Bulletin OEPP/EPPO Bulletin (2013) 43 (1), 21–45 ISSN 0250-8052. DOI: 10.1111/epp.12019

European and Mediterranean Plant Protection Organization Organisation Europeenne et Mediterran eenne pour la Protection des Plantes PM 7/20 (2)

Diagnostics Diagnostic

PM 7/20 (2)* Erwinia amylovora

Specific scope Specific approval and amendment This standard describes a diagnostic protocol for Erwinia This standard was developed under the EU DIAGPRO Pro- amylovora1. ject (SMT 4-CT98-2252) and EUPHRESCO Pilot project (ERWINDECT) by partnership of contractor laboratories. Test performance studies were performed with different laboratories in 2002, 2009 and 2010. Approved as an EPPO Standard in 2003-09. Revised in 2012-09.

Fire blight is probably the most serious disease affect- Introduction ing Pyrus spp. (pear) and Malus spp. (apple) cultivars in Erwinia amylovora is the causal agent of fire blight in most many countries. Although the life cycle of the bacterium species of the subfamily Maloideae of the family Rosaceae. is still not fully understood, it is known that it can sur- The most economically important hosts are Pyrus spp., vive as endophyte or epiphyte for variable periods Malus spp., Cydonia spp., Eriobotrya japonica, Cotoneaster depending on environmental factors (Thomson, 2000). spp., Crataegus spp., Pyracantha spp. and Sorbus spp. Other The development of fire blight symptoms follows the sea- hosts include Chaenomeles, Mespilus and Photinia.Aforma sonal growth development of the host plant. It begins in specialis was described from Rubus spp. (Starr et al., 1951; the spring with production of primary inoculum and Bradbury, 1986). An exhaustive list of affected plants, infection of flowers, continues in summer with infection including those susceptible only after inoculation, was of shoots and fruits, and ends in autumn with the devel- reported by van der Zwet & Keil (1979). It includes more opment of cankers. The pathogen is apparently quiescent than 180 species from 39 genera of the Rosaceae. Erwinia through the dormant period of the host (van der Zwet & amylovora was the first bacterium described as a causal agent Beer, 1995). of a plant disease by Burrill (1883). It was reported in North Flow diagrams describing the diagnostic procedure for America and was later detected in New Zealand in 1920. In E. amylovora in symptomatic and asymptomatic material Europe, fire blight was reported in 1957 in the United King- are presented in Figs 1 and 2. dom and has since been identified in most areas where sus- ceptible hosts are cultivated. Erwinia amylovora is now present in more than 40 countries (van der Zwet, 2002; Identity CABI/EPPO, 2007), but it has not been recorded in South Name: Erwinia amylovora (Burrill) Winslow et al. America, Asia or sub-Saharan African countries. It has been Synonyms: Micrococcus amylovorus Burrill recorded in some North African countries and only once in Bacillus amylovorus (Burrill) Trevisan Australia (Bonn & van der Zwet, 2000). It represents a threat Bacterium amylovorus (Burrill) Chester to the pome fruit industry of all the countries. Details on geo- Erwinia amylovora f. sp. rubi Starr, Cardona & Falson graphical distribution can be found in the EPPO Plant Quar- Taxonomic position: Bacteria, Proteobacteria, c Subdivi- antine Data Retrieval system (PQR, 2012). sion, Enterobacteriales, Enterobacteriaceae 1Use of names of chemicals or equipment in these EPPO Standards implies EPPO code: ERWIAM no approval of them to the exclusion of others that may also be suitable. Phytosanitary categorization: EPPO A2 list no. 52, EU *This protocol number was corrected online on 25th April 2013. Annex designation II/A2

ª 2013 OEPP/EPPO, Bulletin OEPP/EPPO Bulletin 43, 21–45 21 22 Diagnostics

Plants with typical symptoms Asymptomatic sample

Pathogen extraction Pathogen extraction and enrichment

Rapid screening test Enrichment DASI-ELISA, Lateral flow devices, Screening tests IF, PCR, real-time PCR, LAMP (At least two tests based on different biological Enrichment DASI-ELISA, enrichment-PCR, principles) enrichment-real-time PCR, enrichment isolation (At least two tests based on different biological principles)

Some tests Negative positive

Some tests All tests positive negative

Isolation and/or enrichment-isolation

Isolation and Colonies with typical morphology enrichment-isolation

Colonies with typical E. amylovora Negative morphology not detected Positive Negative

Identification tests Positive (At least two tests based on different biological principles) Identification tests (At least two tests based on Negative E. amylovora Positive Negative not detected different biological principles)

E. amylovora identified Positive

Confirm pathogenicity by E. amylovora host test when required confirmed E. amylovora identified Fig. 1 Flow diagram for diagnosis of fire blight in plants with symptoms. Confirm pathogenicity by host test

E. amylovora confirmed Detection Fig. 2 Flow diagram for analysis of Erwinia amylovora in asymptomatic samples. Disease symptoms Symptoms of fire blight on the principal hosts are relatively for some time. Immature fruits (or less frequently mature similar and easily recognized (Figs 3–5). The name of the fruits) have infected parts that appear oily or water-soaked, disease is descriptive of its major characteristic: the brown- becoming brown to black and often exuding droplets of ish appearance of twigs, flowers and leaves as though bacterial ooze. They also remain attached to the tree. Char- burned by fire. Typical symptoms on pome fruit trees are acteristic reddish-brown streaks are often found in the sub- the brown to black colour of leaves on affected branches, cortical tissues when bark is peeled from infected twigs, the production of exudates under humid conditions, and the branches or trunks (van der Zwet & Keil, 1979). Brown to typical ‘shepherd’s crook’ in the shoots. Depending on the black, slightly depressed cankers can develop in the bark of affected plant part, the disease causes blossom blight, shoot twigs or branches, or even the trunk, in autumn and winter. or twig blight, leaf blight, fruit blight, limb and trunk These cankers may later become defined by cracks near the blight, collar or rootstock blight (van der Zwet & Keil, margin of diseased and healthy tissue (Dye, 1983). 1979; van der Zwet & Beer, 1995). Confusion between fire blight and blight- or blast-like In apple and pear, the first symptoms usually appear in symptoms, especially in blossoms and shoots, may occur early spring during warm and humid weather, and can pro- with diseases/disorders caused by other bacteria, fungi, insect gress very quickly under favourable conditions. Flowers damage and physiological disorders, and consequently labo- appear to be water-soaked, then wilt, shrivel and turn pale ratory analysis is always necessary. Other bacteria can cause brown to black. Peduncles may also appear water-soaked, blight-like symptoms, including Erwinia pyrifoliae, causal become dark green, and finally brown or black, sometimes agent of bacterial shoot blight of Pyrus pyrifolia (Asian pear) oozing droplets of sticky bacterial exudates. Leaves wilt (Kim et al., 1999); Erwinia piriflorinigrans, isolated from and shrivel, and entire spurs turn brown in most hosts, or necrotic pear blossoms in Spain (Lopez et al., 2011); Erwinia dark brown to black in pear, but remain attached to the tree sp. and Erwinia uzenensis, causing different types of pear

ª 2013 OEPP/EPPO, Bulletin OEPP/EPPO Bulletin 43, 21–45 Erwinia amylovora 23

AB A B

C D C D

Fig. 3 Symptoms of fire blight on pear trees. (A) necrotic flowers; (B) necrosis on leaves and typical shepherd’s crook; (C) mummified Fig. 4 Typical symptoms of fire blight on: (A) pear branches; (B) immature fruits with small ooze drops; (D) canker after removing bark apple shoot; (C) quince shoot; (D) loquat shoot. showing necrotic inner tissues. symptoms in Japan (Tanii et al., 1981; Matsuura et al., 2011); and Pseudomonas syringae pv. syringae, the causal A B agent of blossom blast.

Detection from symptomatic samples Sampling Symptomatic samples can be processed individually or in small batches combining material from several samples (see C D Appendix 1). Precautions to avoid cross-contamination should be taken when collecting samples and during the extraction process. Samples with symptoms for diagnosis of fire blight should preferably be composed of flowers, shoots or twigs, leaves, fruitlets (all with necrosis and with exu- dates if possible), or the discoloured subcortical tissues Fig. 5 Typical symptoms of fire blight on: (A) Crataegus sp. shoot; (after peeling bark from cankers in twigs, branches, trunk (B) Cotoneaster sp. shoot; (C,D) Pyracantha sp. branches. or collar). Samples should be processed as soon as possible after collection and stored at 4–8°C before analysis. Sam- ples may be cold stored after processing for up to 2 weeks bacteria in such samples is usually high. However, when in case further testing is required. symptoms are advanced or when environmental conditions are not favourable for fire blight symptom expression, the Isolation number of E. amylovora culturable cells can be very low. Fresh sample extracts are necessary for successful isolation. When plates are overcrowded by plant microbiota, the sam- Details on the extraction procedure from plant material are ple should be retested and enrichment according to Appen- given in Appendix 1. Details on isolation are provided in dix 4 performed before isolation, as described in Appendix 5. Isolating E. amylovora from symptomatic Appendix 5. Enrichment is also recommended when the samples is relatively easy because the number of culturable presence of antagonistic bacteria in the sample is suspected.

ª 2013 OEPP/EPPO, Bulletin OEPP/EPPO Bulletin 43, 21–45 24 Diagnostics

A B C

Fig. 6 Typical colony morphology of E. amylovora on: (A) King’s medium; (B) levan (NSA) medium and (C) CCT medium.

For direct isolation, plating on three media is advised for Conventional PCR, real-time PCR and loop-mediated iso- maximum recovery of E. amylovora, in particular when thermal amplification (LAMP) were evaluated in a test per- samples are in poor condition. The efficiency of the differ- formance study in 2010 and are also recommended for the ent media depends on the number and composition of mic- analyses of organs with symptoms after a DNA extraction robiota in the sample. Three media: King’s B, levan and step. The DNA extraction protocols that were evaluated in CCT (Appendix 2) have been validated in a test perfor- a test performance study in 2009 (Dreo et al., 2009) are mance study. Figure 6 shows the typical appearance of indicated in Appendix 6. Some other commercial kits for E. amylovora bacterial cultures in the three media. extracting DNA are available, but have not yet been vali- dated. Amplification protocols for PCR and real-time PCR Rapid screening tests are indicated in Appendices 7–13 and a LAMP protocol is These tests facilitate presumptive diagnosis on plants with included in Appendix 14. symptoms, in samples with more than 105–106 cfu gÀ1 (the minimum concentrations usually present in symptomatic Detection from asymptomatic samples samples). Several tests are described in Appendices 3–14. At least two tests, based on different biological principles, Sampling and sample preparation should be performed: one may be a serological test, prefer- Warning: detection of E. amylovora in asymptomatic plants ably using specific monoclonal antibodies; the other a has been shown to be difficult. PCR-based test. Test performance studies were organized The analyses of asymptomatic plants should be per- and the results are indicated. As differences in analytical formed in summer or early autumn to increase the likeli- sensitivity were observed in such studies, the decision on hood of detecting E. amylovora. Asymptomatic samples the tests selected should be based on a comparative analy- may be processed individually, or bulked (see Appendix 1). sis of the sensitivity and specificity of the different tech- Precautions to avoid cross-contamination should be taken niques in each laboratory, the number of samples to when collecting the samples and during the extraction pro- analyse, etc. cess. Sampling and sample preparation can be performed In areas where the disease is endemic, these tests can be following one of the methods for asymptomatic samples used without further confirmation in routine analyses of described in Appendix 1. samples. Direct analysis of asymptomatic samples is usually nega- • Serological tests tive for E. amylovora due to the low bacterial population. Indirect immunofluorescence (IF), enrichment DASI- Consequently, an enrichment step is advised (Appendix 4). ELISA and lateral flow devices are described for analyses of organs with symptoms. Quality of the antibodies is criti- Screening tests cal for performance of the tests. In test performance studies, Enrichment-isolation, enrichment-DASI ELISA, and several commercial antisera and monoclonal antibodies enrichment followed by conventional PCR or real-time were compared for IF [polyclonal antiserum from Loewe, PCR can be used as screening tests and are described in Biochemica GmbH (Sauerlach, Germany) and monoclonal Appendices 4–13. At least two screening tests should be antibodies from Plant Print Diagnostics S. L. (Faura, performed. Spain)]. For ELISA, a complete kit based on a combination of specific monoclonal antibodies, from Plant Print Diag- Confirmation of positive results of screening tests nostics S.L., was also evaluated. If these screening tests are positive, an attempt should be Two lateral flow devices commercialized by Bioreba, made to isolate the pathogen directly from the extract of Reinach, Switzerland (Ea AgriStrip) and Forsite Diagnos- non-enriched samples (Appendices 1–3), or from the tics, York, UK (Pocket Diagnostics) are available for the enriched samples (Appendices 4 and 5). As little is usually rapid analysis of symptomatic plant material (Braun-Kiew- known about the microbiota present in the samples, the nick et al., 2011). three media (CCT, King’s B, levan) indicated in Appen- Details of the tests are given in Appendix 3. dix 2 should be used to maximize the likelihood of success- • Molecular tests ful direct isolation of E. amylovora. However, plating only

ª 2013 OEPP/EPPO, Bulletin OEPP/EPPO Bulletin 43, 21–45 Erwinia amylovora 25 on CCT medium is advised after enrichment of the samples Table 2 Differences between Erwinia amylovora, Erwinia pyrifoliae in King’s B or in CCT. If the isolation is still negative, and and Erwinia piriflorinigrans cross-reactions or non-desired amplifications can be disre- garded as the controls were correct, it is reasonable to con- Erwinia Erwinia Erwinia Microbiological tests amylovora pyrifoliae piriflorinigrans sider E. amylovora presumptively detected in the sample. Confirmation requires isolation and identification of the Gelatine hydrolysis + ÀÀ bacterium. If necessary, the extract conserved at À80°C Inositol* À ND + * under glycerol (Appendix 2) can also be plated on the three Sorbitol ++À * À + media. Esculin V Melibiose* ÀÀ+ * D-Raffinose ÀÀ+ Identification b-Gentibiose* + À + Pure cultures of presumptive E. amylovora isolates should ND, not determined; V, variable. be identified with at least two tests based on different char- *Oxidation of substrates in API 50 CH (BioMerieux) with a modified protocol from Rosello et al. (2003). More than 90% of strains give the acteristics of the pathogen (e.g. combinations of biochemi- results indicated here. cal, serological or molecular tests) and, when necessary, a pathogenicity test. Two molecular tests may be used if they are based on different DNA sequence targets in the Table 3 Typical results of Erwinia amylovora in API 20E tests after genome and provided that the specificity of the primers has 48 h been evaluated. Known E. amylovora reference strains † should be included for each test performed (see section on Test* Reaction (48 h) Reference material). ONPG Variable ADH À (or weak +) À Biochemical tests LDC ODC À The genus Erwinia has been defined as Gram-negative bac- CIT À teria, facultative anaerobes, motile by peritrichous flagella, SH2 À À rod-shaped, acid produced from glucose, fructose, galactose URE TDA À and sucrose. The phenotypic properties listed in Table 1 IND À (Paulin, 2000), which are universally present or absent in VP + (or variable) E. amylovora, should be determined according to the meth- GEL Variable ods of Jones & Geider (2001). The tests in Table 2, based GLU + mainly on results in API 50 CH strips, allow differentiation MAN Variable of E. amylovora from E. pyrifoliae, causal agent of Asian INO Variable pear blight on Pyrus pyrifolia (Kim et al., 1999, 2001) and SOR Variable RHA À a new Erwinia species, E. piriflorinigrans, isolated from SAC + necrotic pear blossoms in Spain (Lopez et al., 2011). How- MEL À (or weak +) ever, certain physiological and biochemical characteristics AMY À can vary for some strains. For API 50 CH, a suspension of ARA Variable

Table 1 Biochemical tests for identification *Abbreviations used in API 20 E strips. †More than 90% of the strains give the results indicated here. Test Result

Gram staining À OD = 1.0 should be prepared in PBS (Appendix 2), and Levan production* + 1 mL added to 20 mL Ayers’ medium (Appendix 2). The Fluorescent pigment production in King’s B (under UV) À manufacturer’s instructions should be followed for inocula- + Oxidation/fermentation (O/F) test O /F+ tion of the strip. After incubation at 25–26°C in aerobiosis, Kovac’s oxidase test À Reduction of nitrate À the strip should be read after 24 and 48 h. Utilization of Utilization of citrate + the different carbohydrates is indicated by a yellow colour Growth at 39°C À in the wells. Gelatine liquefaction + Urease À Biochemical characterization by API system (BioMerieux, À Indole France) Reducing substances from sucrose + Biochemical identification of E. amylovora can be obtained Acetoin + by specific profile in API 20 E and API 50 CH strips. For *Spontaneous mutants found in nature can be levan-negative. API 20 E, the manufacturer’s instructions should be fol-

ª 2013 OEPP/EPPO, Bulletin OEPP/EPPO Bulletin 43, 21–45 26 Diagnostics lowed for preparing the suspension and inoculating the ELISA tests strip. After incubation at 25–26°C, the strips should be read ELISA tests are described in PM 7/101 ELISA tests for after 24 and 48 h (Table 3). plant pathogenic bacteria (EPPO, 2010). DASI-ELISA for isolate identification can be performed Automated Biolog identification system using the same specific monoclonal antibodies as used for The new version (third generation) Biolog GENIII 96 mi- analysis of plant samples (kit from Plant Print Diagnostics croplate allows rapid identification of isolated bacteria, both S.L.). For DASI-ELISA, a suspension of approximately Gram-negative and Gram-positive, using the same micro- 108 cells mLÀ1 from suspected colonies is prepared in PBS plate. The identification system is based on 94 phenotypic (Appendix 2). The DASI-ELISA procedure (Appendix 3) tests: 71 carbon-source utilization tests and 23 tests for bio- can be followed without prior enrichment for isolate identi- chemical and physiological properties including pH, salt, fication. lactic acid tolerance and antibiotics. Every species tested creates a unique ‘phenotypic fingerprint’ which is automati- Lateral flow immunoassays cally compared with 1200 aerobic species in the database. A suspension of approximately 108 cells mLÀ1 prepared in The microplate and the program are commercially avail- PBS (Appendix 2) from suspected colonies should be used able (Biolog, Omnilog, US). The manufacturer’s instruc- following the manufacturers’ instructions. The two kits tions should be followed for automatic identification of evaluated in a test performance study (Agri-strip and suspected strains of E. amylovora. Pocket Diagnostic) and recommended for analyses of symp- tomatic plants can be used for identification of isolates.

Fatty acid profiling Molecular tests Erwinia amylovora-like colonies should be grown on trypti- case soy agar for 48 h at 28°C, and an appropriate fatty acid Conventional and/or real-time PCR and LAMP are the rec- profiling (FAP) procedure applied. A positive FAP test is ommended molecular tests for rapid identification, but other achieved if the profile of the presumptive culture is identical available techniques are also indicated. to that of the positive control (Sasser, 1990). Commercial software from the MIDI system (Newark, DE, USA) allows Conventional PCR rapid identification of E. amylovora-like colonies. The manu- A suspension of approximately 106 cells mLÀ1 in molecu- facturer’s instructions should be followed for automatic iden- lar-grade water should be prepared from E. amylovora-like tification. Fatty acid composition can be affected by growth colonies. Appropriate PCR procedures should be applied, medium, physiological age of cells and chromatograph sensi- following Appendices 7–11, without DNA extraction, just tivity, but in general E. amylovora strains have a similarity after treatment at 100°C for 10 min. index between 0.6 and 0.9 in this system. Real-time PCR The first protocol for detection of E. amylovora by real-time Serological tests PCR was described by Salm & Geider (2004) and used prim- Performing only two serological tests is not adequate for ers based on sequences of the pEA29 plasmid. However, the identification; at least two tests based on different biologi- sensitivity and specificity of this test were similar to those of cal principles are needed. Different sources of antibodies conventional PCR tests. Since then, two real-time PCR tests should be used for detection (or diagnosis) and identifica- have been published, described in Appendices 12 and 13. tion to reduce the risk of false positives. Colonies can be prepared as for conventional PCR.

Agglutination test Macrorestriction with XbaI and pulse field gel electrophoresis Suspected E. amylovora colonies can be tested for aggluti- Pulse field gel electrophoresis (PFGE) analysis of genomic nation by mixing them in a drop of PBS (Appendix 2) with DNA after XbaI digestion according to Jock et al. (2002) a drop of E. amylovora-specific antiserum (not diluted, or shows six patterns for E. amylovora European strains. This five- or tenfold dilution) on a slide. Monoclonal antibodies method can provide information useful for strain differenti- can be used only if they agglutinate with the reference ation and has been used to analyse the spread of fire blight strains. in Europe.

Immunofluorescence test DNA sequencing methods The IF test is described in PM 7/97 Indirect immunofluores- Comparisons of commercially sequenced PCR products cence test for plant pathogenic bacteria. For identification, amplified from selected housekeeping genes allow differen- IF can be performed using specific monoclonal antibodies tiation of E. amylovora isolates from other members of the from Plant Print Diagnostics S.L. or antiserum from Loewe, Enterobacteriaceae (see EPPO Standard PM 7/XXX on Biochemica GmbH. DNA barcoding as an identification tool for plant pests,

ª 2013 OEPP/EPPO, Bulletin OEPP/EPPO Bulletin 43, 21–45 Erwinia amylovora 27 in preparation). For example, all isolates of E. amylovora Agrarias (IVIA), Carretera Moncada-Naquera km 5, 46113 tested so far are clonally related according to partial recA Moncada, Valencia, Spain; e-mail: [email protected]. gene sequence using the method described by Parkinson T Dreo, National Institute of Biology, Vecna pot 111, et al. (2009). SL-1000, Ljubljana, Slovenia; e-mail: [email protected].

Hypersensitivity and pathogenicity tests Feedback on this diagnostic protocol When necessary, suspected E. amylovora colonies from the If you have any feedback concerning this diagnostic pro- isolation and/or enrichment plates may be inoculated to test tocol or any of the tests included, or if you can provide plants to confirm their pathogenicity. additional validation data for tests included in this proto- The hypersensitive reaction in tobacco leaves can give an col that you wish to share, please contact diagnos- indication of the presence of the hrp pathogenicity genes, but [email protected]. is also positive for many other plant pathogenic bacteria. Tobacco plants of cv. Xanthi or Samsun with more than 5–6 Protocol revision leaves are used. Bacterial suspensions of 108–109 cfu mLÀ1 An annual review process is in place to identify the need (OD at 620 nm = 1.0) are injected into the intercellular space for revision of diagnostic protocols. Protocols identified as of adult leaves with a 25 GA 5/8 0.5 9 16 needle and syr- needing revision are marked as such on the EPPO website. inge. Complete collapse of the infiltrated tissue after 24 h at When errata and corrigenda are in press, this will also be room temperature is recorded as positive. marked on the website. To verify the pathogenicity of suspected E. amylovora colonies, a fire blight host should be inoculated (Appen- dix 15). Acknowledgements This protocol was originally drafted by M. M. Lopez, M. Reference material Keck, P. Llop, M. T. Gorris, J. Penalver,~ V. Donat and M. Cambra, IVIA, Moncada (Valencia) (ES) and the revised The following E. amylovora isolates are recommended for version was prepared by M. M. Lopez (IVIA, ES). T. Dreo use as positive controls: NCPPB683 (type strain) and CFBP (NIB, SL) and R. Gottsberger (AGES, AT) contributed by 1430. The following collections can provide different E. am- preparing the description of two PCR tests. The DNA poly- ylovora reference strains: (i) National Collection of Plant merase used in the test performance study for the PCR tests Pathogenic Bacteria (NCPPB), Fera, Sand Hutton, York described in Appendices 7, 8, 10 and 11 was provided to (GB); (ii) Culture Collection of the Plant Protection Service all laboratories participating by Biotools (B&M labs, S.A., ß (PD), Wageningen (NL); (iii) Collection Francaise de Bacte- Madrid, Spain). ries Phytopathogenes (CFBP), EmerSys – IRHS – INRA Beaucouze (FR). Authenticity of the strains can be guaran- References teed only if obtained directly from the culture collections. Ayers SH, Rupp P & Johnson WT (1919) A study of alkali forming in milk. USDA Bulletin no. 782. USDA, Washington (US). Reporting and documentation Bereswill S, Jock S, Aldridge P, Janse JD & Geider K (1997) Guidelines on reporting and documentation are given in Molecular characterization of natural Erwinia amylovora strains deficient in levan synthesis. Physiological and Molecular Plant Documentation and reporting EPPO Standard PM 7/77 (1) Pathology 51, 215–225. on a diagnosis. Bereswill S, Pahl A, Bellemann P, Zeller W & Geider K (1992) Sensitive and species-specific detection of Erwinia amylovora by polymerase chain reaction analysis. Applied and Environmental Performance criteria Microbiology 58, 3522–3526. If available, performance criteria are provided with the test Bonn WG & van der Zwet T (2000) Distribution and economic importance of fire blight. In: Fire Blight, The Disease and its description. Validation data are also available in the EPPO Causative Agent Erwinia amylovora (Ed. Vanneste J), pp. 37–53. Database on Diagnostic Expertise (http://dc.eppo.int), and it CAB International, Wallingford (GB). is recommended to consult this database as additional infor- Bradbury JF (1986) Guide to Plant Pathogenic Bacteria. CAB mation may be available there (on analytical specificity, full International, Wallingford (GB). validation reports, etc.). Braun-Kiewnick A, Altenbach A, Oberhansli T, Bitterlin W & Duffy B (2011) A rapid lateral-flow immunoassay for phytosanitary detection of Erwinia amylovora and on-site fire blight diagnosis. Journal of Further information Microbiological Methods 987,1–9. Burrill TJ (1883) New species of Micrococcus. American Naturalist 17, Further information on this organism can be obtained from: 319. MMLopez, Bacteriologıa, Centro de Proteccion Vegetal CABI/EPPO (2007) Erwinia amylovora. Distribution Maps of Plant y Biotecnologıa, Instituto Valenciano de Investigaciones Diseases No. 2. CABI/EPPO, Wallingford (GB).

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Dreo T, Duffy B, Lopez M, Paulin JP, Poliakoff F & Reisenzein H (2009) bacteria in plant material by polymerase chain reaction. Journal of Development and validation of innovative diagnostic tools for the Microbiological Methods 37,23–31. detection of fire blight (Erwinia amylovora). EUPHRESCO report. Llop P, Cabrefiga J, Smits THM, Dreo T, Barbe´ S, et al. (2011) Dye DW (1983) Erwinia: the ‘amylovora’ and ‘herbicola’ groups. In: Erwinia amylovora Novel Plasmid pEI70: Complete Sequence, Plant Bacterial Diseases, A Diagnosis Guide (Ed. Fahy PC & Biogeography, and Role in Aggressiveness in the Fire Blight Persley GJ), pp. 67–86. Academic Press, Sydney (AU). Phytopathogen. PLoS ONE 6(12): e28651. doi:10.1371/journal.pone. EPPO (2009) PM 7/097 (1) indirect immunofluorescence test for plant 0028651. pathogenic bacteria. Bulletin OEPP/EPPO Bulletin 39, 413–416. Llop P, Donat V, Rodrıguez M, Cabrefiga J, Ruz L, Palomo JL, EPPO (2010) PM 7/101 (1) ELISA tests for plant pathogenic bacteria. Montesinos E & Lopez MM (2006) An indigenous virulent strain of Bulletin OEPP/EPPO Bulletin 40, 369–372. Erwinia amylovora lacking the ubiquitous plasmid pEA29. EPPO (2012) EPPO plant quarantine data retrieval system. http://www. Phytopathology 96, 900–907. eppo.org/DATABASES/pqr/pqr.htm (accessed on 20 January 2012). Maes M, Garbeva P & Crepel C (1996) Identification and sensitive Gorris MT, Cambra E, Paulin JP, Chartier R, Cambra M & Lopez MM endophytic detection of the fire blight pathogen Erwinia amylovora (1996a) Production and characterization of monoclonal antibodies with 23S ribosomal DNA sequences and the polymerase chain specific for Erwinia amylovora and their use in different serological reaction. Plant Pathology 45, 1139–1149. techniques. Acta Horticulturae 411,47–51. Matsuura T, Mizuno A, Tsukamoto T, Shimizu Y, Saito N, Sato S, Gorris MT, Cambra M, Llop P, Lecomte P, Chartier R, Paulin JP & Kikuchi S, Uzuki T, Azegami K & Sawada H (2011) Erwinia Lopez MM (1996b) A sensitive and specific detection of Erwinia uzenensis sp. nov., a novel pathogen that affects European pear trees amylovora based on the ELISA-DASI enrichment method with (Pyrus communis L.). International Journal of Systematic and monoclonal antibodies. Acta Horticulturae 411,41–45. Evolutionary Microbiology 62, 1799–1803. doi: 10.1099/ijs.1090. Gottsberger RA (2010) Development and evaluation of a real-time PCR 032011-032010. assay targeting chromosomal DNA of Erwinia amylovora. Letters in McManus PS & Jones AL (1995) Detection of Erwinia amylovora by Applied Microbiology 51, 285–292. nested PCR and PCR-dot-blot and reverse blot hybridisations. Guilford PJ, Taylor RK, Clark RG, Hale CN, Forster RLS & Bonn WG Phytopathology 85, 618–623. (1996) PCR-based techniques for the detection of Erwinia Obradovic D, Balaz J & Kevresan S (2007) Detection of Erwinia amylovora. Acta Horticulturae 411,53–56. amylovora by novel chromosomal polymerase chain reaction primers. Ishimaru ES & Klos EJ (1984) New medium for detection of Erwinia Mikrobiologiia 76, 844–852. amylovora and its use in epidemiological studies. Phytopathology 74, Parkinson N, Stead D, Bew J, Heeney J, Tsror L & Elphinstone JG 1342–1345. (2009) Dickeya species relatedness and clade structure determined by Jock S, Donat V, Lopez MM, Bazzi C & Geider K (2002) Following comparison of recA sequences. International Journal of Systematic spread of fire blight in Western, Central and Southern Europe by and Evolutionary Microbiology 59, 2388–2393. molecular differentiation of Erwinia amylovora strains with PFGE Paulin JP (2000) Erwinia amylovora: general characteristics, analysis. Environmental Microbiology 4, 106–114. biochemistry and serology. In: Fire Blight, The Disease and its Jones A & Geider K (2001) II Gram negative bacteria. B. Erwinia and Causative Agent, Erwinia amylovora (Ed. Vanneste J), pp. 87–116. Pantoea. In: Guide for Identification of Plant Pathogenic Bacteria, CAB International, Wallingford (GB). 2nd edn (Ed. Schaad NW, Jones JB & Chum W), pp. 40–55. APS Persen U, Gottsberger RA & Reisenzein H (2011) Spread of Erwinia Press, St Paul (US). amylovora in apple and pear trees of different cultivars after artificial Kim WS, Gardan L, Rhim SL & Geider K (1999) Erwinia pyrifoliae inoculation. Acta Horticulturae 896, 319–330. sp., a novel pathogen that affects Asian pear trees (Pyrus pyrifolia). Pirc M, Ravnikar M, Tomlinson J & Dreo T (2009) Improved fire International Journal of Systematic Bacteriology 49, 899–906. blight diagnostics using quantitative real-time PCR detection of Kim WS, Jock S, Rhim SL & Geider K (2001) Molecular detection Erwinia amylovora chromosomal DNA. Plant Pathology 58, 872– and differentiation of Erwinia pyrifoliae and host range analysis of 881. the Asian pear pathogen. Plant Disease 85, 1183–1188. Powney R, Plummer K, Luck JE, Beer S & Rodoni B (2007) King EO, Ward M & Raney DE (1954) Two simple media for the Evaluation of PCR-based Protocols for the Detection of Erwinia demonstration of pyocyanin and fluorescein. Journal of Laboratory amylovora. XI International Workshop on Fire Blight Conference and Clinical Medicine 44, 301–307. Proceedings published in Acta Horticulturae. Lecomte P, Manceau C, Paulin JP & Keck M (1997) Identification by Salm H & Geider K (2004) Real-time PCR for detection and PCR analysis on plasmid pEA29 of isolates of Erwinia amylovora quantification of Erwinia amylovora, the causal agent of fire blight. responsible of an outbreak in Central Europe. European Journal of Plant Pathology 53, 602–610. Plant Pathology 103,91–98. Sasser M (1990) Identification of bacteria through fatty acid analysis. Lopez MM, Llop P, Gorris MT, Keck M, Penalver~ J, Donat V & In: Methods in Phytobacteriology (Ed. Klement F, Rudolf K & Cambra M (2006) European protocol for diagnosis of Erwinia Sands DC), pp. 199–204. Akademiai Kiado, Budapest (HU). amylovora. Acta Horticulturae 704,99–103. Starr MP, Cardona C & Folsom D (1951) Bacterial fire blight of Lopez MM, Rosello MM, Llop P, Ferrer S, Christen R & Gardan L raspberry. Phytopathology 41, 951–959. (2011) Erwinia piriflorinigrans sp. nov. a novel pathogen that causes Stoger€ A, Schaffer J & Ruppitsch W (2006) A rapid and sensitive necrosis of pear blossoms. International Journal of Systematic and method for direct detection of Erwinia amylovora in symptomatic Evolutionary Microbiology 61, 561–567. and asymptomatic plant tissues by polymerase chain reaction. Llop P, Bonaterra A, Penalver~ J & Lopez MM (2000) Development of Journal of Phytopathology 154, 469–473. a highly sensitive nested-PCR procedure using a single closed tube Tanii A, Tamura O & Ozaki M (1981) The causal agent of a fire for detection of Erwinia amylovora in asymptomatic plant material. blight-like disease. Annals of Phytopathological Society of Japan 47, Applied and Environmental Microbiology 66, 2071–2078. 102. Llop P, Caruso P, Cubero J, Morente C & Lopez MM (1999) A simple Taylor RK, Guilford P, Clark RG, Hal CN & Forster RLS (2001) extraction procedure for efficient routine detection of pathogenic Detection of Erwinia amylovora in plant material using novel

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polymerase chain reaction (PCR) primers. New Zealand Journal of each macerate into three sterile Eppendorf tubes. Use the Crop and Horticultural Science 29,35–43. tube containing 2 mL for the analysis. Store one tube with Temple TN & Johnson KB (2011) Evaluation of loop-mediated 1 mL of each sample at À20°C for subsequent analysis or isothermal amplification for rapid detection of Erwinia amylovora on confirmation; add 30% glycerol (Difco) to the other tube pear and apple fruit flowers. Plant Disease 95, 423–430. À ° Temple TN, Stockwell VO & Johnson K (2008) Development of a and store it at 80 C. rapid detection method for Erwinia amylovora by loop-mediated The isolation should be done on the same day as the isothermal amplification (LAMP). Acta Horticulturae 793, 497– maceration of the samples, as well as the enrichment and 504. the fixation of the slides for immunofluorescence. PCR Thomson SV (2000) Epidemiology of fire blight. In: Fire Blight, The analysis can be performed at earliest convenience, using the Disease and Its Causative Agent, Erwinia amylovora (Ed. Vanneste J), 1 mL stored at À20°C. pp. 9–36. CAB International, Wallingford (GB). van der Zwet T (2002) Present worldwide distribution of fire blight. Acta Horticulturae 590,33–34. Samples from asymptomatic material van der Zwet T & Beer S (1995) Fire blight – its nature, prevention and control. A Practical Guide to Integrated Disease Management. After favourable conditions for multiplication of the causal USDA Agricultural Information Bulletin no. 631. USDA, agent of fire blight have been confirmed, or at least when Washington (US). the average temperature is higher than 18°C (van der Zwet van der Zwet T & Keil HL (1979) Fire Blight: A Bacterial Disease of & Beer, 1995), collect flowers, shoots, fruitlets or stem seg- Rosaceous Plants. USDA Handbook no. 510. USDA, Washington (US). ments in sterile bags or containers. For nursery plants: cut young shoots approximately 20 cm long from the most sus- ceptible hosts available, disinfecting scissors or pruning shears between plants. For plants growing in the field, cut Appendix 1 – Extraction procedures flowers when available, and/or young shoots about 20 cm long, disinfecting between plants. Take flowers or peduncle Samples from symptomatic material and the base of the limb of mature leaves or stem segments of selected plants. If analyses need to be performed in win- The samples may be processed in different buffers according ter, collect 5–10 buds per plant. to the tests to be performed. The use of freshly prepared anti- Direct analysis of asymptomatic samples is usually nega- oxidant maceration buffer (Appendix 2) is required for suc- tive for E. amylovora due to the low bacterial population. cessful enrichment of E. amylovora in plant material (Gorris Consequently it is recommended to enrich the samples et al., 1996b). This buffer has been evaluated in a test perfor- (Appendix 4) in antioxidant buffer (Gorris et al., 1996a) mance study. Sterile phosphate-buffered saline, pH 7.2 (Appendix 2). When analysing asymptomatic material, 10 mM (PBS) (Appendix 2) or sterile water can also be used enrichment should be done for 72 h at approximately for direct isolation, immunofluorescence or PCR. 25°C. Carefully select the plant parts showing the freshest Weigh 0.1–1 g plant material and use for maceration symptoms, with exudates if possible. The leading edge of in antioxidant buffer (Appendix 2) (not in PBS or water), lesions on each organ should be selected for analysis. The in the same amount as for symptomatic material (above). exudates can be processed separately, in 1–4.5 mL sterile It is not advised to analyse larger amounts of plant mate- water or buffer. For shoots, take pieces of symptomatic rial in one sample. Process the samples immediately by shoots, including leaves, at the margin between the necrotic enrichment, followed by DASI-ELISA and/or PCR and/or and healthy tissue. Take one or several flowers, with pedun- isolation, according to the protocols described in the cles. Take one or several leaves and petioles, preferably Appendices 3–13. At the same time, direct isolation can select leaves with vein necrosis, but not fully necrosed. also be performed using the extract, or later with the Take one or several fruits. For stems or trunk, peel off the sample kept at À20°C with glycerol for a short time. external bark of stems showing symptoms using a sterile A sampling procedure for the analysis of twigs of asymp- scalpel and take pieces underneath with typical subcortical tomatic woody material in nurseries is presented. A sample discoloration symptoms. consists of 100 twigs, approximately 10 cm long, from 100 The protocol evaluated in a test performance study was plants. If there are several plant genera in the lot, these as follows: cut 0.1 g shoots, flowers, leaves, stems, trunks should be represented equally in the sample (with a or fruits into pieces and place in plastic bags. Add to each maximum of three genera per sample). From each sample, bag 4.5 mL of the antioxidant maceration buffer described individual twigs are tested or 30 cut twigs are randomly by Gorris et al. (1996a) (Appendix 2). Allow the samples taken and cut into four pieces (120 stem pieces). Place to macerate for at least 5 min. Crush the plant material these for 1.5 h in a rotary shaker at room temperature in slightly in the plastic bag with a rubber hammer, or with a sterile PBS (Appendix 2) with 0.1% Tween 20 in Bioreba homogenizer or similar equipment, avoiding drop- Erlenmeyer flasks. Filter with a paper held in a sintered lets splashing out of the bag. Hold the samples on ice for a glass filter (n2 = 40–100 lm) using a vacuum pump and few minutes and decant approximately 2, 1 and 1 mL of collect the filtrate. Use the filtrate directly for analysis, or

ª 2013 OEPP/EPPO, Bulletin OEPP/EPPO Bulletin 43, 21–45 30 Diagnostics centrifuge it for 20 min at 10 000 g. Suspend the pellet in Media 4.5 mL sterile PBS (Appendix 2). Depending on the sea- son, a similar procedure may be applied for leaves, shoots, Media are sterilized by autoclaving at 120°C for 15 min flowers or buds. unless stated otherwise. Depending on the season of survey, the expected recov- ery of E. amylovora will vary, being high in summer (pro- Ayers’ medium (Ayers et al., 1919) vided weather conditions are favourable to the pathogen) and low in winter. NH4H2PO4 1.0 g Whichever procedure is followed, prepare 3 Eppendorf KCl 0.2 g tubes for each sample with about 2, 1 and 1 mL macerate, MgSO4 0.2 g and use them as for symptomatic material (see above). Bromothymol blue (solution 0.2%) 75 mL Distilled water to 1 L Appendix 2 – Preparation of media and buffers Adjust pH to 7. Buffers CCT medium (Ishimaru & Klos, 1984) Phosphate buffered saline 10 mM, pH 7.2 (PBS) Sucrose 100 g NaCl 8.0 g Sorbitol 10.0 g KCl 0.2 g Niaproof 1.2 mL 2.9 g Crystal violet (sol. 0.1% ethanol) 2 mL Á Na2HPO4 12H2O Nutrient agar 23.0 g KH2PO4 0.2 g Distilled water to 1 L Distilled water to 1 L

– ° Sterilize by filtration. Adjust pH to 7.0 7.2; sterilize by autoclaving at 115 C for 10 min. Then prepare: thallium nitrate 2 mL (1% w/v aqueous solution); 0.05 g cycloheximide. Sterilize by fil- Antioxidant maceration buffer (Gorris et al., 1996a) tration (0.45 lm). Add to 1 L sterile medium (at about 45°C). Polyvinylpyrrolidone (PVP-10) 20.0 g Enrichment media: use CCT medium and King’s B medium Mannitol 10.0 g prepared in liquid form, without agar, for enrichment as Ascorbic acid 1.76 g Reduced glutathion 3.0 g described in Appendix 4. Tubes of at least 5 mL should be PBS 10 mm pH 7.2 1 L used and 0.9 mL medium added.

King’s B medium (King et al., 1954) Adjust pH to 7. Sterilize by filtration. This buffer should be prepared immediately before use. Proteose peptone No. 3 20 g Glycerol 10 mL

K2HPO4 1.5 g Extraction buffer (Llop et al., 1999) 1.5 g MgSO4Á7H2O Agar 15 g Tris HCl 31.52 g Distilled water to 1 L NaCl 14.6 g EDTA 9.3 g SDS 5.0 g Polyvinylpyrrolidone (PVP-10) 20.0 g – Distilled water to 1 L Adjust pH to 7.0 7.2.

Adjust pH to 7.5. Sterilize by filtration.

ª 2013 OEPP/EPPO, Bulletin OEPP/EPPO Bulletin 43, 21–45 Erwinia amylovora 31

Levan medium 2. Enrichment DASI-ELISA Yeast extract 2 g After the enrichment step, the use of validated specific Bactopeptone 5 g monoclonal antibodies is recommended to avoid cross- NaCl 5 g reactions. A complete kit based on polyclonal and mono- Sucrose 50 g + Agar 20 g clonal antibodies (3B 5H IVIA), including extraction Distilled water to 1 L buffer, semi-selective media, ELISA plates and reagents, is available from Plant Print Diagnostics S.L. This com- mercial kit for Enrichment DASI-ELISA (Gorris et al., Adjust pH to 7–7.2. 1996b) has been validated in two test performance studies. It is based on the monoclonal antibodies and technique described in Gorris et al. (1996a,b). As positive controls, use aliquots of a sample extract that previously gave a Appendix 3 – Rapid serological screening negative result on testing, mixed with 108 cells of E. amy- tests lovora per mL. As negative controls, include a sample extract that has previously given a negative result for E. amylovora and a suspension of a non-E. amylovora 1. Immunofluorescence strain in PBS (Appendix 2). Follow the standard instructions described in PM 7/97 Before ELISA, treat the necessary amount of enriched Indirect immunofluorescence test for plant pathogenic extracts and controls in a water bath (or in a thermoblock) ° bacteria. at 100 C for 10 min, ensuring the tubes are not opened. Antibodies to E. amylovora currently used in detection Keep the remaining enriched samples for isolation and/or and identification tests: PCR. Process the boiled samples (once at room tempera- À ° • E. amylovora, polyclonal antibodies, for detection using ture) by ELISA on the same day or store them at 20 C IF test (validated in test performance studies), Loewe Bi- for subsequent analysis. This heat treatment is necessary ochemica GmbH. for optimum sensitivity and specificity using the monoclo- et al. • IVIA EPS 1430, polyclonal antibodies, for detection nal antibodies obtained by Gorris (1996a). Then fol- using IF test (validated in test performance studies), Plant low the instructions for DASI-ELISA given in PM 7/101 ELISA tests for plant pathogenic bacteria Print Diagnostics, S.L. (1) (EPPO, • IVIA Mab 7 A, monoclonal antibodies, for detection 2010) and those of the manufacturers of the commercial using IF test (validated in test performance studies), Plant kit. Print Diagnostics, S.L. Positive ELISA readings in negative control wells Use undiluted macerates and 1:10 and 1:100 dilutions in indicate cross-contaminations or non-specific antibody bind- PBS (Appendix 2) to spot windows of IF slides. Prepare ing. In either case, the test should be repeated or a second one slide for each sample and its dilutions. Use the mono- test based on a different biological principle should be clonal or polyclonal antibodies at the appropriate dilutions performed. Performance criteria available in PBS (Appendix 2). Determination of the contamination 2.1 Analytical sensitivity data level is usually not required. Immunofluorescence is not À1 recommended after enrichment of samples. 10 cfu mL plant extract in King’s B and in CCT Performance criteria available (Gorris et al., 1996b). 10–102 cfu mLÀ1 plant extract in King’s B and 103– 1.1 Analytical sensitivity data 4 À1 103–104 cfu mLÀ1 plant extract 10 cfu mL plant extract in CCT (in the perfor- 1.2 Analytical specificity data mance study in 2010). Not tested for polyclonal antibodies. 2.2 Analytical specificity data + For monoclonal antibody 7A For monoclonal antibodies 3B 5H E. amylovora Target organisms tested: 50 E. amylovora strains. All Target organisms tested: 250 strains. All et al. positive in the test conditions. positive in the test conditions (Gorris , 1996a,b; Non-target organisms tested: 123 unidentified strains and IVIA tests). from E. amylovora hosts, 121 negative and two Er- Non-target organisms tested: 258 unidentified strains E. amylovora winia-related bacteria positive (Erwinia persicina from hosts and 45 strains of other and Dickeya sp.). plant pathogenic bacteria. They were all negative et al. 1.3 Data on repeatability (Gorris , 1996a,b). In IVIA: 100% 2.3 Data on repeatability 1.4 Data on reproducibility In IVIA: 100% In IVIA: 60% 2.4 Data on reproducibility In IVIA: 98%

ª 2013 OEPP/EPPO, Bulletin OEPP/EPPO Bulletin 43, 21–45 32 Diagnostics

5 mL tubes prepared in advance with the same volume of 3. Lateral flow devices each enrichment medium. Do not use Eppendorf tubes, for Two lateral flow devices were evaluated in performance maximum aeration. As additional negative controls, prepare studies in 2009 and 2010 and showed similar results. They three tubes with 0.9 mL maceration buffer (Appendix 2) were appropriate for the analysis of symptomatic plants and add the same volume of the same buffer and of each only, and are based on E. amylovora polyclonal antibodies enrichment medium (Appendix 2). Incubate at 25°C for that are non-specific. Follow the manufacturer’s instructions 48 h without shaking. Incubate for 72 h when very low when performing the analysis. numbers of E. amylovora are expected, as indicated above Performance criteria available for Ea Agri-strip (Bioreba) for asymptomatic samples. 3.1 Analytical sensitivity data (in a test performance study performed in 2010) À Appendix 5 – Isolation 105–106 cfu mL 1 plant extract 3.2 Analytical specificity data 1. Direct isolation Target organisms tested: 39 strains all positive Non-target organisms tested: 61 strains (all negative except Use CCT, King’s B and levan (or nutrient agar sucrose, E. pirifoliae, E. tasmaniensis and E. piriflorinigrans). NAS or NSA) media (Appendix 2). Plating on three media False positive results with E. pirifoliae, E. tasmaniensis is advised for maximum recovery of E. amylovora particu- and E. piriflorinigrans are also reported in AGES (AT) larly when samples are in poor condition. Prepare 1:10 and and Braun-Kiewnick et al. (2011). 1:100 dilutions of each macerate (Appendix 1) in PBS 3.3 Data on repeatability (Appendix 2). Pipette 50 lL of the diluted and undiluted In IVIA: 94% macerates onto separate plates of each medium. Start with 3.4 Data on reproducibility the 1:100 dilution and proceed to the undiluted macerate. In IVIA: 96% Use sterile loops or dip a glass spreader in denatured etha- Performance criteria available for Pocket Diagnostics nol, flame and allow to cool. Carefully spread the pipetted (Forsite Diagnostics, York, UK) volumes by triple streaking. Plate a 103,104 and 3.5 Analytical sensitivity data (in the performance study 105 cfu mLÀ1 dilution of a pure culture of E. amylovora as performed in 2010) a quality control of the media. Incubate the plates at approx- 105–106 cfu mLÀ1 plant extract imately 25°C for 48–72 h. Final reading is at 72–96 h. 3.6 Analytical specificity data Colonies of E. amylovora on CCT appear at about 48 h Non-target organisms tested: false positive results with and are pale violet, circular, highly convex to domed, E. pirifoliae, E. tasmaniensis and E. piriflorinigrans smooth and mucoid after 72 h, showing slower growth than are reported in AGES (AT). on King’s B or levan. CCT medium inhibits most pseudo- 3.7 Data on repeatability monads but not Pantoea agglomerans. Colonies of E. In IVIA: 94% amylovora on King’s B appear at 24 h and are creamy 3.8 Data on reproducibility white, circular, tending to spread and non-fluorescent under In IVIA: 96% UV light at 366 nm after 48 h. This allows distinction from fluorescent pseudomonads. Colonies of E. amylovora on levan medium appear at 24 h and are whitish, circular, Appendix 4 – Enrichment domed, smooth and mucoid after 48 h. Levan-negative col- onies of E. amylovora have also been reported (Bereswill Enrichment is used to multiply the initial population of cul- et al., 1997). Figure 3 shows the appearance of cultures in turable E. amylovora in the sample. It is needed before the three media. detection by ELISA because of the low level of sensitivity Obtain pure cultures from individual suspect colonies of of this technique when using specific monoclonal antibod- each sample by plating on King’s B medium. Identify pre- ies. It should also be used before isolation or before PCR sumptive colonies of E. amylovora as indicated in the Iden- (even in symptomatic samples) when a low number of cul- tification section. Store cultures on nutrient agar slants turable E. amylovora is expected (copper-treated samples, covered with vaseline oil at 10°C or for long-term storage old symptoms, unfavourable weather conditions for fire in 30% glycerol at À80°C or lyophilized. blight, winter, etc.) or when a high level of inhibitory The isolation is negative if no bacterial colonies with mor- organisms are expected. After preparation of the samples in phology similar to E. amylovora are observed after 96 h in the freshly prepared antioxidant buffer, use of two validated any of the three media (provided no inhibition is suspected media is advised [one non-selective (King’s B) and one due to competition or antagonism) and that typical E. amylo- semi-selective (CCT) (Appendix 2)] because the composi- vora colonies are found in the positive controls. The isolation tion and number of microbiota is unknown. is positive if presumptive E. amylovora colonies are isolated As soon as the macerates have been made (Appendix 1), in at least one of the media used and the identification is con- dispense at least 0.9 mL of each sample into two sterile firmed by one of the methods indicated.

ª 2013 OEPP/EPPO, Bulletin OEPP/EPPO Bulletin 43, 21–45 Erwinia amylovora 33

Performance criteria available coloured precipitate (brown or green) at the bottom of the 1.1 Analytical sensitivity data (in a performance study in tubes, carefully take it while discarding the supernatant to 2010): obtain a cleaner DNA. Resuspend the pellet in 200 lLof 103 cfu mLÀ1 in King’s B; 10–102 cfu mLÀ1 in levan water. Use for PCR reaction or store at À20°C. and CCT 2. DNA extraction based on the procedure described 1.2 Analytical specificity data by Taylor et al. (2001) but with minor modifications Not evaluated (elimination of Gene Releaser which was considered 1.3 Data on repeatability unnecessary). In IVIA: 100% Add 200 lL of each macerate and/or of the enriched 1.4 Data on reproducibility macerates in 500 lL of buffer [140 mM NaCl; 50 mM In IVIA: 100% KCl; 0.05% Tween 20; 2% polyvinylpyrrolidone (PVP) 10; 0.4% BSA, distilled water 1 L] for 15 min at room temper- ature. The resulting suspension can be used for PCR reac- 2. Enrichment isolation tion or stored at À20°C. Plate the enrichments only on CCT plates (Appendix 2). 3. DNA extraction using RED-Extract N-Amp T Plant kit Spread 50 lL of each enriched extract and of the 1:10, (Sigma-Aldrich, USA) 1:100 and 1:1000 dilutions prepared in PBS (Appendix 2) Take 100 lL of each macerate and/or the enriched mac- by triple streaking (as for isolations) to obtain isolated colo- erates into an Eppendorf tube. Add 150 lL extraction solu- nies. Incubate at approximately 25°C for 72–96 h. The use tion (kit) supplemented with 0.1% (v/v) Triton X-100 and of only this semi-selective medium and dilutions is advised 0.05% (v/v) Nonidet NP-40 Igepal. Incubate at 95°C for because of the possible abundant multiplication of different 30 min on a heating block. Transfer 50 lL of extract to a bacteria during the enrichment step. new tube and dilute it with 50 lL of the dilution buffer Performance criteria available (kit). Use for PCR reaction or store at À20°C. 2.1 Analytical sensitivity data (in a performance study in The three DNA extraction protocols were validated in test 2010) performance studies in 2009 and 2010, with four PCR proto- 10 cfu mLÀ1 after enrichment in CCT cols (Appendices 8–11), and showed comparable results. 10–102 cfu mLÀ1 after enrichment in King’s B Their efficiency was not improved after diluting the extracts 2.2 Analytical specificity data 1:10, suggesting that no, or few, inhibitors were present. The Not evaluated PCR protocols are detailed in the following appendices. 2.3 Data on repeatability Performance criteria In IVIA: 100% Performance criteria are provided together with the diff- 2.4 Data on reproducibility erent PCR tests. In IVIA: 100%

Conventional PCR protocols Many PCR primers and tests for conventional PCR exist Appendix 6 – DNA extraction and for E. amylovora diagnosis, detection and identification. conventional PCR Some can be used reliably (Guilford et al., 1996; Taylor et al., 2001) but others have shown specificity problems. DNA extraction This is the case for the test described by Maes et al. Two protocols for DNA extraction from plant samples (1996), which also amplifies Erwinia piriflorinigrans iso- (Llop et al., 1999; Taylor et al. 2001) and one commercial lated from necrotic pear blossoms (Lopez et al., 2011). kit have been validated in the test performance studies. Tests described by Bereswill et al. (1992), McManus & Other commercial kits for extracting DNA are available, Jones (1995) and Llop et al. (2000) are based in sequences but they have not been evaluated. of the plasmid pEA29 that is not universal in E. amylovora 1. DNA extraction according to Llop et al. (1999) strains (Llop et al., 2006; Llop et al., 2011). Use 1 mL of each macerate and/or 1 mL of the enriched Two protocols for conventional PCR were validated in a macerates prepared according to Appendices 1 and 4. Cen- test performance study in 2002 and four in test performance trifuge the macerates at 10 000 g for 5 min at room tem- studies conducted in 2009 and 2010. The primers and pro- perature. Discard the supernatant, resuspend the pellet in tocols validated in 2002 were those of Bereswill et al. 500 lL extraction buffer (Llop et al., 1999 Appendix 2) (1992), Llop et al. (2000), with or without previous enrich- and shake for 1 h at room temperature. Centrifuge at ment. The primers and protocols validated in 2009 and 4000 g for 5 min. Take 450 lL of the supernatant and add 2010 were those of Llop et al. (2000), Taylor et al. (2001), the same volume of isopropanol, invert and leave for Stoger€ et al. (2006) and Obradovic et al. (2007). Taking 30 min–1 h at room temperature. Centrifuge at 10 000 g into account the discovery of fully virulent E. amylovora for 5 min, discard the supernatant and dry. If there is still a strains without pEA29 (Llop et al., 2006) and the experi-

ª 2013 OEPP/EPPO, Bulletin OEPP/EPPO Bulletin 43, 21–45 34 Diagnostics ence from different countries (Powney et al., 2007), it is Table (continued) necessary to use two PCR tests, one with primers based on pEA29 sequences and the second based on chromosomal Working Volume per Final sequences. Conventional PCR can be applied using the concentration reaction (lL) concentration primers and conditions validated in test performance stud- MgCl 50 mM 3.00 3 mM ies. Precautions should be taken to avoid contamination of 2 dNTPs 10 mM 1.00 0.2 mM of samples. Prepare positive controls in a laboratory separate each dNTP from the one where the samples will be tested. Primer A 10 lM 0.50 0.1 lM Primer B 10 lM 0.50 0.1 lM Taq polymerase 5 U lLÀ1 0.20 1 U Subtotal 45.00 DNA 5.00 Appendix 7 – PCR according to Bereswill Total reaction 50.00 et al. (1992) volume of a single PCR reaction

1. General information 2.3 PCR cycling conditions: 5 min at 93°C, 40 cycles of 1.1 This test was widely used for many years. The 30 s at 93°C, 30 s at 52°C and 1 min 15 s at 72°C sequences of the primers are based in the plasmid and a final step of 10 min at 72°C. pEA29, and it has been discovered that it is not uni- 2.4 Observations: if the expected target concentration is versal for all E. amylovora strains (Llop et al., 2006; high, i.e. in enriched samples, it is highly recom- Llop et al., 2011). In addition, it frequently shows mended to carry out a tenfold dilution of the purified non-specific banding (see below). DNA solution in water or TE buffer before amplifica- 1.2 The test can be applied to any kind of plant material tion in order to dilute inhibitor compounds. Amplifi- or bacterial colonies. cation is performed on stock solution and the 1.3 The targeted sequences are in the plasmid pEA29. dilution. 1.4 Oligonucleotides: A: 5′-CGG TTT TTA ACG CTG GG-3′ B: 5′-GGG CAA ATA CTC GGA TT-3′ 3. Essential procedural information 1.5 The amplicon size is 900 bp. (Bereswill et al., 3.1 Controls: 1992). However, variations can occur between 900 For a reliable test result to be obtained, the following and 1100 bp (Lecomte et al., 1997), due to the num- (external) controls should be included for each series ber of 8 bp repeat sequences within the fragment of nucleic acid isolation and amplification of the tar- (Jones & Geider, 2001). get organism and target nucleic acid. 1.6 Enzyme: the test performance study was performed • Negative isolation control (NIC) to monitor con- with a DNA polymerase from Biotools. tamination during nucleic acid extraction: nucleic acid extraction and subsequent amplification, pref- 2. Methods erably of a sample of uninfected matrix or, if not available, clean extraction buffer. 2.1 Nucleic acid extraction and purification: • Positive isolation control (PIC) to ensure nucleic For plant material, three DNA extraction methods acid of sufficient quantity and quality is isolated: are described in Appendix 6. These DNA extraction nucleic acid extraction and subsequent amplifica- methods were evaluated in a test performance study. tion of the target organism or a matrix sample that The sensitivity of the tests increases after enrichment contains the target organism (e.g. naturally of the samples in King’s B and CCT (Lopez et al., infected host tissue or host tissue spiked with the 2006). target organism). 2.2 Polymerase chain reaction • Negative amplification control (NAC) to rule out false positives due to contamination during the preparation of the reaction mix: amplification of Working Volume per Final concentration reaction (lL) concentration molecular-grade water that was used to prepare the reaction mix. Molecular-grade 34.80 • Positive amplification control (PAC) to monitor the water efficiency of amplification: amplification of nucleic 9 9 PCR buffer 10 5.00 1 acid of the target organism. This can include nucleic acid extracted from the target organism, total (continued) nucleic acid extracted from infected host tissue,

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whole genome amplified DNA or a synthetic control Appendix 8 – PCR according to Taylor et al. (e.g. cloned PCR product). For PCR tests not per- (2001) formed on bacterial colonies, the PAC should pref- erably be near the limit of detection. 1. General information As an alternative (or in addition) to the external positive controls (PIC and PAC), internal positive controls (IPC) 1.1 This test is universal for all known E. amylovora can be used to monitor each individual sample separately. strains to date. The protocol was validated in a test Positive internal controls may either be genes present in the performance study in 2010. matrix DNA or added to the DNA solutions. 1.2 The test can be applied to any kind of plant material Alternative internal positive controls can include: and to bacterial colonies. • Specific amplification or co-amplification of endogenous 1.3 The targeted sequences are chromosomal (Taylor nucleic acid, using conserved primers that amplify con- et al., 2001). served non-pest target nucleic acid that is also present in 1.4 Oligonucleotides: the sample (e.g. plant cytochrome oxidase gene or G1-F: 5′-CCT GCA TAA ATC ACC GCT GAC eukaryotic 18S rDNA) AGC TCA ATG-3′ • amplification of samples spiked with exogenous nucleic G2-R: 5′-GCT ACC ACT GAT CGC TCG AAT (control sequence) acid that has no relation to the target CAA ATC GGC-3′ nucleic acid (e.g. synthetic internal amplification controls) 1.5 The amplicon size is 187 bp. or amplification of a duplicate sample spiked with the tar- 1.6 Enzyme: the test performance study was performed get nucleic acid. with a DNA polymerase from Biotools. 3.2 Interpretation of results Verification of controls 2. Methods • NIC and NAC should produce no amplicons • PIC and PAC should produce amplicons of 900 bp size. 2.1 Nucleic acid extraction and purification: for plant • If IPCs are used, the amplicons should be of the expected material, three DNA extraction methods are size. described in Appendix 6. These DNA extraction When these conditions are met: methods were evaluated in the test performance • A test will be considered positive if amplicons of 900 bp study. are produced 2.2 Polymerase chain reaction • A test will be considered negative if it produces no band or a band of a different size. • If the test gives a value >900 and  1100 bp, confirma- Working Volume per Final tion with another test is recommended. concentration reaction (lL) concentration • Tests should be repeated if any contradictory or unclear Molecular-grade water 14.3 results are obtained. PCR buffer 109 2.5 19

MgCl2 50 mM 0.75 1.5 mM dNTPs 10 mM 0.25 0.1 mM of 4. Performance criteria available each dNTP G1-F primer 10 lM 1.00 0.4 lM When available, performance criteria are provided for the G2-F primer 10 lM 1.00 0.4 lM À PCR test after enrichment. Taq polymerase 5 U lL 1 0.2 1 U 4.1 Analytical sensitivity data (in the performance study Subtotal 20.00 in 2002, after DNA extraction according to Llop DNA 5.00 Total reaction 25.00 et al., 1999) volume of a single 5– 6 À1 10 10 cfu mL plant extract PCR reaction 102–103 cfu mLÀ1 plant extract after enrichment of the samples in King’s B or CCT. 2.3 PCR cycling conditions: 3 min at 95°C, 40 cycles of 4.2 Analytical specificity data 30 s at 94°C, 30 s at 60°C and 1 min at 72°C, a According to Bereswill et al. (1992) final step of 5 min at 72°C and cooling at 15°C. Target organisms tested: 5 strains all positive 2.4 Observations: if the expected target concentration is Non-target organisms tested: 5 strains all negative high, i.e. in enriched samples, it is highly recom- 4.3 Data on repeatability mended to carry out a tenfold dilution of the purified In IVIA: 92% DNA solution in water or TE buffer before amplifica- 4.4 Data on reproducibility tion in order to dilute inhibitor compounds. Amplifica- In IVIA: 84% tion is performed on stock solution and the dilution.

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3. Essential procedural information When these conditions are met: • A test will be considered positive if amplicons of 3.1 Controls 187 bp are produced For a reliable test result to be obtained, the following • A test will be considered negative if it produces (external) controls should be included for each series no band or a band of a different size. of nucleic acid isolation and amplification of the tar- • Tests should be repeated if any contradictory or get organism and target nucleic acid. unclear results are obtained. • Negative isolation control (NIC) to monitor con- tamination during nucleic acid extraction: nucleic acid extraction and subsequent amplification of a 4. Performance criteria available sample of uninfected host tissue or clean extraction Performance criteria are provided for the PCR test without buffer. enrichment • Positive isolation control (PIC) to ensure nucleic 4.1 Analytical sensitivity data (according to a test perfor- acid of sufficient quantity and quality is isolated: mance study in 2010) nucleic acid extraction and subsequent amplifica- 103–104 cfu mLÀ1 plant extract after DNA extraction tion of the target organism or a matrix sample following Llop et al. (1999) that contains the target organism (e.g. naturally 104–105 cfu mLÀ1 plant extract after DNA extraction infected host tissue or host tissue spiked with the modified after Taylor et al. (2001) target organism). 103–104 cfu mLÀ1 plant extract after DNA extraction • Negative amplification control (NAC) to rule out using RED-Extract N-Amp T Plant kit (Sigma- false positives due to contamination during the Aldrich) preparation of the reaction mix: amplification of 4.2 Analytical specificity data (according to Taylor molecular-grade water that was used to prepare et al., 2001) the reaction mix. Target organisms tested: 69 strains all positive. Neg- • Positive amplification control (PAC) to monitor the ative reaction with strains from Rubus sp. efficiency of amplification: amplification of nucleic Non-target organisms tested: 49 strains all negative acid of the target organism. This can include 4.3 Data on repeatability nucleic acid extracted from the target organism, In IVIA: 100% total nucleic acid extracted from infected host tis- 4.4 Data on reproducibility sue, whole genome amplified DNA or a synthetic In IVIA: 100% control (e.g. cloned PCR product). For PCR tests not performed on bacterial colonies, the PAC should preferably be near the limit of detection. Appendix 9 – PCR according to Stoger€ et al. As an alternative (or in addition) to the external positive (2006) controls (PIC and PAC), internal positive controls (IPC) can be used to monitor each individual sample separately. 1. General information Positive internal controls can either be genes present in the 1.1 This method uses the same primers as the nested matrix DNA or added to the DNA solutions. PCR from Llop et al. (2000), see below. Alternative internal positive controls can include: 1.2 The test can be applied to any kind of plant material • Specific amplification or co-amplification of endoge- or bacterial colonies. nous nucleic acid, using conserved primers that 1.3 The target sequences are located in the plasmid amplify conserved non-pest target nucleic acid that is pEA29. also present in the sample (e.g. plant cytochrome oxi- 1.4 Oligonucleotides: dase gene or eukaryotic 18S rDNA) PEANT 1: 5′-TAT CCC TAA AAA CCT CAG TGC-3′ • amplification of samples spiked with exogenous PEANT 2: 5′-GCA ACC TTG TGC CCT TTA-3′ nucleic (control sequence) acid that has no relation 1.5 The amplicon size is 391 bp. with the target nucleic acid (e.g. synthetic internal 1.6 Enzyme: included in the RED-Extract-N-Amp PCR amplification controls) or amplification of a dupli- Ready+ mix (Sigma-Aldrich). cate sample spiked with the target nucleic acid. 3.2 Interpretation of results Verification of controls 2. Methods • NIC and NAC should produce no amplicons 2.1 Nucleic acid extraction and purification. Stoger€ et al. • PIC and PAC should produce amplicons of (2006) recommended that this method should be 187 bp size. used with DNA extracted with the RED-Extract N- • If IPC are used, the amplicons should be of the Amp T Plant kit (Sigma-Aldrich); details are pro- expected size. vided in Appendix 6.

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2.2 Polymerase chain reaction ular-grade water that was used to prepare the reaction mix. • Positive amplification control (PAC) to monitor the Working Volume per Final efficiency of amplification: amplification of nucleic l concentration reaction ( L) concentration acid of the target organism. This can include Molecular-grade 5.00 nucleic acid extracted from the target organism, water total nucleic acid extracted from infected host tis- RED-Extract- 10.00 sue, whole genome amplified DNA or a synthetic N-Amp control (e.g. cloned PCR product). For PCRs not PCR Ready Mix performed on bacterial colonies, the PAC should (Sigma) preferably be near the limit of detection. MgCl2 Included in Included in master mix master mix As an alternative (or in addition) to the external positive dNTPs Included in Included in controls (PIC and PAC), internal positive controls (IPC) master mix master mix can be used to monitor each individual sample separately. PEANT 1 10 lM 0.5 0.25 lM Positive internal controls can either be genes present in the PEANT 2 10 lM 0.5 0.25 lM matrix DNA or added to the DNA solutions. Taq polymerase Included in Included in Alternative internal positive controls can include: master mix master mix • Specific amplification or co-amplification of endogenous Subtotal 16.00 DNA 4.00 nucleic acid, using conserved primers that amplify con- Total reaction 20.00 served non-pest target nucleic acid that is also present in volume of a the sample (e.g. plant cytochrome oxidase gene or single PCR eukaryotic 18S rDNA) reaction • amplification of samples spiked with exogenous nucleic (control sequence) acid that has no relation with the tar- 2.3 PCR cycling conditions: 95°C for 5 min; 35 cycles get nucleic acid (e.g. synthetic internal amplification con- of: 95°C for 15 s, 58°C for 30 s, and 72°C for 45 s; trols) or amplification of a duplicate sample spiked with and a final step of 72°C, 5 min, and cooling at 15°C. the target nucleic acid. 2.4 Observations: if the expected target concentration is 3.2 Interpretation of results: high, i.e. in enriched samples, it is highly recom- Verification of controls mended to carry out a tenfold dilution of the purified • NIC and NAC should produce no amplicons DNA solution in water or TE buffer before amplifi- • PIC and PAC should produce amplicons of 391 bp cation, to dilute inhibitor compounds. Amplification • If IPC are used, the amplicons should be of the expected is performed on stock solution and the dilution. size When these conditions are met: • A test will be considered positive if amplicons of 391 bp 3. Essential procedural information are produced 3.1 Controls • A test will be considered negative if it produces no band For a reliable test result to be obtained, the follow- or a band of a different size. ing (external) controls should be included for each • Tests should be repeated if any contradictory or unclear series of nucleic acid isolation and amplification of results are obtained. the target organism and target nucleic acid. • Negative isolation control (NIC) to monitor con- 4. Performance criteria available tamination during nucleic acid extraction: nucleic acid extraction and subsequent amplification of a Performance criteria are provided for the PCR test without sample of uninfected host tissue or clean extrac- enrichment tion buffer. 4.1 Analytical sensitivity data (in the performance study • Positive isolation control (PIC) to ensure nucleic in 2010 acid of sufficient quantity and quality is isolated: 104–106 cfu mLÀ1 plant extract after DNA extraction nucleic acid extraction and subsequent amplifica- using RED-Extract N-Amp T Plant kit (Sigma- tion of the target organism or a matrix sample Aldrich) that contains the target organism (e.g. naturally 4.2 Analytical specificity data infected host tissue or host tissue spiked with the Not evaluated target organism). 4.3 Data on repeatability • Negative amplification control (NAC) to rule out In IVIA: 92% false positives due to contamination during prepa- 4.4 Data on reproducibility ration of the reaction mix: amplification of molec- In IVIA: 80%

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Appendix 10 – PCR according to Gottsberger before amplification, to dilute inhibitor compounds. adapted from Obradovic et al. (2007) Amplification is performed on the stock solution and the dilution. 1. General information 1.1 The original protocol and primers from Obradovic 3. Essential procedural information et al. (2007) were modified by Gottsberger for opti- 3.1 Controls mized specificity and maximum sensitivity in plant For a reliable test result to be obtained, the follow- samples. The protocol was validated in the 2010 test ing (external) controls should be included for each performance study. series of nucleic acid isolation and amplification of 1.2 The test can be applied to any kind of plant material the target organism and target nucleic acid. or bacterial colonies. • Negative isolation control (NIC) to monitor con- 1.3 The targeted sequences are chromosomal. tamination during nucleic acid extraction: nucleic 1.4 Oligonucleotides: acid extraction and subsequent amplification of a FER1-F: 5′-AGC AGC AAT TAA TGG CAA GTA sample of uninfected host tissue or clean extrac- TAG TCA-3′ tion buffer rgER2R: 5′-AAA AGA GAC ATC TGG ATT CAG • Positive isolation control (PIC) to ensure nucleic ACA AT-3′ acid of sufficient quantity and quality is isolated: 1.5 The amplicon size is 458 bp. nucleic acid extraction and subsequent amplifica- 1.6 Enzyme: the test performance study was performed tion of the target organism or a matrix sample with a DNA polymerase from Biotools. that contains the target organism (e.g. naturally infected host tissue or host tissue spiked with the 2. Methods target organism). • Negative amplification control (NAC) to rule out 2.1 Nucleic acid extraction and purification: for plant false positives due to contamination during the material, three DNA extraction methods are described preparation of the reaction mix: amplification of in Appendix 6. These DNA extraction methods were molecular-grade water that was used to prepare evaluated in a test performance study. the reaction mix. 2.2 Polymerase chain reaction • Positive amplification control (PAC) to monitor the efficiency of amplification: amplification of nucleic acid of the target organism. This can Working Volume per Final concentration reaction (lL) concentration include nucleic acid extracted from the target organism, total nucleic acid extracted from Molecular-grade 14.3 infected host tissue, whole genome amplified water DNA or a synthetic control (e.g. cloned PCR 19 PCR buffer 109 2.5 19 product). For PCRs not performed on bacterial MgCl2 (or 50 mM 0.75 1.5 mM alternatives, specify) colonies, the PAC should preferably be near the dNTPs 10 mM 0.25 0.1 mM of limit of detection. each of the As an alternative (or in addition) to the external positive dNTP controls (PIC and PAC), internal positive controls (IPC) FER1-F 10 lM 1.00 0.4 lM can be used to monitor each individual sample separately. l l rgER2R 10 M 1.00 0.4 M Positive internal controls can either be genes present in the Taq polymerase 5 U lLÀ1 0.2 1 U matrix DNA or added to the DNA solutions. Subtotal 20.00 DNA 5.00 Alternative internal positive controls can include: Total reaction 25.00 • Specific amplification or co-amplification of volume of a single endogenous nucleic acid, using conserved prim- PCR reaction ers that amplify conserved non-pest target nucleic acid that is also present in the sample (e.g. plant cytochrome oxidase gene or eukary- 2.3 PCR cycling conditions: 3 min at 94°C, 41 cycles of otic 18S rDNA) 10 s at 94°C, 10 s at 60°C and 30 s at 72°C, a final • amplification of samples spiked with exogenous step for 5 min at 72°C and cooling at 15°C. nucleic (control sequence) acid that has no rela- 2.4 Observations: if the expected target concentration tion with the target nucleic acid (e.g. synthetic is high, i.e. in enriched samples, it is highly rec- internal amplification controls) or amplification ommended to carry out a tenfold dilution of the of a duplicate sample spiked with the target purified DNA solution in water or TE buffer nucleic acid.

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3.2 Interpretation of results Internal primers PEANT1: 5′-TAT CCC TAA AAA Verification of controls CCT CAG TGC-3′ and PEANT2: 5′-GCA ACC TTG • NIC and NAC should produce no amplicons TGC CCT TTA-3′ • PIC and PAC should produce amplicons of 458 bp 1.5 The amplicon size is 391 bp. size. 1.6 Enzyme: the test performance study was performed • If IPC are used, the amplicons should be of the with a DNA polymerase from Biotools. expected size. When these conditions are met: 2. Methods • A test will be considered positive if amplicons of 458 bp are produced 2.1 Nucleic acid extraction and purification: for plant • A test will be considered negative if it produces no material three DNA extraction methods are band or a band of a different size. described in Appendix 6. These DNA extraction • Tests should be repeated if any contradictory or methods were evaluated in a test performance unclear results are obtained. study. 2.2 Polymerase chain reaction

4. Performance criteria available Performance criteria are provided for the PCR test without Working Volume per Final l enrichment concentration reaction ( L) concentration 4.1 Analytical sensitivity data (according to the test per- Molecular-grade 36.25 formance study in 2010) water À 103–104 cfu mL 1 plant extract after DNA extrac- PCR buffer 109 5.00 19 tion following Llop et al. (1999) MgCl2 50 mM 3.00 3 mM 104–105 cfu mLÀ1 plant extract after DNA extrac- dNTPs 10 mM 0.50 0.1 mM of tion following Taylor et al. (2001) modified and each dNTP PEANT1 10 lM 1.00 0.2 lM RED-Extract N-Amp T Plant kit (Sigma-Aldrich) PEANT2 10 lM 1.00 0.2 lM 4.2 Analytical specificity data AJ75 0.1 lM 0.32 0.00064 lM According to Obradovic et al. (2007) AJ76 0.1 lM 0.32 0.00064 lM Target organisms tested: 44 strains all positive Taq polymerase 5 U lLÀ1 0.60 3 U Non-target organisms tested: 30 strains all negative Subtotal 48.00 4.3 Data on repeatability DNA 2.00 Total reaction 50.00 In IVIA: 92% volume of a single 4.4 Data on reproducibility PCR reaction In IVIA: 90%

2.3 PCR conditions: 94°C for 4 min followed by 25 cycles of 94°C for 60 s and 72°C for 90 s. This first round PCR is followed in the same thermocycler by Appendix 11 – Nested PCR (Llop et al., 2000) a second denaturation step of 94°C for 4 min and 40 cycles of 94°C for 60 s, 56°C for 60 s, and 72°C for 1. General information 60 s, a final step of 72°C for 10 min and cooling at 1.1 Nested PCR in a single tube (Llop et al., 2000) uses 15°C. two sets of primers placed at the same time. Due to 2.4 Observations: if the expected target concentration is the different annealing temperatures, the two PCR high, i.e. in enriched samples, it is highly recom- reactions are performed consecutively. The external mended to carry out a tenfold dilution of the purified primers were designed by McManus & Jones DNA solution in water or TE buffer before amplifi- (1995); the internal primers are those described by cation, in order to dilute inhibitor compounds. Llop et al. (2000). Both are based on sequences Amplification is performed on stock solution and the from pEA29. dilution. 1.2 The test can be applied to any kind of plant mate- rial or bacterial colonies. 3. Essential procedural information 1.3 The targeted sequences are in the plasmid pEA29. 1.4 Oligonucleotides: 3.1 Controls External primers AJ75: 5′-CGT ATT CAC GGC TTC For a reliable test result to be obtained, the follow- GCA GAT-3′ and AJ76: 5′-ACC CGC CAG GAT AGT ing (external) controls should be included for each CGC ATA-3′ series of nucleic acid isolation and amplification of

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the target organism and target nucleic acid, respec- • A test will be considered negative if it produces no tively. band or a band of a different size. • Negative isolation control (NIC) to monitor con- • Tests should be repeated if any contradictory or tamination during nucleic acid extraction: nucleic unclear results are obtained. acid extraction and subsequent amplification of a sample of uninfected host tissue or clean extrac- 4. Performance criteria available tion buffer. • Positive isolation control (PIC) to ensure nucleic Performance criteria are provided for the PCR test without acid of sufficient quantity and quality is isolated: enrichment nucleic acid extraction and subsequent amplifica- 4.1 Analytical sensitivity data (according to the perfor- tion of the target organism or a matrix sample mance study in 2010) that contains the target organism (e.g. naturally 103–104 cfu mLÀ1 plant extract after DNA extraction infected host tissue or host tissue spiked with the following Llop et al. (1999) and following Taylor target organism). et al. (2001) modified • Negative amplification control (NAC) to rule out 4.2 Analytical specificity data false positives due to contamination during the According to Llop et al. (2000) preparation of the reaction mix: amplification of Target organisms tested: 71 strains all positive molecular-grade water that was used to prepare Non-target organisms tested: 40 strains all negative the reaction mix. 4.3 Data on repeatability • Positive amplification control (PAC) to monitor In IVIA: 98% the efficiency of amplification: amplification of 4.4 Data on reproducibility nucleic acid of the target organism. This can In IVIA: 96% include nucleic acid extracted from the target organism, total nucleic acid extracted from infected host tissue, whole genome amplified DNA or a synthetic control (e.g. cloned PCR Appendix 12 – Real-time PCR (Pirc et al., 2009) product). For PCRs not performed on bacterial colonies, the PAC should preferably be near 1. General information the limit of detection. 1.1 Real-time PCR tests designed by Pirc et al. (2009) As an alternative (or in addition) to the external positive are based on chromosomal sequences. controls (PIC and PAC), internal positive controls (IPC) 1.2 The test can be applied to any kind of plant mate- can be used to monitor each individual sample separately. rial or bacterial colonies. Positive internal controls can either be genes present in the 1.3 The targeted genes are amsC (Ams assay) and 16S- matrix DNA or added to the DNA solutions. 23S rRNA intergenic spacer region (ITS assay). Only Alternative internal positive controls can include: primers from amsC gene were evaluated in the test • Specific amplification or co-amplification of endogenous performance studies in 2009 and 2010. nucleic acid, using conserved primers that amplify con- 1.4 Oligonucleotides: served non-pest target nucleic acid that is also present in Ams116F: 5′-TCC CAC ATA CTG TGA ATC ATC the sample (e.g. plant cytochrome oxidase gene or CA-3′ eukaryotic 18S rDNA) Ams189R: 5′-GGG TAT TTG CGC TAA TTT TAT • amplification of samples spiked with exogenous nucleic TCG-3′ (control sequence) acid that has no relation with the tar- Ams141T: FAM-CCA GAA TCT GGC CCG get nucleic acid (e.g. synthetic internal amplification con- CGT ATA CCG-TAMRA trols) or amplification of a duplicate sample spiked with ITS15F: 5′-TGA GTA ATG AGC GAG CTA the target nucleic acid. AGT GAA G-3′ (3.2) Interpretation of results ITS93R: 5′-CGC AAT GCT CAT GGA CTC AA- Verification of controls 3′ • NIC and NAC should produce no amplicons ITS43T: FAM-AGG CGT CAG CGC GCA GCA • PIC and PAC should produce amplicons of 391 bp AC-TAMRA size. 1.5 Amplicon size in base pairs (including primer • If IPC are used, the amplicons should be of the sequences): Ams primers 74 bp; ITS primers 79 bp. expected size. 1.6 Enzyme: Included in the TaqMan Universal master When these conditions are met: mix (Applied Biosystems, USA). • A test will be considered positive if amplicons of 1.7 Real-time PCR system (ABI PRISM 7900 HT 391 bp are produced Sequence Detection System, Applied Biosystems)

ª 2013 OEPP/EPPO, Bulletin OEPP/EPPO Bulletin 43, 21–45 Erwinia amylovora 41

using the universal cycling conditions for all ampli- 2.3 PCR cycling conditions: 2 min at 50°C, 10 min at cons. 95°C, 40 cycles of 15 s at 95°C and 1 min at 60°C. 2.4 Note: if the expected target concentration is high, i.e. in enriched samples, it is highly recommended 2. Methods to carry out a tenfold dilution of the purified DNA 2.1 Nucleic acid extraction and purification: three solution in water or TE buffer before amplification, DNA extraction methods were used: (i) the silica- in order to dilute inhibitor compounds. Amplifica- column based DNeasy Plant Mini Kit (Qiagen); tion is performed on stock solution and the dilution. (ii) the magnetic bead based QuickPickTM SML Plant DNA Kit (Bio-Nobile, Turku, Finland) with 3. Essential procedural information KingFisherR mL system (Thermo Labsystem); and (iii) a simple extraction method (Llop et al., 3.1 Controls 1999). The DNeasy Plant Mini Kit was used For a reliable test result to be obtained, the follow- according to the manufacturer’s protocol for purifi- ing (external) controls should be included for each cation of total DNA from plant tissue with final series of nucleic acid isolation and amplification of DNA elution into 2 9 50 lL AE buffer. The pro- the target organism and target nucleic acid. tocol for extraction using the QuickPickTM SML • Negative isolation control (NIC) to monitor con- Plant DNA Kit was as follows: 100 lL sample tamination during nucleic acid extraction: nucleic was mixed with 400 lL lysis buffer and 25 lLof acid extraction and subsequent amplification of a proteinase K, incubated for 30 min at 65°C and sample of uninfected host tissue or clean extrac- centrifuged at 6000 g for 1 min. Lysate (300 lL) tion buffer. was transferred to tube 1 of a KingFisher mL tube • Positive isolation control (PIC) to ensure nucleic strip. Strips contained 20 lL of MagaZorbTM Mag- acid of sufficient quantity and quality is isolated: netic Particles and 500 lL binding buffer (tube 1), nucleic acid extraction and subsequent amplifica- 800 lL wash buffer (tubes 2 and 3), 100 lL elu- tion of the target organism or a matrix sample tion buffer (tube 4) and 100 lL water (tube 5). that contains the target organism (e.g. naturally The instrument program Total_RNA_mL_1 in infected host tissue or host tissue spiked with the KingFisherR mL was used with minor modifica- target organism). tion: binding time in well A, 3 9 1 min release • Negative amplification control (NAC) to rule out plus 2 min binding; wash in well B 15 s; wash in false positives due to contamination during the well C, 15 s; elution in well D, 10 min. The sim- preparation of the reaction mix: amplification of ple extraction procedure was performed according molecular-grade water that was used to prepare to the protocol given by Llop et al. (1999), the reaction mix. explained in Appendix 6, except that only 100 lL • Positive amplification control (PAC) to monitor aliquots of crude sample extract were used (Pirc the efficiency of amplification: amplification of et al., 2009). nucleic acid of the target organism. This can 2.2 Polymerase chain reaction (for protocol using Ams include nucleic acid extracted from the target primers) organism, total nucleic acid extracted from infected host tissue, whole genome amplified DNA or a synthetic control (e.g. cloned PCR Working Volume per Final product). For PCRs not performed on bacterial l concentration reaction ( L) concentration colonies, the PAC should preferably be near the Molecular-grade water 1.00 limit of detection. TaqMan universal 29 5.00 19 As an alternative (or in addition) to the external positive master mix (Applied controls (PIC and PAC), internal positive controls can be Biosystems) used to monitor each individual sample separately. Positive l l Ams116F 10 M 0.90 0.9 M internal controls can either be genes present in the matrix l l Ams189R 10 M 0.90 0.9 M DNA or added to the DNA solutions. Ams141T 10 lM 0.20 0.2 lM Subtotal 8.00 Alternative internal positive controls can include: DNA 2.00 • Specific amplification or co-amplification of Total reaction 10.00 endogenous nucleic acid, using conserved primers volume of a that amplify conserved non-pest target nucleic acid single PCR that is also present in the sample (e.g. plant cyto- reaction chrome oxidase gene or eukaryotic 18S rDNA) • amplification of samples spiked with exogenous nucleic (control sequence) acid that has no rela-

ª 2013 OEPP/EPPO, Bulletin OEPP/EPPO Bulletin 43, 21–45 42 Diagnostics

tion with the target nucleic acid (e.g. synthetic Appendix 13 – Real-time PCR (Gottsberger, internal amplification controls) or amplification of 2010) a duplicate sample spiked with the target nucleic acid. 1. General information 3.2 Interpretation of results 1.1 Real-time PCR targeting a hypothetical protein-cod- The cycle cut-off value for this test is set at 38, and was ing gene was designed (Gottsberger, 2010). The accu- obtained using the equipment/materials and chemistry used racy in the 2010 test performance study could not be as described here. When necessary, the C cut-off value t tested with this real-time PCR; however, it was tested should be determined for the required control (e.g. when an by one laboratory in parallel with the real-time PCR internal positive control is used). The cycle cut-off value described in Pirc et al. (2009) and gave the same qual- needs to be verified in each laboratory when implementing itative results with the DNA extraction from Llop the test for the first time. et al. (1999) protocol. Verification of controls 1.2 The test can be applied to any kind of plant mate- • The PIC and PAC amplification curves should be rial or bacterial colonies. exponential. 1.3 The target sequences are located in the chromosome. • NIC and NAC should be negative (Ct  40) 1.4 Oligonucleotides: • PIC and PAC (and if relevant IPC) should have a hpEaF: 5′-CCG TGG AGA CCG ATC TTT TA-3′ Ct value below the relevant cut-off value. hpEaR: 5′-AAG TTT CTC CGC CCT ACG AT- When these conditions are met: 3′ • A test will be considered positive if it produces hpEaP: FAM-TCG TCG AAT GCT GCC TCT CT- an exponential amplification curve, a Ct MGB value <38 1.5 Amplicon size in base pairs (including primer • A test will be considered negative if it produces sequences): 138 bp no exponential amplification curve and a 1.6 Enzyme: included in the TaqMan Universal master Ct  40. mix (Applied Biosystems). • The test should be repeated if any contradictory 1.7 Real-time PCR system (Eppendorf Realplex Master- or unclear results are obtained, or if the Ct value cycler Epgradient S, Eppendorf, Hamburg, Ger- is between 38 and 40. many).

4. Performance criteria available 2. Methods Performance criteria are provided for the PCR test without 2.1 Nucleic acid extraction and purification: several enrichment DNA extraction methods were tested: (i) the silica- 4.1 Analytical sensitivity data column based DNeasy Plant Mini Kit (Qiagen); (ii) Validation data available from the test performance the magnetic bead based QuickPickTM SML Plant study in 2010 À DNA Kit (Bio-Nobile) and (iii) a simple extraction 103–104 cfu mL 1 plant extract after DNA extrac- method (Llop et al., 1999). The DNeasy Plant Mini tion following Llop et al. (1999), Taylor et al. Kit was used according to the manufacturer’s proto- (2001) modified and RED-Extract-N-AmpTkit. col for purification of total DNA from plant tissue Validation data available from the National Biology with final DNA elution into 1 9 100 lL of AE buf- Institute, SL TM À fer. The protocol for extraction using QuickPick 2 9 103 cfu mL 1 DNeasy Plant Mini Kit (Qiagen), À TM SML Plant DNA Kit was performed according to the 9 9 102 cfu mL 1 QuickPick SML Plant DNA manufacturer. The simple extraction procedure was Kit (Bio-Nobile), (data National Biology Institute, performed according to the protocol given by Llop SL) À et al. (1999). Further protocols used are described in 1 9 104 cfu mL 1 following Llop et al. (1999) Stoger€ et al. (2006), Persen et al. (2011). (data National Biology Institute, SL) 2.2 Polymerase chain reaction 4.2 Analytical specificity data Target organisms tested: 423 strains all positive Non-target organisms tested: 97 strains all negative Working Volume per Final 4.3 Data on repeatability concentration reaction (lL) concentration In IVIA: 98% 4.4 Data on reproducibility Molecular-grade water 6.00 9 9 In IVIA: 94% 2 10.00 1 (continued)

ª 2013 OEPP/EPPO, Bulletin OEPP/EPPO Bulletin 43, 21–45 Erwinia amylovora 43

Table (continued) –Positive amplification control (PAC) to monitor the efficiency of amplification: amplification of Working Volume per Final nucleic acid of the target organism. This can concentration reaction (lL) concentration include nucleic acid extracted from the target organism, total nucleic acid extracted from TaqMan Universal infected host tissue, whole genome amplified master mix (Applied Biosystems) DNA or a synthetic control (e.g. cloned PCR

MgCl2 Included in Included in product). For PCRs not performed on bacterial master mix master mix colonies, the PAC should preferably be near the dNTPs Included in Included in limit of detection. master mix master mix As an alternative (or in addition) to the external posi- l l hpEaF 10 M 1.00 0.5 M tive controls (PIC and PAC), internal positive controls hpEaR 10 lM 1.00 0.5 lM (IPC) can be used to monitor each individual sample hpEaP 1 lM 1.00 0.05 lM Taq polymerase Included in Included in separately. Positive internal controls can either be genes master mix master mix present in the matrix DNA or added to the DNA Subtotal 19.00 solutions. DNA 1.00 Alternative internal positive controls can include: Total reaction 20.00 • Specific amplification or co-amplification of volume of a single endogenous nucleic acid, using conserved prim- PCR reaction ers that amplify conserved non-pest target nucleic acid that is also present in the sample 2.3 PCR cycling conditions: 2 min at 50°C, 10 min at (e.g. plant cytochrome oxidase gene or eukary- 95°C, 50 cycles of 15 s at 95°C and 1 min at otic 18S rDNA) • 60°C). amplification of samples spiked with exogenous 2.4 Observations: if the expected target concentration is nucleic (control sequence) acid that has no rela- high, i.e. in enriched samples, it is highly recom- tion with the target nucleic acid (e.g. synthetic mended to carry out a tenfold dilution of the purified internal amplification controls) or amplification DNA solution in water or TE buffer before amplifica- of a duplicate sample spiked with the target tion, in order to dilute inhibitor compounds. Amplifi- nucleic acid. cation is performed on stock solution and the dilution. 3.2 Interpretation of results The cycle cut-off value for this test is set at 48, and was obtained using the equipment/materials and chemistry used 3. Essential procedural information as described here. When necessary the Ct cut-off value 3.1 Controls should be determined for the required control (e.g. internal For a reliable test result to be obtained, the following positive control). The cycle cut-off value needs to be veri- (external) controls should be included for each series of fied in each laboratory when implementing the test for the nucleic acid isolation and amplification of the target organ- first time. ism and target nucleic acid. Verification of controls • –Negative isolation control (NIC) to monitor con- The PIC and PAC amplification curves should tamination during nucleic acid extraction: nucleic be exponential. •  acid extraction and subsequent amplification of a NIC and NAC should be negative (Ct 50) • sample of uninfected host tissue or clean extrac- PIC and PAC (and if relevant IPC) should have tion buffer. a Ct value below the relevant cut-off value. –Positive isolation control (PIC) to ensure that When these conditions are met: • nucleic acid of sufficient quantity and quality is A test will be considered positive if it produces isolated: nucleic acid extraction and subsequent an exponential amplification curve, a Ct value amplification of the target organism or a matrix <48 • sample that contains the target organism (e.g. nat- A test will be considered negative if it produces urally infected host tissue or host tissue spiked no exponential amplification curve and a  with the target organism). Ct 50. • –Negative amplification control (NAC) to rule out The test should be repeated if any contradictory false positives due to contamination during the or unclear results are obtained, or if the Ct preparation of the reaction mix: amplification of value is between 48 and 50. molecular-grade water that was used to prepare the reaction mix.

ª 2013 OEPP/EPPO, Bulletin OEPP/EPPO Bulletin 43, 21–45 44 Diagnostics

Table (continued) 4. Performance criteria available (from AGES, AT, 2010)

This test was not evaluated in the test performance studies Working Volume per Final 4.1 Analytical sensitivity data (according to Gottsberger, concentration reaction (lL) concentration 2010) À dNTPs 10 mM 5.00 1.0 mM 2 9 103 cfu mL 1 MgSO4 100 mM 2.00 4.0 mM 4.2 Analytical specificity data BSA 10 mg mLÀ1 2.00 0.4 mg mLÀ1 According to Gottsberger (2010) ALB FIP 100 lM (1.2) 2.4 lM Target organisms tested: 71 strains all positive ALB BIP 100 lM (1.2) 2.4 lM Non-target organisms tested: 41 strains all negative ALB F 10 lM 1.00 0.2 lM 4.3 Data on repeatability ALB B 10 lM 1.00 0.2 lM l À1 In AGES: 100% Bst DNA polymerase 8 U L 2.00 16 U per reaction 4.4 Data on reproducibility Subtotal 45.0 In AGES: 100% DNA 5.00 Total reaction volume 50.0 of a single PCR reaction Appendix 14 – Loop-mediated isothermal amplification (LAMP) Prior to starting the LAMP reaction, set a water bath at 65°C or a thermal cycler at 65°C for 55 min. Prepare the 1. General information mix and pipette 24.6 lL molecular-grade water into each The test was developed by Temple et al. (2008), Temple & individual 0.2 mL PCR reaction tube, then pipette 18.4 lL Johnson (2011), and was evaluated in a test performance of the master mix (see table above) into each individual study in 2010 because it was considered appropriate for PCR reaction tube, then pipette 2 lL Bst DNA polymerase laboratories that do not have PCR equipment, and is simple into each individual PCR reaction tube. Finally, pipette and easy to perform for analysis of symptomatic plants as 5 lL template DNA. Spin down tubes for just 30 s. Place well as for bacterial identification. However, the sequences tubes in water bath (65°C) in a holder so the reaction end are based on those of the pEA29 plasmid and this test lacks is submerged in water bath, or place in a thermocycler the appropriate sensitivity for the analysis of samples with (set at 65°C) for 55 min. Remove tubes and allow them to low bacterial populations below 105 cfu mLÀ1 plant cool for approximately 10 s at room temperature. Observe extract. tubes for the presence of visual precipitate, a cloudy tube The test can be applied to any kind of plant material or a solid white precipitate at the bottom of the tube (indi- after a DNA extraction, as indicated in Appendix 6, and to cating a positive reaction). A clear solution is a negative bacterial colonies, without DNA extraction. reaction. LAMP primers to detect amsL B: ALB Fip: 5′-CTG CCT GAG TAC GCA GCT GAT 3. Essential procedural information TGC ACG TTT TAC AGC TCG CT-3′; ALB Bip: 5′-TCG TCG GTA AAG TGA TGG GTG 3.1 Controls CCC AGC TTA AGG GGC TGA AG-3′; For a reliable test result to be obtained, the following ALB F: 5′-GCC CAC ATT CGA ATT TGA CC-3′; (external) controls should be included for each series of ALB B: 5′-CGG TTA ATC ACC GGT GTC A-3′. nucleic acid isolation and amplification of the target organ- ism and target nucleic acid. • Negative isolation control (NIC) to monitor 2. Methods contamination during nucleic acid extraction: Melting temperatures for primers were between 58 and nucleic acid extraction and subsequent amplifi- 60°C. cation of a sample of uninfected host tissue LAMP reaction mix: (when working with plant material) or clean extraction buffer (when working with pure cul- ture); 1 per DNA extraction series. Working Volume per Final • Positive isolation control (PIC) to ensure concentration reaction (lL) concentration nucleic acid of sufficient quantity and quality is Molecular-grade water 24.60 isolated: nucleic acid extraction and subsequent 109 ThermoPol buffer 109 5.00 19 amplification of the target organism or a matrix sample that contains the target organism (e.g. (continued) naturally infected host tissue or host tissue

ª 2013 OEPP/EPPO, Bulletin OEPP/EPPO Bulletin 43, 21–45 Erwinia amylovora 45

spiked with the target organism); 1 per DNA 4. Performance criteria available extraction series. • Negative amplification control (NAC) to rule 4.1 Analytical sensitivity data (according to the results out false positives due to contamination during obtained in a performance study in 2010): the preparation of the reaction mix: amplifica- 105–106 cfu mLÀ1 plant extract after DNA extraction tion of molecular-grade water that was used to following Taylor et al. (2001). prepare the reaction mix; 1 per LAMP run. 4.2 Analytical specificity data • Positive amplification control (PAC) to monitor According to Temple et al. (2011) the efficiency of amplification: amplification of Target organisms tested: 10 strains all positive nucleic acid of the target organism. This can (except pEA29 free strains) include nucleic acid extracted from the target Non-target organisms tested: 30 strains all negative organism, total nucleic acid extracted from 4.3 Data on repeatability infected host tissue, whole genome amplified In IVIA: 96% DNA or a synthetic control (e.g. cloned PCR 4.4 Data on reproducibility product); 1 per LAMP run. For PCRs not per- In IVIA: 90% formed on bacterial colonies, the PAC should preferably be near the limit of detection. Appendix 15 – Pathogenicity tests As an alternative (or in addition) to the external positive controls (PIC and PAC), internal positive controls (IPC) Inoculation of fruitlets (of susceptible cultivars of pear, can be used to monitor each individual sample separately. apple or loquat) can be performed on whole disinfected Positive internal controls can either be genes present in the immature fruits or on slices of them, using 10 lLof matrix DNA or added to the DNA solutions. 109 cfu mLÀ1 suspensions of colonies in PBS (Appen- Alternative internal positive controls can include: dix 2). Include a positive and negative control. Incubate in • Specific amplification or co-amplification of a humid chamber at 25°C for 3–7 days. A positive test on endogenous nucleic acid, using conserved prim- fruit is shown by browning around the wounding site and ers that amplify conserved non-pest target oozing of bacteria in 3–7 days (provided the negative con- nucleic acid that is also present in the sample trol gives no lesion or only a necrotic lesion). (e.g. plant cytochrome oxidase gene or eukary- For whole plant inoculation, use susceptible cultivars of otic 18S rDNA) pear, apple or loquat, or susceptible species of Crataegus, • amplification of samples spiked with exogenous Cotoneaster or Pyracantha. To inoculate a potted plant, cut nucleic (control sequence) acid that has no rela- a young leaf from a young shoot to the main vein with tion with the target nucleic acid (e.g. synthetic scissors dipped into a 109 cfu mLÀ1 suspension of each test internal amplification controls) or amplification colony prepared in PBS (Appendix 1). of a duplicate sample spiked with the target Detached young shoots from glasshouse-grown plants nucleic acid. can also be inoculated in the same way, after disinfection 3.2 Interpretation of results for 30 s with 70% ethanol and three washings with sterile Verification of controls distilled water, and kept in tubes with sterile 1% agar. • NIC and NAC should produce no turbidity. Maintain the plants or tubes at 20–25°Cat80–100% rela- • PIC and PAC (and if relevant IPC) should pro- tive humidity with 16 h light. Read results after 3, 7 and duce the expected turbidity. 15 days. Typical E. amylovora symptoms include wilting, When these conditions are met: discoloration, necrotic tissue and ooze. • A test will be considered positive if it produces E. amylovora-like colonies should be re-isolated from turbidity. inoculated fruitlets, plants or shoots showing typical symp- • A test will be considered negative if it produces toms and their identity confirmed. no turbidity. • Tests should be repeated if any contradictory or unclear results are obtained.

ª 2013 OEPP/EPPO, Bulletin OEPP/EPPO Bulletin 43, 21–45