PM 7/31 (2) Citrus tristeza virus

Abstract

EPPO BulletinEarly View EPPO STANADARD ON DIAGNOSTICSFree Access PM 7/031 (2) Citrus tristeza virus First published: 05 February 2023 https://doi.org/10.1111/epp.12908AboutSectionsPDF 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 onFacebookTwitterLinkedInRedditWechat Specific scope: This Standard describes a diagnostic protocol for citrus tristeza virus.1 This Standard should be used in conjunction with PM 7/76 Use of EPPO diagnostic protocols. Specific approval and amendment: First approved in 2003–09. First revision approved in 2022–11. This revision was initially prepared to align the EPPO Diagnostic Protocol to the IPPC Diagnostic Protocol adopted in 2016 (Annex 15 of ISPM 27. Citrus tristeza virus (FAO, 2016)). However, it also includes other tests evaluated in the framework of the EU funded project VALITEST. 1 INTRODUCTION Citrus tristeza virus (CTV) causes one of the most damaging diseases of citrus, devastating epidemics οf which have changed the course of the citrus industry (Moreno et al., 2008). The term ‘tristeza’, refers to the decline seen in many citrus species when grafted on Citrus aurantium (sour orange) or Citrus limon (lemon) rootstocks. Although tristeza is predominantly a bud union disease (Román et al., 2004), some CTV isolates induce other syndromes, including stem pitting, stunting, reduced productivity and impaired fruit quality of many commercial cultivars, even when they are grafted on CTV tolerant rootstocks. CTV probably originated from South-East Asia, the putative area of origin of citrus, and it has been disseminated to almost all citrus-growing countries through the movement of infected plant material. Subsequent local spread by aphid vector species has created major epidemics. Tree losses on sour orange rootstock were first reported in South Africa in the early twentieth century, and in Argentina and Brazil in the 1930s. CTV-induced tree decline has killed or rendered unproductive trees grafted on sour orange rootstock (Bar-Joseph et al., 1989; Cambra et al., 2000). CTV outbreaks have been observed in the United States, some Caribbean countries and some Mediterranean countries (especially Italy and Morocco). Detailed information on the distribution of CTV can be found in EPPO Global Database (EPPO, 2021a). Like all viruses, CTV is a quasi-species, which implies that infected plants contain a population of different genotypes. In the case of CTV, these genotypes can even belong to different phylogenetic groups, which hampers the establishment of an unambiguous relationship between genotype and pathogenic characteristics (Harper, 2013). Moreover, establishing such relation can be further complicated by recombination. It should also be noted that the term ‘strain’ has been used in literature both as a synonym for ‘isolate’ and to group isolates on their molecular and/or biological properties (EFSA, 2017). Therefore, in this protocol the Panel on Diagnostics in Virology and Phytoplasmology decided to use the concept of phylogenetic group in relation to genetic characteristics and strain in relation to pathogenic characteristics. To date, six major CTV phylogenetic groups have been described: T36 (Karasev et al., 1995), T3 (Hilf et al., unpublished), VT (Mawassi et al., 1996), T30 (Albiach-Marti et al., 2000), RB (Harper et al., 2010) and T68 (Harper, 2013) based on their genomic features. In the EPPO region, three of the six major phylogenetic groups are either absent (T68) and/or have a limited distribution (RB & T36) (Cevik et al., 2013; Ghosh et al., 2022). This implies that CTV isolates present in the EPPO region represent only a fraction of the biological and genetic diversity present in CTV isolates throughout the world. Consequently, introduction and further spread of genotypes belonging to these three ‘foreign’ phylogenetic groups will increase the genetic diversity and may affect the impact of the virus. Therefore, it is important to be able to identify these isolates at phylogenetic-group level in order to prevent their introduction and/or further spread within the region. Although sequence variants genetically similar to those of the stem pitting-inducing non-European CTV isolates have been detected in Europe (i.e. VT, T3) and have even been involved in outbreaks with severe tristeza decline symptoms, stem pitting symptoms in sweet orange have not been observed in surveys. Biological indexing of these isolates resulted in rare occurrence of inconspicuous symptoms on indicator plants. Outside of Europe, in the main citrus producing countries of the world, CTV isolates causing stem pitting appear to be present (EFSA, 2017). CTV is naturally transmitted by some aphid species in a semi-persistent manner. Worldwide, the most efficient vector of CTV is Toxoptera citricida (Kirkaldy). Aphis gossypii Glover is the main vector in Spain, Israel, some citrus growing areas in California (United States) and areas where T. citricida is absent (Cambra et al., 2000; Marroquín et al., 2004; Yokomi et al., 1989). Other aphid species have also been described as CTV vectors (Moreno et al., 2008), including Aphis spiraecola Patch, Aphis aurantii (Boyer de Fonsicolombe), Myzus persicae (Sulzer), Aphis craccivora Koch and Uroleucon jaceae (Linnaeus). Information on CTV vectors is available in the EPPO datasheet (EPPO, 2022). CTV is also graft-transmitted, but not transmitted through seed. Under natural conditions, CTV readily infects most species of Citrus and Fortunella and some species in genera known as citrus-relatives of the family Rutaceae. A list of CTV host species can be found in the Global Database (EPPO, 2021a). Routine testing for CTV focusses on plant material and testing of vectors is not covered in this Standard. A flow diagram describing the diagnostic procedure for citrus tristeza virus in plant material is presented in Figure 1. FIGURE 1Open in figure viewerPowerPoint Flow diagram describing the diagnostic procedure for citrus tristeza virus in plant samples. 2 IDENTITY Preferred name: Citrus tristeza virus Other names: Citrus tristeza closterovirus Acronym: CTV Taxonomic position: Viruses, Riboviria, Closteroviridae, Closterovirus EPPO code: CTV000 Phytosanitary categorization: EPPO A2 list n°93, EU A1 Quarantine pest (Annex II) A for non EU isolates, EU PZ Quarantine pest (Annex III) EU isolates, EU RNQP (Annex IV) EU isolates Note on the phytosanitary categorization: CTV non-EU isolates are able to cause severe symptoms on a range of citrus crops that EU isolates do not induce. For this reason, non-EU CTV isolates have been evaluated as Union quarantine pests (EFSA, 2017). Throughout this document, CTV isolates that can cause severe symptoms are referred to as ‘severe isolates’. 3 DETECTION 3.1 Symptoms Symptoms and symptom expression in CTV-infected citrus hosts is highly variable and is influenced by environmental conditions, host species and isolate. In general, Citrus reticulata (mandarin) plants infected with CTV do not show symptoms. Citrus sinensis (sweet orange), C. aurantium (sour orange, as a seedling and not as grafted rootstock), C. jambhiri (rough lemon) and C. limonia (mandarin lime) are usually symptomless, but symptoms can be observed when infected by some severe CTV isolates. Citrus hosts that develop symptoms include C. aurantiifolia (lime), C. macrophylla (alemow), C. paradisi (grapefruit and some cultivars of pomelo), some citrus hybrids and some citrus relatives of the family Rutaceae. Depending on the CTV isolate and citrus species or rootstock/scion combination, the virus may cause no symptoms, tristeza, stem pitting or seedling yellows (Dawson et al., 2013; Moreno & Garnsey, 2010). Typically, mild CTV isolates belonging to the T30 or RB phylogenetic groups produce no noticeable symptoms on most commercial citrus species, and citrus species grafted on C. aurantium remain symptomless for many years. Types of symptoms and pathogenicity associated with the six major phylogenetic groups are presented in Table 1. TABLE 1. Symptoms and pathogenicity associated with the six major phylogenetic groups. Phylogenetic group T36 T68 RB T3 VT T30 Type of symptoms currently described in the field SY, QD, SP SP NNa SY, SP SP, SY, QD SD Pathogenicity Mild and severe Severe Mild Severe Mild and severe Mild Abbreviations: NN, generally no noticeable symptoms; QD, quick decline; SD, slow decline; SP, stem pitting; SY, seedling yellows. a The RB isolate present in the island of Crete is inducing slow decline in trees grafted on sour orange. It is also important to note that in areas where CTV and ‘Candidatus Liberibacter’ spp. (huanglongbing) are present, co-infection by both pathogens is common and can lead to increased disease severity due to synergism (Fu et al., 2017). The same stands for other pathogens [e.g. Phytophthora spp., citrus psorosis virus (CPsV) in Argentina, citrus sudden death associated virus (CSDaV) in Brazil, citrus exocortis viroid (CEVd) on sensitive rootstocks] and environmental conditions. In the Mediterranean basin in particular, drought is greatly contributing to the tristeza syndrome on infected trees grafted on sour orange rootstock (M. Cambra, pers. comm.). 3.1.1 Tristeza (decline syndrome) Tristeza is a bud union disease that develops only in susceptible rootstocks-scion combinations. The vast majority of CTV isolates cause a decline syndrome in different citrus species such as C. sinensis, C. reticulata, C. paradisi, Fortunella spp. and C. aurantiifolia when grafted on rootstocks of C. aurantium or C. limon. The decline can be extremely rapid (‘quick decline’), with wilting and death of trees occurring within a few days or weeks, or it can be a slower process (‘slow decline’), with no symptoms or symptoms appearing over months or even years (EFSA, 2014). Decline symptoms resemble those caused by root injury. These symptoms include thinning of foliage, twig defoliation and dieback, delayed growth and possibly tree collapse. Trees that decline slowly generally have a bulge above the bud union, a brown line just at the point of bud union, and inverse pinhole pitting (honeycombing) on the inner face of sour orange rootstock bark. On susceptible hosts, stunting, leaf cupping, vein clearing, chlorotic leaves, stem pitting and reduced fruit size are symptoms commonly observed. 3.1.2 Stem pitting Stem pitting syndrome (caused by severe isolates within the T3, T68 and occasionally VT phylogenetic groups) occurs in susceptible species regardless of the rootstock used and can affect both rootstock and grafted varieties (Moreno et al., 2008). Severe CTV isolates can seriously affect trees, inducing stem pitting on the trunk and branches of lime, grapefruit and sweet orange. However, it should be noted that most CTV isolates seriously affect rootstocks of Citrus macrophylla by causing stem pitting that results in reduced tree vigour. The stem pitting syndrome on inoculated C. paradisi and/or C. sinensis seedlings may sometimes cause a bumpy or ropy appearance of the trunks and limbs of adult trees, deep pits in the wood under depressed areas of the bark, and a reduction in fruit quality and yield. 3.1.3 Seedling yellows The seedling yellows syndrome (caused by isolates within the T36, T3, VT phylogenetic groups) is observed in young plants of sour orange, grapefruit and lemon, most notably under greenhouse conditions (20–26°C) rather than in field situations. The seedling yellows syndrome is characterized by stunting, production of chlorotic or pale leaves, development of a reduced root system, and stops the growth of the trees grafted on C. aurantium, and also stops the growth of C. aurantium, C. limon and C. paradisi seedlings. Figures 2-12 show the main symptoms caused by CTV. FIGURE 2Open in figure viewerPowerPoint Decline: Leaf chlorosis (a, b) and twig defoliation (b on top) of CTV infected sweet orange trees grafted on sour orange rootstock. (a) healthy tree on the left. (b) healthy trees on the right. Courtesy: Varveri C, BPI, Greece. FIGURE 3Open in figure viewerPowerPoint Bud-union of sweet orange CTV-infected tree grafted on sour orange rootstock, and pin holing or honeycombing in the inner face of the bark of the sour orange rootstock below the bud union of the CTV-infected tree. Courtesy: Navarro L and Moreno P, IVIA, Spain. FIGURE 4Open in figure viewerPowerPoint Seedlings of Duncan grapefruit inoculated with a CTV strain inducing seedling yellows syndrome. Healthy seedling on the right. Courtesy: Yokomi R, ARS-USDA Parlier, Parlier, USA. FIGURE 5Open in figure viewerPowerPoint Seedling of sour orange inoculated with a CTV strain inducing seedling yellows syndrome. Courtesy: Yokomi R, ARS-USDA Parlier, Parlier, USA. FIGURE 6Open in figure viewerPowerPoint Seedling yellows symptoms in Duncan grapefruit. Courtesy: Harper SJ, Washington State University, US. FIGURE 7Open in figure viewerPowerPoint Seedling yellows symptoms in Mexican lime. Courtesy: Harper SJ, Washington State University, US. FIGURE 8Open in figure viewerPowerPoint Seedling yellows symptoms in sour orange. Courtesy: Harper SJ, Washington State University, US. FIGURE 9Open in figure viewerPowerPoint Severe CTV isolate induced small fruits (compared with a normal fruit on the hand) and stem pitting in branches and trunk of a grapefruit tree in Uruguay. Courtesy: Cambra M, IVIA, ES. FIGURE 10Open in figure viewerPowerPoint Leaf cupping symptoms in Mexican lime. Courtesy Harper SJ, Washington State University (US). FIGURE 11Open in figure viewerPowerPoint Vein clearing symptoms in Mexican lime. Courtesy: Harper SJ Washington State University (US) FIGURE 12Open in figure viewerPowerPoint Severe stem pitting symptoms in alemow. Courtesy: P Moreno (formerly IVIA, ES) 3.2 Test sample requirements General guidance on sampling methodologies is described in ISPM 31Methodologies for sampling of consignments2 and in Cambra et al. (2002) specifically for CTV sampling. Procedures for sample preparation are described in Appendix 1. 3.2.1 Plant material Collection of plant material by hand is recommended to avoid mechanical contamination (e.g. by using scissors). Samples (shoots or fully expanded leaves and peduncles) can be taken all year round from grapefruit, lemon, mandarin and sweet orange in temperate Mediterranean climates. Spring and autumn are the optimal sampling periods because the highest CTV titres are observed in the plant during these seasons. During summer, reduced CTV titres are observed when temperatures rise above 35°C. The decision to test samples (e.g., shoots, leaves, petioles) from individual or multiple plants by serological or molecular methods depends on the expected virus concentration in the plants, the prevalence of CTV in the area (Vidal et al., 2012), and the level of confidence required by the NPPO. Specific examples of testing of multiple plants are given below. Samples usually consist of leaves and shoots. Fruits and flowers can also be tested. Samples can be stored at 4°C for up to 7 days before processing. Fruits can be stored for 1 month at 4°C. Samples can also be stored at −20°C for up to 3 months and at −80°C for longer periods. 3.2.1.1 Leaves The best tissue for testing is the main leaf vein and the petiole. In orchards, the standard sample for adult trees consists of ten fully expanded leaves collected throughout the canopy of an individual tree including different scaffold branches. In Spain, leaf material from up to 5 trees are pooled in one sample when using molecular tests. In Greece, leaf material from up to 4 trees is pooled when using ELISA and up to 25 trees when using molecular tests (Sambade et al., 2002). For nursery plants, the standard sample is composed of four leaves per plant. Experience with serological tests shows that samples can be prepared by pooling leaves from up to five nursery plants. 3.2.1.2 Shoots In orchards, the standard sample for an adult tree is five young shoots collected throughout the canopy including different scaffold branches. For nursery plants, the standard sample is composed of two young shoots per plant. Shoots from up to 10 nursery plants can be pooled when serological methods are used for detection. From woody shoots phloem scrapings are taken. 3.2.1.3 Fruits A standard sample for an adult tree consists of five fruits or fruit peduncles collected throughout the canopy including different scaffold branches. Tissue from the fruit peduncle (taken at the junction between the peduncle and the fruit), or from the columella (Figure 13) is the best tissue for testing. FIGURE 13Open in figure viewerPowerPoint Columella (courtesy Petter F, EPPO) 3.2.1.4 Flowers The standard sample for an adult tree consists of five flowers collected throughout the canopy including different scaffold branches. 3.3 Screening tests 3.3.1 Serological tests Polyclonal and monoclonal antibodies are available and can be used in double-antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA), double antibody sandwich indirect (DASI) or triple-antibody sandwich enzyme-linked immunosorbent assay (TAS-ELISA) and direct tissue print ELISA. A commercial kit Immunostrip Flashkit is available from Agdia. Instructions on how to perform an ELISA test are described in the EPPO Standard PM 7/125 ELISA tests for viruses (EPPO, 2015) and further information is provided in Appendix 2. 3.3.2 Molecular tests Procedures for RNA extraction are described in Appendix 3. The following molecular tests which have been evaluated in the framework of the EU funded VALITEST project (https://www.valitest.eu/) are recommended to detect CTV: Conventional reverse transcription PCR (RT-PCR) (Olmos et al., 1999), see Appendix 4. Real-time RT-PCR (Saponari et al., 2008), see Appendix 5 Real-time RT-PCR kit from Ipadlab, based on Bertolini et al. (2008), see Appendix 6. RT-LAMP (Wang et al., 2013), see Appendix 7. ‘Immunocapture (IC)-RT-PCR’ and ‘IC-nested RT-PCR in a single closed tube’ are not recommended in this protocol as they are no longer commonly used. An in silico analysis (19 isolates) showed that the tests of Saponari et al. (2008) and Wang et al. (2013) were able to detect all phylogenetic groups/isolates included, unlike the protocols of Bertolini et al. (2008) and Olmos et al. (1999) which missed one isolate (GenBank acc. no MF595989) (Varveri, pers. comm.). The real-time RT-PCR (Saponari et al., 2008), the real-time RT-PCR kit developed by Ipadlab based on Bertolini et al. (2008) and the RT-LAMP (Wang et al., 2013), were also evaluated with tissue prints (Cambra et al., 2019) (see respective appendices). 3.3.3 Biological indexing Biological indexing (see Appendix 8) is commonly used in the framework of certification programmes or post entry quarantine for Citrus fruit trees. It is considered a sensitive and reliable method for the detection and characterization of new and/or unusual isolates. However, it has some disadvantages: it is time consuming (symptom development requires up to 6 months post-inoculation); it requires dedicated containment facilities such as temperature-controlled insect-proof greenhouses; and it requires experienced staff who can accurately interpret disease symptoms that can be confused with symptoms of other graft-transmissible agents. In addition, CTV isolates that do not induce symptoms (latent isolates) are not detectable on indicator plants (e.g. the CTV “strain K” described by Albertini et al. (1988) and for which no molecular data is available). Consequently, biological indexing should always be used in combination with another test. 4 IDENTIFICATION 4.1 Identification of CTV The tests described in Section 3.3 allow both detection and identification of CTV. However, in the case of findings in the EPPO region, it is important to be able to identify isolates containing genotypes which are not present or have a limited distribution in the EPPO region and are (potentially) able to cause severe symptoms (stem pitting) in citrus orchards or break resistance. 4.2 Assignment of isolates to phylogenetic groups and/or strains3 The assignment of isolates (genotypes) to phylogenetic groups and strains is described in Sections 4.2.1 and 4.2.2, respectively. Table 2 provides an overview of CTV phylogenetic groups, their pathogenicity (assessed by biological indexing) and the recommended tests. Since molecular tests alone appear of limited value for the prediction of pathogenic properties of CTV isolates (Bar-Joseph et al., 2010; Harper et al., 2010), a combination of molecular and/or biological tests is needed for a conclusive characterization of the genetic and pathogenic characteristics of a CTV isolate. TABLE 2. Available tests to characterize CTV isolates Phylogenetic group T36a T68a RBa,1 T3b VTb T30b Method/Test Biological indexing (Appendix 8) (assignment to strains) SY, QD, SP SP Potentially all types of symptoms SY, SP All types of symptoms Leaf yellowing, vein clearing, leaf cupping HTS ✓ ✓ ✓ ✓ ✓ ✓ Multiplex conventional RT-PCR (Roy et al., 2010) ✓2 ✓ ✓2 ✓ ✓ ✓ Multiplex real-time RT-PCR (Yokomi et al., 2010) ✓ ✓3 ✓ ✓3 ✓3 ✓4 Conventional RT-PCR (Roy et al., 2013) – – ✓ – – – Conventional RT-PCR (Cook et al., 2016)5 ✓ – ✓ – – – Two-step conventional RT-PCR (Saponari et al., 2019) – – ✓ – – – One-step real-time RT-PCR (Saponari et al., 2019) – – ✓ – – – Abbreviations: QD, quick decline; SP, stem pitting; SY, seedling yellows; ✓, test (or combination of tests) considered suitable to assign isolates to phylogenetic groups; –, test does not detect isolates from the phylogenetic group. 1 Able to overcome Citrus trifoliata resistance. 2 The test will detect T36 and RB isolates, but these cannot be distinguished. 3 The test will detect T68, T3 and VT isolates, but these cannot be distinguished. 4 Results obtained from the four different probes should be combined to assign an isolate to the T30 phylogenetic group. 5 Two specific RT PCR tests are described in this publication. Note: a and b are additional information that relate to the presence of the phylogenetic groups in EU countries or not. a Not present in EU countries. b Present in EU and also in non-EU countries. 4.2.1 Molecular tests For the assignment of CTV isolates to particular phylogenetic groups, the molecular tests included in Table 2 can be used. Validation data for these tests is currently not available. Their analytical specificity (inclusivity-exclusivity) needs to be evaluated using representative isolates of the different phylogenetic groups. The molecular tests include: Conventional simplex and multiplex RT-PCR tests followed by sequencing and sequence analysis of the amplicon. Simplex and multiplex real-time RT-PCR tests using specific primers (and probes) for particular phylogenetic groups. High-Throughput Sequencing (HTS) and analysis of the sequences obtained. Conventional RT-PCR tests followed by Sanger sequencing of amplicons or HTS analysis (Bester et al., 2021) can be used for assignment of an isolate to a phylogenetic group when the virus concentration allows (Ruiz-García et al., 2019). Obtaining a ‘near’ complete genome sequence is preferable. Sequence analysis should follow the guidelines described in Appendices 7 and 8 of the EPPO Standard PM 7/129 DNA barcoding as an identification tool for a number of regulated pests (EPPO, 2021b). 4.2.2 Biological indexing Biological indexing is recommended for the characterization of the pathogenic properties of CTV isolates. Although biological methods are time consuming and can be performed only for a limited number of samples, biological indexing is the only method to assess the pathogenic features of CTV isolates. Further information is given in Appendix 8. 5 REFERENCE MATERIAL CTV-infected and healthy citrus controls, and CTV-specific oligonucleotide primer sequences are available for non-profit institutions from Instituto Valenciano de Investigaciones Agrarias (IVIA), Centro Protección Vegetal y Biotecnología, Carretera de Moncada-Náquera km 5, 46113 Moncada, Valencia, Spain. A Olmos (aolmos@ivia.es) and DSMZ Leibniz-Institut DSMZ-Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH Inhoffenstraβe 7 B 38124 Braunschweig (DE). 6 REPORTING AND DOCUMENTATION Guidelines on reporting and documentation are given in EPPO Standard PM 7/77 Documentation and reporting on a diagnosis. 7 PERFORMANCE CHARACTERISTICS When performance characteristics are available, these are provided with the description of the test. Validation data are also available in the EPPO Database on Diagnostic Expertise (http://dc.eppo.int), and it is recommended to consult this database as additional information may be available there (e.g. more detailed information on analytical specificity, full validation reports, etc.). 8 FURTHER INFORMATION Further information on this organism can be obtained from: Instituto Valenciano de Investigaciones Agrarias (IVIA), Centro Protección Vegetal y Biotecnología, Carretera de Moncada-Náquera km 5, 46113 Moncada (Valencia). Spain. E-mail: aolmos@ivia.es. Council for Agronomic Research and the bioeconomy – Research Centre for Plant Protection and Certification, Via C. G. Bertero 22–00156 Rome. Italy. E-mail: luca.ferretti@crea.gov.it. 9 FEEDBACK ON THIS DIAGNOSTIC STANDARD If you have any feedback concerning this Diagnostic Protocol, or any of the tests included, or if you can provide additional validation data for tests included in this protocol that you wish to share please contact diagnostics@eppo.int. 10 STANDARD REVISION An annual review process is in place to identify the need for revision of diagnostic protocols. Protocols identified as needing revision are marked as such on the EPPO website. When errata and corrigenda are in press, this will also be marked on the website. ACKNOWLEDGEMENTS This protocol was originally drafted by: Cambra M, Olmos A and Gorris MT, Valencian Institute for Agricultural Research (IVIA, Spain). This revision was prepared by: Varveri C, Benaki Phytopathological Institute (BPI, Greece), Faggioli F and Ferretti L, Council for Agricultural Research and Economics, Research Centre for Plant Protection and Certification (CREA, Italy) and Olmos A, Valencian Institute for Agricultural Research (IVIA, Spain). It was reviewed by the EPPO Panel on Diagnostics in Virology and Phytoplasmology. APPENDIX 1: SAMPLE PREPARATION 1 1. Preparation of tissue prints for serological and molecular tests The freshly cut sections of young shoots, leaf petioles, fruit peduncles or flower ovaries are carefully pressed against a nitrocellulose or cellulose-ester membrane (0.45 mm) and prints are allowed to dry for 2–5 min. For routine serological and molecular testing, at least two prints should be made per selected shoot (one from each end of the shoot) or peduncle and one per leaf petiole or flower ovary. Printed membranes can be kept for several months in a dry and dark place. 2 2. Preparation of plant extracts for serological and molecular tests For serological testing, 0.2–0.5 g fresh plant material (leaf midribs, petioles or phloem scrapings) is cut into small pieces with disposable razor blades or bleach-treated scissors and placed into a suitable tube or plastic bag. The sample is homogenized thoroughly in 2–10 mL (from 1:10 to 1:20 w/v) extraction buffer (PBS with DIECA see below) using an electrical tissue homogenizer, a manual roller, a hammer or a similar tool. For molecular testing, fresh plant material, 0.2 g for samples from individual trees up to 2 g for pooled samples (pooled samples consisting of equal amounts of each tree), is cut into small pieces as described above, placed into individual plastic bags and homogenized thoroughly in 1–20 mL (from 1:10 to 1:5 w/v) extraction buffer (PBS with DIECA, see below). Using a 1:5 w/v ratio was evaluated in a small experiment at the Benaki Phytopathological Institute (GR) and showed to yield a higher amount of RNA compared to the ratio 1:10 w/v ratio used for serological testing (Varveri, pers. comm.). Extraction buffer for leaf, bark tissues, tissue prints and squashes, Phosphate-buffered saline (PBS) with DIECA. NaCl 8.0 g KCl 0.2 g Na2HPO4.12H2O 2.9 g KH2PO4 0.2 g Distilled water to 1 L Adjust pH to 7.2–7.4 The extraction buffer is supplemented with sodium diethyl dithiocarbamate (DIECA) just before use to give a final concentration of 0.2% (2 g/L). APPENDIX 2: SEROLOGICAL TESTS Instructions on how to p