PM 7/20 (3) Erwinia amylovora

Abstract

EPPO BulletinEarly View EPPO STANDARD ON DIAGNOSTICSFree Access PM 7/20 (3) Erwinia amylovora First published: 24 April 2022 https://doi.org/10.1111/epp.12826AboutSectionsPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinked InRedditWechat Specific scope This Standard describes a diagnostic protocol for Erwinia amylovora.11 Use of names of chemicals or equipment in these EPPO Standards implies no approval of them to the exclusion of others that may also be suitable. It should be used in conjunction with PM 7/76 Use of EPPO diagnostic protocols. Specific approval and amendment This Standard was developed under the EU DIAGPRO Project (SMT 4-CT98-2252) and EUPHRESCO Pilot project (ERWINDECT) by a partnership of contractor laboratories. Test performance studies were performed with different laboratories in 2002, 2009 (Reisenzein et al., 2010), 2010 (López et al., 2010) and 2019 (Alič et al., 2020). Approved as an EPPO Standard in 2003–09. Revised in 2012–09 and 2021–09. Authors and contributors are given in the Acknowledgements section. 1 INTRODUCTION Erwinia amylovora is the causal agent of fire blight, a disease that affects most species of the subfamily Maloideae of the family Rosaceae (Spiraeoideae). It was the first bacterium described as the causal agent of a plant disease (Burrill, 1883). E. amylovora is considered to be native to North America and was first detected outside North America in New Zealand in 1920. Fire blight was reported in England in 1957 and since then the bacterium has been detected in most areas of Europe where susceptible hosts are cultivated. E. amylovora is now present in more than 50 countries. It has not been recorded in South America and most African and Asian countries (except for countries surrounding the Mediterranean Sea), and it has been eradicated in Australia after a first report there (van der Zwet, 2004). It represents a threat to the pome fruit industry of all these countries (Bonn & van der Zwet, 2000). The most important host plants from both economic and epidemiological viewpoints are in the genera Chaenomeles, Cotoneaster, Crataegus, Cydonia, Eriobotrya, Malus, Mespilus, Pyracantha, Pyrus, Sorbus and Stranvaesia (Bradbury, 1986). The E. amylovora strains isolated from Rubus sp. in the United States are distinct from the strains on other hosts (Starr et al., 1951; Powney et al., 2011). Details on geographic distribution and host plants can be found in the EPPO Global Database (EPPO, 2021a). Fire blight is probably the most serious bacterial disease affecting Pyrus communis (pear) and Malus domestica (apple) cultivars in many countries. Epidemics are sporadic and are dependent on several factors, including favourable environmental conditions, sufficient inoculum level present in the orchard and host susceptibility. The disease is easily dispersed by birds, insects, rain or wind (Thomson, 2000). The development of fire blight symptoms follows the seasonal growth development of the host plant. The disease begins in spring with the production of the primary inoculum from bacteria overwintering in cankers (Thomson, 2000) causing blossom infection driven by the activity of pollinating insects (van der Zwet & Keil, 1979) and other climatic factors (e.g. rain, wind, and hail), continues into summer with shoot and fruit infection, and ends in winter with the development of cankers. The pathogen appears quiescent through the dormant period of the host (van der Zwet & Beer, 1995), but the experience in Portugal is that it may remain active during winter (L. Cruz, personal communication). Flow diagrams describing the diagnostic procedure for E. amylovora in symptomatic and asymptomatic material are presented in Figures 1 and 2. FIGURE 1Open in figure viewerPowerPoint Flow diagram for diagnosis of Erwinia amylovora in symptomatic samples FIGURE 2Open in figure viewerPowerPoint Flow diagram for analysis of Erwinia amylovora in asymptomatic samples 2 IDENTITY Name: Erwinia amylovora (Burrill, 1882) Winslow et al., 1920 Other scientific names: Micrococcus amylovorus (Burrill, 1882), Bacillus amylovorus (Burrill, 1882) Trevisan, 1889, Bacterium amylovorus (Burrill, 1882) Chester, 1897, Erwinia amylovora f. sp. rubi (Starr, 1951), Cardona & Falson Taxonomic position: Bacteria, Proteobacteria, γ Subdivision, Enterobacteriales, Enterobacteriaceae EPPO Code: ERWIAM Phytosanitary categorization: EPPO A2 list no. 52, EU Protected Zone Quarantine pest (Annex III), EU Regulated Non-Quarantine Pest (Annex IV) 3 DETECTION 3.1 Disease symptoms Symptoms of fire blight on the most common hosts such as P. communis (pear), M. domestica (apple), Cydonia spp. (quince), Eriobotrya japonica (loquat), Cotoneaster spp. (cotoneaster), Pyracantha spp. (pyracantha) and Crataegus spp. (hawthorn) are relatively similar and easily recognized (Figures 3-5). The name of the disease is descriptive of its major characteristic: the brownish to blackish necrotic appearance of twigs, flowers and leaves, as though they had been burned by fire. The typical symptoms are the brown to black colour of leaves on affected branches, the production of exudates, and the characteristic ‘shepherd's crook’ of terminal shoots. Depending on the affected plant part and phenological stage, the disease symptoms may include blossom blight, shoot or twig blight, leaf blight, fruit blight, limb or trunk blight, or collar or rootstock blight (van der Zwet & Keil, 1979; van der Zwet & Beer, 1995). FIGURE 3Open in figure viewerPowerPoint Symptoms of fire blight on pear trees: (a) necrotic flowers, (b) necrosis on leaves and typical shepherd's crook, (c) mummified immature fruits with small ooze drops and (d) canker after removing bark showing necrotic inner tissues FIGURE 4Open in figure viewerPowerPoint Typical symptoms of fire blight on (a) pear branches, (b) apple shoot, (c) quince shoot and (d) loquat shoot FIGURE 5Open in figure viewerPowerPoint Typical symptoms of fire blight on (a) Crataegus sp. shoot, (b) Cotoneaster sp. shoot and (c, d) Pyracantha sp. branches In apple and pear trees the first symptoms usually appear in early spring when the average temperature rises above 15°C, during humid weather. Infected blossoms become soaked with water, then wilt, shrivel and turn orange or brown to black. Peduncles may also appear water-soaked, become dark green, and finally brown or black, sometimes oozing droplets of sticky bacterial exudates. Infected leaves wilt and shrivel, and entire spurs turn brown in apples and dark brown to black in pears but remain attached to the tree for some time. On infection young fruitlets turn brown but also remain attached to the tree. Immature fruit lesions appear oily or water-soaked, become brown to black and often ooze droplets of bacterial exudate. Characteristic running reddish-brown streaks are often found in the subcortical tissues when the bark is peeled from infected limbs or twigs (van der Zwet & Keil, 1979). Brown to black slightly depressed cankers form in the bark of twigs, branches or the trunk of infected trees. These cankers later become defined by cracks near the margin of diseased and healthy tissue (Thomson, 2000). Additionally, the epidermis may roll up, resembling papyrus paper (L. Cruz, personal communication). Confusion may occur between fire blight and blight- or blast-like symptoms – especially in blossoms and buds – caused by other pathogens including bacteria (Figure 6a,b) and fungi (Figure 6c,d), insect damage (Figure 6e) and physiological disorders. FIGURE 6Open in figure viewerPowerPoint Confusing symptoms caused by (a) Erwinia piriflorinigrans in Pyrus, (b) Pseudomonas syringae pv. syringae in Pyrus, (c) Stemphylium vesicarium in Pyrus, (d) Monilia laxa in Prunus persicae (similar mumification caused by monilia may be observed) and (e) Janus compressus (Hymenoptera) (note oviposition punctures at the base of the crook) Other bacteria that cause fire blight-like symptoms include Erwinia pyrifoliae, the causal agent of bacterial shoot blight of Pyrus pyrifolia (Asian pear) (Kim et al., 1999), Erwinia piriflorinigrans (Figure 6a), isolated from necrotic pear blossoms in Spain (López et al., 2011) and Iran (Moradi Amirabad & Khodakaramian, 2017; Moradi-Amirabad et al., 2020), Erwinia uzenensis, recently described in Japan (Matsuura et al., 2012), other Erwinia spp. reported in Japan that cause bacterial shoot blight (Tanii et al., 1981; Kim, Hildebrand, et al., 2001; Kim, Jock, et al., 2001; Palacio-Bielsa et al., 2012), and Pseudomonas species such as Pseudomonas syringae pv. syringae, the causal agent of blossom blast (Figure 6b). 3.2 Detection from symptomatic samples 3.2.1 Test sample requirements Symptomatic samples can be processed individually or in small batches combining material from several samples (see 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 (with necrosis and/or with exudates), or the discoloured subcortical tissues (after peeling bark from cankers in branches, trunk or collar). Samples should be processed as soon as possible after collection but can be kept at 4–8°C for up to 1 week before analysis, if necessary. 3.2.2 Screening tests At least two tests, based on different biological principles or targeting different parts of the genome, should be performed. For areas where the pest is established one screening test may be sufficient to declare that E. amylovora is detected or not detected in the sample. However, in case of doubt a second test should be performed. In addition, if isolation of colonies with typical morphology and identification of E. amylovora is positive, a second screening test is not necessary. Isolation Fresh sample extracts are necessary for successful isolation. Details on the extraction procedure from plant material are given in Appendix 1. Details of isolation are provided in Appendix 5. Isolating E. amylovora from symptomatic samples is relatively easy because the number of culturable bacteria in such samples is usually high. However, when phytosanitary treatments with bacteriostatic products are used, when symptoms are very advanced or when the environmental conditions after infection are not favourable for bacterial multiplication, the number of culturable E. amylovora cells can be very low. Isolation under these conditions can result in plates with few cells of the pathogen that can be overgrown with saprophytic and antagonistic bacteria. If this is suspected, the sample should be re-tested and/or enriched before isolation. The induction of the reversible viable but non-culturable state (VBNC) has been shown for E. amylovora in vitro using copper treatments and in fruits (Ordax et al., 2009), and it can be the cause of false-negative isolation results. Indeed, in this VBNC state, bacteria do not grow in the solid culture media but remain pathogenic. This state is reversible, and the bacteria can become culturable and pathogenic again (Ordax et al., 2006). If E. amylovora is found in the samples in the VBNC state, the results of isolation will be negative, but the bacteria will still be potentially pathogenic and can be detected by Enrichment-ELISA and PCR-based methods. When plates are overcrowded by plant microbiota, the sample should be retested and enrichment (according to Appendix 4) performed before isolation (as described in Appendix 5). For direct isolation, plating on at least two different media in parallel (to be chosen depending on the sample) is recommended for maximum recovery of E. amylovora, in particular when samples are in poor condition. The efficiency of the different media depends on the number and composition of microbiota in the sample. Three media, King's B, NSA and CCT (Appendix 2), have been validated in a test performance study. Colonies of E. amylovora on CCT appear at about 48 h and are pale violet, circular, highly convex to domed, smooth and mucoid after 72 h, showing slower growth than on King's B or NSA. CCT medium inhibits most pseudomonads but not enterobacteria such as Pantoea agglomerans. Colonies of E. amylovora on King's B appear at approximately 24 h and are creamy white, circular, tending to spread and non-fluorescent under UV light at 366 nm after 48 h. This allows distinction from fluorescent pseudomonas. Colonies of E. amylovora on NSA medium appear at about 24 h and are whitish, circular, domed, smooth and mucoid after 48 h. NSA-negative colonies of E. amylovora have also been reported (Bereswill et al., 1997). Figure 7 shows the typical appearance of E. amylovora bacterial cultures in the three media after incubation at 25°C for 24, 48 and 72 h. Colonies that were in VBNC state may appear later. Very rarely, E. amylovora colonies may exhibit a pink colour because of the presence of other bacteria affecting bacterial iron acquisition. When re-streaked and purified, the E. amylovora colonies lose the pink colouration (Stockwell et al., 2008; personal communication, Tanja Dreo, NIB). FIGURE 7Open in figure viewerPowerPoint Typical colony morphology of Erwinia amylovora on CCT medium (left), NSA medium (middle) and King's B medium (right) after incubation at 25°C for (a) 24 h, (b) 48 h and (c) 72 h Pure cultures from individual suspect colonies of each sample should be obtained by plating on King's B medium and presumptive colonies of E. amylovora should be identified as indicated in the identification section. The isolation is negative if no bacterial colonies with morphology similar to E. amylovora are observed after 96 h in any of the media (provided no inhibition is suspected due to competition or antagonism) and typical E. amylovora colonies are found in the positive controls. The isolation is positive if presumptive E. amylovora colonies are isolated in at least one of the media used and the identification is confirmed by one of the methods indicated. On the mentioned media and under the same incubation conditions, E. piriflorinigrans colonies are similar to E. amylovora in terms of size and morphology but appear in general faster than E. amylovora. E. pyrifoliae colonies are more mucoid and fluid compared to the E. amylovora colonies under the same incubation conditions and on the same media (Figure 8). Pseudomonas syringae pv. syringae grows faster on NSA medium and colonies are fluorescent under King's B medium. FIGURE 8Open in figure viewerPowerPoint Typical colony morphology of Erwinia piriflorinigrans on (a) CCT medium, (b) NSA medium and (c) King's B medium after incubation at 25°C for 48 h Other screening tests These tests facilitate the presumptive diagnosis of plants with fresh pronounced symptoms. Several tests are described in Appendices 3–11. Test performance studies were conducted, and the results are indicated. Serological tests Indirect immunofluorescence (IF), enrichment DASI-ELISA and lateral flow devices are described for analyses of organs with symptoms. Instructions for performing an IF test are provided in EPPO Standard PM 7/97 Indirect immunofluorescence test for plant pathogenic bacteria (EPPO, 2009) and those for performing ELISA are provided in EPPO Standard PM 7/101 ELISA tests for plant pathogenic bacteria (EPPO, 2010). Quality of the antibodies is critical for the performance of the tests. In test performance studies, several commercial antisera and monoclonal antibodies were compared for IF [polyclonal antiserum from Loewe Biochemica GmbH (Sauerlach, Germany) and monoclonal antibodies from Plant Print Diagnostics S.L. (Faura, Spain)]. For ELISA, a complete kit based on a combination of specific monoclonal antibodies, from Plant Print Diagnostics S.L., was also evaluated. Two lateral flow devices commercialized by Bioreba, Reinach, Switzerland (Ea AgriStrip) and Abingdon Health, York, UK (Pocket Diagnostics) are available for the rapid analysis of symptomatic plant material (Braun-Kiewnick et al., 2011). Details of the tests are given in Appendix 3. Molecular tests Many tests for conventional PCR have been developed for E. amylovora but some have shown a lack of analytical specificity, e.g. cross-reaction with Erwinia piriflorinigrans (Maes et al., 1996), or do not detect all strains (Bereswill et al., 1992; McManus & Jones, 1995 and Llop et al., 2000). It has also been observed that the nested PCR (Llop et al., 2000) generates several false-positive results which cannot be confirmed with any other test or subsequent testing or symptom observation (Tanja Dreo, personal communication, NIB). Two conventional PCR tests (Taylor et al. (2001), and an adaptation from Obradovic et al. (2007), two real-time PCR tests (Pirc et al., 2009 and Gottsberger, 2010) and one loop-mediated isothermal amplification (LAMP) test are recommended in this diagnostic protocol and are described in Appendices 7–11. All tests were evaluated in test performance studies in 2009 (Reisenzein et al., 2010), 2010 (López et al., 2010) and/or 2019 (Alič et al., 2020; Trontin et al., 2021), and are recommended for the analyses of organs with symptoms after a DNA extraction step. The DNA extraction protocols that were evaluated in a test performance study in 2009 and/or 2019 are indicated in Appendix 6. 3.3 Detection from asymptomatic samples 3.3.1 Test sample requirements Warning: Detection of E. amylovora in asymptomatic plants is difficult. Whenever possible, testing of asymptomatic plants should be performed in summer or early autumn to increase the likelihood of detecting E. amylovora. Asymptomatic samples may be processed individually or bulked (see Appendix 1). Precautions to avoid cross-contamination should be taken when collecting the samples and during the extraction process. Sampling and sample preparation can be performed following one of the methods described in Appendix 1 for asymptomatic samples. Direct analysis of asymptomatic samples is usually negative for E. amylovora due to the low bacterial population. Consequently, an enrichment step is recommended (Appendix 4). 3.3.2 Screening tests Enrichment-isolation, enrichment-DASI ELISA and enrichment conventional PCR or enrichment real-time PCR can be used as screening tests and are described in Appendices 4–10. At least two screening tests should be performed. 3.4 Confirmation of positive results of screening tests In critical cases (EPPO, 2017) and for asymptomatic samples, if two of the screening tests are positive, an attempt should be made to isolate the pathogen directly from the extract of non-enriched samples (Appendices 1 and 5), or from the enriched samples (Appendices 4 and 5). As little is usually known about the microbiota present in the samples, at least two different media (CCT, King's B, NSA) described in Appendix 2 should be used to maximize the likelihood of successful direct isolation of E. amylovora. However, plating on CCT only is sufficient after enrichment of the samples in King's B or CCT. If necessary, the extract is conserved at approximately −20 or −80°C under glycerol (Appendix 1). 4 IDENTIFICATION Pure cultures of presumptive E. amylovora isolates should be identified with at least two tests based on different characteristics of the pathogen (e.g. combinations of biochemical, 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 genome and provided that the specificity of the primers has been evaluated. Known E. amylovora reference strains should be included for each test performed (see the section on Reference material). 4.1 Serological tests Different sources of antibodies should be used for identification to reduce the risk of false positives. 4.1.1 Agglutination test Suspected E. amylovora colonies can be tested for agglutination by mixing them in a drop of PBS (Appendix 2) with a drop of E. amylovora-specific antiserum (not diluted, or five- or tenfold dilution) on a slide. Monoclonal antibodies can be used only if they agglutinate with the reference strains. Colonies grown on media promoting the production of polysaccharides (e.g. NSA) should be washed three times in saline solution. 4.1.2 Immunofluorescence test Instructions for performing an IF test are provided in EPPO Standard PM 7/97 Indirect immunofluorescence test for plant pathogenic bacteria (EPPO, 2009). For identification, IF can be performed using specific monoclonal antibodies from Plant Print Diagnostics S.L. or antiserum from Loewe Biochemica GmbH. 4.1.3 ELISA tests Instructions for performing ELISA are provided in EPPO Standard PM 7/101 ELISA tests for plant pathogenic bacteria (EPPO, 2010). DASI-ELISA for isolate identification can be performed using the same specific monoclonal antibodies as used for the analysis of plant samples (kit from Plant Print Diagnostics S.L.). For DASI-ELISA, a suspension of approximately 108 cells/mL from suspected colonies is prepared in PBS (Appendix 2). The DASI-ELISA procedure (Appendix 3) can be followed without prior enrichment for isolate identification. 4.1.4 Lateral flow immunoassays A suspension of approximately 108 cells/mL prepared in PBS (Appendix 2) from suspected colonies should be used following the manufacturers’ instructions. The two kits evaluated in a test performance study (Agri-strip and Pocket Diagnostic) and recommended for analyses of symptomatic plants can be used for identification of isolates. 4.2 Molecular tests Conventional and/or real-time PCR and LAMP are the recommended molecular tests for rapid identification, but other available techniques are also indicated. 4.2.1 Conventional PCR A suspension of approximately 106 cells/mL in molecular-grade water should be prepared from E. amylovora-like colonies. Appropriate PCR procedures should be applied, following Appendices 7 or 8, without DNA extraction, just after treatment at approximately 95–100°C for approximately 8–10 min. 4.2.2 Real-time PCR Two published real-time PCR tests, described in Appendices 9 and 10, are recommended. Colonies can be prepared as for conventional PCR (section 4.2.1). 4.2.3 LAMP One LAMP test described in Appendix 11 is recommended. Suspensions can be prepared as for conventional PCR (section 4.2.1). 4.2.4 DNA sequencing methods Comparisons of sequenced PCR products amplified from selected housekeeping genes allow differentiation of E. amylovora isolates from other members of the Enterobacteriaceae. For example, all isolates of E. amylovora tested so far are clonally related according to partial recA gene sequence (Waleron et al., 2002) using the method described by Parkinson et al. (2009). These data are also confirmed by comparative genome analysis (Mann et al., 2013; Zeng et al., 2018; Parcey et al., 2020). Sequence analysis should follow the guidelines described in Appendices 7 and 8 of the EPPO Standard PM 7/129 DNA barcoding as an identification tool for a number of regulated pests (EPPO, 2021b). 4.3 Matrix-assisted laser desorption/ionization-time of flight mass spectrometry A matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) mass spectrometry method for proteomic analysis has been described by Sauer et al. (2008) and Wensing et al. (2011). This allows rapid, reliable and robust identification of E. amylovora isolates from plant samples. For their routine identification all individual isolates should be included in duplicate by directly depositing harvested 3-day-old bacterial cells from nutrient agar (NA) plates onto a stainless plate, without any prior formic acid treatment. All spectra should be obtained in linear positive-ion mode with an m/z range of 2000–20 000 Da. Validation data of the MALDI-TOF as an identification test of E. amylovora isolates is already available. 4.4 Pathogenicity tests and hypersensitivity When necessary, suspected E. amylovora colonies from the isolation and/or enrichment plates may be inoculated in plants to confirm their pathogenicity, preferably on detached organs of a fire blight host (Appendix 12). The hypersensitive reaction in tobacco leaves can give an indication of the presence of the hrp pathogenicity genes, but is also positive for many other plant pathogenic bacteria and can be difficult to interpret (Appendix 12). 4.5 Other tests 4.5.1 Biochemical tests The genus Erwinia has been defined as Gram-negative bacteria, facultative anaerobes, motile by peritrichous flagella, rod-shaped, acid produced from glucose, fructose, galactose and sucrose. The phenotypic properties are described in Paulin (2000) and should be determined according to the methods of Jones and Geider (2001). The tests in Table 1, based mainly on results in API 50 CH strips (BioMérieux, France), allow differentiation of E. amylovora from E. pyrifoliae, causal agent of Asian pear blight on Pyrus pyrifolia (Kim et al., 1999; Rosello et al., 2006) and E. piriflorinigrans (López et al., 2011; Moradi Amirabad & Khodakaramian, 2017; Moradi-Amirabad et al., 2020). However, certain physiological and biochemical characteristics can vary for some strains. TABLE 1. Differences between Erwinia amylovora, Erwinia pyrifoliae and Erwinia piriflorinigrans Microbiological tests Erwinia amylovora Erwinia pyrifoliae Erwinia piriflorinigrans Gelatine hydrolysis + − − Inositola a Oxidation of substrates in API 50CH (BioMérieux) with a modified protocol from Roselló et al. (2006). More than 90% of strains gave the results indicated here. − + + Sorbitola a Oxidation of substrates in API 50CH (BioMérieux) with a modified protocol from Roselló et al. (2006). More than 90% of strains gave the results indicated here. + + − Esculina a Oxidation of substrates in API 50CH (BioMérieux) with a modified protocol from Roselló et al. (2006). More than 90% of strains gave the results indicated here. V − + Melibiosea a Oxidation of substrates in API 50CH (BioMérieux) with a modified protocol from Roselló et al. (2006). More than 90% of strains gave the results indicated here. + − + d-Raffinosea a Oxidation of substrates in API 50CH (BioMérieux) with a modified protocol from Roselló et al. (2006). More than 90% of strains gave the results indicated here. − − + β-Gentibiosea a Oxidation of substrates in API 50CH (BioMérieux) with a modified protocol from Roselló et al. (2006). More than 90% of strains gave the results indicated here. + − + Abbreviation: V, variable a Oxidation of substrates in API 50CH (BioMérieux) with a modified protocol from Roselló et al. (2006). More than 90% of strains gave the results indicated here. Biochemical characterization by API system (BioMérieux, France) Biochemical identification of E. amylovora can be obtained by specific profile in API 20 E and API 50 CH strips. For API 20 E, the manufacturer's instructions should be followed for preparing the suspension and inoculating the strip. After incubation at 25–26°C, the strips should be read after 24 and 48 h (Table 2). For API 50 CH, a suspension of OD = 1.0 should be prepared in PBS (Appendix 2), and 1 mL added to 20 mL of Ayers’ medium (Appendix 2). The manufacturer's instructions should be followed for inoculation of the strip. After incubation at 25–26°C in aerobiosis, the strip should be read after 72 h. TABLE 2. Typical readings of Erwinia amylovora in API 20E tests after 48 h Test Reaction (48 h) ONPG Variable ADH – (or weak +) LDC – ODC – CIT – SH2 – URE – TDA – IND – VP + (or variable) GEL Variable GLU + MAN Variable INO Variable SOR Variable RHA – SAC + MEL – (or weak +) AMY – ARA + (some −) Automated Biolog identification system The new version (third-generation) Biolog GENIII 96 microplate allows rapid identification of isolated bacteria, both Gram-negative and Gram-positive, using the same microplate. The microplate and the program are commercially available (Biolog, Omnilog, USA). The manufacturer's instructions should be followed for automatic identification of suspected strains of E. amylovora. 4.5.2 Fatty acid profiling Erwinia amylovora-like colonies should be grown on Trypticase Soy Broth Agar (TSA) for 24 h at 28°C, and an appropriate fatty acid profiling (FAP) procedure applied. A positive FAP test is achieved if the profile of the presumptive culture is identical to that of the positive control (Sasser, 1990). Commercial software from the MIDI system (Newark, DE, USA) allows