Bulletin OEPP/EPPO Bulletin (2013) 43 (1), 21–45 ISSN 0250-8052. DOI: 10.1111/epp.12019 European and Mediterranean Plant Protection Organization Organisation Europeenne et Mediterran eenne pour la Protection des Plantes PM 7/20 (2) Diagnostics Diagnostic PM 7/20 (2)* Erwinia amylovora Specific scope Specific approval and amendment This standard describes a diagnostic protocol for Erwinia This standard was developed under the EU DIAGPRO Pro- amylovora1. ject (SMT 4-CT98-2252) and EUPHRESCO Pilot project (ERWINDECT) by partnership of contractor laboratories. Test performance studies were performed with different laboratories in 2002, 2009 and 2010. Approved as an EPPO Standard in 2003-09. Revised in 2012-09. Fire blight is probably the most serious disease affect- Introduction ing Pyrus spp. (pear) and Malus spp. (apple) cultivars in Erwinia amylovora is the causal agent of fire blight in most many countries. Although the life cycle of the bacterium species of the subfamily Maloideae of the family Rosaceae. is still not fully understood, it is known that it can sur- The most economically important hosts are Pyrus spp., vive as endophyte or epiphyte for variable periods Malus spp., Cydonia spp., Eriobotrya japonica, Cotoneaster depending on environmental factors (Thomson, 2000). spp., Crataegus spp., Pyracantha spp. and Sorbus spp. Other The development of fire blight symptoms follows the sea- hosts include Chaenomeles, Mespilus and Photinia.Aforma sonal growth development of the host plant. It begins in specialis was described from Rubus spp. (Starr et al., 1951; the spring with production of primary inoculum and Bradbury, 1986). An exhaustive list of affected plants, infection of flowers, continues in summer with infection including those susceptible only after inoculation, was of shoots and fruits, and ends in autumn with the devel- reported by van der Zwet & Keil (1979). It includes more opment of cankers. The pathogen is apparently quiescent than 180 species from 39 genera of the Rosaceae. Erwinia through the dormant period of the host (van der Zwet & amylovora was the first bacterium described as a causal agent Beer, 1995). of a plant disease by Burrill (1883). It was reported in North Flow diagrams describing the diagnostic procedure for America and was later detected in New Zealand in 1920. In E. amylovora in symptomatic and asymptomatic material Europe, fire blight was reported in 1957 in the United King- are presented in Figs 1 and 2. dom and has since been identified in most areas where sus- ceptible hosts are cultivated. Erwinia amylovora is now present in more than 40 countries (van der Zwet, 2002; Identity CABI/EPPO, 2007), but it has not been recorded in South Name: Erwinia amylovora (Burrill) Winslow et al. America, Asia or sub-Saharan African countries. It has been Synonyms: Micrococcus amylovorus Burrill recorded in some North African countries and only once in Bacillus amylovorus (Burrill) Trevisan Australia (Bonn & van der Zwet, 2000). It represents a threat Bacterium amylovorus (Burrill) Chester to the pome fruit industry of all the countries. Details on geo- Erwinia amylovora f. sp. rubi Starr, Cardona & Falson graphical distribution can be found in the EPPO Plant Quar- Taxonomic position: Bacteria, Proteobacteria, c Subdivi- antine Data Retrieval system (PQR, 2012). sion, Enterobacteriales, Enterobacteriaceae 1Use of names of chemicals or equipment in these EPPO Standards implies EPPO code: ERWIAM no approval of them to the exclusion of others that may also be suitable. Phytosanitary categorization: EPPO A2 list no. 52, EU *This protocol number was corrected online on 25th April 2013. Annex designation II/A2 ª 2013 OEPP/EPPO, Bulletin OEPP/EPPO Bulletin 43, 21–45 21 22 Diagnostics Plants with typical symptoms Asymptomatic sample Pathogen extraction Pathogen extraction and enrichment Rapid screening test Enrichment DASI-ELISA, Lateral flow devices, Screening tests IF, PCR, real-time PCR, LAMP (At least two tests based on different biological Enrichment DASI-ELISA, enrichment-PCR, principles) enrichment-real-time PCR, enrichment isolation (At least two tests based on different biological principles) Some tests Negative positive Some tests All tests positive negative Isolation and/or enrichment-isolation Isolation and Colonies with typical morphology enrichment-isolation Colonies with typical E. amylovora Negative morphology not detected Positive Negative Identification tests Positive (At least two tests based on different biological principles) Identification tests (At least two tests based on Negative E. amylovora Positive Negative not detected different biological principles) E. amylovora identified Positive Confirm pathogenicity by E. amylovora host test when required confirmed E. amylovora identified Fig. 1 Flow diagram for diagnosis of fire blight in plants with symptoms. Confirm pathogenicity by host test E. amylovora confirmed Detection Fig. 2 Flow diagram for analysis of Erwinia amylovora in asymptomatic samples. Disease symptoms Symptoms of fire blight on the principal hosts are relatively for some time. Immature fruits (or less frequently mature similar and easily recognized (Figs 3–5). The name of the fruits) have infected parts that appear oily or water-soaked, disease is descriptive of its major characteristic: the brown- becoming brown to black and often exuding droplets of ish appearance of twigs, flowers and leaves as though bacterial ooze. They also remain attached to the tree. Char- burned by fire. Typical symptoms on pome fruit trees are acteristic reddish-brown streaks are often found in the sub- the brown to black colour of leaves on affected branches, cortical tissues when bark is peeled from infected twigs, the production of exudates under humid conditions, and the branches or trunks (van der Zwet & Keil, 1979). Brown to typical ‘shepherd’s crook’ in the shoots. Depending on the black, slightly depressed cankers can develop in the bark of affected plant part, the disease causes blossom blight, shoot twigs or branches, or even the trunk, in autumn and winter. or twig blight, leaf blight, fruit blight, limb and trunk These cankers may later become defined by cracks near the blight, collar or rootstock blight (van der Zwet & Keil, margin of diseased and healthy tissue (Dye, 1983). 1979; van der Zwet & Beer, 1995). Confusion between fire blight and blight- or blast-like In apple and pear, the first symptoms usually appear in symptoms, especially in blossoms and shoots, may occur early spring during warm and humid weather, and can pro- with diseases/disorders caused by other bacteria, fungi, insect gress very quickly under favourable conditions. Flowers damage and physiological disorders, and consequently labo- appear to be water-soaked, then wilt, shrivel and turn pale ratory analysis is always necessary. Other bacteria can cause brown to black. Peduncles may also appear water-soaked, blight-like symptoms, including Erwinia pyrifoliae, causal become dark green, and finally brown or black, sometimes agent of bacterial shoot blight of Pyrus pyrifolia (Asian pear) oozing droplets of sticky bacterial exudates. Leaves wilt (Kim et al., 1999); Erwinia piriflorinigrans, isolated from and shrivel, and entire spurs turn brown in most hosts, or necrotic pear blossoms in Spain (Lopez et al., 2011); Erwinia dark brown to black in pear, but remain attached to the tree sp. and Erwinia uzenensis, causing different types of pear ª 2013 OEPP/EPPO, Bulletin OEPP/EPPO Bulletin 43, 21–45 Erwinia amylovora 23 AB A B C D C D Fig. 3 Symptoms of fire blight on pear trees. (A) necrotic flowers; (B) necrosis on leaves and typical shepherd’s crook; (C) mummified Fig. 4 Typical symptoms of fire blight on: (A) pear branches; (B) immature fruits with small ooze drops; (D) canker after removing bark apple shoot; (C) quince shoot; (D) loquat shoot. showing necrotic inner tissues. symptoms in Japan (Tanii et al., 1981; Matsuura et al., 2011); and Pseudomonas syringae pv. syringae, the causal A B agent of blossom blast. Detection from symptomatic samples Sampling Symptomatic samples can be processed individually or in small batches combining material from several samples (see C D Appendix 1). Precautions to avoid cross-contamination should be taken when collecting samples and during the extraction process. Samples with symptoms for diagnosis of fire blight should preferably be composed of flowers, shoots or twigs, leaves, fruitlets (all with necrosis and with exu- dates if possible), or the discoloured subcortical tissues Fig. 5 Typical symptoms of fire blight on: (A) Crataegus sp. shoot; (after peeling bark from cankers in twigs, branches, trunk (B) Cotoneaster sp. shoot; (C,D) Pyracantha sp. branches. or collar). Samples should be processed as soon as possible after collection and stored at 4–8°C before analysis. Sam- ples may be cold stored after processing for up to 2 weeks bacteria in such samples is usually high. However, when in case further testing is required. symptoms are advanced or when environmental conditions are not favourable for fire blight symptom expression, the Isolation number of E. amylovora culturable cells can be very low. Fresh sample extracts are necessary for successful isolation. When plates are overcrowded by plant microbiota, the sam- Details on the extraction procedure from plant material are ple should be retested and enrichment according to Appen- given in Appendix 1. Details on isolation are provided in dix 4 performed before isolation, as described in Appendix 5. Isolating E. amylovora from symptomatic Appendix 5. Enrichment is also recommended when the samples is relatively easy because the number of culturable presence of antagonistic bacteria in the sample is suspected. ª 2013 OEPP/EPPO, Bulletin OEPP/EPPO Bulletin 43, 21–45 24 Diagnostics A B C Fig. 6 Typical colony morphology of E. amylovora on: (A) King’s medium; (B) levan (NSA) medium and (C) CCT medium. For direct isolation, plating on three media is advised for Conventional PCR, real-time PCR and loop-mediated iso- maximum recovery of E. amylovora, in particular when thermal amplification (LAMP) were evaluated in a test per- samples are in poor condition.