PM 4/17 (3) Certification scheme for olive trees and rootstocks

Abstract

EPPO BulletinVolume 52, Issue 3 p. 590-601 EPPO STANDARD ON SCHEMES FOR THE PRODUCTION OF HEALTHY PLANTS FOR PLANTINGFree Access PM 4/17 (3) Certification scheme for olive trees and rootstocks First published: 20 October 2022 https://doi.org/10.1111/epp.12883AboutSectionsPDF 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 the production of certified pathogen-tested olive trees and rootstocks. Specific approval and amendment: First approved in 1996–09. Revised in 2005–09 and in 2022–09. Authors and contributors are given in the Acknowledgements section. 1 INTRODUCTION The certification scheme for pathogen-tested trees and rootstocks of olive (Olea europaea) provides detailed guidance on the production of propagated varieties to be grown on their own roots, of vegetatively propagated or seedling rootstocks, and of grafted trees. Although the production of grafted plants is covered in this scheme, it should be noted that, in practice, olive plants are mainly produced by rooting shoots in appropriate rooting conditions. Plant material produced according to this certification scheme is derived from nuclear-stock plants that have been tested and found to be free from the following pathogens: Arabis mosaic virus (ArMV), Cherry leaf roll virus (CLRV), Strawberry latent ringspot virus (SLRSV), Verticillium dahliae and Xylella fastidiosa and produced under conditions minimizing infestation by other pests. Certified olive material for export should in any case satisfy the phytosanitary regulations of importing countries, especially with respect to any of the pathogens covered by the scheme which are also quarantine pests. The scheme is presented according to the general sequence proposed by the EPPO Panel on Certification of Fruit Crops and adopted by EPPO Council (EPPO, 1992). Definitions such as ‘candidate nuclear stock’, ‘certification scheme’, ‘certified material’, ‘nuclear stock’ and ‘propagation stock’ are included in the general introduction to the Series PM 4 (EPPO, 2009a). Other terms are used as defined in ISPM 5 (IPPC, 2021), such as ‘field’, ‘free from’, ‘inspection’, ‘lot’, ‘official’, ‘pest’, ‘plants’, ‘quarantine pest’, ‘regulated non-quarantine pest’, ‘test’. 2 OUTLINE OF THE SCHEME For the production of certified olive varieties and rootstocks the following successive steps should be taken. Selection for pomological quality or other specific desired traits: individual plants of each variety and rootstock to be taken into the scheme are selected. Production of nuclear stock: candidates for nuclear-stock plants are propagated by seedlings, cuttings or grafts. For grafted candidates, rootstocks of nuclear-stock status should be used. The candidate plants are kept isolated from the nuclear stock. The candidate plant is tested. The candidate for nuclear stock is kept under conditions ensuring freedom from infestation. Only candidate plants that have met all requirements are promoted to nuclear-stock plants. Maintenance of nuclear stock: nuclear-stock plants are maintained under conditions ensuring freedom from infection, with re-testing as appropriate. The plants should be grown in containers of sterilized or pest-free growing medium, isolated from the soil. Production of propagation stock: propagation stock is produced from nuclear-stock material in as few steps as possible under conditions ensuring freedom from infection. Production of certified plants: certified plants (varieties, rootstocks or grafted trees) are produced in nurseries from the propagation stock. For grafted trees, rootstocks fulfilling conditions of at least propagation-stock should be used. Throughout the whole procedure, care should be taken to maintain the pomological characters of the originally selected plants. The scheme is represented diagrammatically in Fig. 1. FIGURE 1Open in figure viewerPowerPoint Diagram of the stages in the olive certification scheme. : main line for production of olive plant reproductive material is through rooting shoots in appropriate rooting conditions. #: although this is not illustrated in the diagram, propagation stock can also be grown in pots. Frame in bold: always under insect proof conditions. Remarks: Propagation stock can be obtained from nuclear stock material or by multiplication from other propagation stock mother plant. Certified mother plants can be obtained from nuclear stock material or from propagation stock material. The nursery stage corresponds to ‘certified mother plants’ and ‘certified materials’. In the scheme, a specific terminology has been used for the successive stages of multiplication and certification: ‘candidate nuclear stock’, ‘nuclear stock’ and ‘propagation stock’. These terms have been defined for all EPPO certification schemes (EPPO, 2009a) and can be related as follows to alternative terms used by e.g. the EU (EU, 2014): candidate nuclear stock corresponds to ‘candidate pre-basic mother plant’, nuclear stock corresponds to ‘pre-basic mother plants and pre-basic materials’ and propagation stock corresponds to ‘basic mother plants and basic materials’. The certification scheme should be carried out by an official organization or by an officially registered, specialized nursery or laboratory satisfying defined criteria (see EPPO Standard PM 4/7 Nursery requirements - recommended requirements for establishments participating in certification of fruit or ornamental crops). All tests and inspections during production should be recorded. If the stages of the certification scheme are conducted by a registered nursery, certification will be granted by the official organization on the basis of the records of the tests and inspections performed during production, and of visual inspections to verify the apparent health of the stock. 3 SELECTION OF CANDIDATES FOR NUCLEAR STOCK Varieties A number of productive trees bearing typical characters of each variety or clone to be taken into the scheme (i.e. true to type), and showing the same genetic profile (when available), should be selected in different orchards and/or pomological trial fields. Seedling rootstocks Healthy-looking, vigorous, uniformly growing, and well rooted individual plants of each rootstock type to be taken into the scheme should be selected in different germination beds. Trees for production of seeds for seedling rootstocks Vigorous productive trees of each rootstock type to be taken into the scheme should be selected in different orchards or plantations. Selected trees should show no apparent symptoms of virus infection, and be affected as little as possible by other pathogens causing infectious graft-transmissible diseases. The selected trees should be known, as far as possible, to produce uniformly growing and true-to-type progeny, or else this should be checked. 4 PRODUCTION OF NUCLEAR STOCK Propagation material should be collected from the authenticated selected trees. This material should be budded or grafted onto pathogen-tested rootstocks, or one-year-old young shoots should be rooted in a substrate (either inert or sterilized) under 100% humidity conditions, in an isolated, suitably designed, aphid proof house fulfilling the requirements of EPPO Standard PM 5/8 Guidelines on the phytosanitary measure ‘Plants grown under physical isolation’, and separately from the nuclear stock. When produced in an area where X. fastidiosa is present, conditions in Appendix 1 should be fulfilled (including a 5-m wide surrounding zone around the structure kept free from any vegetation). Well rooted shoots (2 months are generally enough to obtain rooted plantlets) should be transplanted into suitable containers. The plants should be grown in inert or sterilized growing medium, in containers isolated from the soil to avoid any type of contamination. To date, 17 viruses have been detected from olive trees (Appendix 2). Testing is recommended for the following pathogens that can be transmitted on propagation material: ArMV, CLRV, SLRSV, V. dahliae and X. fastidiosa. (Table 1). Tests are specified in Appendices 3 and 4. For X. fastidiosa see EPPO Standard PM 7/24 Xylella fastidiosa (EPPO, 2019). Plants giving negative results in all tests can be promoted to nuclear stock and transplanted into the nuclear stock plot. Nuclear stock material and propagation material can be propagated from the nuclear stock plants. TABLE 1. Pests covered by the scheme Pest Categorization Geographical distribution Main transmission modes (in addition to plants for planting) Bacteria Pseudomonas savastanoi pv. savastanoia Worldwide Wind driven rain, agricultural practices (incl. machinery) Xylella fastidiosa EPPO A2 List The Americas, Europe, Iran, Taiwan, Israel Insect vectors (xylem feeders) Fungi Verticillium dahliae Worldwide Soil (incl. Soil in irrigation water or on agricultural machinery) Venturia oleaginea a Worldwide Rain droplets, wind driven rain and insects Insects Euzophera pinguis a Bulgaria, Spain, Tunisia Saissetia oleae a Worldwide Nematodes Meloidogyne arenaria Worldwide Soil Meloidogyne incognita Worldwide Soil Meloidogyne javanica Worldwide Soil Pratylenchus vulnus Worldwide Soil Xiphinema diversicaudatum Europe, New-Zealand, South Africa, USA Soil Viruses Arabis mosaic virus (ArMV) Europe, Egypt, Lebanon, Syria, USA Xiphinema diversicaudatum, seed?b Cherry leaf roll virus (CLRV) Europe, Egypt, Lebanon, Syria, Tunisia, USA Pollen, seed Olive leaf yellowing associated virus (OLYaV)a Europe, Egypt, Lebanon, Slovenia Syria, Tunisia, USA Pseudococcids Olive vein yellowing-associated virus (OVYaV)a Italy Olive yellow mottling and decline associated virus (OYMDaV)a Italy Strawberry latent ringspot virus (SLRSV) Europe, Egypt, Lebanon, Syria, Tunisia, USA Xiphinema diversicaudatum, seed?b a Pest only covered in the scheme by visual examination and/or conditions minimizing infestation. b These viruses are known to be often transmitted through infected seeds (EFSA PLH, 2013), but seed transmission of ArMV and SRLSV in olive plants has not been demonstrated (Albanese et al., 2012; Lister & Murant, 1967). Note: Most of the viruses in Appendix 1 have been isolated from symptomless trees and reported only in one or a very limited number of trees (e.g. Olive latent ringspot virus (OLRSV), Olive semilatent virus (OSLV)). Only the viruses which were assessed as fulfilling the minimum criteria for listing as regulated non-quarantine pests (RNQPs) are recommended for testing in this certification scheme: SLRSV in olive trees has been well investigated. It was reported for the first time in 1979 in Central Italy and then its role in causing ‘bumpy fruit’ disease on cv. ‘Ascolana tenera’ was ascertained. In addition, its effect on some morphological parameters of cv. ‘Raggiola’ has been clearly demonstrated, confirming its pathological and economic importance among olive viruses. Even though CLRV is asymptomatic in olive, infected olives of cv. ‘Frantoio’ were shown to produce a lower oil yield and maturity index; and the quality of the virgin oil obtained was reduced in terms of richness in o-diphenols, and oleic/linoleic (the latter could lower the oxidative stability during the oil storage) (Godena et al., 2012). It should be noted that, such results would need confirmation on a broader scale and in other olive varieties (same impact was not observed in cv. ‘Ascolana tenera’). SLRSV, CLRV and ArMV are reported to have an economic impact in other host plants (e.g. Juglans, Vitis, Rubus) which may be produced at the same place of production, and be infected via tools and machinery. Other olive viruses have been found associated with specific symptoms. For example, Olive vein yellowing associated virus (OVYaV), Olive yellow mottling and decline associated virus (OYMDaV) and Olive leaf yellowing associated closterovirus (OLYaV) have been associated with leaf-yellowing complex disease. OLYaV is most often asymptomatic and the presence of OVYaV as well as OYMDaV in olive is very scarce. These yellowing viruses are not considered to cause any unacceptable economic impact in olive. OSLV has been associated with vein clearing and Tobacco mosaic virus (TMV) with vein banding. There is no clear evidence, however, of the etiological involvement of these viruses with the diseases. 5 MAINTENANCE OF NUCLEAR STOCK Nuclear-stock plants should be kept in a suitably designed insect proof house fulfilling the requirements of Standard PM 5/8 and containing only nuclear-stock plants. They should be maintained under the same conditions as candidate nuclear stock, with the exception that soil can be tested for nematodes in Table 1 and for V. dahliae instead of using sterilized or inert soil. A limited number of nuclear-stock plants (at least 2) should be maintained for each source of each variety, rootstock type or tree for seed production included in the scheme and checked for trueness to type. Initially, all the sources of each variety, rootstock type or tree for seed production should be maintained. However, the number of sources may be reduced when the pomological comparisons have been made and the best sources are known. Each nuclear-stock plant should be routinely tested for V. dahliae and X. fastidiosa (each plant should be tested at least every 10 years, with some plants tested every year, for testing for X. fastidiosa see PM 7/24). In addition, all plants should be inspected for symptoms associated to these pests (see EPPO Standard PM 3/82 for X. fastidiosa) and to OLYaV, OVYaV, OYMDaV, SLRSV1 Venturia oleaginea, Pseudomonas savastanoi pv. savastanoi, and for infestations by Euzophera pinguis and Saissetia oleae. 6 PRODUCTION OF PROPAGATION STOCK The nuclear stock is multiplied in as few steps as possible to obtain the required quantity of propagation stock: For rooting shoots and grafted plants only one multiplication step should be allowed. For grafted plants, nuclear-stock material should be budded or grafted onto rootstocks of equivalent certification status or onto seedling rootstocks produced under propagation stock conditions. Multiplication in vitro beginning with axillary buds from nuclear stock plants can also be used (Appendix 6) for both varieties and rootstocks. To avoid mutations, in vitro multiplication of chimeric clones is not allowed. Care should be taken to limit the number of propagation steps (e.g. maximum of 10 subcultures for the propagation phase). The total duration of propagation steps should not exceed 4 years and cold storage is not allowed for more than 12 months. Care should also be taken to prevent the formation of callus and any in vitro cultures showing morphological abnormalities (e.g. fasciations) should be eliminated. The propagation material is kept either under insect proof conditions or outdoors. In areas where X. fastidiosa is present, the propagation material is kept under insect proof conditions fulfilling the requirements of Standard PM 5/8 for vectors of X. fastidiosa (see Appendix 1, including a surrounding zone of 5 m wide around the structure kept free from any vegetation). The propagation material is kept either in containers of sterilized growing medium or in soil that has been tested and found free from Xiphinema diversicaudatum (see EPPO Standard PM 4/35), Meloidogyne arenaria, M. incognita, M. javanica, Pratylenchus vulnus and V. dahliae (Appendix 5). In the field, the material should be separated by at least 20 m from other olive material, and reasonably isolated from sources of infection (e.g. irrigation water should be filtered and a surrounding zone 2 m wide around the plot should be kept free from any vegetation). Propagation stock should be kept under continuous surveillance and sprayed regularly with appropriate plant protection products, to control the normal pests of olive. General precautions against infection should be maintained, and appropriate control measures should be taken if any pests are observed. The propagation stock should be inspected each year for virus symptoms and for the other pests mentioned above (see EPPO Standard PM 3/82 for X. fastidiosa). Any symptomatic plant should be removed. If there is an indication that infestation may have derived from the previous generation, it is recommended to remove all the plants in the lot and to retest the possible source plant. For quarantine pests (e.g. X. fastidiosa), eradication measures should be applied. For the production of rootstock not belonging to a variety, coming from seed, and intended to be propagation stock, the plants growing from the seeds should be tested for viruses (see Table 1). Trueness to type must be checked by examining morphological characteristics or testing the genetic profile. 7 PRODUCTION OF CERTIFIED PLANTS For the production of certified grafted olive trees, the scion material should be grafted or budded onto rootstocks of equivalent (i.e. propagation-stock) or higher certification status only. Certified mother plants and plants to be certified should be kept in nurseries in soil which has been tested and found free from Xiphinema diversicaudatum, Meloidogyne arenaria, M. incognita, M. javanica, Pratylenchus vulnus and Verticillium dahliae (or which has been fumigated and retested to ensure freedom), separated by at least 4 m from other olive material, and reasonably isolated from sources of infection (e.g. irrigation water should be filtered and a surrounding zone 2 m wide around the plot should be kept free from any vegetation). In areas where X. fastidiosa is present, the propagation material should be kept under insect proof conditions fulfilling the requirements of Standard PM 5/8 for vectors of X. fastidiosa (see Appendix 1, including a surrounding zone of 5 m wide around the structure kept free from any vegetation). To be certified, the plants should be inspected by the official organization for symptoms of nematodes, viruses, virus-like disease or any of the pests mentioned above (see EPPO Standard PM 3/82 for X. fastidiosa). Any plants showing symptoms should be removed and certification may be granted to the remainder. For the production of rootstock not belonging to a variety, coming from seed, and intended to be certified plant, the plants growing from the seeds should be tested for viruses (see Table 1). Trueness to type must be checked by examining morphological characteristics or testing the genetic profile. 8 ADMINISTRATION OF THE CERTIFICATION SCHEME Monitoring of the scheme An official organization should be responsible for the administration and monitoring of the scheme. If officially registered nurseries carry out the different stages of the scheme, the official organization should confirm that all necessary tests and inspections have been performed during production, and should verify the general health status of the plants in the scheme by visual inspections. If these requirements are not met, certification will not be granted and/or the plants concerned will not be permitted to continue in the certification scheme. Control on the use and status of certified material Throughout the certification scheme, the origin of each plant should be known so that any problems of health or trueness to-type may be traced. The use of propagation material in nurseries to produce certified plants should be checked by an official or officially authorized organization which controls the health, origin and amount of such material on the basis of field inspections and of the records and documents presented by the nursery. The nursery plant protection programme and the inspections should also take account of other important pests that can affect quality, so that the certified plants delivered to the olive tree grower are substantially free from these pests. Certified material for export should in any case satisfy the phytosanitary regulations of importing countries. Certified plants leaving the scheme should carry an official certificate (which may be a label) indicating the certifying authority, the plant producer and the certification status of the plants. ACKNOWLEDGEMENTS This Standard was originally developed and reviewed by the Panel on the Certification of Pathogen-tested Fruit Crops and revised in 2005 to include new detection methods and viruses. It was extensively revised in 2021 by an Expert Working Group (EWG) composed of Mr D. Boscia (CNR, Italy), Mr F. Faggioli (CREA, IT), Ms R. Félix (UEVORA, PT), Ms C. Martinez (MAPAMA, ES), Mr P. Morello (University of Cordoba, ES), Ms M. Saponari (CNR, IT), Mr C. Trapero (University of Cordoba, ES) and Ms C. Varveri (BPI, GR); Mr D. Boscia, Ms G. Bottalico (University of Bari, IT) and Mr M. Micheli (University of Perugia, IT) extensively revised Appendix 6 on the In vitro maintenance and multiplication of olive. The Standard was reviewed by the Panel on Phytosanitary Measures. APPENDIX 1: INSECT PROOF MAINTENANCE CONDITIONS IN AREAS WHERE X. FASTIDIOSA IS PRESENT The following conditions are based on recommendations provided during task 9.4 of the XF-Actors Project (EU H2020 research project on X. fastidiosa) Description of the structure - Materials can be either rigid (e.g. glass, Plexiglas) or flexible (plastic, insect proof nets). - Fixation between the soil and the structure, as well as between flexible and rigid parts should be airtight. - The insect proof facilities should have a double door entrance. Dimensions of this entrance should be compatible with the size of the largest machinery to be used. Size net and properties - Insect proof nets should either cover the whole production site or be installed at the vents of structures, including windows. - The nets used should have a suitable mesh size to exclude the potential vectors, and a sufficient resistance to wind. - It is required that the mesh size is not larger than 1 mm. - Nets should be knitted. Entrance, dedicated clothes and controlled access (movement controls) - Any windows and doors should be locked shut when not in use. The two doors should never be open at the same time. - Considering the risk of entry and movement of vector(s) with the personnel working in the structure, dedicated outerwear should be used. Other plants/material entering the insect proof facility - Any plant for planting that enters the structure (even if it is not a known host plant for X. fastidiosa) should be treated (if possible) against the vectors and found to be free of the vectors. - Freedom of vectors should be verified prior to introduction. Vector control - Examination for the presence of vectors in the insect proof facilities should preferably be performed visually. These inspections should be recorded. - In case of the finding of a known vector of X. fastidiosa in the insect proof structure, an inspection of the structure should be carried out, the entire protected production site should be treated against the vector and the need for additional corrective actions should be considered. The vector should be tested to know whether it was infected with X. fastidiosa (see EPPO Standard PM 7/24 (EPPO, 2019)). - In case of the finding of an unexpected insect (including other xylem-feeding species), an inspection of the structure should be carried out and the need for other corrective actions should be considered (including treatment). Buffer zone It is recommended to control plants (including weeds) over a distance of at least 5 m around the protected production site, to prevent the entry of non-adult vectors which are considered to be less mobile. Specific traceability requirements: A register for personal should be made available at the entrance of the insect proof structure (name, date). APPENDIX 2: VIRUSES OCCURRING IN OLIVE (ALBANESE ET AL., 2012; MARTELLI, 2011) Virus Countries where the virus is reported in olive Frequency in olive References Arabis mosaic virus (ArMV) Egypt, Greece, Italy, Lebanon, Portugal, Syria, Turkey, USA Widely distributed Caglayan et al. (2004), Savino et al. (1979) Cherry leafroll virus (CLRV) Croatia, Egypt, Greece, Italy, Lebanon, Portugal, Spain, Syria, Tunisia, Turkey, USA Widely distributed Bertolini et al. (2001), Caglayan et al. (2004), Mathioudakis et al. (2020), Rei et al. (1993), Savino and Gallitelli (1981) Cucumber mosaic virus (CMV) Croatia, Greece, Italy, Portugal, Slovenia, Spain, Syria, Tunisia, Turkey, USA Widely distributed Bertolini et al. (2001), Caglayan et al. (2004), Mathioudakis et al. (2020), Rei et al. (1993), Savino and Gallitelli (1983), Viršček Marn and Mavrič Pleško (2018), Xylogianni et al. (2021) Olive latent ringspot virus (OLRSV) Italy, Portugal, Syria, Tunisia At low prevalence Rei et al. (1993), Savino et al. (1983) Olive latent virus 1 (OLV-1) Egypt, Italy, Jordan, Lebanon, Portugal, Syria, Turkey, Tunisia, USA At low prevalence Gallitelli and Savino (1985), Martelli et al. (1995) Olive latent virus 2 (OLV-2) Italy At low prevalence Savino et al. (1984) Olive latent virus 3 (OLV-3) Italy At low prevalence Alabdullah et al. (2010) Olive leaf yellowing-associated virus (OLYaV) Albania, Croatia, Egypt, Greece, Israel, Italy, Lebanon, Slovenia, Spain, Syria, Tunisia, USA Widely distributed Campos et al. (2019), Savino et al. (1996), Sbanadzovic et al. (1999), Mathioudakis et al. (2020), Viršček Marn and Mavrič Pleško (2018) Olive mild mosaic virus (OMMV) Portugal, Tunisia At low prevalence Campos et al. (2019), Cardoso et al. (2005) Olive semilatent virus (OSLV) Italy At low prevalence Materazzi et al. (1996) Olive vein yellowing associated virus (OVYaV)a Italy At low prevalence Faggioli and Barba (1995) Olive virus T (OlVT) Greece At low prevalence Xylogianni et al. (2021) Olive yellow mottling and decline associated virus (OYMDaV)a Italy At low prevalence Savino et al. (1996) Strawberry latent ringspot virus (SLRSV) Albania, Croatia, Egypt, Greece, Italy, Lebanon, Portugal, Spain, Syria, Tunisia, Turkey, USA, At low prevalence Bertolini et al. (1998), Caglayan et al. (2004), Henriques et al. (1990, 1992), Marte et al. (1986), Mathioudakis et al. (2020), Savino et al. (1979) Tobacco mosaic virus (TMV) Italy At low prevalence Triolo et al. (1996) Tobacco necrosis virus (TNV) Portugal At low prevalence Felix and Clara (2002) Tobacco necrosis virus D (TNV-D) Portugal, Tunisia At low prevalence Campos et al. (2019), Cardoso et al. (2004) a OYMDaV and OVYaV are not confirmed by the International Committee of Taxonomy of Viruses (ICTV). APPENDIX 3: GUIDELINES ON TESTING PROCEDURES FOR VIRUSES Testing on olive indicators No information is available at the moment on the use of woody indicators to detect the viruses listed in Table 1. Inoculation on herbaceous hosts Bioassays on herbaceous hosts were used in the past before the introduction of PCR-based tests for the identification of plant viruses but are no longer used since the introduction of PCR-based tests and are consequently not described in this Standard. ELISA testing Suitable antisera and ELISA procedures are not currently available for olive. According to Martelli (1999) and Bertolini et al. (2001), serological tests were not considered reliable when olive material was analysed by ELISA. Molecular testing RT-PCR tests (in two-step or one-step format) using specific primers (Table A1) are used for virus detection in olive (Loconsole et al., 2010). Optimal results are obtained when phloem tissues from 1- to 2-year-old twigs, collected from late winter to early spring (end of February – end of April), are tested. TABLE A1. Examples of sequences of specific primers which may be used for the detection of olive tree viruses Target virus Primer name F/R/Pa Size of product Primer sequence Test References ArMV ArMV-5 A F 302 5′-TACTATAAGAAACCGCTCCC-3′ One step RT-PCR Faggioli et al. (2005) ArMV-3 A R 5′-CATCAAAACTCATAACCCAC-3′ ArMV1 F 340 5′-CGGATTGGGAGTTCGTTGTCG-3′ Multiplex Nested RT-PCR in a Single Tube Bertolini et al. (2003) ArMV 2 R 5′-CCGTTCCATTCACTAACAACTC-3′ ArMV i1 F 203 5′-AATTATATGCTGAGTTTGAG-3′ ArMV i2 R 5′-AAAATTATACACCTTATGAGTA-3′ ArMVb P 5′-ATCCCACCACTGGAATATGACTTAAGTGCAACCAG-3′ ArMV-F F 504 5′-TTGGTTAGTGAATGGAACGG-3′ RT-PCR Grieco et al. (2000) ArMV-R R 5′-TCAACTCACCCTCCAAATCCC-3′ CLRV CLRV-5 F 416 5′-TGGCGACCGTGTAACGGCA-3′ One step RT-PCR Faggioli et al. (2005) (primers Werner et al. (1997)) CLRV-3 R 5′-GTCGGAAAGATTACGTAAAAGG-3′ CLRV 1 F 283 5′-CATTTCCATGCGACCGGTCTT-3′ Multiplex Nested RT-PCR in a single tube Bertolini et al. (2003) CLRV 2 R 5′-AGTCCGACACTCATACAATAAGC CLRV i1 F 171 5′-GTTAACGAATATCTACTGC-3′ CLRV i2 R 5′-CAAATATTGCTAAACAACC-3′ CLRVb P 5′-AAGCCCAAGAATTTAGGGGGTTATGTGGGTAGATAGCGTT-3′ CLRV-F F 431 5′-TTGGCGACCGTGTAACGGCA-3′ RT-PCR Grieco et al. (2000) CLRV-R R 5′-GTCGGAAAGA′ITACGTAAAAGG-3′ SLRSV SLRSV-5D F 293 5′-CCCTTGGTTACTTTTACCTCCTCATTGTCC-3′ One step RT-PCR Faggioli et al. (2005) SLRSV-3D R 5′-AGGCTCAAGAAAACACAC-3′ SLRSV 1 F 181 5′-GTTACTT