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Blackwell Publishing, Ltd. Organisation Européenne et Méditerranéenne pour la Protection des Plantes European and Mediterranean Plant Protection Organization

Diagnostic protocols for regulated pests Protocoles de diagnostic pour les organismes réglementés

PM 7/20

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155

156 Diagnostic protocols

Approval ¥ laboratory procedures presented in the protocols may be adjusted to the standards of individual laboratories, provided EPPO Standards are approved by EPPO Council. The date of that they are adequately validated or that proper positive and approval appears in each individual standard. In the terms of negative controls are included. Article II of the IPPC, EPPO Standards are Regional Standards for the members of EPPO. References Review EPPO/CABI (1996) Quarantine Pests for Europe, 2nd edn. CAB Interna- tional, Wallingford (GB). EPPO Standards are subject to periodic review and amendment. EU (2000) Council Directive 2000/29/EC of 8 May 2000 on protective The next review date for this EPPO Standard is decided by the measures against the introduction into the Community of organisms EPPO Working Party on Phytosanitary Regulations harmful to plants or plant products and against their spread within the Community. Official Journal of the European Communities L169, 1Ð112. FAO (1997) International Plant Protection Convention (new revised text). Amendment record FAO, Rome (IT). IPPC (1993) Principles of plant quarantine as related to international trade. Amendments will be issued as necessary, numbered and dated. ISPM no. 1. IPPC Secretariat, FAO, Rome (IT). The dates of amendment appear in each individual standard (as IPPC (2002) Glossary of phytosanitary terms. ISPM no. 5. IPPC Secretariat, appropriate). FAO, Rome (IT). OEPP/EPPO (2003) EPPO Standards PM 1/2 (12): EPPO A1 and A2 lists of quarantine pests. EPPO Standards PM1 General phytosanitary meas- Distribution ures, 5Ð17. OEPP/EPPO, Paris. EPPO Standards are distributed by the EPPO Secretariat to all EPPO member governments. Copies are available to any Definitions interested person under particular conditions upon request to Regulated pest: a quarantine pest or regulated non-quarantine pest. the EPPO Secretariat. Quarantine pest: a pest of potential economic importance to the area endangered thereby and not yet present there, or present but not widely distributed and being officially controlled. Scope EPPO Diagnostic Protocols for Regulated Pests are intended to be used by National Plant Protection Organizations, in their Outline of requirements capacity as bodies responsible for the application of phyto- EPPO Diagnostic Protocols for Regulated Pests provide all the sanitary measures to detect and identify the regulated pests of information necessary for a named pest to be detected and the EPPO and/or European Union lists. positively identified by an expert (i.e. a specialist in In 1998, EPPO started a new programme to prepare diagnostic entomologist, mycology, virology, bacteriology, etc.). Each protocols for the regulated pests of the EPPO region (including the protocol begins with some short general information on the pest EU). The work is conducted by the EPPO Panel on Diagnostics (its appearance, relationship with other organisms, host range, and other specialist Panels. The objective of the programme is to effects on host, geographical distribution and its identity) and develop an internationally agreed diagnostic protocol for each then gives details on the detection, identification, comparison regulated pest. The protocols are based on the many years of experi- with similar species, requirements for a positive diagnosis, list ence of EPPO experts. The first drafts are prepared by an assigned of institutes or individuals where further information on that expert author(s). They are written according to a ‘common format organism can be obtained, references (on the diagnosis, and content of a diagnostic protocol’ agreed by the Panel on Dia- detection/extraction method, test methods). gnostics, modified as necessary to fit individual pests. As a general rule, the protocol recommends a particular means of detection or Existing EPPO Standards in this series identification which is considered to have advantages (of reliabil- ity, ease of use, etc.) over other methods. Other methods may also Nineteen EPPO standards on diagnostic protocols have already be mentioned, giving their advantages/disadvantages. If a method been approved and published. Each standard is numbered in the not mentioned in the protocol is used, it should be justified. style PM 7/4 (1), meaning an EPPO Standard on Phytosanitary The following general provisions apply to all diagnostic Measures (PM), in series no. 7 (Diagnostic Protocols), in this protocols: case standard no. 4, first version. The existing standards are: ¥ laboratory tests may involve the use of chemicals or appar- PM 7/1 (1) Ceratocystis fagacearum. Bulletin OEPP/EPPO atus which present a certain hazard. In all cases, local safety Bulletin 31, 41Ð44 procedures should be strictly followed PM 7/2 (1) Tobacco ringspot nepovirus. Bulletin OEPP/EPPO ¥ use of names of chemicals or equipment in these EPPO Bulletin 31, 45Ð51 Standards implies no approval of them to the exclusion of PM 7/3 (1) Thrips palmi. Bulletin OEPP/EPPO Bulletin 31, others that may also be suitable 53Ð60

EPPO Standards 157

PM 7/4 (1) Bursaphelenchus xylophilus. Bulletin OEPP/ PM 7/16 (1) Fusarium oxysporum f. sp. albedinis. Bulletin EPPO Bulletin 31, 61Ð69 OEPP/EPPO Bulletin 33, 265Ð270 PM 7/5 (1) Nacobbus aberrans. Bulletin OEPP/EPPO Bulletin PM 7/17 (1) Guignardia citricarpa. Bulletin OEPP/EPPO 31, 71Ð77 Bulletin 33, 271Ð280 PM 7/6 (1) Chrysanthemum stunt pospiviroid. Bulletin OEPP/ PM 7/18 (1) Monilinia fructicola. Bulletin OEPP/EPPO EPPO Bulletin 32, 245Ð253 Bulletin 33, 281Ð288 PM 7/7 (1) Aleurocanthus spiniferus. Bulletin OEPP/EPPO PM 7/19 (1) Helicoverpa armigera. Bulletin OEPP/EPPO Bulletin 32, 255Ð259 Bulletin 33, 289Ð296 PM 7/8 (1) Aleurocanthus woglumi. Bulletin OEPP/EPPO Several of the Standards of the present set result from a differ- Bulletin 32, 261Ð265 ent drafting and consultation procedure. They are the output PM 7/9 (1) Cacoecimorpha pronubana. Bulletin OEPP/EPPO of the DIAGPRO Project of the Commission of the European Bulletin 32, 267Ð275 Union (no. SMT 4-CT98-2252). This project involved four PM 7/10 (1) Cacyreus marshalli. Bulletin OEPP/EPPO ‘contractor’ diagnostic laboratories (in England, Netherlands, Bulletin 32, 277Ð279 Scotland, Spain) and 50 ‘intercomparison’ laboratories in many PM 7/11 (1) Frankliniella occidentalis. Bulletin OEPP/EPPO European countries (within and outside the European Union), Bulletin 32, 281Ð292 which were involved in ring-testing the draft protocols. The PM 7/12 (1) Parasaissetia nigra. Bulletin OEPP/EPPO DIAGPRO project was set up in full knowledge of the parallel Bulletin 32, 293Ð298 activity of the EPPO Working Party on Phytosanitary PM 7/13 (1) Trogoderma granarium. Bulletin OEPP/EPPO Regulations in drafting diagnostic protocols, and covered Bulletin 32, 299Ð310 regulated pests which were for that reason not included in the PM 7/14 (1) Ceratocystis ﬁmbriata f. sp. platani. Bulletin EPPO programme. The DIAGPRO protocols have been OEPP/EPPO Bulletin 33, 249Ð256 approved by the Council of EPPO as EPPO Standards in series PM 7/15 (1) Ciborinia camelliae. Bulletin OEPP/EPPO PM7. They will in future be subject to review by EPPO Bulletin 33, 257Ð264 procedures, on the same terms as other members of the series.

EuropeanBlackwell Publishing, Ltd. and Mediterranean Plant Protection Organization PM 7/20(1) Organisation Européenne et Méditerranéenne pour la Protection des Plantes

Diagnostic protocols for regulated pests1 Protocoles de diagnostic pour les organismes réglementés

Erwinia amylovora

Speciﬁc scope Speciﬁc approval and amendment This standard describes a diagnostic protocol for Erwinia This Standard was developed under the EU DIAGPRO Project amylovora. (SMT 4-CT98-2252) by partnership of contractor laboratories and intercomparison laboratories in European countries. Approved as an EPPO Standard in 2003-09.

depending of environmental factors (Thomson, 2000). The Introduction development of fireblight symptoms follows the seasonal Erwinia amylovora is the causal agent of fireblight in most growth development of the host plant. It begins in the spring species of subfamily Maloideae of the family Rosaceae. The with production of primary inoculum and infection of flowers, most economically important hosts are Pyrus spp., Malus spp., continues in summer with infection of shoots and fruits, and Cydonia spp., Eriobotrya japonica, Cotoneaster spp., ends in autumn with the development of cankers. The pathogen Crataegus spp., Pyracantha spp. and Sorbus spp. Other hosts is apparently quiescent through the dormant period of the host include Chaenomeles, Mespilus and Photinia. A forma (van der Zwet & Beer, 1995). specialis was described from Rubus spp. (Starr et al., 1951; EPPO/CABI, 1997). An exhaustive list of affected plants, Identity including those susceptible after artificial inoculation, was reported by van der Zwet & Keil (1979) and Bradbury (1986). Name: Erwinia amylovora (Burrill) Winslow et al. It includes more than 180 species from 39 genera of the Synonyms: Micrococcus amylovorus Burrill Rosaceae. E. amylovora was the first bacterium described as Bacillus amylovorus (Burrill) Trevisan causal agent of a plant disease by Burrill (1883). It was Bacterium amylovorus (Burrill) Chester considered to be native to North America and later detected in Erwinia amylovora f.sp. rubi Starr, Cardona & Falson New Zealand in 1920. Fireblight was reported in 1957 in Taxonomic position: Bacteria, Gracilicutes, Proteobacteria, γ England and has since been detected in most areas in Europe Subdivision, Enterobacteriales, Enterobacteriaceae where susceptible hosts are cultivated, except in Portugal (in the Bayer computer code: ERWIAM EU). E. amylovora is now present in 43 countries (van der Phytosanitary categorization: EPPO A2 list no. 52, EU Zwet, 2002), but it has not been recorded either in South Annex designation II/A2 America, or in most African and Asian countries (with the exception of some Mediterranean countries), and only once in Detection Australia (Bonn & van der Zwet, 2000). It represents a threat to the pome fruit industry of all these countries. Details on Symptoms geographical distribution can be found in EPPO/CABI (1998). Fireblight is probably the most serious disease affecting pear Symptoms of fireblight on the principal hosts are relatively or apple cultivars in many countries. Although the life cycle of similar and easily recognized (Web Figs 1 and 2). The name of the bacterium is still not fully understood, it is known that it can the disease is descriptive of its major characteristic: the survive as an endophyte or epiphyte for variable periods of time brownish aspect of twigs, flowers and leaves as though burned by fire. The typical symptoms on pome fruit trees are the brown to black colour of leaves on affected branches, the production 1The Figures in this Standard marked ‘Web Fig.’ are published on the EPPO of exudates and the typical ‘shepherd’s crook’ in the shoots. website www.eppo.org. Depending on the affected plant part, the disease causes

160 Erwinia amylovora blossom blight, shoot or twig blight, leaf blight, fruit blight, the sample should be re-tested and/or enrichment used before limb and trunk blight, collar or rootstock blight (van der Zwet isolation, according to Appendix IV. & Keil, 1979; van der Zwet & Beer, 1995). Plating on three media is advised for maximum recovery of In apple and pear, the first symptoms usually appear in early E. amylovora specially when samples are not in the best condi- spring, during warm and humid weather. Flowers appear to be tion. Depending on the number and composition of the micro- water-soaked, then wilt, shrivel, and turn brown to black. biota of the sample, each is more or less efficient. Three media Peduncles may also appear water-soaked, become dark green, (CCT, Levan and King’s B) have been validated in the ring test. and finally brown or black, sometimes oozing droplets of sticky Levan had the highest recovery (Appendix I). bacterial exudates. Leaves wilt, shrivel and entire spurs turn When analysing symptomatic samples good correlation is brown in apples or dark brown to black in pears, but remain expected between isolation, immunofluorescence, enrichment attached to the tree for some time. Immature fruits (or less fre- DASI-ELISA and PCR. quently mature fruits) show infected parts appearing oily or water-soaked, becoming brown to black and often exuding Identification in asymptomatic samples droplets of bacterial ooze. They also remain attached to the tree. Characteristic reddish-brown streaks are often found in the Sampling and sample preparation subcortical tissues when the bark is peeled from the infected limb or twig (van der Zwet & Keil, 1979). Brown to black, Asymptomatic samples can be processed individually or in slightly depressed cankers develop in the bark of twigs or groups of up to 100 samples (OEPP/EPPO, 1992). Where surveys branches or even the trunk. These cankers may later become are performed, they should be based on statistically representative defined by cracks near the margin of diseased and healthy tissue samples. Samples taken from stored material can be considered (Dye, 1983). random whereas those from the orchards or nurseries may not be. Precautions to avoid cross contamination should be taken when collecting the samples and during the extraction process. Identification on host plants with symptoms Sampling and sample preparation can be performed following one of the methods for asymptomatic samples in Appendix II. Sampling The direct analysis of asymptomatic samples is normally Symptomatic samples can be processed individually or in small negative for E. amylovora due to the low bacterial population. groups. Precautions to avoid cross contamination should be Consequently, it is advised to perform a previous enrichment of taken when collecting the samples and during the extraction the samples (Appendix IV) prepared in the antioxidant buffer process. The samples for diagnosis of fireblight on plants with (Gorris et al., 1996a) (Appendix I). When analysing asympto- symptoms should preferably be flowers, shoots or twigs, leaves, matic material, the enrichment should be performed for 72 h at fruitlets (with necrosis and exudates if possible) or the about 25 °C. discoloured subcortical tissues after peeling the bark from cankers in twig, branches, trunk or collar. They should be Screening tests processed as soon as possible after being collected and conserved at 4Ð8 °C until then. The samples can be cold-stored At least two screening tests should be performed. For IF, one after processing for further verification for up to a few weeks. sample extract should be used before or after concentration by For further details of sample preparation, see Appendix II. centrifugation (but non-enriched) per IF slide window. Decimal dilutions of the sample should also be fixed, following the protocol described in Appendix III. For enrichment isolation, Rapid screening tests enrichment ELISA, and enrichment-PCR, the procedures in These tests facilitate presumptive diagnosis. At least two Appendix III should be followed. In the last case, 500 µL of serological tests and another PCR-based should be positive for samples should be used, enriched in King’s B and in CCT for E. amylovora positive detection in the samples. Details of the DNA extraction following Llop et al. (2000) or other tests are given in Appendix III. appropriate protocols or kits. If any of the screening tests are positive, an attempt should be made to isolate the pathogen from the extract (Appendix V) or Isolation the enriched samples (Appendix IV). If three or four tests are Freshly prepared sample extracts are necessary for successful positive and the isolation is negative or not done, it is reasonable isolation. The isolation of E. amylovora from symptomatic to consider E. amylovora presumptively detected in the sample, samples is relatively easy because the number of culturable but confirmation requires isolation and identification of the bac- bacteria in them is usually high. However, when symptoms are terium. If necessary, the extract conserved at Ð80 °C under very advanced or when the environmental conditions are not glycerol (Appendix II) should be spread. The three media favourable for fireblight expression, the number of E. (CCT, King’s B, Levan) indicated in Appendix I should be used amylovora culturable cells can be very low. Furthermore, if to have a maximum possibility of isolating E. amylovora, when plates are overcrowded or antagonistic bacteria are suspected no information about the microbiota of the samples is available.

Diagnostic protocols 161

Table 1 Nutritional and enzymatic identiﬁcation tests Table 3 Typical readings of Erwinia amylovora in API 20E tests after 48 h

Test Expected result Test Reaction (48 h)

Gram staining Ð ONPG Variable Levan production + ADH Ð (or weak +) Fluorescent pigment production in King’s B (under UV) Ð LDC Ð Oxidation/Fermentation (O/F) test O +/F + ODC Ð Kovac’s oxidase test Ð CIT Ð

Reduction of nitrate Ð SH2 Ð Utilization of citrate + URE Ð Growth at 39 °CÐTDA Ð Gelatine liquefaction + IND Ð Urease Ð VP + (or variable) Indole Ð GEL Variable Reducing substances from sucrose + GLU + Acetoin + MAN Variable INO Variable SOR Variable RHA Ð Confirmation SAC + MEL Ð (or weak +) Pure cultures of presumptive E. amylovora isolates should be AMY Ð identified using at least two tests related to two different ARA + (some −) characteristics of the pathogen (nutritional, fatty acids, serological or molecular). An appropriate host test should be included as final confirmation of pathogenicity. Known E. amylovora reference strains should be included where et al., 2002), although some physiological and biochemical appropriate for each test performed (Appendix I). characteristics may vary for some strains.

Biochemical characterization by API system Nutritional and enzymatic identification tests (BioMérieux, France) The genus Erwinia was defined for Gram-negative bacteria, Biochemical identification of E. amylovora can be obtained by facultative anaerobes, motile by peritrichous flagella, rod- specific profile in API 20 E and API 50 CH strips. For API 20 shaped and acid produced from glucose, fructose, galactose and E, the manufacturer’s instructions should be followed for sucrose. The phenotypic properties of Table 1 (Paulin, 2000), preparing the suspension and inoculating the strip. After that are universally present or absent in E. amylovora, should be incubation at 25Ð26 °C, the strips should be read after 24 and determined according to the methods of Jones & Geider (2001). 48 h (Table 3). For API 50 CH, a suspension of OD = 1.0 should The tests in Table 2 allow differentiation of E. amylovora from be prepared in PBS (Appendix I), and 1 mL added to 20 mL of E. pyrifoliae, causal agent of Asian pear blight on Pyrus Ayers medium (Appendix I). The manufacturer’s instructions pyrifolia (Kim et al., 1999; Kim et al., 2001) and a new Erwinia should be followed for inoculation of the strip. After incubation sp. isolated from necrotic pear blossoms in Spain (Roselló at 25Ð26 °C in aerobiosis, the strip should be read after 24 and

Table 2 Differences between Erwinia amylovora, Erwinia pyrifoliae and Erwinia sp. isolated from necrotic pear ﬂowers

Microbiological tests Erwinia amylovora Erwinia pyrifoliae Erwinia sp.

Gelatine hydrolysis + Ð Ð Inositol1 ÐND3 + Sorbitol1 ++Ð Esculin1 V4 Ð+ Melibiose1 ÐÐ+ D-Rafﬁnose1 ÐÐ+ β-Gentibiose1 +Ð+ Ampliﬁcation2 with EP16A/EPI62CCPS1/CPS2C Ð + ND3

1 = From Roselló et al. (2003). Oxidation of substrates in API 50 CH (BioMérieux) with a modiﬁed protocol. 2 = According to Kim et al. (2001). 3 = ND: Not determined. 4 = V: Variable.

162 Erwinia amylovora

48 h. Utilization of the different carbohydrates is indicated by European strains. It can provide information useful for strain a yellow colour in the well. differentiation and has been used to analyse the spread of fireblight in Europe. Automated Biolog identification system An identification system based on utilization of 95 carbon Bioassay sources in a microtiter plate is commercially available (Biolog, US). The manufacturer’s instructions should be followed for Suspected E. amylovora colonies from the isolation and automatic identification of suspected strains of E. amylovora. enrichment plates should be inoculated to test plants to prove their pathogenicity. The hypersensitive reaction in tobacco leaves can give an indication of the presence of the hrp genes, Fatty acid profiling (FAP) but is also positive for many plant pathogenic bacteria. Tobacco Levan-positive, non-fluorescent colonies should be grown on plants of cv. Xanthi or Samsun with more than 5Ð6 leaves are trypticase soy agar for 48 h at 28 °C, and an appropriate FAP used. Bacterial suspensions of 109 cfu mL−1 (OD at 620 nm procedure applied. A positive FAP test is achieved if the profile = 1.0) are injected into the intracellular space of adult leaves of the presumptive culture is identical to that of the positive with a 25 GA 5/8 0.5 × 16 needle and syringe. Complete collapse control (Sasser, 1990). of the infiltrated tissue after 24 h at room temperature is recorded as positive. To verify the pathogenicity of suspected E. amylovora colonies, Serological identification a fireblight host should be inoculated. A positive control should Agglutination test always be included (pure culture of a known E. amylovora Suspected E. amylovora colonies which are levan-positive, strain) and a negative control with sterile PBS (Appendix I). non-fluorescent in King’s B medium can be tested for slide Plants inoculated with positive and negative controls should be agglutination by mixing them in a drop of PBS (Appendix I) kept apart from other test plants, but under the same conditions. with a drop of E. amylovora-specific antiserum (not diluted or Inoculation methods are given in Appendix VI. E. amylovora- only at 5 or 10 fold dilution) on a slide. Monoclonal antibodies like colonies should be re-isolated from the inoculated fruitlets, can be used only if they agglutinate with the reference strains. plants or shoots showing typical E. amylovora symptoms.

IF test Possible confusion with similar species A suspension of approximately 106 cells per mL is prepared in PBS (Appendix I) from levan-positive, non-fluorescent colonies, Symptoms of fireblight caused by E. amylovora may be con- and the IF procedure described in Appendix III is applied. fused with those caused by Erwinia pyrifoliae, causal agent of bacterial shoot blight of Asian pear (Pyrus pyrifolia) (Kim ELISA tests et al., 1999) and with those caused by a new Erwinia species DASI-ELISA and indirect-ELISA for isolate identification can isolated from necrotic pear blossoms in Spain (Roselló et al., be performed using specific monoclonal antibodies. A mixture 2002). Definitive diagnosis should always be obtained through of monoclonal antibodies has been validated in a ring test. For laboratory analysis. DASI-ELISA, a suspension of approximately 108 cells per mL is prepared in PBS (Appendix I) from suspected colonies. The Reference material DASI-ELISA procedure (Appendix III) can be followed without previous enrichment. If only two identification tests and The following E. amylovora isolates are recommended for use inoculation are being performed, another serological test should as positive controls: NCPPB683 and CFBP 1430. The not be used in addition to this method. For indirect ELISA, the following collections can provide different E. amylovora separate procedure in Appendix III should be followed reference strains: (1) National Collection of Plant Pathogenic Bacteria (NCPPB), Central Science Laboratory, York (GB); (2) Culture Collection of the Plant Protection Service (PD), Molecular identification Wageningen (NL); (3) Collection Française de Bactéries PCR Phytopathogènes (CFBP), INRA Station Phytobactériologie, A suspension of approximately 106 cells per mL in molecular- Angers, France. Authenticity of the strains can be guaranteed grade sterile water should be prepared from levan-positive, only if directly obtained from the culture collections. non-fluorescent colonies. Appropriate PCR procedures should be applied, following Appendix III, without DNA extraction. Requirements for a positive diagnosis Macrorestriction with Xba I and Pulse Field Gel Electrophore- Figures 3 and 4 summarize the testing schemes for E. amylovora sis (PFGE) in symptomatic and asymptomatic material, respectively. PFGE analysis of genomic DNA after Xba I digestion according When E. amylovora is diagnosed for the first time, or in crit- to Jock et al. (2002) shows 6 patterns for E. amylovora ical cases (import/export) the following requirements apply:

Fig. 3 Detection scheme for the detection and identiﬁcation of Erwinia amylovora in host plants with symptoms.

Fig. 4 Detection scheme for the detection and identiﬁcation of Erwinia amylovora in asymptomatic samples.

• the disease symptoms, morphological, biochemical, and References molecular characteristics of the pathogen and its pathogenic properties should be in accordance with the descriptions in Ayers SH, Rupp P & Johnson WT (1919) A Study of Alkali Forming in Milk. the protocol USDA Bulletin no. 782. Washington (US). • for isolation of the bacterium and description of morpholo- Bereswill S, Bugert P, Bruchmuller I & Geider K (1995) Identification of the fireblight pathogen, Erwinia amylovora by PCR assays with chromosomal gical, biochemical, molecular, and pathogenic characteristics, DNA. Applied and Environmental Microbiology 61, 2636Ð2642. the procedures and requirements of the protocol should be Bereswill S, Jock S, Aldridge P, Janse JD & Geider K (1997) Molecular followed characterization of natural Erwinia amylovora strains deficient in levan • in the case of latent infections, after an initial screening test, synthesis. Physiological and Molecular Plant Pathology 51, 215Ð225. the pathogen should be isolated and correctly identified, Bereswill S, Pahl A, Bellemann P, Zeller W & Geider K (1992) Sensitive including a pathogenicity test with the pure culture and species-specific detection of Erwinia amylovora by polymerase chain • the original sample (with labels, if applicable), sample reaction analysis. Applied and Environmental Microbiology 58, 3522Ð 3526. extract, and pure culture should be kept under proper condi- Bonn WG & van der Zwet T (2000) Distribution and economic importance tions with correct registration and should be available in case of fireblight. In: Fireblight, the Disease and its Causative Agent Erwinia of doubt. amylovora (Ed. Vanneste, J). CAB International, Wallingford (GB). Bradbury JF (1986) Guide to Plant Pathogenic Bacteria. CAB International, Wallingford (GB). Report on the diagnosis Burrill TJ (1883) New species of Micrococcus. American Naturalist 17, 319. Dye DW (1983) Erwinia: the ‘amylovora’ and ‘herbicola’ groups. In: Plant A report on the execution of the protocol should include: Bacterial Diseases, a Diagnosis Guide (Ed. Fahy PC & Persley GJ). Aca- • information and documentation on the origin of the infected demic Press, Sydney (AU). material; EPPO/CABI (1997) Erwinia amylovora. Quarantine Pests for Europe, 2nd • a description of the disease symptoms (if applicable and pre- edn, pp. 1001Ð1007. CAB International, Wallingford (GB). ferably with pictures); EPPO/CABI (1998) Map 257. In: Distribution Maps of Quarantine Pests for • a description of the morphological, biochemical and patho- Europe. CAB International, Wallingford (GB). genic characteristics of the bacterium; EU (1998) Council Directive 98/57 EC of 20 July 1998 on the control of Ralstonia solanacearum. Official Journal of the European Communities • information on the magnitude of the infection; L235, 1Ð39. • comments as appropriate on the certainty or uncertainty of Gorris MT, Cambra M, Llop P, López MM, Lecomte P, Chartier R & the identification. Paulin JP (1996b) A sensitive and specific detection of Erwinia amylovora based on the ELISA-DASI enrichment method with monoclonal antibodies. Acta Horticulturae no. 411, 41Ð45. Further information Gorris MT, Cambra E, López MM, Paulin JP, Chartier R & Cambra M Further information on this organism can be obtained from: (1996a) Production and characterization of monoclonal antibodies specific for Erwinia amylovora and their use in different serological tech- M. M. López ([email protected]), Bacteriología, Department niques. Acta Horticulturae no. 411, 47Ð51. Protección Vegetal y Biotecnología, Instituto Valenciano de Guilford PJ, Taylor RK, Clark RG, Hale CN, Forster RLS & Bonn WG Investigaciones Agrarias (IVIA), Carretera Moncada-Náquera (1996) PCR-based techniques for the detection of Erwinia amylovora. km 5, 46113 Moncada (Valencia), Spain; M. Keck, Bundesamt Acta Horticulturae no. 411, 53Ð56. und Forschungszentrum für Landwirtschaft, Spargelfeldstrasse Ishimaru ES & Klos EJ (1984) New medium for detection of Erwinia amy- 191, A-1226 Vienna PO Box 400, Austria. lovora and its use in epidemiological studies. Phytopathology 74, 1342Ð 1345. Jock S, Donat V, López MM, Bazzi C & Geider K (2002) Following spread Acknowledgements of fireblight in Western, Central and Southern Europe by molecular differentiation of Erwinia amylovora strains with PFGE analysis. Environ- This protocol was originally drafted by: M. M. López, M. Keck, mental Microbiology 4, 106Ð114. P. Llop, M. T. Gorris, J. Peñalver, V. Donat and M. Cambra., Jones A & Geider K (2001) II Gram negative bacteria. B. Erwinia and IVIA, Moncada (Valencia) (ES). Pantoea. In: Guide for Identification of Plant Pathogenic Bacteria (Ed. This protocol was ring-tested in different European Schaad NW, Jones JB & Chum W), 2nd edn. APS Press, St Paul (US). Kim WS, Gardan L, Rhim SL & Geider K (1999) Erwinia pyrifoliae sp., a laboratories2. novel pathogen that affects Asian pear trees (Pyrus pyrifolia). Interna- tional Journal of Systematic Bacteriology 49, 899Ð906. Kim WS, Jock S, Rhim SL & Geider K (2001) Molecular detection and dif- 2J. Janse, Plant Protection Service, Wageningen, NL; M. Keck, Bundesamt ferentiation of Erwinia pyrifoliae and host range analysis of the Asian und Forschungszentrum für Landwirtschaft, Vienna, AT; A. Sletten, Plant pear pathogen. Plant Disease 85, 1183Ð1188. Protection Centre, Ås, NO; M. A. Cambra, Centro de Protección Vegetal, King EO, Ward M & Raney DE (1954) Two simple media for the demon- Zaragoza, ES; J. L. Palomo, Centro Regional de Diagnóstico, Aldearrubia stration of pyocyanin and fluorescein. Journal of Laboratory and Clinical Salamanca, ES; S. Simpkins, Central Science Laboratory, Sand Hutton, Medicine 44, 301Ð307. York, GB; T. T. Duarte, Direcção Geral de Protecção das Culturas, Lisbon, Lecomte P, Manceau C, Paulin JP & Keck M (1997) Identification by PCR PT; F. Poliakoff, LNPV Unité Bactériologie, Angers, FR; J. Van analysis on plasmid pE29 of isolates of Erwinia amylovora responsible of Vaerenbergh, Rijkstation voor Plantenziekten, Merelbeke, BE; M. M. an outbreak in Central Europe. European Journal of Plant Pathology 103, López, Instituto Valenciano de Investigaciones Agrarias, Valencia, ES. 91Ð98.

Llop P, Bonaterra A, Peñalver J & López MM (2000) Development of a Substrate buffer for alkaline phosphatase: diethano- highly sensitive nested-PCR procedure using a single closed tube for lamine 97 mL; dilute in 800 mL of distilled water. Adjust pH to detection of Erwinia amylovora in asymptomatic plant material. Applied 9.8 with concentrated HCl. Adjust to 1000 mL with distilled and Environmental Microbiology 66, 2071Ð2078. water. Llop P, Caruso P, Cubero J, Morente C & López MM (1999) A simple extraction procedure for efficient routine detection of pathogenic bacteria Precipitating substrate buffer for alkaline phosphatase: in plant material by polymerase chain reaction. Journal of Microbiologi- Sigma Fast 5-bromo-4-chloro-3-indolyl phosphate/nitro blue cal Methods 37, 23Ð31. tetrazolium tablets (BCIP-NBT) Ð Cat No. B-5655 Sigma Maes M, Garbeva P & Crepel C (1996) Identification and sensitive endo- Aldrich GmbH (Stenheim), Germany. phytic detection of the fireblight pathogen Erwinia amylovora with 235 Extraction buffer (Llop et al., 1999): Tris HCl, pH 7.5 24.2 g; ribosomal DNA sequences and the polymerase chain reaction. Plant NaCl 14.6 g; EDTA 9.3 g; SDS 5 g; polyvinylpyrrolidone Pathology 45, 1139Ð1149. McManus PS & Jones AL (1995) Detection of Erwinia amylovora by nested PVP-10 20 g; distilled water to 1 L. Sterilize by filtration. PCR and PCR-dot-blot and reverse blot hybridisations. Phytopathology 50X TAE buffer: Tris 242 g; 0.5 m Na2EDTA pH 8.0 100 mL; 85, 618Ð623. glacial acetic acid 57.1 mL; distilled water to 1 L. OEPP/EPPO (1992) EPPO Standards PM 3/40 Erwinia amylovora. Loading buffer: Bromophenol blue 0.025 g; glycerol 3 g; Sampling and test methods. Bulletin OEPP/EPPO Bulletin 22, 225Ð distilled water 10 mL. 231. Paulin JP (2000) Erwinia amylovora: general characteristics, biochemistry and serology. In: Fireblight, the Disease and its Causative Agent, Erwinia Media amylovora (Ed. Vanneste, J). CAB International, Wallingford (GB). Roselló M, García-Vidal S, Tarín A, Llop P, Gorris MT, Donat V, Peñalver J, CCT medium (Ishimaru & Klos, 1984): sucrose 100 g; sorbitol Chartier R, Paulin JP, Gardan L & López MM (2002) Characterization of 10 g; Niaproof 1.2 mL; crystal violet 2 mL (sol. 0.1% ethanol); an Erwinia sp. isolated from necrotic pear blossoms in Valencia, Spain. nutrient agar 23 g; distilled water to 1 L. Adjust pH to 7.0Ð7.2; Acta Horticulturae no. 590, 139Ð142. sterilize by autoclaving at 115 °C for 10 min. Then prepare: Roselló M, Peñalver J, Llop P, Gorris MT, Charter R, Cambra M & thallium nitrate 2 mL (1% w/v aqueous solution); cyclohexi- López MM (2003) Identification of an Erwinia sp., different from Erwinia mide 0.05 g (highly toxic reagents, handle with care). Sterilize amylovora which is responsible for necrosis on pear blossoms. Authors to µ be contacted (submitted). by filtration (0.45 m). Add to 1 L of the sterile medium (at Sasser M (1990) Identification of bacteria through fatty acid analysis. In: about 45 °C). Methods in Phytobacteriology (Ed. Klement F, Rudolf K & Sands DC), King’s B medium (King et al., 1954): proteose peptone N°3 pp. 199Ð204. Akademiai Kiado, Budapest (HU). 20 g; glycerol 10 mL; K2HPO4 1.5 g; MgSO4 á7H2O 1.5 g; agar Starr MP, Cardona C & Folsom D (1951) Bacterial fire blight of raspberry. 15 g; distilled water to 1 L. Adjust pH to 7.0Ð7.2. Sterilize by Phytopathology 41, 951Ð959. autoclaving at 120 °C for 15 min. Thomson SV (2000) Epidemiology of fireblight. In: Fireblight, the Disease Levan medium and its Causative Agent, Erwinia amylovora (Ed. Vanneste, J). CAB : yeast extract 2 g; Bactopeptone 5 g; NaCl International, Wallingford (GB). 5 g; sucrose 50 g; agar 20 g; distilled water to 1 L. Adjust pH to van der Zwet T (2002) Present worldwide distribution of fire blight. Acta 7Ð7.2. Sterilize by autoclaving at 120 °C for 15 min.

Horticulturae no. 590, 33Ð34. Ayers Medium (Ayers et al., 1919): NH4H2PO4 1 g; KCl 0.2 g; van der Zwet T & Beer S (1995) Fire blight Ð its nature, prevention and MgSO4 0.2 g; bromothymol blue 75 mL (solution 0.2%); control. A Practical Guide to Integrated Disease Management. USDA distilled water to 1 L. Adjust pH to 7. Sterilize by autoclaving Agricultural Information Bulletin no. 631. Washington (US). at 120 °C for 15 min. van der Zwet T & Keil HL (1979) Fire blight: a bacterial disease of rosa- ceous plants. USDA Handbook no. 510. Washington (US). Primers Oligonucleotide primer sequences (Bereswill et al., 1992): Appendix I Materials Primer A: 5′-CGG TTT TTA ACG CTG GG Primer B: 5′-GGG CAA ATA CTC GGA TT Buffers Oligonucleotide primer sequences for nested PCR in a single Phosphate buffered saline 10 mM, pH 7.2 (PBS): NaCl 8 g; closed tube (Llop et al., 2000):

KCl 0.2 g; Na2HPO4á12H2O 2.9 g; KH2PO4 0.2 g; distilled External primers water to 1 L. AJ75: 5′-CGT ATT CAC GGC TTC GCA GAT Antioxidant maceration buffer (Gorris et al., 1996a): poly- AJ76: 5′-ACC CGC CAG GAT AGT CGC ATA vynilpyrrolidone (PVP-10) 20 g; mannitol 10 g; ascorbic acid Internal primers 1.76 g; reduced glutathione 3 g; PBS 10 mm pH 7.2-1 L. Adjust PEANT1: 5′-TAT CCC TAA AAA CCT CAG TGC pH to 7. Sterilize by ﬁltration. PEANT2: 5′-GCA ACC TTG TGC CCT TTA

Carbonate buffer pH 9.6: Na2CO3 1.59 g; NaHCO3 2.93 g; distilled water to 1 L. Adjust pH to 9.6. Commercially available standardized control materials Washing buffer (PBS, pH 7.2–7.4 supplemented with 0.05%

Tween 20): NaCl 8 g; KCl 0.2 g; Na2HPO4á12H2O 2.9 g; Antibodies to E. amylovora currently recommended for use in µ KH2PO4 0.2 g; Tween 20 500 L; distilled water to 1 L. detection and identiﬁcation tests:

• E. amylovora, polyclonal, recommended for detection using leaves, at the margin between the necrotic and healthy tissue. IF test (validated in ring tests), Loewe Biochemica GmbH, For flowers, take one or several, with peduncles. For leaves, Mühlweg 2a, D-82054 Sauerlach (DE). take one or several leaves and petioles; preferably select leaves • IVIA EPS 1430, polyclonal, recommended for detection with vein necrosis, but not fully necrosed. For fruits, take one using IF test (validated in ring tests), Plant Print Diagnostics, or several fruits. For stems: peel off the external bark of stems S.L., De la March 36, 46512 Faura, Valencia (ES). with symptoms using a sterile scalpel and take pieces under- • IVIA Mab 7 A, monoclonal, recommended for detection neath with typical subcortical discoloration symptoms. In all using IF test (validated in ring tests), Plant Print Diagnostics cases, the sample should total about 0.1 g of material. (ES); Cut the shoots, flowers, leaves, stems or fruits into pieces into • IVIA Mab 8B + 5H, monoclonals, recommended for detec- plastic bags. Add to each bag 4.5 mL of the antioxidant maceration using Enrichment DASI-ELISA test (validated in ring tion buffer described by Gorris et al. (1996a) (Appendix I). tests), Plant Print Diagnostics (ES). Let the samples macerate at least 5 min. Slightly crush the plant Note: After an enrichment step the use of validated specific material in the plastic bag with a rubber hammer or with a monoclonal antibodies is recommended to avoid cross reactions. Bioreba stomacher or similar equipment, avoiding droplets splashing out of the bag. Hold the samples on ice for few minutes, then transfer about Enrichment DASI-ELISA 2 mL, 1 mL and 1 mL of each macerate into three sterile Complete kit based on polyclonal and monoclonal antibodies Eppendorf tubes by decantation. Store one tube with 1 mL of (8B + 5H IVIA), including extraction buffer, semiselective each sample at −20 °C for subsequent analysis or confirmation media, ELISA plates and reagents is commercially available and the other with 30% glycerol (Difco) at −80 °C. Use the from: PLANT PRINT Diagnostics, S.L., De la March 36, remaining tube with 2 mL on ice for analysis. 46512 Faura, Valencia (ES). The isolation should be done on the same day as the maceration of the samples, as well as the enrichment and the fixation of the slides for immunofluorescence. PCR analysis can be per- Direct Tissue print-ELISA formed at earliest convenience, using the Eppendorf tubes Kit based on specific monoclonal antibodies including positive stored at −20 °C. and negative preprinted controls, nitrocellulose membranes, alkaline phosphatase-conjugated antibodies and substrate: Samples from asymptomatic material PLANT PRINT Diagnostics, S.L., De la March 36, 46512 Faura, Valencia (ES). E-mail: [email protected] Collect flowers, shoots, fruitlets or stem segments in sterile bags or containers in summer or early autumn, after favourable conditions for fireblight have been confirmed (van der Zwet & Immunoglobulins Beer, 1995). For nursery plants: cut young shoots about 20 cm • Goat antimouse immunoglobulins (alkaline phosphatase- long from the most susceptible hosts available, disinfecting linked) Sigma, Steinhein (DE); scissors or pruning shears between plants. If analyses need to be • Goat antimouse immunoglobulins (fluorescein-labelled). performed in winter, collect 5Ð10 buds per plant. For plants Bio-Rad, Marnes-la-Coquette (FR); growing in the field, cut flowers when available and/or young • Goat antirabbit immunoglobulins (fluorescein-labelled). Bio- shoots about 20 cm long, disinfecting scissors or pruning shears Rad, Marnes-la-Coquette (FR). between plants. Take flowers, or peduncle and base of the limb of several leaves, or stem segments, of the selected plants. Weigh 0.1Ð1 g of plant material and use for maceration in Appendix II Sample preparation for testing antioxidant buffer (Appendix I) in amounts as for symptomatic material (above). Process the samples immediately by enrich- Samples from symptomatic material ment followed by DASI-ELISA and/or PCR and/or isolation, The samples are processed in different buffers according to the following the protocols described for symptomatic samples. IF techniques used. Antioxidant maceration buffer (Appendix I) is should be done directly with the extracts, without enrichment. strictly required for optimal enrichment of E. amylovora in The EPPO phytosanitary procedure (OEPP/EPPO, 1992) plant material (Gorris et al., 1996b). Samples can also be includes a sampling procedure for the analysis of twigs of processed in sterile phosphate-buffered saline, pH 7.2 10 mm asymptomatic woody material in nurseries. A sample consists (PBS) (Appendix I) or sterile water for direct isolation, of 100 twigs about 10 cm in length from 100 plants. If there are inmunofluorescence or direct PCR. several plant genera in the lot, these should be equally repre- Carefully select the plant parts showing the freshest symp- sented in the sample (with a maximum of three genera per toms, with exudates if possible. Select for processing the lead- sample). From each sample, randomly take 30 cut twigs and cut ing edge of lesions on each organ to analyse. The exudates can them into four pieces (120 stem pieces). Place them for 1.5 h in be processed separately, in 1Ð4.5 mL of sterile water or buffer. a rotary shaker at room temperature in sterile PBS (Appendix I) For shoots, take pieces of symptomatic shoots, including with 0.1% Tween 20 in Erlenmeyer flasks. Filter with a paper

© 2004 OEPP/EPPO, Bulletin OEPP/EPPO Bulletin 34, 159 –171 Diagnostic protocols 167 held in a sintered glass filter (n°2 = 40Ð100 µm) using a vacuum (manually or mechanically) with a larger amount of buffer for pump and collect the filtrate. Use the filtrate directly for analy- 5 min. Eliminate washing buffer and repeat the operation twice. sis or centrifuge it for 20 min at 10.000 g. Suspend the pellet in Prepare BCIP-NBT (Sigma Fast) substrate buffer (Appendix I). 4.5 mL sterile PBS (Appendix I). Pour over the membranes and incubate until appearance of A similar protocol can be applied for leaves, shoots, flowers purple-violet colour in the positive control (approx. 10 min). or buds. Depending on the season of survey the expected recovery Stop the reaction by washing the membranes with tap water. of E. amylovora will vary, being high in summer (provided weather Dry membrane by spreading out on absorbent paper. Observe conditions are favourable to E. amylovora) and low in winter). the prints at low power magnification (X10 or X20). Whichever procedure is followed, prepare for each sample 3 The test is positive when purple-violet precipitates appear in Eppendorf tubes with about 2 mL, 1 mL and 1 mL of macerate the sections of plant material and in the positive control, and not and use them as for symptomatic material (above). Direct ana- in the negative control. If exudates or colonies are printed they lysis of asymptomatic samples is normally negative for E. amy- should appear violet when positive. When using the commer- lovora due to the low bacterial population. It is accordingly cial kit (Appendix 1), preprinted membranes include positive advisable to enrich the samples (Appendix IV) in antioxidant and negative controls. The test is negative when no purple- buffer (Gorris et al., 1996a) (Appendix I). When analysing violet precipitates appear, as in the negative control. asymptomatic material, the enrichment should be done for 72 h at about 25 °C. Enrichment DASI-ELISA The only commercial kit for Enrichment DASI-ELISA (Gorris Appendix III Rapid screening tests et al., 1996b) has been validated in the ring tests. It is based on the monoclonal antibodies and technique described in Gorris Direct tissue-print ELISA et al. (1996a,b). As positive controls, use aliquots of a sample This method may facilitate presumptive diagnosis on extract that previously gave a negative result on testing, mixed symptomatic plant material but it needs further confirmation with 108 cells of E. amylovora per mL. As negative controls, because of specificity problems. The method can also be used include a sample extract that has previously given a negative to spot bacterial colonies or exudates as an additional rapid test result for E. amylovora and a suspension of a non-E. amylovora of serological identification. The use of antibodies of well strain in PBS (Appendix I). known specificity is required. The only complete kit based on Boil the necessary amount of the enriched extracts and controls specific commercially available monoclonal antibodies is in a water bath (or in a thermoblock) at 100° for 10 min before indicated in Appendix I. ELISA, making sure that the tubes are not opened. Keep the For preparation of plant-tissue prints or blots (membrane remaining enriched samples for isolation and/or PCR. Process by printing), clean cross-cuts should be made on symptomatic ELISA the boiled samples (once at room temperature) on the shoots, flowers, leaves or fruitlets in the field or in the lab. The same day or freeze them at −20 °C for subsequent analysis. This heat freshly made sections should be pressed carefully against the treatment is necessary for optimum sensitivity and specificity nitrocellulose membrane. Exudates or bacterial colonies on using the monoclonal antibodies obtained by Gorris et al. (1996a). solid medium can also be printed or spotted. The cutting tool Prepare the appropriate dilution of rabbit anti-E. amylovora should be disinfected after use to avoid contaminations. Posit- polyclonal immunoglobulins in carbonate buffer, pH 9.6 ive and negative controls should be printed, using an inoculated (Appendix I). Add 200 µL to each well of a Nunc Polysorp (or host plant with fireblight symptoms and a healthy plant of the same equivalent) ELISA plate. Incubate at 37 °C for 4 h or at 4 °C for species. The trace or print should be left to dry for a few minutes. 16 h. Wash the wells three times with washing buffer (Appen- The membranes should be handled with care. Printed membranes dix 1). Add 200 µL per well of the plant macerates previously can be kept for several months in a dry place at room temperature. enriched in the two media and boiled. Use two wells per sample Prepare a solution of 1% bovine serum albumin (BSA) in dis- enriched on each medium, and two of the positive and negative tilled water (stirring with a stick). Place the membranes in an controls. It is also necessary to place negative controls of the appropriate container (tray, hermetic container, plastic bag). extraction buffer and of the enrichment media used (previously Pour, covering them, the albumin solution and incubate for 1 h prepared as additional negative controls of the enrichment). at room temperature, or overnight at 4 °C. Slight agitation is Incubate for 16 h at 4 °C. Wash the wells three times with wash- beneficial. The incubation time elapsed, discard the albumin ing buffer (as above). solution, keeping the membranes in the same container. Prepare the appropriate dilution of specific E. amylovora Dilute specific E. amylovora monoclonal antibodies (alkaline monoclonal antibodies (Appendix 1) in PBS plus 0.5% bovine phosphatase-conjugated) in PBS (Appendix I) to the appropriate serum albumin (BSA) and add 200 µL to each well. Incubate at dilution. Pour the solution onto the membranes, covering them. 37 °C for 2 h. Wash the wells three times with PBS-Tween. Pre- Incubate for 2Ð3 h by shaking at room temperature and discard pare the appropriate dilution of goat antimouse immunoglobu- the solution. lins conjugated with alkaline phosphatase (Sigma) (Appendix Prepare washing buffer (Appendix I). Rinse the mem- 1) in PBS. Add 200 µL to each well. Incubate at 37 °C for 2 h. branes and the container with washing buffer. Wash by shaking Wash the wells three times as above. Prepare a 1 mg mL−1

© 2004 OEPP/EPPO, Bulletin OEPP/EPPO Bulletin 34, 159 –171 168 Erwinia amylovora alkaline phosphatase substrate (p-nitrophenylphosphate) in adding 1 mL of PBS (Appendix I), centrifuge for 15 min at substrate buffer (Appendix I). Add 200 µL to each well. Incubate 7000 g, discard supernatant and resuspend in PBS. at room temperature and read at 405 nm after 30, 45, and 60 min. For each set of tests, prepare a positive control slide using a The ELISA test is negative if the average optical density (OD) suspension of 106 cells mL−1 of a known pure culture of E. amy- reading from duplicate sample wells is < 2x OD of that in the neg- lovora. For large-scale survey work, it is recommended to ative sample extract control wells (provided the OD for the positive include blind positive control slides. Use PBS and an aliquot of controls are above 1.0 after 60 min incubation and are greater a sample extract, which was negative by several techniques, as than twice the OD obtained for negative sample extracts). The negative controls. ELISA test is positive if the average OD readings from duplicate Use undiluted macerates and 1 : 10 and 1 : 100 dilutions in sample wells is > 2x OD in the negative sample extract wells PBS (Appendix I) to spot windows of the IF slides. Prepare one provided that 2x average OD readings in all negative control slide for each sample and its dilutions. Allow to air-dry and fix wells are lower those in the positive control wells. Negative ELISA by flaming or by absolute or 95° ethanol according to the char- readings in positive control wells indicate that the test has not acteristics of the antibodies used. Store slides at −20 °C until been performed correctly and/or the reagents were not well pre- required. Use the monoclonal or polyclonal antibodies at the pared. Positive ELISA readings in negative control wells indicate appropriate dilutions in PBS (Appendix I). Spot 25Ð30 µL per cross-contaminations or non-specific antibody binding has well. Incubate slides in a moist chamber for 30 min at room occurred. In either case, the test should be repeated or a second test temperature. Use of two dilutions of the antibodies is advised based on a different biological principle should be performed. when working with polyclonal antibodies, to detect cross reactions with other bacteria. Shake droplets off the slide and rinse slides carefully with PBS (Appendix I). Wash 10 min with the Indirect ELISA same buffer. Carefully remove excess moisture. Use 200 µL aliquots of pure cultures of the suspected isolates Dilute the appropriate FITC conjugates in PBS: antimouse (after being treated at 100 °C for 10 min, in a water bath or for the monoclonal antibodies (GAM-FITC) (Appendix I) and heating block, to reduce non-specific reactions). Add an equal antirabbit (GAR-FITC) (Appendix I) or antigoat (Appendix I). volume of carbonate buffer (Appendix I). Apply 200 µL Cover the windows of all slides with the corresponding diluted aliquots to at least 2 wells of a Nunc-Polysorp microtitre plate conjugate and incubate in a moist chamber for 30 min at room or equivalent. Use as positive control a 109 cfu mL−1 heat- temperature. Repeat the washing step. Pipette 5Ð10 µL 0.1 m treated suspension of a pure culture of E. amylovora and as phosphate-buffered glycerol mountant with antifading (0.5% p- negative control a similar suspension of another species. phenylenediamine or other) on each window and apply a cover Incubate for 1 h at 37 °C or overnight at 4 °C. Flick out extracts slip. View slides under oil immersion at 500Ð1000× magnifica- from the wells. Wash the wells the three times with washing tion by scanning windows across 2 diameters at right angles and buffer (Appendix I), leaving the last washing solution in the around the perimeters. Calculate the number of cells per mL = l wells for at least 5 min. of the sample, according to EU (1998). Prepare the appropriate dilution of anti E. amylovora anti- The test is negative if green fluorescing cells with morphology bodies using the recommended dilutions for commercial anti- typical of E. amylovora are observed in positive controls but not bodies (Appendix I). Add 200 µL to each well and incubate for in sample windows. The test is positive if green fluorescing cells 1 h at 37 °C. Flick out the antibody solution from the wells and with typical morphology are observed in positive control and wash as before. Prepare the appropriate dilution of secondary sample windows, but not in negative control windows. As a antibody-alkaline phosphatase conjugate (GAM-AP) (Appen- population of 103 cells per mL is considered the limit of reliable dix I) in PBS + 0.5% BSA. Add 200 µL to each well and incub- detection by the IF test, for samples with > 103 cells per mL, the ate for 1 h at 37 °C. Flick out conjugated antibody from wells IF test is considered positive. For samples with < 103 cells per ml, and wash as before. Prepare a 1 mg mL−1 alkaline phosphatase the result of the IF test may be considered doubtful. In such a substrate (p-nitrophenylphosphate) in substrate buffer (Appen- case, further testing or re-sampling should be performed. Samples dix I). Add 200 µL alkaline phosphatase substrate solution to with large numbers of incomplete or weakly fluorescing cells each well. Incubate in the dark at room temperature and read at compared to the positive control need further testing, with dif- 405 nm at regular intervals within 90 min. ferent dilutions of antibody or pellet or a second source of antibodies.

Immunoﬂuorescence (IF) PCR Follow the standard protocol described in EU (1998). Use Use validated PCR reagents and protocols. Take every pre- a validated source of antibodies to E. amylovora. Three caution to avoid contamination of samples. Use two positive commercial antibodies have been validated in the ring test. It is controls with aliquots of sample extract of the same host (which recommended that the titre and dilution of use are determined previously gave a negative test result) to which suspensions of for each new batch of antibodies. The IF test should be 104 and 106 cells mL−1 of a known E. amylovora strain have performed on freshly prepared sample extracts. If the extracts been added and a suspension of 104 cells mL−1 of E. amylovora. stored at −80 °C under glycerol are used, remove glycerol it by Prepare positive controls in a laboratory separate from the one

© 2004 OEPP/EPPO, Bulletin OEPP/EPPO Bulletin 34, 159 –171 Diagnostic protocols 169 where the samples will be tested. As negative controls, use at Primer A: 5′-CGG TTT TTA ACG CTG GG least one sample extract whch previously gave a negative test Primer B: 5′-GGG CAA ATA CTC GGA TT result for E. amylovora by several techniques and a sample of The PCR mix is as follows: water (PCR grade) 20 µL; buffer × µ µ µ the ultra-pure water (PCR reagent) used. Include more negative 10 5 L; MgCl2 50 mm 1.5 L; dNTPs 10 mm 1 L; 2- controls for each step of the DNA extraction and amplification mercaptoethanol 1.4 µL; bovine serum albumin 1 µg µL−1 if contamination is suspected. Perform the DNA extraction 8 µL; DMSO 2.5 µL; Tween 20 0.5 µL; Primer A 10 pmol µL−1 from the positive and negative controls as well as from the 2.5 µL; Primer B 10 pmol µL−1 2.5 µL; Tth polymerase 5 U samples. µL−1 0.1 µL. Sample volume: add 5 µL to 45 µL mix. One protocol for DNA extraction from plant samples (Llop The reaction conditions are: a denaturation step of 93 °C for et al., 1999) and two amplification protocols (Bereswill et al., 2 min followed by 37 cycles of 93 °C for 1 min, 52 °C for 2 min, 1992 and Llop et al., 2000) have been validated in the ring test. and 72 °C for 2 min. A final step of 72 °C for 10 min finishes the Some commercial kits for extracting DNA are available, but reaction. The amplicon size is 900 bp size according to Bereswill they have not been validated. et al. (1992) although variations in size can occur between 900 and 1100 bp (Lecomte et al., 1997), due to the number of 8 bp repeats sequences within the fragment (Jones & Geider, 2001). DNA extraction A simpler PCR mix gave the same results as the original: µ × µ µ Take directly 1 mL of each macerate and/or 1 mL of the water (PCR grade) 33.1 L; buffer 10 5 L; MgCl2 50 mm 3 L enriched macerates to follow the protocol for extracting the 3 mm; formamide 2 µL 4% (v/v); dNTPs 10 mm 0.5 µL; Primer DNA according to Llop et al. (1999). Centrifuge the macerates A 10 pmol µL−1 0.5 µL; Primer B 10 pmol µL−1 0.5 µL; Taq poly- at 13.000 rev min−1 for 5 min at room temperature. Discard the merase 5 U µL−1 0.4 µL. Sample volume: add 5 µL to 45 µL mix. supernatant, and resuspend the pellet in 500 µL of extraction The reaction conditions are: a denaturation step of 93 °C for buffer (Appendix I) and shake for 1 h at room temperature. 5 min followed by 40 cycles of 93 °C for 30 s, 52 °C for 30 s, Centrifuge the tubes at 5.000 rev min−1 for 5 min. Take 450 µL and 72 °C for 1 m 15 s. A final step of 72 °C for 10 min finishes of the supernatant and place it into a new Eppendorf tube. Add the reaction. Single components of the mix can be replaced by the same volume of isopropanol, invert several times and leave Ready Mix Taq with MgCl2 or Ready Mix Red Taq PCR Reac- −1 for 1 h at room temperature. Centrifuge at 13.000 rev min for tion Mix with MgCl2 (Sigma). 5 min, discard the supernatant and dry on bench at room temperature. If there is still a coloured precipitate (brown or Nested PCR green) at the bottom of the tubes, carefully take it while discarding the supernatant, thus obtaining a cleaner DNA. The nested-PCR in a single tube (Llop et al., 2000) uses two Normally, the DNA sticks to the wall of the Eppendorf tube sets of primers added at the same time. Due to their different more than to the bottom. Resuspend the pellet in 200 µL of annealing temperatures, the two PCR reactions are performed water. Use 5 or 1 µL for the PCR reaction depending on the consecutively. The external primers are those of McManus & amplification performed. Jones (1995), while the internal ones are those of Llop et al. (2000). The sequences are the following (Appendix I): External primers Amplification AJ75: 5′-CGT ATT CAC GGC TTC GCA GAT There are many PCR primers and protocols described for AJ76: 5′-ACC CGC CAG GAT AGT CGC ATA E. amylovora detection and identification. Most of them can Internal primers be used for specific identification (Bereswill et al., 1992; PEANT1: 5′-TAT CCC TAA AAA CCT CAG TGC Bereswill et al., 1995; Guilford et al., 1996; McManus & PEANT2: 5′-GCA ACC TTG TGC CCT TTA Jones, 1995). Some have specificity problems, like the primers The PCR mix is as follows: water (PCR grade) 34.76 µL; × µ µ µ of Maes et al. (1996) which amplified an Erwinia sp. isolated buffer 10 5 L; MgCl2 50 mm 3 L; formamide 2 L; dNTPs from necrotic pear blossoms and different from E. amylovora 10 mm 1 µL; Primer AJ75 0.1 pmol µL−1 0.32 µL; Primer AJ76 (Roselló et al., 2002). The primers and protocols validated were 0.1 pmol µL−1 0.32 µL; PEANT1 10 pmol µL−1 1 µL; those of Bereswill et al. (1992) and Llop et al. (2000) because PEANT2 10 pmol µL−1 1 µL; polymerase 5 U µL−1 0.6 µL. these were the conventional nested PCRs found to be most Sample volume: add 1 µL to 49 µL PCR mix. sensitive and robust in preliminary experiments with artificially The reaction conditions are: a denaturation step of 94 °C for contaminated plant material. Nevertheless, PCR analysis 4 min followed by 25 cycles of 94 °C for 30 s and 72 °C for following Guilford et al. (1996) or McManus & Jones (1995) is 1 min. This first-round PCR is followed in the same thermocycler also good, in the case of tests on bacterial suspensions. by a second denaturation step of 94 °C for 4 min and 40 cycles of 94 °C for 30 s, 56 °C for 30 s, and 72 °C for 45 s. A final step Conventional PCR of 72 °C for 10 min finishes the reaction. The amplicon size is The validated conventional (or single) PCR uses the primers 391 bp, although some variations in size can occur. and conditions described by Bereswill et al. (1992). The primer The amplification conditions are optimized for the Perkin- sequences are the following (Appendix I): Elmer 9600 apparatus, so small modifications of the protocol

© 2004 OEPP/EPPO, Bulletin OEPP/EPPO Bulletin 34, 159 –171 170 Erwinia amylovora could be necessary for other thermocyclers. When using the As soon the macerates have been made (Appendix II), dis- PCR reactions to confirm the isolated colonies, use 1 U of Taq pense 0.9 mL of each sample into two sterile 3Ð5 mL tubes pre- polymerase for Bereswill et al. (1992) protocol or 2 U with pared in advance with the same volume of each enrichment Llop et al., 2000) (instead of 2 or 3 U as for plant material). medium. Prepare, as additional negative controls, three tubes with 0.9 mL of maceration buffer (Appendix I) and add the same volume of the same buffer and of each enrichment medium Electrophoresis (CCT and King’s B sterile liquid media) (Appendix I). Incubate After either PCR, prepare a 1.5% agarose gel in TAE buffer at 25 °C for 48 h without shaking. Incubate for 72 h when low 0.5 X (Appendix I). Place 3 µL droplets of loading buffer numbers of E. amylovora are expected, as indicated above. (Appendix I) on parafilm, mix 20 µL of PCR product by gentle aspiration with the pipette before loading. Load wells of gel and Appendix V Isolation include positive and negative controls. Include DNA marker 100 bp ladder in the first and last well of the gel. Run the gel for Direct isolation 20 min at 120 V (medium gel tray: 15 × 10 cm) or 40 min at 160 V (big gel tray or electrophoresis tank: 15 × 25 cm). Soak Use CCT, King’s B and Levan (or Nutrient Agar Sucrose) the gel in ethidium bromide solution for 20 min. Visualize the media (Appendix I). Prepare 1 : 10 and 1 : 100 dilutions of each amplified DNA fragments by UV trans-illumination. macerate (Appendix II) in PBS (Appendix I). Pipette 50 µL of The amplicons obtained with the primers of Bereswill et al. the diluted and undiluted macerates onto separate plates of each (1992) and the amplicons obtained with the nested PCR in a medium. Start with the 1 : 100 dilution and proceed to the single tube (Llop et al., 2000) can be treated with Dra I and Sma undiluted macerate. Use sterile loops or spreader or dip a glass I endonucleases, and the restriction patterns obtained used to spreader in denatured ethanol, flame and allow cooling. confirm the PCR analysis. Carefully spread the pipetted volumes by triple streaking. Plate a 103, 104 and 105 cfu mL−1 dilution of a pure culture of E. amylovora as a quality control of the media. Incubate the plates Interpretation of the PCR results at about 25 °C for 48Ð72 h. Final reading is at 72Ð96 h. The PCR test is negative if the E. amylovora-specific amplicon Colonies of E. amylovora on CCT appear at about 48 h and of expected size and restriction pattern is not detected for the are pale-violet, circular, high convex to domed, smooth and sample in question but is detected for all positive control samples. mucoid after 72 h, showing slower growth than on King’s B or The PCR test is positive if the E. amylovora-specific amplicon Levan. CCT medium inhibits most pseudomonads but not Pantoea of expected size is detected, provided that it is not amplified agglomerans. Colonies of E. amylovora on King’s B appear at from any of the negative control samples and the restriction 24 h and are creamy white, circular, tending to spread and non- enzyme pattern is identical with that derived from the positive fluorescent under UV light at 366 nm after 48 h. This allows control strain. If one of the negative controls shows a band of distinction from fluorescent pseudomonads. Colonies of E. the size expected for E. amylovora, repeat the PCR with a new amylovora on Levan medium appear at 24 h and are whitish, circular, mix and include several negative controls that allow detection domed, smooth and mucoid after 48 h. Levan-negative colonies of contaminants. Reliable confirmation of a positive result can be of E. amylovora have also been reported (Bereswill et al., 1997). obtained by repeating the test with a second set of PCR primers. Obtain pure cultures from individual suspect colonies of each Inhibition of the PCR may be suspected if the expected amplicon sample by plating on King’s B medium. Identify presumptive is obtained from the positive control sample containing E. colonies of Erwinia amylovora by inoculation on the available amylovora in water but negative results are obtained from E. amylovora host, by DASI-ELISA or PCR, or by any other test positive controls with E. amylovora in plant extract. (biochemical tests, IF, fatty acids profile, etc.) as indicated. Store cultures in Nutrient agar slants covered with mineral oil at 10 °C or for long-term in 30% glycerol at −80 °C or lyophilized. Appendix IV Enrichment The isolation is negative if no bacterial colonies with mor- Enrichment is used to multiply the initial population of phology similar to E. amylovora are observed after 96 h in any culturable E. amylovora in the sample. It is needed before of the three media (provided that no inhibition is suspected due detection by ELISA, because of the low level of sensitivity of to competition or antagonism) and that typical E. amylovora this technique when using specific monoclonal antibodies. It colonies are found in the positive controls. The isolation is should also be used before isolation or before PCR (even in positive if presumptive E. amylovora colonies are isolated in at symptomatic samples) when a low number of culturable E. least one of the media used and the identification is confirmed amylovora is expected (copper-treated samples, old symptoms, by one of the methods indicated. unfavourable weather conditions for fireblight, winter, etc.) or when a high amount of inhibitors is expected. Use of two Enrichment isolation validated media, one non-selective (King’s B) and one semi-selective (CCT) (Appendix I), is advised because the Plate the enrichments only on CCT plates (Appendix I). Spread composition and number of the microbiota is unknown. 50 µL of each enriched extract and of the 1 : 10, 1 : 100 and

1 : 1000 dilutions prepared in PBS (Appendix I) by triple wounding site and oozing of bacteria in 3Ð7 days (provided that streaking (as for isolations) to obtain isolated colonies. Incubate the negative control gives only a necrotic lesion). at about 25 °C for 72Ð96 h. The use of only semi-selective For whole plant inoculation, use susceptible cultivars of pear, medium and dilutions is advised because of the abundant apple or loquat, or susceptible species of Crataegus, Cotoneaster multiplication of different bacteria during the enrichment step. or Pyracantha. To inoculate a potted plant, cut a young leaf on a young shoot to the main vein with scissors dipped into a 109 cfu mL−1 suspension of each test colony prepared in PBS (Appendix I). Appendix VI Bioassay Detached young shoots from glasshouse-grown plants can be Inoculation of fruitlets (of susceptible cultivars of pear, apple or inoculated in the same way, after disinfection for 30 s with 70% loquat) can be performed on whole immature fruits or on slices ethanol and 3 washings with sterile distilled water, and kept in of them, using 10 µL of 109 cfu mL−1 suspensions of colonies tubes with sterile 1% agar. Maintain the plants at 20Ð25 °C at in PBS (Appendix I). Include a positive and negative control as 80Ð100% relative humidity and the tubes at 20Ð25 °C with 16 indicated below. Incubate in a humid chamber at 25 °C for 3Ð h light. Read results after 3, 7 and 15 days. Typical E. amylovora 5 days. A positive test on fruit is shown by browning around the symptoms include wilting, discoloration, necrotic tissue and ooze.

A B

C D

Fig. 1 A: Symptoms of fire blight in pear blossoms (M. Cambra, IVIA, Spain); B: Symptoms of fire blight in an apple shoot (M.A.Cambra, D.G.Aragón, Spain); C: Canker caused by E. amylovora on pear trunk (M. Cambra, IVIA, Spain); D: Symptoms of fire blight in pear shoots (M.M.López, IVIA, Spain)

A B

Fig. 2 A: Symptoms of E. amylovora in Pyracantha (M.M.López, IVIA, Spain) ; B: Inoculation on leaves of detached pear shoots: left, negative control; right, E. amylovora (P. Llop, IVIA, Spain); C: Symptoms of E. amylovora in immature pear fruits after inoculation (V. Donat, IVIA, Spain); D: Colonies of E. amylovora on CCT (M.M.López, IVIA, Spain).