PM 7/20 (3) <i>Erwinia amylovora</i>

EPPO BulletinVolume 52, Issue 2 p. 198-224 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 full-text accessPlease review our Terms and Conditions of Use check box below share version article.I have read accept the Wiley Online Library UseShareable LinkUse link a this article with your friends colleagues. Learn more.Copy URL Share linkShare onFacebookTwitterLinkedInRedditWechat Specific scope This Standard describes diagnostic protocol for amylovora.1 It should be used in conjunction 7/76 protocols. approval amendment was developed under EU DIAGPRO Project (SMT 4-CT98-2252) EUPHRESCO Pilot project (ERWINDECT) by partnership contractor laboratories. Test performance studies were performed different laboratories 2002, 2009 (Reisenzein et al., 2010), 2010 (López 2010) 2019 (Alič 2020). Approved as an 2003–09. Revised 2012–09 2021–09. Authors contributors are given Acknowledgements section. 1 INTRODUCTION is causal agent fire blight, disease that affects most species subfamily Maloideae family Rosaceae (Spiraeoideae). first bacterium described plant (Burrill, 1883). E. considered native North America detected outside New Zealand 1920. Fire blight reported England 1957 since then has been areas Europe where susceptible hosts cultivated. now present more than 50 countries. not recorded South African Asian countries (except surrounding Mediterranean Sea), it eradicated Australia after report there (van der Zwet, 2004). represents threat pome fruit industry all these (Bonn & van 2000). The important host plants from both economic epidemiological viewpoints genera Chaenomeles, Cotoneaster, Crataegus, Cydonia, Eriobotrya, Malus, Mespilus, Pyracantha, Pyrus, Sorbus Stranvaesia (Bradbury, 1986). strains isolated Rubus sp. United States distinct on other (Starr 1951; Powney 2011). Details geographic distribution can found Global Database (EPPO, 2021a). probably serious bacterial affecting Pyrus communis (pear) Malus domestica (apple) cultivars many Epidemics sporadic dependent several factors, including favourable environmental conditions, sufficient inoculum level orchard susceptibility. easily dispersed birds, insects, rain or wind (Thomson, development symptoms follows seasonal growth plant. begins spring production primary bacteria overwintering cankers 2000) causing blossom infection driven activity pollinating insects Zwet Keil, 1979) climatic factors (e.g. rain, wind, hail), continues into summer shoot infection, ends winter cankers. pathogen appears quiescent through dormant period Beer, 1995), but experience Portugal may remain active during (L. Cruz, personal communication). Flow diagrams describing procedure symptomatic asymptomatic material presented Figures 2. FIGURE 1Open figure viewerPowerPoint diagram diagnosis samples 2Open analysis IDENTITY Name: 1882) Winslow 1920 Other scientific names: Micrococcus amylovorus 1882), Bacillus Trevisan, 1889, Bacterium Chester, 1897, f. rubi (Starr, 1951), Cardona Falson Taxonomic position: Bacteria, Proteobacteria, γ Subdivision, Enterobacteriales, Enterobacteriaceae Code: ERWIAM Phytosanitary categorization: A2 list no. Protected Zone Quarantine pest (Annex III), Regulated Non-Quarantine Pest IV) 3 DETECTION Disease Symptoms common such P. (pear), M. (apple), Cydonia spp. (quince), Eriobotrya japonica (loquat), Cotoneaster (cotoneaster), Pyracantha (pyracantha) Crataegus (hawthorn) relatively similar recognized (Figures 3-5). name descriptive its major characteristic: brownish blackish necrotic appearance twigs, flowers leaves, though they had burned fire. typical brown black colour leaves affected branches, exudates, characteristic ‘shepherd's crook’ terminal shoots. Depending part phenological stage, include twig leaf limb trunk collar rootstock 1979; 1995). 3Open pear trees: (a) flowers, (b) necrosis shepherd's crook, (c) mummified immature fruits small ooze drops (d) canker removing bark showing inner tissues 4Open Typical apple shoot, quince loquat 5Open (c, d) branches In trees usually appear early when average temperature rises above 15°C, humid weather. Infected blossoms become soaked water, wilt, shrivel turn orange black. Peduncles also water-soaked, dark green, finally black, sometimes oozing droplets sticky exudates. wilt shrivel, entire spurs apples pears attached tree some time. On young fruitlets tree. Immature lesions oily often exudate. Characteristic running reddish-brown streaks subcortical peeled infected limbs twigs 1979). Brown slightly depressed form trees. These later defined cracks near margin diseased healthy tissue Additionally, epidermis roll up, resembling papyrus paper Confusion occur between blight- blast-like – especially buds caused pathogens (Figure 6a,b) fungi 6c,d), insect damage 6e) physiological disorders. 6Open Confusing piriflorinigrans Pseudomonas syringae pv. Stemphylium vesicarium Monilia laxa Prunus persicae (similar mumification monilia observed) (e) Janus compressus (Hymenoptera) (note oviposition punctures at base crook) cause blight-like pyrifoliae, pyrifolia (Asian pear) (Kim 1999), 6a), Spain 2011) Iran (Moradi Amirabad Khodakaramian, 2017; Moradi-Amirabad 2020), uzenensis, recently Japan (Matsuura 2012), (Tanii 1981; Kim, Hildebrand, 2001; Jock, Palacio-Bielsa syringae, blast 6b). Detection 3.2.1 sample requirements Symptomatic processed individually batches combining (see Appendix 1). Precautions avoid cross-contamination taken collecting extraction process. Samples preferably composed shoots (with and/or exudates), discoloured (after peeling collar). soon possible collection kept 4–8°C up week before analysis, if necessary. 3.2.2 Screening tests At least two tests, based biological principles targeting parts genome, performed. For established one screening test declare sample. However, case doubt second addition, isolation colonies morphology identification positive, Isolation Fresh extracts necessary successful isolation. 1. provided 5. Isolating easy because number culturable high. phytosanitary treatments bacteriostatic products used, very advanced conditions multiplication, cells low. result plates few overgrown saprophytic antagonistic bacteria. If suspected, re-tested enriched induction reversible viable non-culturable state (VBNC) shown vitro using copper (Ordax 2009), false-negative results. Indeed, VBNC state, do grow solid culture media pathogenic. reversible, pathogenic again 2006). results will negative, still potentially Enrichment-ELISA PCR-based methods. When overcrowded microbiota, retested enrichment (according 4) (as 5). direct isolation, plating parallel (to chosen depending sample) recommended maximum recovery amylovora, particular poor condition. efficiency depends composition microbiota Three media, King's B, NSA CCT (Appendix 2), validated study. Colonies about 48 h pale violet, circular, highly convex domed, smooth mucoid 72 h, slower B NSA. medium inhibits pseudomonads enterobacteria Pantoea agglomerans. approximately creamy white, tending spread non-fluorescent UV light 366 nm h. allows distinction fluorescent pseudomonas. whitish, NSA-negative (Bereswill 1997). Figure 7 shows cultures three incubation 25°C 24, later. Very rarely, exhibit pink presence iron acquisition. re-streaked purified, lose colouration (Stockwell 2008; communication, Tanja Dreo, NIB). 7Open colony (left), (middle) (right) Pure individual suspect each obtained presumptive identified indicated negative no observed 96 any (provided inhibition suspected due competition antagonism) positive controls. confirmed methods indicated. mentioned same terms size general faster amylovora. pyrifoliae fluid compared 8). grows medium. 8Open medium, facilitate fresh pronounced symptoms. Several Appendices 3–11. conducted, Serological Indirect immunofluorescence (IF), DASI-ELISA lateral flow devices analyses organs Instructions performing IF 7/97 2009) those ELISA 7/101 2010). Quality antibodies critical tests. studies, commercial antisera monoclonal [polyclonal antiserum Loewe Biochemica GmbH (Sauerlach, Germany) Plant Print Diagnostics S.L. (Faura, Spain)]. ELISA, complete kit combination specific antibodies, S.L., evaluated. Two commercialized Bioreba, Reinach, Switzerland (Ea AgriStrip) Abingdon Health, York, UK (Pocket Diagnostics) available rapid (Braun-Kiewnick 3. Molecular Many conventional PCR lack analytical specificity, e.g. cross-reaction (Maes 1996), detect 1992; McManus Jones, 1995 Llop nested (Llop generates false-positive which cannot subsequent testing symptom observation (Tanja (Taylor al. (2001), adaptation Obradovic (2007), real-time (Pirc Gottsberger, loop-mediated isothermal amplification (LAMP) 7–11. All evaluated 2020; Trontin 2021), DNA step. protocols study 6. 3.3.1 Warning: difficult. Whenever possible, autumn increase likelihood detecting Asymptomatic bulked Sampling preparation following samples. Direct low population. Consequently, step 4). 3.3.2 Enrichment-isolation, enrichment-DASI 4–10. Confirmation cases 2017) samples, attempt made isolate directly extract non-enriched (Appendices 5), 4 As little known (CCT, NSA) maximize only CCT. necessary, conserved −20 −80°C glycerol IDENTIFICATION isolates characteristics combinations biochemical, serological molecular tests) and, pathogenicity test. sequence targets genome specificity primers Known reference included section Reference material). Different sources reduce risk false positives. 4.1.1 Agglutination Suspected tested agglutination mixing them drop PBS 2) amylovora-specific (not diluted, five- tenfold dilution) slide. Monoclonal agglutinate strains. grown promoting polysaccharides washed times saline solution. 4.1.2 Immunofluorescence 2009). identification, GmbH. 4.1.3 (kit S.L.). DASI-ELISA, suspension 108 cells/mL prepared 2). 3) followed without prior identification. 4.1.4 Lateral immunoassays A manufacturers’ instructions. kits (Agri-strip Pocket Diagnostic) isolates. Conventional LAMP techniques 4.2.1 106 molecular-grade water amylovora-like colonies. Appropriate procedures applied, 8, extraction, just treatment 95–100°C 8–10 min. 4.2.2 Real-time published 9 10, recommended. (section 4.2.1). 4.2.3 One 11 Suspensions 4.2.4 sequencing Comparisons sequenced amplified selected housekeeping genes allow differentiation members Enterobacteriaceae. example, so far clonally related according partial recA gene (Waleron 2002) method Parkinson (2009). data comparative (Mann 2013; Zeng 2018; Parcey Sequence follow guidelines 8 7/129 barcoding tool regulated pests 2021b). Matrix-assisted laser desorption/ionization-time flight mass spectrometry matrix-assisted (MALDI-TOF) proteomic Sauer (2008) Wensing (2011). rapid, reliable robust their routine duplicate depositing harvested 3-day-old nutrient agar (NA) onto stainless plate, formic acid treatment. spectra linear positive-ion mode m/z range 2000–20 000 Da. Validation MALDI-TOF already available. Pathogenicity hypersensitivity inoculated confirm pathogenicity, detached 12). hypersensitive reaction tobacco give indication hrp genes, difficult interpret 4.5.1 Biochemical genus Gram-negative bacteria, facultative anaerobes, motile peritrichous flagella, rod-shaped, produced glucose, fructose, galactose sucrose. phenotypic properties Paulin (2000) determined Jones Geider (2001). Table 1, mainly API CH strips (BioMérieux, France), 1999; Rosello 2006) 2011; Moradi certain biochemical vary TABLE Differences Microbiological Gelatine hydrolysis + − Inositola Sorbitola Esculina V Melibiosea d-Raffinosea β-Gentibiosea Abbreviation: V, variable Oxidation substrates 50CH (BioMérieux) modified Roselló (2006). More 90% gave here. characterization system France) profile 20 E strips. E, manufacturer's instructions preparing inoculating strip. After 25–26°C, (Table CH, OD = 1.0 mL added Ayers’ inoculation 25–26°C aerobiosis, strip readings 20E Reaction (48 h) ONPG Variable ADH (or weak +) LDC ODC CIT SH2 URE TDA IND VP variable) GEL GLU MAN INO SOR RHA SAC MEL AMY ARA (some −) Automated Biolog new (third-generation) GENIII microplate Gram-positive, microplate. program commercially (Biolog, Omnilog, USA). automatic 4.5.2 Fatty profiling Trypticase Soy Broth Agar (TSA) 28°C, appropriate fatty (FAP) applied. FAP achieved identical control (Sasser, 1990). Commercial software MIDI (Newark, DE, USA) age chromatograph sensitivity, similarity index 0.6 0.9 system. 5 REFERENCE MATERIAL use controls: NCPPB683 (type strain) CFBP 1430. collections provide strains: National Collection Pathogenic Bacteria (NCPPB), Fera, Sand Hutton, York (GB); https://www.fera.co.uk/ncppb International Center Microbial Ressources French Plant-associated (CIRM-CFBP), IRHS INRAE Beaucouzé (FR); https://www6.inrae.fr/cirm/CFBP-Bacteries-associees-aux-Plantes Leibniz Institute DSMZ German Microorganisms Cell Cultures, Braunschweig (DE); www.dsmz.de/dsmz Laboratorium voor Microbiologie Bacterial (LMG), Universiteit Gent (BE); http://bccm.belspo.be/ Authenticity guaranteed collections. 6 REPORTING AND DOCUMENTATION Guidelines reporting documentation 7/77 (1) Documentation diagnosis. PERFORMANCE CHARACTERISTICS available, description Diagnostic Expertise (http://dc.eppo.int), consult database additional information detailed validation reports, etc.). FURTHER INFORMATION Further informat