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PM 7/143 (1) ‘Candidatus Liberibacter solanacearum’

2020; Wiley; Volume: 50; Issue: 1 Linguagem: Catalão

10.1111/epp.12611

1365-2338

A wide range of symptoms similar to potato purple top and psyllid yellows, including chlorosis, twisted stems with a zigzag appearance, proliferation of axillary buds, shortened internodes, swollen nodes, aerial tubers, vascular discoloration, and leaf scorching and wilting are associated with ', L. solanacearum' in potato crops Fig., The specific symptoms in tubers consist of collapsed stolons, browning of vascular tissue and medullary rays throughout the entire length of the tuber Fig., A. ', L. solanacearum' has been shown to severely disrupt carbohydrate flow in potato plants, leading to zebra chip symptoms. This is usually very visible when tuber slices are fried Fig., B) but can also be observed in a transversal cut of the tubers in the field or in storage. The …,

Mosquito-borne diseases and control

EPPO BulletinVolume 50, Issue 1 p. 49-68 DiagnosticFree Access PM 7/143 (1) 'Candidatus Liberibacter solanacearum' First published: 14 March 2020 https://doi.org/10.1111/epp.12611Citations: 8AboutSectionsPDF 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 Abstract Specific scope This Standard describes a diagnostic protocol for 'Candidatus Liberibacter solanacearum', and for its detection in the psyllid vectors Bactericera cockerelli, Trioza apicalis and Bactericera trigonica.1 This Standard should be used in conjunction with PM 7/76(5) Use of EPPO diagnostic protocols. Specific approval and amendment 2019–09 This Diagnostic Protocol was prepared in parallel to the IPPC Diagnostic Protocol adopted in 2017 on 'Candidatus Liberibacter solanacearum' (Annex 21 to ISPM 27; IPPC, 2017). The EPPO Diagnostic Protocol differs in terms of format but it is consistent with the content of the IPPC Standard. With regard to molecular methods, one real-time PCR test used in the EPPO region is included, and more information on the different haplotypes is included as well as additional information on reference accessions. 1 Introduction 'Candidatus Liberibacter solanacearum' is a Gram-negative bacterium. It is restricted to the host plant's phloem and psyllid vector's hemolymph, alternating its life cycle between host plants and insect vectors. 'Ca. L. solanacearum' has not been cultivated in axenic medium yet. In North and Central America and Oceania it primarily infects solanaceous crops and weeds, including Solanum tuberosum (potato), Solanum lycopersicum (tomato), Capsicum annuum (pepper), Solanum betaceum (tamarillo), Nicotiana tabacum (tobacco), Solanum melongena (eggplant), Physalis peruviana (tomatillo), Solanum elaeagnifolium (silverleaf nightshade), Solanum ptychanthum (black nightshade), Lycium barbarum (wolfberry) and other crops or weeds in the family Solanaceae (EPPO 2013; Haapalainen, 2014). In the EPPO region, 'Ca. L. solanacearum' has been associated with symptoms in species of the Apiaceae family, including Daucus carota (carrot), Apium graveolens (celery), Pastinaca sativa (parsnip), Petroselinum crispum (parsley), Anthriscus cerefolium (chervil) and Foeniculum vulgare (fennel) (EPPO 2013; Alfaro-Fernández et al., 2014, 2017 ; Teresani et al., 2014; Hajri et al., 2017). In addition, 'Ca. L. solanacearum' was found on Urtica dioica (stinging nettle) in Finland (Haapalainen et al., 2018a). 'Ca. Liberibacter solanacearum' is transmitted by different psyllids species in a propagative, circulative and persistent manner. The tomato/potato psyllid Bactericera cockerelli has been described as the vector of haplotypes A and B in solanaceous crops (Munyaneza et al., 2007; Buchman et al., 2011; Sengoda et al., 2014). Evidence of effective transovarial transmission of 'Ca. L. solanacearum' has been provided in B. cockerelli (Hansen et al., 2008). 'Ca. L. solanacearum' is transmitted to apiaceous species by Trioza apicalis (Nissinen et al., 2014) and Bactericera trigonica (Antolínez et al., 2016, 2017; Teresani et al., 2017). T. apicalis has been reported as a vector of haplotype C (Nissinen et al., 2014), and B. trigonica as a vector of haplotypes D and E in Spain (Nelson et al., 2012; Teresani et al., 2015, 2017; Antolínez et al., 2016, 2017). There are also reports of 'Ca. L. solanacearum' detection, but not transmission, in Bactericera tremblayi collected from carrots (Teresani et al., 2015; Antolínez et al., 2017). 'Ca. L. solanacearum' was also detected in B. nigricornis, Trioza urticae and Trioza anthrisci in Spain, Finland, Germany and the United Kingdom (Teresani et al., 2015; Haapalainen et al., 2018a; Sjölund et al., 2017, 2018), but no study was conducted to determine their potential as a vector of the bacterium. In addition, 'Ca. L. solanacearum' can be transmitted by propagative plant material and, as shown in experimental setup, it can also be transmitted by Cuscuta campestris (dodder) to Catharanthus roseus (periwinkle) and other herbaceous plants (Bertolini et al., 2015). Although the presence of the bacterium has been detected in seeds of chili pepper (Camacho-Tapia et al., 2011), parsley (Monger & Jeffries, 2016) and parsnip (Morán et al., unpublished data), bacterial seed transmission has only been suggested for carrot seeds and only in one study (Bertolini et al., 2015). Those results have never been reproduced (Loiseau et al., 2017a,b).1 Seven haplotypes of 'Ca. L. solanacearum' have been described (Nelson et al., 2011, 2012; Teresani et al., 2014; Haapalainen et al., 2018a; Swisher Grimm & Garczynski, 2018). Three haplotypes (A, B and F) are known so far to be associated with diseases caused by this bacterium in potatoes and other solanaceous crops, whereas the C, D and E haplotypes are known to be associated with apiaceous species. In 2018, a new haplotype U was described on Urtica dioica (stinging nettle). Haplotype A has been detected primarily from Honduras and Guatemala through Western Mexico to the USA (Arizona, California, Oregon, Washington and Idaho) and in New Zealand. Haplotype B has been detected in Mexico and the USA. Haplotype F has been detected in the USA on a single tuber (Swisher Grimm & Garczynski, 2018). Haplotype C was detected in Finland, Sweden, Norway, Germany and Austria (Haapalainen, 2014; Munyaneza et al., 2015; EPPO Global Database, 2019). Haplotypes D and E have been detected in the Canary Islands and in mainland Belgium, Spain, France, Greece, Tunisia, Morocco and Portugal (Haapalainen, 2014; Tahzima et al., 2014; Teresani et al., 2014; Hajri et al., 2017; Holeva et al., 2017; Ben Othmen et al., 2018; EPPO, 2019). Haplotype D has been detected in Israel (EPPO, 2017). An outbreak was detected in Italy (Sicily) and haplotype D was detected in one sample (Catara, pers. comm., 2019). Finally, an outbreak has been reported in Estonia (EPPO, 2018; haplotype not known). Information can also be retrieved from the EPPO Global Database (EPPO, 2019). 'Ca. Liberibacter solanacearum' (mainly haplotype D) was detected in old commercial seed (the earliest dating from 1973) from countries not previously reporting the presence of this bacterium in Apiaceae species: the Czech Republic, Denmark, Egypt, Japan, Netherlands, the Soviet Union, Syria, the United Kingdom and the USA (Monger & Jeffries, 2018). Haplotypes D and E of 'Ca. Liberibacter solanacearum' have also been detected in commercial carrot seeds lots (Ilardi et al., 2016). The discovery in Finland of asymptomatic potato volunteers infected with haplotype C (Haapalainen et al., 2018b) and symptomatic ware potato tubers infected with haplotype E in Spain (Palomo et al., 2014) would suggest that all haplotypes can infect potato, but transmission is limited between the different plant families because of the lack of a vector that is able to feed efficiently on plants in both families. In addition, with regards to the detection in ware tubers, Palomo et al. (2014) state that 'these data would indicate that this haplotype could have sporadically infected the potato and would not have any repercussion epidemiological or economic'. Similarly, the solanaceous-infecting haplotype B can infect carrot (Munyaneza et al., 2016). Consequently, although EPPO recommends regulation of Solanaceae haplotypes of 'Ca. L. solanacearum' only (EPPO, 2012), testing for other hosts than Solanaceae is included in this Diagnostic Protocol. Detailed information on the distribution of 'Ca. Liberibacter solanacearum' can be found in Global Database (EPPO, 2019). A flow diagram describing the diagnostic procedure for 'Ca. Liberibacter solanacearum' is presented in Fig. 1. Figure 1Open in figure viewerPowerPoint Flow diagram for the detection and identification of 'Candidatus Liberibacter solanacearum' (Part A) and haplotype determination (Part B). [Colour figure can be viewed at wileyonlinelibrary.com] 2 Identity Name: 'Candidatus Liberibacter solanacearum' (Liefting et al., 2009b) Synonyms: 'Candidatus Liberibacter psyllaurous' (Hansen et al., 2008) Taxonomic position: Bacteria, Proteobacteria, Alphaproteobacteria, Rhizobiales, Rhizobiaceae, 'Candidatus Liberibacter' EPPO Code: LIBEPS Phytosanitary categorization: EPPO A1 list no. 365 (for Solanaceae haplotypes) 3 Detection The symptoms associated with 'Ca. L. solanacearum' are not always easy to distinguish from those associated with phytoplasmas, Spiroplasma citri or other biotic factors as well as from those associated with abiotic factors. Several tests have been developed for the detection of 'Ca. L. solanacearum' in plant material and vectors, and include conventional PCR (Hansen et al., 2008; Li et al., 2009; Liefting et al., 2009a,b; Lin et al., 2009, 2011; Munyaneza et al., 2009; Secor et al., 2009; Wen et al., 2009, 2011; Crosslin et al., 2011; Pitman et al., 2011; Ravindran et al., 2011) and real-time PCR (Li et al., 2009; Teresani et al., 2014). The most widely used reagents and PCR tests are presented in Appendices 4–8. 3.1 Disease symptoms The descriptions of symptoms provided below are according to Munyaneza (2012), Haapalainen (2014) and Teresani et al. (2014). Symptoms (such as deformations) can be seen on all or parts of the plants, but plants may remain asymptomatic. 3.1.1 Symptoms on Solanum tuberosum (potato): zebra chip A wide range of symptoms similar to potato purple top and psyllid yellows, including chlorosis, twisted stems with a zigzag appearance, proliferation of axillary buds, shortened internodes, swollen nodes, aerial tubers, vascular discoloration, and leaf scorching and wilting are associated with 'Ca. L. solanacearum' in potato crops (Fig. 2). The specific symptoms in tubers consist of collapsed stolons, browning of vascular tissue and medullary rays throughout the entire length of the tuber (Fig. 3A). 'Ca. L. solanacearum' has been shown to severely disrupt carbohydrate flow in potato plants, leading to zebra chip symptoms. This is usually very visible when tuber slices are fried (Fig. 3B) but can also be observed in a transversal cut of the tubers in the field or in storage. The optimum development of zebra chip symptoms was observed at a daily temperature regime of 27–32°C. Figure 2Open in figure viewerPowerPoint Potato zebra chip symptoms. Chlorosis, twisted stems with a zigzag appearance, proliferation of axillary buds, shortened internodes, swollen nodes, aerial tubers, vascular discoloration, and leaf scorching and wilting. Courtesy of G. Secor. Figure 3Open in figure viewerPowerPoint Potato zebra chip symptoms. (A) Browning of vascular tissue and medullary rays throughout the entire length of the tuber. (B) Necrotic flecking and streaking of the medullary ray tissue symptoms in processed chips or fries. Courtesy of G. Secor. 3.1.2 Symptoms on Solanum lycopersicum (tomato): psyllid yellows On tomato, the symptoms associated with 'Ca. L. solanacearum' are named psyllid yellows. The symptoms are similar to those caused by a toxin associated with feeding by the psyllid nymphal instars. Symptoms include spiky, chlorotic apical growth, general mottling of the leaves, curling of the midveins, overall stunting of the plants and in some cultivars fruit deformation (some of the symptoms are shown in Fig. 4). The severity of damage can vary between tomato cultivars and levels of disease prevalence. Figure 4Open in figure viewerPowerPoint Tomato psyllid yellows symptoms. Spiky, chlorotic apical growth, general mottling of the leaves, curling of the midveins. Courtesy of J.E. Munyaneza. 3.1.3 Symptoms on Daucus carota (carrot): yellow declines On carrot the symptoms associated with 'Ca. L. solanacearum' are named yellow decline. Symptoms include leaf curling, yellowish, bronze and purplish discoloration of leaves, stunting of the carrot shoots and roots, and proliferation of secondary roots. The symptoms collectively resemble those caused by leafhopper-transmitted 'Candidatus Phytoplasma' and Spiroplasma citri (Fig. 5) (Cebrián et al., 2010; Munyaneza et al., 2011). Figure 5Open in figure viewerPowerPoint Carrot yellow decline symptoms. (A) Leaf curling, yellowish, bronze and purplish discoloration of leaves, (B) and (C) stunting of the carrot shoots and roots, and (C) proliferation of secondary roots. Courtesy of IVIA. 3.1.4 Symptoms on Apium graveolens (celery) and Pastinaca sativa (parsnip): vegetative disorders Vegetative disorders are the syndrome associated to 'Ca. L. solanacearum' in infected celery plants. They show an abnormal number of shoots, curling of stems and yellowing (Fig. 6). Severe stunting was described on A. graveolens var. rapaceum (Mill) (Teresani et al., 2014). On parsnip the vegetative disorders include yellowish, leaf proliferation, root deformation and early senescence as well as proliferation of secondary roots (Alfaro-Fernández et al., 2017). Figure 6Open in figure viewerPowerPoint Celery vegetative disorders: abnormal number of shoots, curling of stems, and yellowing. Courtesy of IVIA. 3.2 Sampling for laboratory testing General guidance on sampling methodologies is provided in ISPM 31 (Methodologies for sampling of consignments), which provides useful information on the number of plants to be sampled2 . 3.2.1 Sampling of plants The distribution of 'Ca. L. solanacearum' in plant parts may be heterogeneous depending on the plant species and consequently appropriate sampling is required to improve detection. It should be noted that 'Ca. L. solanacearum' may not be detectable by molecular tests until three weeks after infective psyllids have fed on the plants (Levy et al., 2011). In experiments conducted in Spain (under both field and greenhouse conditions) it has been shown that both symptoms and bacterial titre of haplotypes D and E can decrease at temperatures higher than 26ºC (Lopez, pers. comm., 2019, unpublished results). 3.2.1.1 Plant material (except seeds) Plant material (leaves, petioles, midribs, stems, tubers and roots) is collected from any potential host. Care should be taken to avoid cross-contamination between samples (hand collection or disinfected tools). When typical foliar symptoms are present it is recommended to collect three to five leaves and/or stems from symptomatic parts of the plant. Experiments conducted in the framework of the POnTE project in 2017 have shown that for carrot and celery no significant differences were recorded when testing different parts of the plants, indicating that in these plants 'Ca. L. solanacearum' is homogenously distributed (Loiseau et al., 2018). In asymptomatic plants, leaves and/or stems from five to ten different parts of the plant should be sampled and should include newly developing leaves (Levy et al., 2011; Teresani et al., 2014; Cooper et al., 2015). Belowground plant parts such as tubers, roots and stolons can also be used to detect 'Ca. L. solanacearum'. Potato tubers showing obvious zebra chip symptoms should be tested individually. The tuber is cut and symptomatic tissue from the vascular area and the heel end is sampled. Detection from asymptomatic potato tubers will be less reliable and is not recommended, even if aboveground symptoms are present, as not all tubers from an infected plant will become infected by 'Ca. L. solanacearum' (Buchman et al., 2011). Before extraction, all plant material is subsampled so that the material used contains as much vascular tissue as possible (e.g. petioles, leaf midribs, cambium and the heel end or vascular ring of potato tubers). 3.2.1.2 Seeds Insufficient data exists to recommend a sample size and bulking rate for seed testing. In their study on seed transmission, Bertolini et al. (2015) detected 'Ca. L. solanacearum' in samples of 500 carrot seeds. The International Seed Federation (ISF, 2015) recommends testing samples of 20 000 carrot seeds composed of two subsamples of 10 000 seeds. 3.2.2 Sampling of vectors for testing Adults of B. cockerelii, B. trigonica and T. apicalis reported as vectors (see Fig. 7A–C), or of other psyllids suspected to be vectors, can be collected from symptomatic or asymptomatic plants. Some of these vectors overwinter as adults and can be collected during winter on conifers or weeds (Wenninger et al., 2017; Čermák & Lauterer, 2008; Kristofferson & Anderbrant, 2007). Whenever possible, psyllids should be identified before testing for 'Ca L. solanacearum'. Morphological identification is possible based on keys published by Ossiannilsson (1992) or Ouvard (2017). Figure 7Open in figure viewerPowerPoint (A) Bactericera cockerelli newly emerged adult, (B) adult female Bactericera trigonica and (C) adult female Trioza apicalis. Courtesy A: Oregon State University; B and C I Malenovský (Masaryk University). The bacterium is present in several organs and tissues of its vectors, including the alimentary canal, salivary glands, hemolymph and bacteriomes (Cooper et al., 2013). Crosslin et al. (2011) determined that 'Ca. L. solanacearum' can be reliably detected by conventional and real-time PCR in bulk samples of 30 laboratory-reared adult B. cockerelli. However, experience in the EPPO region shows that it is best to limit bulking to ten psyllids if they are sampled from the field by either sticky traps or hand collection. If the insects are collected from sticky traps, it is not necessary to remove the glue before DNA extraction. If desired, the glue may be removed before testing as described by Bertolini et al. (2014) and Teresani et al. (2014). 'Ca. L. solanacearum' can be reliably detected in infected psyllids for up to ten months on sticky traps stored inside at room temperature (Crosslin et al., 2011). For long-term storage before testing, psyllids are preserved in 70% ethanol. 3.3 Sample preparation Details on sample preparation are provided in Appendix 2. 3.4 Screening tests Two real-time PCR tests are recommended for the detection of 'Ca. Liberibacter solanacearum' in both asymptomatic and symptomatic plant material or in vectors. Teresani et al. (2014) is described in Appendix 4 and can be used for direct testing of crude extracts without DNA extraction. The test has been validated by the Instituto Valenciano de Investigaciones Agrarias (IVIA) in national and international test performance studies. Li et al. (2009) is described in Appendix 5. Validation data for this test has been generated by Anses-FR. The three conventional PCR tests described in section 4 for haplotype identification (see Appendices 6–8) can also be used as screening tests. 3.5 Comparison of the screening tests An international test performance study involving 26 laboratories from 14 countries (including non-EPPO countries) was organized in the framework of the following projects: POnTE (H2020), PhyLib II (Euphresco) and CaLiso (French funded project). All molecular tests included in this Diagnostic Protocol have been evaluated on DNA extracted and five different concentration levels (see table below). Five positive duplicate DNA samples including the five haplotypes known in 2017 and five negative duplicate DNA samples were provided to participants (Table 1). Table 1. The performance characteristics of the different tests included in this protocol in terms of performance criteria (unpublished data) Real-time PCR, Teresani et al. (2014) (Appendix 4) Real-time PCR adapted from Li et al. (2009) (see Appendix 5) PCR, Li et al. (2009) and Jagoueix et al. (1996) (Appendix 6) PCR, Ravindran et al. (2011) (Appendix 7) PCR, Munyaneza et al. (2009) (Appendix 8) Accuracy 97.3% 96.3% 93.7% 96.0% 97.3% Diagnostic sensitivity 96.2% 100.0% 89.2% 93.4% 94.7% Diagnostic specificity 98.5% 92.6% 98.0% 98.5% 100.0% Analytical sensitivity* 7.9 × 104 95% 100% 60% 88% 74% 1.7 × 104 93% 98% 45% 80% 56% 8.2 × 103 90% 100% 33% 78% 35% 1.6 × 103 17% 98% 8% 40% 6% 2.1 × 102 7% 50% 3% 8% 3% Average repeatability 96.8% 97.5% 95.2% 97.1% 96.2% Reproducibility 88.3% 94.3% 78.7% 84.2% 83.8% *The probability of detection of the target at the different levels of dilution. The level is given as the concentration of bacteria for 1 g of biological material. A national test performance study of a diagnostic protocol for 'Ca. L. solanacearum' in carrot seed was organized in Italy. This study was based on an adapted version of Li et al. (2009), different from the version recommended in this protocol (primer concentration and DNA input). The test involved 11 Italian laboratories that received both the samples (seeds and DNA). The results of this evaluation have been posted on the EPPO database on diagnostic expertise (section validation http://dc.eppo.int/validationlist.php) and published in Ilardi et al. (2018). 4 Identification The minimum identification requirement for 'Ca. L. solanacearum' is a positive result from one of the PCR tests described in this diagnostic protocol. Confirmation is recommended for critical cases, as described in PM 7/76 (EPPO, 2018), after 'Ca. L. solanacearum' has been detected by one rapid screening test. A conventional PCR should be performed and the product should be sequenced. For the sequence to be considered as the same species as 'Ca. L. solanacearum', it should be ≥98% identical to the sequence from the reference isolate (GenBank accession number EU834130). 4.1 Determination of haplotypes The haplotype can be determined by amplifying and sequencing up to three genomic regions2 . The tests are: Li et al. (2009) targeting the 16S rRNA gene region (Appendix 6). Ravindran et al. (2011) targeting a region of the 16–23S rRNA intergenic spacer (IGS) (Appendix 7). Please note that these primers will fail to amplify the 16S-23S rRNA IGS region containing the last five SNP differences between haplotypes. Munyaneza et al., 2009: a region of the rplL-rplJ gene region (50S rRNA) (Appendix 8). Amplicons should be sequenced to determine the species and the haplotype of the bacterium in suspect samples. Haplotypes can be identified following the table based on data from Nelson et al. (2012) and Teresani et al.(2014). The sequence of the unknown haplotype is aligned with the reference sequences for the 16S rRNA and 16S-23S rRNA IGS region (GenBank acc. number EU812559) and for the 50S rRNA (EU834131). The haplotype is determined by comparing the sequence at each nucleotide position listed in Table 2. Table 2. Single-nucleotide polymorphism differences between haplotypes of 'Candidatus Liberibacter solanacearum'. Source: Adapted from Nelson et al. (2013), Teresani et al. (2014), Swisher Grimm & Garczynski (2018); Haapalainen et al. (2018a). - = deletion Region (gene/position of reference sequence EU812559, EU834131) Haplotype A B C D E F U 16S rRNA 16S rRNA/115 A A A A G A A 16S rRNA/116 C C C T C C C 16S rRNA/151 A A A A G G A 16S rRNA/212 T G T T T T T 16S rRNA/359 A A A A A C A 16S rRNA/524 G G G G G A G 16S rRNA/581 T C T T T C T 16S rRNA/959 C C C C T C C 16S rRNA/1039 A A G G A G G 16S rRNA/1073 G G G A G G G 16S-23S IGS 16S-23S rRNA IGS/1620 A A A A G Unknown Unknown 16S-23S rRNA IGS/1632 G G G G A Unknown G 16S-23S rRNA IGS/1648 G G G G A Unknown G 16S-23S rRNA IGS/1689 A A A A A Unknown G 16S-23S rRNA IGS/1742 A A A G A Unknown A 16S-23S rRNA IGS/1748 C C C T C Unknown C 16S-23S rRNA IGS/1858 A G G A A Unknown A 16S-23S rRNA IGS/1859delT T T T - T Unknown T 16S-23S rRNA IGS/1867delT T T - T T Unknown T 16S-23S rRNA IGS/1873 A A A A G Unknown A 16S-23S rRNA IGS/1920 T T C T T Unknown T 16S-23S rRNA IGS/1943 G A G G Unknown Unknown G 16S-23S rRNA IGS/2055 C T C C Unknown Unknown C 16S-23S rRNA IGS/2081 G G G A Unknown Unknown G 16S-23S rRNA IGS/2220 G A G G Unknown Unknown G 16S-23S rRNA IGS/2262 C T C C Unknown Unknown C 50S rplJ-rplL 50S rplJ-rplL/558 T T T T T G T 50S rplJ-rplL/583 G G C G G G G 50S rplJ-rplL/622 A A A G A A A 50S rplJ-rplL/640 C C T C C C C 50S rplJ-rplL/669 G C G G G G G 50S rplJ-rplL/689 C C C T T C T 50S rplJ-rplL/691 G T T G G T G 50S rplJ-rplL/695 G G G G G A G 50S rplJ-rplL/697 A A A A A G A 50S rplJ-rplL/700 A A A G A A A 50S rplJ-rplL/712 G T G G G T G 50S rplJ-rplL/722 G G G G A G G 50S rplJ-rplL/749 C C C A C C C 50S rplJ-rplL/779_780delA A A A A A - A 50S rplJ-rplL/780_781insA - - A A A - A 50S rplJ-rplL/785 G A G G G G G 50S rplJ-rplL/849 T T T C C T T 50S rplJ-rplL/909 T C C C C C C 50S rplJ-rplL/919_920ins[C/T]TG – – CTG – – TTG – 50S rplJ-rplL/938 C C C C C C T 50S rplJ-rplL/955 G G T G G G G 50S rplJ-rplL/961 G G G G G G T 50S rplJ-rplL/987 T G G G G G G 50S rplJ-rplL/993 A A G A A A A 50S rplJ-rplL/1005 T T T T T C T 50S rplJ-rplL/1041 G A A G G A A 50S rplJ-rplL/1042 A A A A A G A 50S rplJ-rplL/1049 A G A A A A A 50S rplJ-rplL/1068 C C C T C C C 50S rplJ-rplL/1107 G A G G G A G 50S rplJ-rplL/1110 C C C C C C T 50S rplJ-rplL/1111_1112insC – – C – – – – 50S rplJ-rplL/1122 G A A A A A A 50S rplJ-rplL/1137 A A A A A G A 50S rplJ-rplL/1143 G A G G G A G There is currently no consensus in the scientific community about the delimitation of a new haplotype. Haplotypes are considered to be stable, but when 100% agreement with the SNPs of a known haplotype is not reached using the tests recommended in this protocol, it is recommended to repeat the test or to resample. 5 Reporting and documentation Guidelines on reporting and documentation are given in EPPO standard PM 7/77 Documentation and reporting of a diagnosis. 6 Performance criteria When performance criteria 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/validationlist.php) and it is recommended that this database is consulted as additional information may be available there (e.g. more detailed information on analytical specificity, full validation reports, etc.). 7 Reference material Paper immobilized positive controls or DNA extracts can be obtained from Plant Print Diagnostics, Valencia, Spain. Potato and tomato plant tissue infected with 'Ca. L. solanacearum', infected plant matrix extracts (inactivated by heat treatment) or extracted DNA from those matrices can be obtained from the Bacteriology Department, The National Reference Centre (NRC), Netherlands Food and Consumer Product Safety Authority (NVWA) Wageningen, the Netherlands. 8 Further information Further information on this organism can be obtained from E. Marco, Instituto Valenciano de Investigaciones Agrarias (IVIA), Carretera Moncada-Náquera Km. 4.5, 46113, Moncada, Valencia (ES); e-mail: emarco@ivia.es, Tjou-Tam-Sin N.N.A, National Reference Centre, NPPO-NL. P.O. Box 9102, 6700 HC. Wageningen (NL); e-mail: n.tjou-tam-sin@nvwa.nl, Loiseau M., Laboratoire de la santé des végétaux de l'ANSES (ANSES-LSV), 7 rue Jean Dixméras, 49044 Angers cedex 01 (FR); e-mail: marianne.loiseau@anses.fr. 9 Feedback on this Diagnostic Protocol 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 Protocol 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 has been prepared by E. Bertolini, M.M. López, F. Morán, S. Barbé, I. Navarro, M.T. Gorris, G.R. Teresani, and M. Cambra from Instituto Valenciano de Investigaciones Agrarias (IVIA), Valencia (ES); Tjou-Tam-Sin N.N.A (Leon), department National Reference Centre, team Bacteriology, Netherlands Food and Consumer Product Safety Authority (NVWA). Wageningen (NL) and M. Loiseau ANSES-LSV, Angers (FR). Appendix 1: Buffers CTAB buffer (as used in IVIA): Tris HCl 1 M pH 8.0 0.1 L NaCl 81.82 g EDTA 0.5 M pH 8.0 0.1 L CTAB 20 g PVP-10 10 g Optional: β-mercaptoethanol 2 mL Distilled water to 1 L Alternative recipes have not affected the test result, for example β-mercaptoethanol can be replaced by 30 mM ascorbic acid (Ilardi et al., 2018). Phosphate buffer (PB)10 mM, pH approx. 7.2 (PB): Na2HPO4.12H2O 2.15 g KH2PO4.2H2O 0.544 g Distilled water to 1 L Sterilize by filtration. Phosphate buffered saline (PBS) 10 mM, pH approx. 7.2: NaCl 8.0 g KCl 0.2 g Na2HPO4·12H2O 2.9 g KH2PO4 0.2 g Distilled water to 1 L Sterilize by filtration. Extraction buffer: PBS buffer supplemented with 2 g of sodium diethyl dithiocarbamate (DIECA) and 20 g of polyvinylpyrrolidone (PVP-10) per 1 L. Tris-HCl 1 M pH 8 (121.14 g/mol) Tris – 60.57 g Add water tol 400 mL Dissolve under shaking Add water to 500 mL Adjust to pH 8 Store at room temperature EDTA 0.5 M pH 8 (372.24 g/mol) EDTA – 186.12 g Add water to 800 mL Dissolve under shaking Add water to 1 L Adjust to pH 8 Store at room temperature Appendix 2: Sample preparation for testin