al., 1993; Cambra et al., 2000b) for CTV detection in imprinted sections of plant material on nitrocellulose membranes, allowed the sensitive testing of thousands of samples simply and without the need to prepare extracts. PCR-based assays have been developed based on immunocapture (Nolasco et al., 1993) or print or squash capture (Olmos et al., 1996; Cambra et al., 2000c). A simple procedure has been described to perform nested PCR in a single closed tube (Olmos et al., 1999) which allowed CTV detection in single aphids and in plant tissues. A co-operational PCR system (Co-PCR) using a universal probe for hybridization with PCR products (Olmos et al., 2002) has been described, giving sensitivity similar to that of nested PCR. Sampling Appropriate sampling is critical for serological or molecular detection of CTV. The standard sample for adult trees involves 5 young shoots (from the last flush) or fruit peduncles, or 10 fully expanded leaves, or 5 flowers or fruits, collected around the canopy of each individual tree from each scaffold branch. Samples (shoots or fully expanded leaves and peduncles) can be taken at any time of year from sweet orange, mandarin, lemon and grapefruit in the Mediterranean area, but springtime give the highest CTV titres. A reduced CTV titre is observed in Satsuma mandarin during summer. Consequently, the recommended period for sampling includes all vegetative seasons except summer (July–August in the Mediterranean Basin). Flowers or fruits (when available) are also suitable materials for testing (Cambra et al., 2002). Standard sampling for nursery plants involves 2 young shoots or 4 leaves. Samples (shoots, leaf petioles, fruit peduncles and flowers) can be stored at 4 °C for not more than 7 days before processing. Fruits can be stored for 1 month at 4 °C. Sample preparation Preparation of tissue prints for testing Tender shoots, leaf petioles, fruit peduncles or flowers are cleanly cut. The fresh cut sections are carefully pressed against a nitrocellulose membrane (0.45 mm), and the trace or print is allowed to dry for a few minutes. For routine testing, at least two prints should be made per selected shoot or peduncle and one per leaf petiole or flower (see sampling). Printed membranes can be kept for several years in a dry place. Preparation of plant extracts for testing About 1 g of plant material is weighed, cut in small pieces and placed in a suitable tube or plastic bag for processing. About 20 volumes of extraction buffer are added and the sample is homogenized in tubes using a Polytron (Kinematica) or similar blender. Alternatively, the sample may be homogenized in the plastic bag, using Homex 6 machine (Bioreba) or any manual roller, hammer, or similar tool. The extraction buffer is phosphate-buffered saline (PBS) pH 7.2–7.4 (Appendix 1) supplemented with 0.2% sodium diethyl dithiocarbamate (DIECA) or 0.2% mercapto-ethanol. Samples for serological testing can be prepared in tubes or in plastic bags. Samples for molecular testing should only be prepared in appropriate individual plastic bags. Screening tests Biological testing The object of testing is to detect the presence of CTV in plant accessions or selections, or in samples whose sanitary status is being assessed, and to estimate the aggressiveness of the isolate on Citrus aurantifolia (Mexican lime). The indicator is graft-inoculated according to conventional methods and held under standard conditions (Roistacher, 1991), with 4–6 replicates. Symptom onset is compared with that of positive and negative control plants. Serological tests Tissue print or immunoprinting ELISA, or direct tissue blot immunoassay (DTBIA), is performed according to Garnsey et al. (1993) and Cambra et al. (2000b) using the detailed protocol described in Appendix 2 and materials described in Appendix 1. Double Antibody Sandwich ELISA (DAS-ELISA), in the conventional or biotin/streptavidin system, is performed according to Garnsey & Cambra (1991), using the detailed protocol described in Appendix 2 and materials described in Appendix 1. Molecular tests Immunocapture RT-PCR (IC-RT-PCR) is performed according to Wetzel et al. (1992), Nolasco et al. (1993) and Rosner et al. (1998) using the detailed protocol described in Appendix 2 and materials described in Appendix 1. Immunocapture nested RT-PCR in a single closed tube is performed according to Olmos et al. (1999) using the detailed protocol described in Appendix 2 and materials described in Appendix 1. Reference material Standard CTV-infected and healthy citrus controls, CTV-specific monoclonal antibodies (in addition to the commercially available ones of Appendix 1) and CTV-specific oligonucleotide primer sequences are available for non-profit institutions from Instituto Valenciano de Investigaciones Agrarias (IVIA), Department Protección Vegetal y Biotecnología, Carretera de Moncada-Náquera km 5, 46113 Moncada (Valencia). Spain. Possible confusion with similar species None. Requirements for a positive diagnosis The procedures for detection and identification described in this protocol and in the decision scheme (Fig. 2) should have been followed and appropriate controls should have been included. When CTV is diagnosed for the first time, or in critical cases (import/export), a combination of two different screening methods should be used, based on biological testing (inoculation of Mexican lime) and on serological or molecular detection (with the validated protocols and reagents). Figure 2Open in figure viewerPowerPoint Detection scheme for the detection and identification of Citrus tristeza closterovirus. Report on the diagnosis The report on the execution of the protocol should include: • results obtained by the recommended procedures • information and documentation on the origin of the infected material • a description of the disease symptoms (with photographs if possible) • an indication of the magnitude of the infection • comments as appropriate on the certainty or uncertainty of the identification. The original sample (with labels, if applicable) should be kept under proper conditions as long as possible. Sample extract and PCR amplification product should be kept at −80 °C for 3 months (or longer for legal purposes). Printed tissue sections on nitrocellulose (see sample preparation) and the developed membrane after reading should be kept at room temperature for 6 months. Further information Further information on this organism can be obtained from: Instituto Valenciano de Investigaciones Agrarias (IVIA), Department Protección Vegetal y Biotecnología, Carretera de Moncada-Náquera km 5, 46113 Moncada (Valencia). Spain. E-mail: mcambra@ivia.es. Footnotes 1 The Figures in this Standard marked ‘Web Fig.’ are published on the EPPO website http://www.eppo.org. 2 C. Varveri (Benaki Phytopathological Institute, GR); D. Boscia & O. Potere (Istituto di Virologia Vegetale del CNR, Bari, IT); K. Djelouah & A. M. D’Onghia (Istituto Agronomico Mediterraneo, Valenzano, IT); R. Flores & C. Muñoz Noguera (Laboratorio de Sanidad Vegetal de Sevilla, ES); M. A. Cambra & M. L. Palazón (Centro de Protección Vegetal, Zaragoza, ES); J. Serra-Aracil (Servicio de Sanidad y Certificación, Valencia, ES); P. Moreno, R. Albiach-Martí & M. E. Martínez (Instituto Valenciano de Investigaciones Agrarias, Laboratorio de Virologia, ES); E. Bertolini & M. C. Martínez (Instituto Valenciano de Investigaciones Agrarias Laboratorio de Serología, ES); M. T. Gorris & A. Olmos (Instituto Valenciano de Investigaciones Agrarias Laboratorio de Virología e Immunología, ES). Acknowledgements This protocol was originally drafted by: M. Cambra, A. Olmos and M. T. Gorris, Instituto Valenciano de Investigaciones Agrarias (IVIA), Department Protección Vegetal y Biotecnología, Carretera de Moncada-Náquera km 5, 46113 Moncada (Valencia). Spain. Tel. #34 961391000 Fax #34 961390240 This protocol was ring-tested in different European laboratories22 C. Varveri (Benaki Phytopathological Institute, GR); D. Boscia & O. Potere (Istituto di Virologia Vegetale del CNR, Bari, IT); K. Djelouah & A. M. D’Onghia (Istituto Agronomico Mediterraneo, Valenzano, IT); R. Flores & C. Muñoz Noguera (Laboratorio de Sanidad Vegetal de Sevilla, ES); M. A. Cambra & M. L. Palazón (Centro de Protección Vegetal, Zaragoza, ES); J. Serra-Aracil (Servicio de Sanidad y Certificación, Valencia, ES); P. Moreno, R. Albiach-Martí & M. E. Martínez (Instituto Valenciano de Investigaciones Agrarias, Laboratorio de Virologia, ES); E. Bertolini & M. C. Martínez (Instituto Valenciano de Investigaciones Agrarias Laboratorio de Serología, ES); M. T. Gorris & A. Olmos (Instituto Valenciano de Investigaciones Agrarias Laboratorio de Virología e Immunología, ES). . Appendices Appendix 1. Materials Standard CTV-infected and healthy citrus controls and CTV specific monoclonal antibodies (in addition to the commercially available ones indicated below) are available for non-profit purposes at Instituto Valenciano de Investigaciones Agrarias (IVIA). Carretera de Moncada-Náquera km 5. 46113 Moncada (Valencia). Spain. Materials for serological testing Tissue print-ELISA A complete kit (validated in ring tests) based on 3DF1 + 3CA5 CTV-specific monoclonal antibodies, including preprinted membranes with positive and negative controls and all reagents, buffers and substrate, is available from PLANT PRINT Diagnòstics, S.L., De la March 36, Bajo, 46512 Faura, Valencia. Spain. E-mail: plantprint@wanadoo.es DAS-ELISA kits Complete kits based on 3DF1 + 3CA5 specific monoclonal antibodies to CTV are commercially available, for the DAS-ELISA biotin/streptavidin system, from: 1) INGENASA (validated in ring tests), Hermanos García Noblejas 41, 2a planta, 28037 Madrid (ES), http://www.ingenasa.es; (2) REAL, CE Durviz S.L., Parque Tecnológico de Valencia, Leonardo Da Vinci, 10, 46980 Paterna (Valencia) (ES), http://www.durviz.com. For conventional DAS-ELISA, they are available from: Agdia Incorporated, 30380 County Road 6, 46514 Elkart (US). http://www.agdia.com Complete kits based on polyclonal antibodies to CTV are commercially available from: (1) Adgen Limited, Nellies Gate, Auchincruive, Ayr KA6 5HW (GB), http://www.adgen.co.uk; (2) BIORAD Laboratories-SANOFI, Rue Raimond Poincaré 3-BD, 92430 Marnes La Coquette (FR), http://www.bio-rad.com; (3) Bioreba, Chr. Merian-Ring 7, CH 4153 Reinach BL1 (CH), http://www.bioreba.ch. Streptavidin alkaline phosphatase-linked ELISA (Cat no. 1089 161) is available from Roche Diagnostics GmbH, Mannheim (DE). Buffers PBS, pH 7.2–7.4: NaCl 8 g; KCl 0.2 g; Na2HPO4·12H2O 2.9 g; KH2PO4 0.2 g; distilled water 1 L. Carbonate buffer pH 9.6: Na2CO3 1.59 g; NaHCO3 2.93 g; distilled water 1 L. Washing buffer (PBS, pH 7.2–7.4, with 0.05% Tween 20): NaCl 8 g; KCl 0.2 g; Na2HPO4·12H2O 2.9 g; KH2PO4 0.2 g; Tween 20 500 µL; distilled water 1 L. Colorimetric substrate buffer for alkaline phosphatase: diethanolamine 97 mL; dilute in 800 mL of distilled water. Adjust to pH 9.8 with concentrated HCl and make up to 1000 mL with distilled water. Precipitating substrate buffer for alkaline phosphatase: Sigma Fast 5-bromo-4-chloro-3-indolyl phosphate/nitro blue tetrazolium tablets (BCIP-NBT) – Cat No. –B-5655 – Sigma Aldrich GmbH, Stenheim (DE). Materials for molecular tests Oligonucleotide primer sequences (validated in ring-test): PEX1: 5′–3′ TAA ACA ACA CAC ACT CTA AGG PEX2: 5′–3′ CAT CTG ATT GAA GTG GAC PIN1: 5′–3′ GGT TCA CGC ATA CGT TAA GCC TCA CTT PIN2: 5′–3′ TAT CAC TAG ACA ATA ACC GGA TGG GTA Buffers Carbonate buffer pH 9.6: Na2CO3 1.59 g; NaHCO3 2.93 g; distilled water 1 L. Washing buffer (PBS, pH 7.2–7.4, with 0.05% Tween 20): NaCl 8 g; KCl 0.2 g; Na2HPO4·12H2O 2.9 g; KH2PO4 0.2 g; Tween 20 500 µL; distilled water 1 L. 50X TAE buffer: Tris 242 g; 0.5 m Na2EDTA pH 8.0 100 mL; glacial acetic acid 57.1 mL; distilled water to 1 L. Loading buffer: 0.25% bromophenol blue; 30% glycerol in H2O. Appendix 2. Detailed protocols Tissue print ELISA The method follows Garnsey et al. (1993) and Cambra et al. (2000b). Prepare 1% solution of bovine serum albumin (BSA) in distilled water. Place the membranes (recommended size about 7 × 13 cm) in an appropriate container (tray, hermetic container, plastic bag … ). Cover with albumin solution and incubate for 1 h at room temperature, or overnight at 4 °C. Slight agitation is recommended during this step. Discard the albumin solution and keep the membranes in the same container. Prepare a solution of CTV-specific 3DF1 + 3CA5 monoclonal antibodies linked to alkaline phosphatase (about 0.1 µg mL−l of each monoclonal antibody in PBS) (Appendix 1) or of 3DF1 scFv-AP/S + 3CA5 scFv-AP/S fusion proteins expressed in E. coli (appropriate dilution in PBS). Pour onto the membranes, covering them and incubate for 2–3 h at room temperature, then discard the conjugate solution. Rinse the membranes and the container with washing buffer (Appendix 1). Wash by shaking (manually or mechanically) for 5 min. Discard the washing buffer and repeat the process twice. Pour the alkaline phosphatase substrate buffer over the membranes (Appendix 1) and incubate until a purple-violet colour appears in positive controls (about 10–15 min). Stop the reaction by washing the membranes with tap water. Spread the membranes on absorbent paper and let them dry. Observe the prints using a low-power magnification (X10–X20). Presence of purple-violet precipitates in the vascular region of plant material reveals the presence of Citrus tristeza virus. DAS-ELISA The method follows Garnsey & Cambra (1991), by the conventional or biotin/streptavidin systems. Prepare an appropriate dilution of polyclonal antibodies or monoclonal antibodies 3DF1 + 3CA5 (Appendix 1) (usually 1–2 µg mL−1) in carbonate buffer pH 9.6 (Appendix 1). Add 200 µL to each well. Incubate at 37 °C for 4 h or at 4 °C for 16 h. Wash the wells three times with PBS-Tween (washing buffer) (Appendix 1). Add 200 µL per well of the plant extract (see sample preparation). Use two wells of the plate for each sample or positive controls and at least two wells for negative controls. Incubate at 4 °C for 16 h. Wash as before. Prepare specific polyclonal or monoclonal antibodies (3DF1 + 3CA5) linked with alkaline phosphatase or biotin (Appendix 1) at appropriate dilution (about 0.1 µg mL−1 in PBS with 0.5% bovine serum albumin-BSA added). Add 200 µL to each well. Incubate at 37 °C for 3 h. Wash as before. If antibodies are linked with biotin, use an appropriate dilution of streptavidin-alkaline phosphatase conjugated (Appendix 1). Add 200 µL to each well. Incubate at 37 °C for 30 min and wash as before. For both methods (conventional or biotin/streptavidin), prepare 1 mg mL−1 alkaline phosphatase solution (p-nitrophenyl phosphate) in substrate buffer. Add 200 µL to each well. Incubate at room temperature and read at 405 nm after 30, 60 and 90 min. The ELISA test is negative if the absorbance of the sample is less than twice the absorbance of the healthy control, and positive if the absorbance of the sample is equal to or greater than twice that value. IC-RT-PCR Immunocapture phase (IC) Immunocapture follows Wetzel et al. (1992), Nolasco et al. (1993) or Rosner et al. (1998). Prepare a dilution (1 µg mL−l) of CTV-specific polyclonal antibodies or a dilution (0.5 µg mL−1 + 0.5 µg mL−1) of monoclonal antibodies (3DF1 + 3CA5) in carbonate buffer pH 9.6 (Appendix 2). Dispense 100 µL of the diluted antibodies into the Eppendorf tubes. Incubate at 37 °C or on ice for 3 h. Wash twice with 150 µL of sterile washing buffer (Appendix 1). Clarify 100 µL plant extract previously obtained (see extract preparation) by centrifugation (5 min at 13 000 rev min−1), and submit sample to an Immunocapture phase for 2 h on ice (Rosner et al., 1998) or alternatively at 37 °C (Wetzel et al., 1992), in coated Eppendorf tubes. After the immunocapture phase, wash Eppendorf tubes three times with 150 µL of sterile washing buffer. Amplification by RT-PCR CTV detection PIN1-PIN2 primers Olmos et al., 1999) (Appendix 2): PIN1: 5′–3′ GGT TCA CGC ATA CGT TAA GCC TCA CTT PIN2: 5′–3′ TAT CAC TAG ACA ATA ACC GGA TGG GTA Cocktail reaction: H2O 14.30 µL; 10X-Taq Polymerase Buffer 2.5 µL; 25 mm MgCl2 1.5 µL (1.5 mm); 5 mm dNTPs 1.25 µL (250 µm); 4% Triton X-100 2 µL (0.3%); 25 µm primer PIN1: 1 µL (1 µm); 25 µm primer PIN2 1 µL (1 µm); DMSO 1.25 µL (5%); 10 U µL−1 AMV 0.1 µL; 5 U µL−1 Taq Polymerase 0.1 µL. Add the 25 µL of cocktail reaction mixture directly to the washed tubes. Conditions for RT-PCR: 42 °C for 45 min; 92 °C for 2 min; 40 cycles of 92 °C for 30 s, 60 °C for 30 s, 72 °C for 1 min; finally 72 °C for 10 min. Hold at 4 °C. IC nested RT-PCR in a single closed tube The method follows Olmos et al. (1999). Immunocapture phase (IC) as above for IC-RT-PCR. Amplification by nested RT-PCR CTV detection PEX1, PEX2, PIN1, PIN2 primers (Olmos et al., 1999) (Appendix 1): PEX1: 5′–3′ TAA ACA ACA CAC ACT CTA AGG PEX2: 5′–3′ CAT CTG ATT GAA GTG GAC PIN1: 5′–3′ GGT TCA CGC ATA CGT TAA GCC TCA CTT PIN2: 5′–3′ TAT CAC TAG ACA ATA ACC GGA TGG GTA The device for compartmentalization of a 0.5-mL Eppendorf tube for nested RT-PCR in a single closed tube is according Olmos et al. (1999) (Web Fig. 3). Cocktail A (dropped in the bottom of the Eppendorf tube): H2O 15.8 µL; 10X-Taq Polymerase Buffer 3 µL; 25 mm MgCl2 3.6 µL (3 mm); 5 mm dNTPs 2 µL (300 µm); 4% Triton X-100 2.2 µL (0.3%); 25 µm primer PEX1 0.6 µL (0.5 µm); 25 µm primer PEX2 0.6 µL (0.5 µm); DMSO 1.5 µL (5%); 10 U µL−1 AMV 0.2 µL; 5 U µL−l Taq Polymerase 0.5 µL. Cocktail B (placed in the cone): H2O 2.6 µL; 10X-Taq Polymerase Buffer 1 µL; 25 µm primer PIN1 3.2 µL (8 µm); 25 µm primer PIN2 3.2 (8 µm). Conditions for RT-PCR: 42 °C for 45 min; 92 °C for 2 min; 25 cycles of 92 °C for 30 s, 45 °C for 30 s, 72 °C for 1 min. After this first step, vortex the tube and centrifuge (6000 g × 5 s) to mix cocktail B with products of first amplification. Place the tubes on the thermal cycler and proceed as follows: 40 cycles of 92 °C for 30 s, 60 °C for 30 s, 72 °C for 1 min; finally 72 °C for 10 min. Electrophoresis of PCR products Prepare 2% agarose gel in TAE buffer 0.5 × (Appendix 2). Place droplets of about 3 µL of loading buffer (Appendix 2) on parafilm, mix 20 µL of PCR product by gentle aspiration with the pipette before loading. Load wells of gel and include positive and negative controls. Include DNA marker 100 bp in the first well of the gel. Run the gel for 20 min at 120 V (medium gel tray: 15 × 10 cm) or 40 min at 160 V (big gel tray or electrophoresis tank: 15 × 25 cm). Soak the gel in ethidium bromide solution for 20 min. Visualize the amplified DNA fragments by UV trans-illumination. Observe specific amplicons of 131 bp. References Albiach MR, Rubio L, Guerri J, Moreno P, Laigret F & Bové JM (1995) [Differentiation of races of Citrus tristeza virus by molecular hybridization.]Investigacion Agraria. Producción Y Protección Vegetales 10, 263– 274 (in Spanish). Google Scholar Albiach-Marti MR, Mawassi M, Gowda S, Satyanarayana T, Hilf ME, Shanker S, Almira EC, Vives MC, Lopez C, Guerri J, Flores R, Moreno P, Garnsey SM & Dawson WO (2000) Sequences of Citrus tristeza virus separated in time and space are essentially identical. Journal of Virology 74, 6856– 6865. CrossrefCASPubMedWeb of Science®Google Scholar Ayllón MA, Rubio L, Moya A, Guerri J & Moreno P (1999) The haplotype distribution of two genes of citrus tristeza virus is altered after host change or aphid transmission. Virology 255, 32– 39. CrossrefCASPubMedWeb of Science®Google Scholar Bar-Joseph Garnsey SM, Gonsalves D, Moscovitz M, Purcifull DE, Clark MF & Loebenstein G (1979) The use of enzyme-linked immunosorbent assay for detection of citrus tristeza virus.