1 SMT PROJECT SMT-4-CT98-2252 DIAGNOSTIC PROTOCOLS FOR ORGANISMS HARMFUL TO PLANTS

DIAGNOSIS OF Erwinia amylovora

PROTOCOL FOR THE DIAGNOSIS OF QUARANTINE ORGANISM Erwinia amylovora

Identity Name: Erwinia amylovora (Burrill) Winslow et al.

Synonyms: Micrococcus amylovorus Burrill. Bacillus amylovorus (Burrill) Trevisan. Bacterium amylovorus (Burrill) Chester. Erwinia amylovora f.sp. rubi Starr, Cardona and Falson. Common name: Fire blight. Taxonomic position: Proteobacteria, Subdivision, orden Enterobacteriales, family Enterobacteriaceae, genus Erwinia. Quarantine status: EPPO A2 list, EU Annex II/A2. Bayer computer code: ERWIAM

2 DIAGNOSTIC PROTOCOLS FOR ORGANISMS HARMFUL TO PLANTS. SMT PROJECT SMT4-CT98-2252 Diagnostic protocol for Erwinia amylovora (Burrill) Winslow et al.

1. INTRODUCTION Erwinia amylovora is the causal agent of fire blight in most species of subfamily Maloideae of the family Rosaceae. The most economically important hosts are Pyrus spp., Malus spp., Cydonia spp., Eriobotrya japonica, Cotoneaster spp., Crataegus spp., Pyracantha, spp. and Sorbus spp. A forma specialis was described from Rubus spp. (Starr et al, 1951). An exhaustive list of affected plants, including those susceptible after artificial inoculation, was reported by van der Zwet and Keil (1979) and Bradbury (1986). It includes more than 180 species from 39 genus among the Rosaceae. E. amylovora was the first bacterium described as causal agent of a plant disease by Burrill (1893). It was considered to be native to North America and later detected in New Zealand in 1920. Fire blight was reported in 1957 in England and since them it has been detected elsewhere in Europe, in most areas where susceptible hosts are cultivated, except in Portugal, among the UE countries. E. amylovora is now present in 43 countries (van der Zwet, 2002), but it has not been recorded either in South America or in most African and Asiatic countries (with the exception of most of those surrounding the Mediterranean sea) and only once in Australia (Bonn and van der Zwet, 2000). It represents a threat to the pome fruit industry of all these countries. Details on geographical distribution can be found in EPPO/CABI Distribution Maps (1998). Fire blight is probably the most serious disease affecting pear or apple cultivars in many countries. Although the life cycle of the bacterium is still not well understood, it is known that it can survive as endophyte or epiphyte for variable periods of time depending of environmental factors (Thomson, 2000). The development of fire blight symptoms follows the seasonal growth development of the host plant. It begins in the spring with the production of the primary inoculum and the blossoms infection, continuing on summer with the shoots and fruits infection and ending in fall with the development of cankers. The pathogen is apparently quiescent through the dormant period of the host (van der Zwet and Beer, 1995).

3 1.1. Principal hosts The principal hosts are in the sub-family Maloideae of the family Rosaceae. The most important hosts from both economic and epidemiological points of view are in the genera Chaenomeles, Cotoneaster, Crataegus, Cydonia, Eriobotrya, Malus, Mespilus, Pyracantha, Pyrus, Sorbus and Stranvaesia. E. amylovora isolated from Rubus sp. in the USA appears distinct from the strains from other hosts (EPPO-CABI, 1997). 1.2. Symptoms Symptoms of fire blight on the most common hosts (pear, apple, quince, loquat, cotoneaster, hawthorn, pyracantha) are relatively similar and easily recognized. The name of the disease is descriptive of its major characteristic: the 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 of exudates and the typical shepherds crook in the shoots. Depending on the affected plant part the disease produces blossom blight, shoot or twig blight, leaf blight, fruit blight, limb and trunk blight, collar or rootstock blight (van der Zwet and Keil, 1979, van der Zwet and Beer, 1995). In apple and pear trees the first symptoms usually appear in early spring, during warm and humid weather. Blossoms appear to be water-soaked, then wilt, shrivel, and turn brown to black. Peduncles may also appear water-soaked, become dark green, and finally brown or black, sometimes oozing droplets of sticky bacterial exudates. Leaves wilt, shrivel and entire spurs turn brown in apples or dark brown to black in pears, but remain attached to the tree for some time. Immature fruit (or less frequently mature fruit) show infected parts appearing oily or water-soaked, becoming brown to black and often exuding droplets of bacterial ooze. They also remain attached to the tree. Characteristic reddish-brown streaks are often found in the subcortical tissues when the bark is peeled from the infected limb or twig (van der Zwet and Keil, 1979). Brown to black, slightly depressed cankers develop in the bark of twigs or branches or even the trunk. These cankers may later become defined by cracks near the margin of diseased and healthy tissue (Dye, 1983).

C
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.Aragn, Spain); C: Canker caused by E. amylovora on pear trunk (M. Cambra, IVIA, Spain); D: Symptoms of fire blight in pear shoots (M.M.Lpez, IVIA, Spain)

D
A: Symptoms of E. amylovora in Pyracantha (M.M.Lpez, 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 loquat fruits after inoculation (V. Donat, IVIA, Spain); D: Colonies of E. amylovora on CCT (M.M.Lpez, IVIA, Spain)

6 FLOW DIAGRAM FOR DIAGNOSIS OF FIRE BLIGHT (Erwinia amylovora) ON HOST PLANTS WITH SYMPTOMS.

Plants with typical symptoms1 RAPID SCREENING TESTS2 Direc Tissue print-ELISA3, IF 4, Enrichment DASI-ELISA5, PCR6.

ISOLATION7 or ENRICHMENT-ISOLATION8 All Negative Colonies with typical morphology10 Yes IDENTIFICATION TESTS12 Confirm identity of pure culture as E. amylovora, including pathogenicity No Yes No11 Positive9

E. amylovora not detected

E. amylovora confirmed

E. amylovora detected13

1. 2. 3. 4.

5. 6. 7. 8. 9.

10. 11.

12.

13.

Description of symptoms and sample preparation are in Section 1.2, 2.2 and 2.3 Rapid screening tests facilitate presumptive diagnosis. Direct Tissue print-ELISA is described in Section 2.5.1 IF (Immunofluorescence) test on bacterial ooze suspended in water or symptomatic tissue extracts is described in Section 2.5.3. ELISA test on bacterial ooze suspended in water or symptomatic tissue extracts is described in Section 2.5.2. PCR test on bacterial ooze suspended in water or symptomatic tissue extracts is described in Section 2.5.4. The pathogen is usually easily isolated from symptomatic plant material by dilution plating as described in Section 3. Enrichment - isolation is described in Section 3.2. Positive by at least two serological tests (3, 4, 5) using different validated method and antibodies and by a validated PCR protocol (6). If only one or two tests are positive try isolation following Section 3. Typical colony morphology is described in Section 3.1. Culturing may fail from advanced stages of infection due to competition or overgrowth by saprophytic bacteria. If disease symptoms are typical, but the isolation test is negative, then the isolation must be repeated. Reliable identification of pure cultures of presumptive E. amylovora isolates is achieved using tests described in Sections 6 and 7. The rapid detection scheme of E. amylovora is advised for routine surveys but not in the case of new findings.

7 2. DETAILED PROTOCOL FOR DIAGNOSIS

2.1. Sampling The symptomatic samples can be processed individually or in small groups. Precautions to avoid cross contaminations should be taken when collecting the samples and during the extraction process. The samples for diagnosis of fire blight on plants with symptoms can be preferently flowers, shoots or twigs, leaves, fruitlets (with necrosis and exudates if possible) or the discoloured subcortical tissues after peeling the bark from cankers in twig, branches, trunk or collar. They should be processed as soon as possible after being collected and conserved at 4-8 C until that. The samples can be cold stored after processing for further verifications, for no longer than few weeks. 2.2. Plant tissue prints or blottings For preparation of the plant tissue prints or blottings (membrane printing) make crossclean cuts on symptomatic shoots, blossoms, leaves or fruitlets in the field or in the lab. Press carefully freshly made sections against the nitrocellulose membrane. The exudates or bacterial colonies in solid medium can also be printed or spotted. Disinfect the cutting tool after using to avoid contaminations. Print a positive and negative controls using an inoculated host plant with fire blight symptoms and a healthy plant of the same species to analyse. Let dry the trace or print for a few minutes. Handle the membranes with care. Printed membranes can be kept for several months in a dry place at room temperature. 2.3. Sample preparation The samples could be processed in a different buffer depending on the techniques to follow. The use of an antioxidant maceration buffer (appendix 1) is strictly required for an optimal enrichment of E. amylovora in plant material (see 2.4), as indicated by Gorris et al (1996b). The samples can also be processed in sterile phosphate buffered saline, pH 7.2 10mM (PBS) (appendix 1) or sterile water for direct isolation, inmunofluorescence or direct PCR. Carefully select the plant parts showing the most fresh symptoms and with exudates when possible. Select for processing the leading edge of lesions on each organ to analyse. The exudates can be processed separately, in 1-4.5 ml of sterile water or buffer. For shoots: take pieces of the symptomatic shoots, including leaves, at the margin between the necrotic and healthy tissue, to reach ca. 0.1 g of material. Put it into a plastic bag and proceed with the maceration as indicated below. For blossoms: take one or several flowers and peduncles until reaching ca 0.1 g and proceed as below. For leaves: take one or several leaves and petioles until reaching ca. 0.1 g and proceed as below. Preferently select leaves with vein necrosis and not fully necrosed. For fruits: take one or several fruits until reaching ca.0.1g and proceed as below. For stems: peel off the external bark of the stems with symptoms, using a sterile scalpel and take pieces underneath with typical subcortical discolouration symptoms until reaching ca. 0.1 g of material. Proceed as follows with the different types of samples:

8 a) Cut the shoots, flowers, leaves, stems, fruits, etc. in some pieces into plastic bags (preferently with a heavy net). Add to each bag 4.5 ml of the antioxidant maceration buffer described by Gorris et al., (1996) (appendix 1). Let stand the samples in maceration at least 5 min. Slightly crush the plant material in the plastic bag with a rubber hammer or with stomacher Bioreba or similar equipment, avoiding droplets splashing out of the bag. b) Let stand the samples on ice for few minutes. Transfer ca. 2 ml, 1 ml and 1 ml of each macerate to three sterile Eppendorf tubes by decantation. Store one tube with 1 ml of each sample at 20C for subsequent analysis or confirmation and another with 30% glycerol (Difco) at 20C. Keep the remaining tube with 2 ml on ice for analysis. The same day of the maceration of the samples the isolation must be done, as well as the enrichment and the fixation of the slides for immunofluorescence. The PCR analysis can be performed at the earliest convenience using the Eppendorf tubes stored at 20C.

2.4. Enrichment The enrichment is used to multiply the initial population of culturable E. amylovora in the sample. It is necessary to perform it before detection by ELISA, due to the low level of sensitivity obtained by such technique when using specific monoclonal antibodies. It should also be used before isolation or before PCR (even in symptomatic samples) when a low number of culturable E. amylovora is expected (copper treated samples, old symptoms, non favourable weather conditions for fire blight, winter season, etc) or when high amount of inhibitors is expected. The use of two validated media, one non selective (Kings B) and one semi-selective, (CCT) (appendix 1) is advised because the composition and number of the microbiota is unknown. a) Dispense, as soon the macerates (see 2.3) have been made, 0.9 ml of each sample in two sterile 3-5 ml tubes that have been prepared in advance with the same volume of each enrichment medium. b) Place, as additional negative controls, three tubes with 0.9 ml of maceration buffer (appendix 1) in use and add the same volume of the same buffer and of each enrichment medium (CCT and Kings B sterile liquid media) (appendix 1). Incubate at 25C for 48 h without shaking. Incubate for 72 h when low numbers of E. amylovora are expected, as indicated above. 2.5. Rapid screening tests These tests facilitate presumptive diagnosis. At least two serological tests and another PCR-based should be positive for E. amylovora positive detection in the samples. 2.5.1. Direct tissue print-ELISA This method may facilitate presumptive diagnosis on symptomatic plant material but it needs further confirmation because specificity problems. The method can also be used to spot bacterial colonies or exudates as an additional rapid test of serological identification. The use of antibodies of well known specificity is required. The only complete kit based on specific monoclonal antibodies, commercially available is indicated in appendix 2.

9 a) For performing plant tissue-prints and blottings, proceed as indicated in 2.2. Use always a positive and negative control. b) Prepare a solution of 1% bovine serum albumin (BSA) in distilled water (stirring up with a stick). Place the membranes in an appropriate container (tray, hermetic container, plastic bag). Pour, covering them, the albumin solution and incubate for 1h at room temperature, or overnight at 4C. Slight agitation is beneficial. The incubation time elapsed, discard the albumin solution, keeping the membranes in the same container. c) Dilute specific E. amylovora monoclonal antibodies alkaline phosphatase conjugated in PBS (appendix 2) at the appropriate dilution. Pour the solution on the membranes, covering them. Incubate for 2-3 h by shaking at room temperature and discard the solution. d) Prepare washing buffer (appendix 2). Rinse the membranes and the container with washing buffer. Wash by shaking (manually or mechanically) with a larger buffer amount for 5 min. Eliminate washing buffer and repeat the operation twice. e) Prepare BCIP-NBT (Sigma Fast) substrate buffer (appendix 2). Pour over the membranes and let incubate until appearance of purple-violet colour in the positive control (approx. 10 min). Stop the reaction by washing the membranes with tap water. Let dry, spreading them out on absorbent paper. f) Observe the printings by using a low power magnification (X10 or X20). Interpretation of Tissue print-ELISA results: The test is positive when purple-violet precipitates appear in the sections of plant material and in the positive control, and not in the negative control. If exudates or colonies are printed they should appear violet when positive. When using the commercially available kit (see appendix 2), pre-printed membranes include positive and negative controls. The test is negative when no purple-violet precipitates appear as in the negative control. 2.5.2. Enrichment DASI-ELISA (Gorris et al., 1996 b) The only commercial kit for Enrichment DASI-ELISA has been validated in the ring tests (appendix 2). It is based on the monoclonal antibodies and technique described in Gorris et al (1996 a and 1996 b). As positive control use aliquots of a sample extract that previously tested negative, mixed with 108 cells of E. amylovora per ml. As negative control include a sample extract that have been previously tested negative for E. amylovora and a suspension of a non E. amylovora strain in PBS (appendix 1). a) Boil the necessary amount of the enriched extracts and controls in a water bath (or in a thermoblock) at 100 for 10 min before being processed by ELISA, minding the tubes not to be opened. Keep the remaining enriched samples for isolation and/or PCR. Process by ELISA the boiled samples (once at room temperature) the same day or freeze them at 20C for subsequent analysis. This heat treatment is necessary for optimum sensitivity and specificity using the monoclonal antibodies obtained by Gorris et al (1996a).

10

b) Prepare the appropriate dilution of rabbit anti-E. amylovora polyclonal immunoglobulins in carbonate buffer, pH 9.6 (appendix 2). Add 200 l to each well of a Nunc Polysorp (or equivalent) ELISA plates. Incubate at 37C for 4 h or at 4C for 16 h. Wash the wells three times with washing buffer (appendix 2). c) Add 200 l per well of the plant macerates previously enriched in the two media and boiled. Use two wells per sample enriched on each medium, and two of the positive and negative controls. It is also necessary to place negative controls of the extraction buffer and of the enrichment media used (previously prepared as additional negative controls of the enrichment). Incubate for 16 h at 4C. Wash the wells three times with washing buffer (as above). d) Prepare the appropriate dilution of specific E. amylovora monoclonal antibodies (appendix 2) in PBS plus 0.5% bovine serum albumin (BSA) and add 200 l to each well. Incubate at 37C for 2 h. Wash the wells three times with PBS-Tween. e) Prepare the appropriate dilution of goat anti-mouse immunoglobulins conjugated with alkaline phosphatase (Sigma) (appendix 2) in PBS. Add 200 l to each well. Incubate at 37C for 2 h. Wash the wells three times as above. f) Prepare a 1 mg/ml alkaline phosphatase substrate (p-nitrophenylphosphate) in substrate buffer (appendix 1). Add 200 l to each well. Incubate at room temperature and read at 405 nm after 30, 45, and 60 min. Interpretation of DASI-ELISA test results: The ELISA test is negative if the average optical density (OD) reading from duplicate sample wells is <2x OD of that in the negative sample extract control wells (providing the OD for the positive controls are above 1.0 after 60 minutes incubation and are greater than twice the OD obtained for negative sample extracts). The ELISA test is positive if the average OD readings from duplicate sample wells is >2x OD in the negative sample extract wells provided that 2x average OD readings in all negative control wells are lower those in the positive control wells. Negative ELISA readings in positive control wells indicate that the test has not been performed correctly and/or the reagents were not well prepared. Positive ELISA readings in negative control wells indicate cross-contaminations or non-specific antibody binding has occurred. In either case, the test should be repeated or a second test based on a different biological principal should be performed. 2.5.3. Immunofluorescence (IF) Follow the standard protocol for described in Anonymous (1998). Use a validated source of antibodies to E. amylovora. Three commercial antibodies have been validated in the ring test (appendix 2). It is recommended that the titre and the dilution of use are determined for each new batch of antibodies. IF test should be performed on freshlyprepared sample extracts. When the extracts stored at 80C under glycerol are used, remove glycerol it by adding 1 ml of PBS (appendix 1), centrifuge for 15 min at 7000 g, discard supernatant and resuspend in PBS.

11 For each set of tests prepare a positive control slide using a suspension of 106 cells/ml of a known pure culture of E. amylovora. For large-scale survey work it is recommended to include blind positive control slides. Use PBS and an aliquot of a sample extract, which was negative by several techniques, as negative controls. a) Use undiluted macerates and 1:10 and 1:100 dilutions in PBS, (appendix 1) to spot windows of the IF slides. Prepare one slide for each sample and its dilutions. b) Allow to air dry and fix by flaming or by absolute or 95 ethanol according to the characteristics of the antibodies used. Store slides at 20C until required. c) Use the monoclonal or polyclonal antibodies at the appropriate dilutions in PBS (appendix 1). Spot 25-30 l per well. Incubate slides in moist chamber for 30 minutes at room temperature. Use of two dilutions of the antibodies is advised when working with polyclonal antibodies, to detect cross reactions with other bacteria. d) Shake droplets off the slide and rinse slides carefully with PBS (appendix 1). Wash 10 minutes with the same buffer. Carefully remove excess moisture. e) Dilute the appropriate FITC conjugates in PBS: anti-mouse for the monoclonal antibodies (GAM-FITC) (appendix 2) and anti-rabbit (GAR-FITC) (appendix 2) or anti-goat (appendix 2). Cover the windows of all slides with the corresponding diluted conjugate and incubate in moist chamber for 30 minutes at room temperature. Repeat the washing step. f) Pipette 5-10 l 0.1M phosphate buffered glycerol mountant with anti-fadding (0.5% pphenylendiamine or other) on each window and apply a cover slip. g) View slides under oil immersion at 500-1000X magnification by scanning windows across 2 diameters at right angles and around the perimeters. Calculate the number of cells per ml of the sample, according to Anonymous (1998). Interpretation of the IF test results: The test is negative if green fluorescing cells with morphology typical of E. amylovora are observed in positive controls but not in sample windows. The test is positive if green fluorescing cells with typical morphology are observed in positive control and sample windows, but not in negative control windows. As a population of 103 cells per ml is considered the limit of reliable detection by the IF test, for samples with > 103 cells per ml, the IF test is considered positive. For samples with <103 cells per ml, the result of the IF test may be considered doubtful. In such case further testing or re-sampling should be performed. Samples with large numbers of incomplete or weakly fluorescing cells compared to the positive control would need further testing, with different dilutions of antibody or pellet or a second source of antibodies. 2.5.4. PCR Use validated PCR reagents and protocols. Take as many precautions as possible to avoid contamination of samples with target DNA. Use two positive controls with aliquots of sample extract of the same host previously tested negative to which suspensions of 104 and 106 cells/ml of a known E. amylovora strain have been added and a suspension of 104

12 cells/ml of E. amylovora. Prepare positive controls in a separate laboratory where samples will be tested. As negative controls use at least one sample extract previously tested negative for E. amylovora by several techniques and a sample of the ultra pure water (PCR reagent) used. Include more negative controls for each step of the DNA extraction and amplification if contaminations are suspected. Perform the DNA extraction from the positive and negative controls as well as from the samples. One protocol for DNA extraction from plant samples (Llop et al, 1999) and two amplification protocols (Bereswill et al, 1992 and Llop et al, 2000) have been validated in the ring test. Some commercial kits for extracting DNA are available, but they have not been validated. 2.5.4.1. DNA extraction (Llop et al., 1999) Take directly 1 ml of each macerate and/or 1 ml of the enriched macerates to follow the protocol for extracting the DNA according to Llop et al, (1999). a) Centrifuge the macerates at 13.000 rpm for 5 min at room temperature. Discard the supernatant, and resuspend the pellet in 500 l of extraction buffer (appendix 3) and shake for 1 h at room temperature. b) Centrifuge the tubes at 5.000 rpm for 5 min. Take 450 l of the supernatant and place it into a new Eppendorf tube. Add the same volume of isopropanol, invert several times and leave for 1 h at room temperature. c) Centrifuge at 13.000 rpm for 5 min, discard the supernatant and dry on bench at room temperature. If there is still a coloured precipitate (brown or green) at the bottom of the tubes, carefully take it while discarding the supernatant, thus obtaining a cleaner DNA. Normally, the DNA sticks at the wall of the Eppendorf tube more than at the bottom. Resuspend the pellet in 200 l of water. Use 5 or 1 l for the PCR reaction depending on the amplification performed. 2.5.4.2.Amplification There are many PCR primers and protocols described for E. amylovora detection and identification. Most of them can be used for specific identification (Bereswill et al, 1992; Bereswill et al, 1995; Guilford et al, 1996; McManus and Jones, 1995). Some have specificity problems as the primers of Maes et al, (1996) because they also amplified an Erwinia sp. isolated from necrotic pear blossoms and different from E. amylovora (Rosell et al, 2002). The primers and protocols validated were those of Bereswill et al (1992) and Llop et al (2000) because they were the conventional and nested PCRs most sensitive and robust in preliminary experiments with spiked plant material. Nevertheless, for confirming bacterial suspensions, PCR analysis with Guilford et al, (1996) and McManus and Jones (1995), are also good options. a) The validated conventional or single PCR uses the primers and conditions described by Bereswill et al, (1992). The primer sequences are the following (appendix 3):

13

Primer A: Primer B: PCR mix Reagent

5 CGG TTT TTA ACG CTG GG 5 GGG CAA ATA CTC GGA TT

Initial concentration Volume 20 l 5 l 1.5 l 1 l 1.4 l 8 l 2.5 l 0.5 l 2.5 l 2.5 l 0.1 l

Final concentration

-Water (PCR grade) -Buffer -MgCl2 -dNTPs -2-mercaptoethanol -Bovine serum albumin -DMSO -Tween 20 -Primer A -Primer B -Tth polymerase

10x 50 mM 10 mM 1 g/l

10 pmol/l 10 pmol/l 5 U/l

1x 1.5 mM 0.2 mM 10 mM 160 g/ml 5 % (v/v) 1 % (v/v) 25 pmol 25 pmol 0.5 U

- Sample volume: add 5 l to 45 l mix The reaction conditions are: a denaturation step of 93C for 2 min followed by 37 cycles of 93C for 1 min, 52C for 2 min, and 72C for 2 min. A final step of 72C for 10 min finishes the reaction. The amplicon size is 900 bp size according to Bereswill et al, (2002) 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 and Geider, 2001). Also, a more simple PCR mix was assayed with the same results as the original, as shown below: Reagent Initial concentration Volume 33.1 l 5 l 3 l 2 l 0.5 l 0.5 l 0.5 l 0.4 l Final concentration

- Water (PCR grade) - Buffer - MgCl2 - Formamide - dNTPs - Primer A - Primer B - Taq polymerase

10x 50 mM 10 mM 10 pmol/l 10 pmol/l 5 U/l

1x 3 mM 4% (v/v) 0.1 mM 5 pmol 5 pmol 2U

- Sample volume: add 5 l to 45 l mix

14 The reaction conditions are: a denaturation step of 93C for 5 min followed by 40 cycles of 93C for 30 s, 52C for 30 s, and 72C for 1 m 15 s. A final step of 72C for 10 min finishes the reaction. Single components of the mix can be replaced by Ready Mix Taq with Cl2 Mg or Ready Mix Red Taq PCR Reaction Mix with MgCl2 (Sigma). b) The nested-PCR in a single tube (Llop et al, 2000) uses two sets of primers placed at the same time, and due to the different annealing temperatures the two PCR reactions are performed consecutively. The external primers are the same designed by McManus and Jones (1995), whilst the internal are the ones described by Llop et al (2000). The sequences are the following (see appendix 3): External primers AJ75: AJ76: Internal primers 5 CGT ATT CAC GGC TTC GCA GAT 5 ACC CGC CAG GAT AGT CGC ATA

PEANT1: 5 TAT CCC TAA AAA CCT CAG TGC PEANT2: 5 GCA ACC TTG TGC CCT TTA

PCR mix Reagent - Water (PCR grade) - Buffer - MgCl2 - Formamide - dNTPs - Primer AJ75 - Primer AJ76 - PEANT1 - PEANT2 - Polymerase Initial concentration Volume 34.76 l 5 l 3 l 2 l 1 l 0.32 l 0.32 l 1 l 1 l 0.6 l Final concentration

10x 50 mM 10 mM 0.1 pmol/l 0.1 pmol/l 10 pmol/l 10 pmol/l 5 U/l

1x 3 mM 4 % (v/v) 0.2 mM 0.03 pmol 0.03 pmol 10 pmol 10 pmol 3U

- Sample volume: add 1 l to 49 l PCR mix The reaction conditions are: a denaturation step of 94C for 4 min followed by 25 cycles of 94C for 30 s and 72C for 1 min. This first round PCR is followed in the same thermocycler by a second denaturation step of 94C for 4 min and 40 cycles of 94C for 30 s, 56C for 30 s, and 72C for 45 s. A final step of 72C for 10 min finishes the reaction. The amplicon size is 391 bp, although some variations in size can occur. The amplification conditions are optimised for the Perkin-Elmer 9600 apparatus, so small modifications of the protocol could be necessary in other thermocyclers.

15 When using the PCR reactions to confirm the isolated colonies, use 1 U of Taq polymerase for Bereswill et al (1992) protocol or 2 U with the Nested PCR of Llop et al (2000), (instead of 2 or 3 U as for plant material). c) After both PCRs, prepare a 1.5 % agarose gel in TAE buffer 0.5 X (appendix 1). Place ca. 3 l droplets of loading buffer (appendix 1) on parafilm, mix 20 l of PCR product by gentle aspiration with the pipette before loading. Load wells of gel and include positive and negative controls. Include DNA marker 100 bp ladder in the first and last well of the gel. Run the gel for 20 min at 120 V (medium gel tray: 15x10 cm) or 40 min at 160 V (big gel tray or electrophoresis tank: 15x25 cm). Soak the gel in ethidium bromide solution for 20 minutes. Visualise the amplified DNA fragments by UV transillumination. d) The restriction pattern of the amplicons obtained with the primers of Bereswill et al, (1992) and of the amplicons obtained with the nested PCR in a single tube (Llop et al, 2000) can be performed with Dra I and Sma I endonucleases, to confirm the PCR analysis. Interpretation of the PCR results: The PCR test is negative if the E. amylovora specific amplicon of expected size and restriction pattern is not detected for the sample in question but is detected for all positive control samples. The PCR test is positive if the E. amylovora specific amplicon of expected size is detected, providing that it is not amplified from any of the negative control samples and the restriction enzyme pattern is identical with that derived from the positive control strain. If one of the negative controls shows a band of the size expected for E. amylovora, repeat the PCR with a new mix and include several negative controls that allow the detection of the contaminations. Reliable confirmation of a positive result can be obtained by repeating the test with a second set of PCR primers. Inhibition of the PCR may be suspected if the expected amplicon is obtained from the positive control sample containing E. amylovora in water but negative results are obtained from positive controls with E. amylovora in plant extract. 3. Isolation Freshly prepared sample extracts are necessary for successful isolation. The isolation of E. amylovora from symptomatic samples is relatively easy because the number of culturable bacteria in them is usually high. However, when symptoms are very advanced or when the environmental conditions are not favourable for fire blight expression, the number of E. amylovora culturable cells can be very low. Furthermore, if plates are overcrowded or antagonistic bacteria are suspected the sample should be retested and/or enrichment used before isolation, according to 2.4. Plating on three media is advised for maximum recovery of E. amylovora specially when samples are not in the best conditions. Depending on the number and composition of the microbiota of the sample, each one is more or less efficient. Three media (CCT, Levan and Kings B) have been validated in the ring test being Levan the one with the highest recovery (appendix 1). When analysing symptomatic samples good correlation is expected between isolation, immunofluorescence, enrichment DASI-ELISA and PCR.

16 3.1. Direct isolation a) Use CCT, Kings B and Levan (or Nutrient Agar Sucrose) media (appendix 1). Prepare 1:10 and 1:100 dilutions of each macerate (obtained following 2.3) in PBS (appendix 1). b) Pipette 50 l of the diluted and undiluted macerates onto separate plates of each medium. Start with the 1:100 dilution and proceed to the undiluted macerate. Use sterile loops or spreader or dip a glass spreader in denatured ethanol, flame and allow cooling. Carefully spread the pipetted volumes by triple streaking. c) Plate a 103, 104 and 105 cfu/ml dilution of a pure culture of Erwinia amylovora as a quality control of the media. Incubate the plates at ca 25C for 48-72 h. Final reading is at 72-96 h. d) Colonies of E. amylovora on CCT appear at about 48 h and are pale-violet, circular, high convex to domed, smooth and mucoid after 72 h, showing slower growth than on Kings B or Levan. CCT medium inhibits most pseudomonads but not Pantoea agglomerans. Colonies of E. amylovora on Kings B appear at 24 h and are creamy white, circular, intending to spread and non-fluorescent under UV light at 366 nm after 48 h. This allows the distinction from fluorescent pseudomonads. Colonies of E. amylovora on Levan medium appear at 24 h and are whitish, circular, domed, smooth and mucoid after 48 h. Levan negative colonies of E. amylovora have also been reported (Bereswill et al, 1997). e) Obtain pure cultures from individual suspect colonies of each sample by plating on Kings B medium. Identify presumptive colonies of Erwinia amylovora by inoculation on the available E. amylovora host, by DASI-ELISA or PCR, or by any other test (biochemical tests, IF, fatty acids profile, etc) as indicated. Store cultures in Nutrient agar slants covered with mineral oil at 10C or for long term in 30% glycerol at 80C or lyophilised. Interpretation of isolation results: The isolation is negative if no bacterial colonies with morphology similar to E. amylovora are observed after 96 h in any of the three media (provided that no inhibition is suspected due to competition or antagonism) and that 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 they are confirmed as indicated in Section 6.

3.2. Enrichment-isolation Plate the enrichments only on CCT plates (appendix 1). Spread 50 l of each enriched extract and of the 1:10, 1:100 and 1:1000 dilutions prepared in PBS (appendix 1) by triple streaking (as for isolations) to obtain isolated colonies. Incubate at ca. 25C for 72-96 h. The use of only semi-selective medium and dilutions is advised because of the abundant multiplication of different bacteria during the enrichment step.

17 4. FLOW DIAGRAM FOR THE DIAGNOSIS OF Erwinia amylovora IN ASYMPTOMATIC SAMPLES.

Asymptomatic sample1 Pathogen extraction, optative concentration and enrichment2 SCREENING TESTS3 IF , enrichment DASI-ELISA5, enrichment-PCR6
4

All three tests positive

At least one test positive ISOLATION TESTS Perform direct isolation7 and enrichment isolation8 Colonies with typical morphology9 Yes IDENTIFICATION TESTS12 Confirm identity of pure culture as E. amylovora and pathogenicity test13 Yes

Tests all negative

E. amylovora detected

No10

E. amylovora not detected11

No

E. amylovora confirmed
1. 2. 3.

4. 5. 6. 7. 8. 9. 10.

11. 12.

Sampling is described in Section 4.1. Pathogen extraction, enrichment and concentration are described in Sections 2.3, 2.4 and 5.1, 5.2. The use of more than one screening test is recommended. Isolation, ELISA and PCR should be performed after previous enrichment of the samples. Additional tests should be based on different biological principals. IF test is described in Section 2.5.3. Enrichment-ELISA tests are described in Section 2.5.2. Enrichment and PCR tests are described in Sections 2.4. and 2.5.4. Direct isolation is described in Section 3.1. Enrichment-isolation test is described in Section 3.2. Typical colony morphology is described in Section 3.1. Culturing can fail due to competition or inhibition by saprophytic bacteria. If positive results are obtained in screening tests, but the isolation tests are negative, then repeat the isolation. A negative result does not always guarantee the absence of the pathogen. Reliable identification of pure cultures of presumptive E.amylovora isolates is achieved using the tests described in Section 6 and 7.

18 5. METHODS FOR DETECTION AND IDENTIFICATION OF Erwinia amylovora IN ASYMPTOMATIC SAMPLES. 5.1. Sampling and sample preparation The asymptomatic samples can be processed individually or in groups of up to 100 samples (EPPO, 1992). Where surveys are performed they should be based on statistically representative samples. Samples taken from stored material can be considered 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. Sampling and sample preparation can be performed following one of these protocols: a) Collect blossoms, shoots, fruitlets or stem segments in sterile bags or containers in summer or early autumn, after favourable conditions for fire blight confirmed (van der Zwet and Beer, 1995), according to the following sampling procedures: Nursery plants: Cut young shoots of ca. 20 cm from E. amylovora more susceptible hosts available, disinfecting scissors or pruning shears among plants. If analyses need to be performed in winter collect 5-10 buds per plant. Field growing plants: Cut blossoms when available and/or young shoots of ca. 20 cm., disinfecting scissors or pruning shears among plants. Take blossoms, or peduncle and base of the limb of several leaves, or stem segments of the selected plants. Weight ca. 0,1 1 g of plant material and use for maceration the antioxidant buffer (appendix 1) at the appropriate amount following the protocol described in Section 2.3. Process the samples immediately by performing enrichment followed by DASIELISA and/or PCR and/or isolation, following the protocols described for symptomatic samples (Sections 2.5.2, 2.5.4 and 3). IF must be done directly with the extracts, without enrichment (Section 2.5.3.). b) The quarantine procedure N 40 of the EPPO (1992) includes a sampling procedure for the analysis of twigs of asymptomatic woody material from nurseries. A sample consists of 100 twigs about 10 cm in length from 100 plants. If there are several plant genera in the lot, these should be equally represented in the sample (with a maximum of three genera per/sample). b.1) From each sample randomly take 30 cut twigs and cut them in four pieces (120 stem pieces). Place them for 1,5 h in a rotary shaker at room temperature in sterile PBS (appendix 1) additioned with 0,1 % Tween 20 in Erlenmeyer flasks. Filter with a paper held in a sintered glass filter (n 2= 40-100 m) using a vacuum pump and collect the filtrate. b.2.) Use directly the filtrate for analysis or centrifuge it for 20 min at 10.000 g. Suspend the pellet in 4.5 ml sterile PBS (appendix 1). Perform the techniques indicated below.

19 b.3.) A similar protocol can be applied for leaves, shoots, flowers or buds. Depending on the season of survey the expected recovery of E. amylovora will be more or less high being maximum in summer (provided that weather conditions being favourable to E. amylovora and minimum in winter). When following a) or b), prepare for each sample 3 Eppendorf tubes with ca. 2 ml, 1 ml of macerate an use them as indicated in section 2.3. 5.2. Enrichment The direct analysis of asymptomatic samples is normally negative for E. amylovora due to the low bacterial population. Consequently, it is advised to perform a previous enrichment of the samples (Section 2.4) prepared in the antioxidant buffer (Gorris et al.,1996) (appendix 1) as indicated in 2.3. When analysing asymptomatic material perform the enrichment for 72 h at ca. 25C. 5.3. Screening tests Perform at least two screening tests: a) IF. Use one sample extract before or after concentration by centrifugation (but non enriched) per IF slide window. Fix also decimal dilutions of the sample and follow the protocol described in Section 2.5.3. b) Enrichment-isolation. Follow the procedure indicated in Section 2.4. c) Enrichment-ELISA. Follow the procedure indicated in Section 2.5.2. d) Enrichment-PCR. Use 500 l of the samples enriched in Kings B and in CCT for DNA extraction following Llop et al. (2000) or other appropriate protocols or kits. Follow the amplification protocols described in Section 2.5.4. If any of the screening tests are positive, attempt to isolate the pathogen from the extract or the enriched samples. When three or four tests are positive and the isolation is negative or not done, it is reasonable to consider E. amylovora presumptively detected in the sample, but confirmation needs the isolation and identification of the bacterium. If necessary, spread the extract (Section 2.3.) conserved at 80C. Use the three media (CCT, Kings B, Levan) indicated in appendix 1 for having maximum possibilities, to isolate E. amylovora, when no information about the microbiota of the samples is available.

20 6. IDENTIFICATION & CONFIRMATION Identify pure cultures of presumptive E. amylovora isolates using at least two tests related to two different characteristics of the pathogen (nutritional, fatty acids, serological or molecular). Include an appropriate host test as final confirmation of pathogenicity. Include known E. amylovora reference strains where appropriate for each test performed (appendix 1). 6.1. Nutritional and enzymatic identification tests. The genus Erwinia was defined for Gram-negative bacteria, facultative anaerobes, motile by peritrichous flagella, rod shaped and acid produced from glucose, fructose, galactose and sucrose. Determine the following phenotypic properties (Paulin, 2000) that are universally present or absent in E. amylovora, according to the methods of Jones and Geider (2001). Test Gram staining Levan production Fluorescent pigment production in Kings B (under UV) Oxidation/Fermentation (O/F) test Kovacs oxidase test Reduction of nitrate Utilisation of citrate Growth at 39C Gelatine liquefaction Urease Indol Reducing substances from sucrose Acetoin Expected result + O+/F+ + + + +

21 The following tests allow to differentiate E. amylovora from E. pyrifoliae, causal agent of Asian pear blight on Pyrus pyrifolia, (Kim et al., 1999, Kim et al., 2001) and a new Erwinia sp. isolated from necrotic pear blossoms in Spain (Rosell et al., 2002), although some physiological and biochemical characteristics may vary for some strains. Differences among Erwinia amylovora, Erwinia pyrifoliae and Erwinia sp. isolated from necrotic pear blossoms. Microbiological tests Gelatine hydrolysis Inositol1 Sorbitol1 Esculin1 Melibiose1 D-Raffinose1 -Gentibiose1 Amplification with2 EP16A/EPI62C CPS1/CPS2C + ND3 Erwinia amylovora + + V4 + Erwinia pyrifoliae ND3 + Erwinia sp. + + + + +

1= From Rosell et al., (submitted). Oxidation of substrates in API 50 CH (bioMrieux) with a modified protocol. 2= According to Kim et al., (2001). 3= ND: Not determined 4= V: Variable 6.1.1 Biochemical characterisation by API system (BioMrieux, France) Biochemical identification of E. amylovora can be obtained by specific profile in API 20 E and API 50 CH strips. a) API 20 E. Follow manufacturers instructions for preparing the suspension and inoculating the strip. Incubate at 25-26C and read after 24 and 48 h. The readings after 48 h should be as indicated for a typical E. amylovora culture.

22 Reaction of E. amylovora in API 20E tests Test ONPG ADH LDC ODC CIT SH2 URE TDA IND VP GEL GLU MAN INO SOR RHA SAC MEL AMY ARA
1

Reaction (48 h)1 Variable - (or weak +) + (or variable) Variable + Variable Variable Variable + - (or weak +) + (some -)

= Common reactions of 90 % strains of E. amylovora analysed (Donat et al., unpublished results). b) API 50 CH. Prepare a suspension of OD=1.0 in PBS (appendix 1): Add 1 ml of the suspension to 20 ml of Ayers medium (appendix 1). Follow the manufacturers instructions for inoculation of the strip. Incubate at 25-26C in aerobiosis and read after 24 and 48. Utilisation of the different carbohydrates is observed by a yellow colour in the well.

23 The reading after 72 h should be as indicated for a typical E. amylovora culture: Test1 L-arabinose Ribose D-xilose Galactose D-glucose D-fructose D-manose Mannitol Sorbitol N-acetylglucosamine Melibiose Sucrose Trehalose -gentiobiose
1

Reaction + + Variable Variable (mostly +) + + Variable + + + Variable + + +

= The remaining sugars are not utilised by E. amylovora but some strains can utilise glycerol and D-fucose (Donat et al., unpublished results).

c) Automated Biolog identification system. An identification system based on 95 carbon sources utilisation in a microtiter plate is commercially available (Biolog, CA, USA). Follow the manufacturers instructions for automatic identification of E. amylovora suspected strains. 6.2. Fatty acid profiling (FAP) Grow the levan-positive, non-fluorescent colonies, on trypticase soy agar for 48 hours at 28 C. Apply an appropriate FAP procedure. A positive FAP test is achieved if the profile of the presumptive culture is identical to that of the positive control.

6.3. Serological identification 6.3.1. Agglutination test Suspected E. amylovora colonies levan-positive, non fluorescent in Kings B medium, can be tested for slide agglutination mixing them in a drop of PBS (appendix 1) with a drop of E. amylovora specific antiserum (not diluted or only at 5 or 10 fold dilution) on a slide. Monoclonal antibodies can be used only provided they agglutinate with the reference strains.

24

If performing only two identification tests and inoculation do not use another serological test in addition to this method.

6.3.2. IF test Prepare a suspension of approximately 106 cells per ml in PBS (appendix 1) from levan-positive, non fluorescent colonies and apply the IF procedure described in 2.5.3. If performing only two identification tests and inoculation, do not use another serological test in addition to this method.

6.3.3. ELISA tests DASI-ELISA (described in Section 2.5.2.) and indirect-ELISA (see below) for isolates identification can be performed using specific monoclonal antibodies. A mixture of monoclonal antibodies has been validated in a ring test (appendix 2). Prepare a suspension of approximately 108 cells per ml in PBS (appendix 1) from suspected colonies. DASI-ELISA procedure (described in Section 2.5.2.) can be follow without previous enrichment. If performing only two identification tests and inoculation, do not use another serological test in addition to this method.

6.3.3.1. Indirect-ELISA a) Use 200 l aliquots of pure cultures of the suspected isolates (after being treated at 100C for 10 min, in a waterbath or heating block, for reducing non-specific reactions). Add an equal volume of carbonate buffer (appendix 1). Apply 200 l aliquots to at least 2 wells of a microtitre plate NuncPolysorp or equivalent. Use as positive control a 109 cfu/ml heat treated suspension of a pure culture of E. amylovora and as negative control a similar suspension of another species. Incubate for 1 hour at 37 C or overnight at 4 C. Flick out extracts from the wells. Wash the wells the three times with washing buffer (appendix 1), leaving the last washing solution in the wells for at least 5 minutes. b) Prepare the appropriate dilution of anti E. amylovora antibodies using the recommended dilutions for commercial antibodies (appendix 2). Add 200 l to each well and incubate for 1 hour at 37 C. Flick out the antibody solution from the wells and wash as before. c) Prepare the appropriate dilution of secondary antibody-alkaline phosphatase conjugate (GAM-AP) (appendix 2) in PBS + 0.5% BSA. Add 200 l to each well and incubated for 1 hour at 37 C. Flick out conjugated antibody from wells and wash as before.

25 d) Prepare a 1mg/ml alkaline phosphatase substrate (p-nitrophenylphosphate) in substrate buffer (appendix 1). Add 200 l alkaline phosphatase substrate solution to each well. Incubate in the dark at room temperature and read at 405 nm at regular intervals within 90 min.

6.4. Molecular identification 6.4.1. PCR Prepare a suspension of approximately 106 cells per ml in molecular grade sterile water from levan-positive, non fluorescent colonies. Apply appropriate PCR procedures, following Section 2.5.4 (without DNA extraction).

6.4.2. Macrorestriction with Xba I and Pulse Field Gel Electrophoresis (PFGE) PFGE analysis of genomic DNA after Xba I digestion according to Jock et al, (2002) shows 6 patterns for E. amylovora European strains. It can provide information useful for strain differentiation and has been applied to understand the spread of fire blight in Europe.

7. INOCULATION Suspected E. amylovora colonies from the isolation and enrichment plates should be inoculated to prove their pathogenicity. Hypersensitive reaction in tobacco leaves can give an indication of the presence of the hrp genes, but is also positive for many plant pathogenic bacteria. Use tobacco plants of cv. Xanthi or Samsun with more than 5-6 leaves. Prepare bacterial suspensions of 109 cfu/ml (OD at 620 nm =1.0) and inject them into the intracellular space of the adult leaves with a 25 GA 5/8 0.5 x 16 needle and syringe. Complete collapse of the infiltrated tissue after 24 h at room temperature is recorded as positive. To verify the pathogenicity of the suspected E. amylovora colonies it is necessary to perform inoculations in a fire blight host. Include always a positive control using a pure culture of a known E. amylovora strain and a negative control with sterile PBS (appendix 1). Plants inoculated with positive and negative controls should be kept apart from other test plants, but at the same conditions. Reisolate E. amylovora-like colonies from the inoculated fruitlets, plants or shoots showing typical E. amylovora symptoms. a) Inoculation of fruitlets (pear, apple or loquat susceptible cultivars) can be performed on whole immature fruits or on slices of them, using 10 l of 109 cfu/ml suspensions of the colonies in PBS (appendix 1). Include a positive and negative control as indicated below. Incubate in humid chamber at 25C for 3-5 days. A positive test on fruit is evident by a browning coloration around the

26 wounding site and oozing of bacteria in about 3-7 days, provided that only a necrotic lesion is observed in the negative control. b) Use pear, apple or loquat susceptible cultivars, or Crataegus, Cotoneaster or Pyracantha susceptible species for whole plant inoculation. In potted plants use young shoots for inoculation by cutting a young leaf until the main vein with scissors dipped into a 109 cfu/ml suspension of each colony prepared in PBS (appendix 1). Disinfected detached young shoots (30 sec ethanol 70%; 3 washings with sterile distilled water) from greenhouse growing plants can also be inoculated in the same way and kept in tubes with sterile 1% agar. Maintain the plants at 20-25C at 80-100 % relative humidity and the tubes at 20-25C with 16 h. light. Read results after 3, 7 and 15 days. Typical E. amylovora symptoms include wilting, and/or discolouration and/or necrotic tissue and/or ooze.

8. COMPARISON WITH SIMILAR ORGANISMS Symptoms of fire blight caused by E. amylovora may be confused with those caused by E. pyrifoliae, causal agent of bacterial shoot blight of Asian pear (Pyrus pyrifolia) (Kim et al, 1999 ) and with those caused by a new Erwinia species isolated from necrotic pear blossoms in Spain (Rosell et al, 2002). Definitive diagnosis should always be obtained through laboratory analysis. 9. REQUIREMENTS FOR A POSITIVE DIAGNOSIS When E. amylovora is diagnosed for the first time, or in critical cases (import/export) the following should be performed and provided: Disease symptoms, morphological, biochemical, and molecular characteristics of the pathogen and their pathogenic properties must be in accordance with the descriptions in the protocol. For isolation of the bacterium and descriptions of morphological, biochemical, molecular, and pathogenic characteristics the procedures and requirements of the protocol must be followed. In the case of latent infections, after an initial screening test the pathogen should be isolated and correctly identified, including a pathogenicity test with the pure culture. The original sample (with labels, if applicable), sample extract, and pure culture should be kept under proper conditions with correct registration and should be available in case of doubts.

27 10. REPORT ON THE DIAGNOSIS A report on the execution of the protocol should include: Information and documentation on the origin of the infected material. A description of the disease symptoms (if applicable and preferably with pictures). A description of the morphological, biochemical and pathogenic characteristics of the bacterium. The magnitude of the infection. Comments on the certainty or doubts about identification.

11. CONTACT POINT FOR PROTOCOL. NAME OF AUTHOR AND ORIGINATING LABORATORY Further information on this organism can be obtained from: Mara M. Lpez (mlopez@ivia.es) Bacteriologa. Dept. Proteccin Vegetal y Biotecnologa Instituto Valenciano de Investigaciones Agrarias (IVIA), Carretera Moncada-Nquera km 5, 46113 Moncada (Valencia). Spain. Phone 34963424000, Fax 34-963424001 Marianne Keck Bundesamt und Forschungszentrum fuer Landwirtschaft Spargelfeldstrasse 191, A-1226 Vienna P.O. BOX 400 Austria Diagnostic protocol prepared by Mara M. Lpez, Marianne Keck, Pablo Llop, Mara Teresa Gorris, Javier Pealver, Victoria Donat and Mariano Cambra. IVIA, 46113 Moncada (Valencia). Spain.

12. REFERENCES Anonymous (1998). Council Directive 98/57 EC of 20 July 1998 on the control of Ralstonia solanacearum (Smith) Yabuuchi et al. Official Journal of the European Communities L235: 1-39. Ayers SH, Rupp P, Johnson WT (1919). A study of alkali forming in milk. U.S. Dept. Agric. Bull. 782. Bereswill S, Pahl A, Bellemann P, Zeller W & Geider K (1992). Sensitive and species-specific detection of Erwinia amylovora by polymerase chain reaction analysis. Appl. Environ. Microbiol. 58: 3522-3526. Bereswill S, Bugert P, Bruchmuller I, Geider K (1995). Identification of the fire blight pathogen, Erwinia amylovora by PCR assays with chromosomal DNA. Appl. Environ. Microbiol. 61: 2636-2642.

28

Bereswill S, Jock S, Aldridge P, Janse JD, Geider K (1997). Molecular characterization of natural Erwinia amylovora strains deficient in levan synthesis. Physiol. Mol. Plant Pathol. 51: 215-225. Bonn WG & van der Zwet T (2000). Distribution and economic importance of fire blight. In J. Vanneste: Fire blight, the disease and its causative agent Erwinia amylovora. CABI Wallingford, UK. Bradbury JF (1986). Guide to plant pathogenic bacteria. CAB International Mycological Institute, Kew, Surrey, UK. Burrill TJ (1883) New species of Micrococcus. Am. Naturalist. 17:319. Dye DW (1983). Erwinia: The amylovora and herbicola groups. In P.C. Fahy and GJ Persley ed. Plant bacterial diseases. A diagnosis guide. Academic Press, Sydney. EPPO (1992) Quarantine procedure no.40. Erwinia amylovora. Sampling and test methods. Bull. OEPP 22: 225-231. EPPO/CABI (1997) Erwinia amylovora. In Quarantine Pests for Europe(2nd edn), pp.1001-1007. CAB International, Wallingford (UK) EPPO/CABI (1998) Map 257. In Distribution maps of quarantine pests for Europe CAB International, Wallingford (UK) Gorris MT, Cambra E, Lpez MM, Paulin JP, Chartier R, Cambra M (1996a). Production and characterization of monoclonal antibodies specific for Erwinia amylovora and their use in different serological techniques. Acta Horticulturae 411: 47-51. Gorris MT, Cambra M, Llop P, Lpez MM, Lecomte P, Chartier R, Paulin JP (1996b) A sensitive and specific detection of Erwinia amylovora based on the ELISA-DASI enrichment method with monoclonal antibodies. Acta Horticulturae 411: 41-45 Guilford PJ, Taylor RK, Clark RG, Hale CN, Forster RLS, Bonn WG (1996). PCRbased techniques for the detection of Erwinia amylovora. Acta Horticulturae. 411: 53-56. Ishimaru ES, Klos EJ (1984) New medium for detection of Erwinia amylovora and its use in epidemiological studies. Phytopathology 74: 1342-1345. Jock S, Donat V, Lpez MM, Bazzi C, Geider K (2002). Following spread of fire blight in Western, Central and Southern Europe by molecular differentiation of Erwinia amylovora strains with PFGE analysis. Envir. Microbiol. 4: 106-114.

29 Jones A, Geider K. (2001). II Gram negative bacteria. B. Erwinia and Pantoea. In: Guide for identification of plant pathogenic Bacteria, 2nd edition, Schaad NW, Jones JB, Chum W, APS Press, St Paul, USA. Kim WS, Gardan L, Rhim S.L, Geider K (1999) Erwinia pyrifoliae sp., a novel pathogen that affects Asian pear trees (Pyrus pyrifolia Nakai) Int. J. Systematic Bact. 49: 899-906. Kim WS, Jock S, Rhim S-L, Geider K (2001). Molecular detection and differentiation of Erwinia pyrifoliae and host range analysis of the Asian pear pathogen. Plant Dis. 85: 1183-1188.

King EO, Ward M, Raney DE (1954). Two simple media for the demonstration of pyocyanin and fluorescein. J. Lab. Clin. Med. 44: 301-307. Lecomte P, Manceau C, Paulin JP, Keck (1997). Identification by PCR analysis on plasmid pE29 of isolates of Erwinia amylovora responsible of an outbreak in Central Europe. Eur. J. Plant Path. 103: 91-98. Llop P, Caruso P, Cubero J, Morente C, Lopez MM (1999) A simple extraction procedure for efficient routine detection of pathogenic bacteria in plant material by polymerase chain reaction. J. Microbiol. Meth. 37: 23-31. Llop P, Bonaterra A, Pealver J, Lopez MM (2000) Development of a highly sensitive nested-PCR procedure using a single closed tube for detection of Erwinia amylovora in asymptomatic plant material. Appl. Environ. Microbiol. 66: 20712078. Maes M, Garbeva P, Crepel C (1996). Identification and sensitive endophytic detection of the fire blight pathogen Erwinia amylovora with 235 ribosomal DNA sequences and the polymerase chain reaction. Plant Pathol. 45: 1139-1149. Mc Manus PS, Jones AL (1995). Detection of Erwinia amylovora by nested. PCR and PCR-dot-blot and reverse blot hybridisations. Phytopathology 85: 618-623. Paulin JP (2000) Erwinia amylovora: general characteristics, biochemistry and serology, in J. Vanneste: Fire blight, the disease and its causative agent, Erwinia amylovora. CABI, Wallingford, UK. Rosell M, Garca-Vidal S, Tarn A, Llop P, Gorris M.T, Donat V, Pealver J, Chartier, R, Paulin JP, Gardan L, Lpez MM (2002). Characterization of an Erwinia sp. isolated from necrotic pear blossoms in Valencia, Spain. Acta Horticulturae 590: 139-142.

30 Rosell M, Pealver J, Llop P, Gorris MT, Charter R, Cambra M, Lpez MM. Identification of an Erwinia sp., different from Erwinia amylovora which is responsible for necrosis on pear blossoms, (submitted). Starr MP, Cardona C, Folsom D (1951). Bacterial fire blight of raspberry. Phytopathology 41: 9515-59. Sasser M (1990) Identification of bacteria through fatty acid analysis. In: Klement F, Rudolf K, Sands DC (eds), Methods in Phytobacteriology, 199-204. Akademiai Kiado, Budapest. Thomson SV (2000). Epidemiology of fire blight. In J. Vanneste: Fire blight, the disease and its causative agent Erwinia amylovora. CABI, Wallingford, UK.

Van der Zwet (2002). Present world wide distribution of fire blight. Acta Horticulturae 590: 33-34. van der Zwet T, Keil HL (1979) Fire blight: A bacterial disease of rosaceous plants. United States Department Agriculture Handbook 510, Washington DC, USA. van der Zwet T, Beer S (1995). Fire blight its nature, prevention and control. A practical guide to integrated disease management, USDA. Agricultural Information Bulletin No. 631, Washington DC, USA.

31 APPENDIX 1 Buffers:

Phosphate buffered saline 10mM, pH 7.2 (PBS) NaCl KCl Na2HPO412H2O KH2PO4 Distilled water 8g 0.2 g 2.9 g 0.2 g 1l

Antioxidant maceration buffer (Gorris et al., 1996) Polyvinilpyrrolidone (PVP-10) Mannitol Ascorbic acid Reduced glutathion PBS 10mM pH 7,2 Adjust pH to 7 Sterilise by filtration Carbonate buffer pH 9.6 Na2CO3 NaHCO3 Distilled water Adjust pH to 9.6 1.59 g 2.93 g 1l 20 g 10 g 1.76 g 3g 1l

Washing buffer (PBS, pH 7.2-7.4 supplemented with 0.05% Tween 20) NaCl KCl Na2HPO412H2O KH2PO4 Tween 20 Distilled water 8g 0.2 g 2.9 g 0.2 g 500 l 1l

Substrate buffer for alkaline phosphatase Diethanol amine 97 ml Dilute in 800 ml of distilled water Adjust pH 9.8 with concentrated HCl Adjust at 1000 ml with distilled water

32

Precipitating substrate buffer for alkaline phosphatase Sigma Fast 5-bromo-4-chloro-3-indolyl phosphate/nitro blue tetrazolium tablets (BCIPNBT) Cat No. B-5655 Sigma Aldrich GmbH (Stenheim), Germany Extraction buffer (Llop et al., 1999) Tris HCl, pH 7.5 NaCl EDTA SDS Polyvinilpyrrolidone PVP-10 Distilled water Sterilised by filtration 50X TAE buffer Tris 0.5 M Na2EDTA pH 8.0 Glacial acetic acid Distilled water Loading buffer Bromophenol blue Glycerol Distilled water Media: CCT medium (Ishimaru and Klos, 1984) Part 1 Sucrose 100 g Sorbitol 10 g Niaproof 1,2 ml Crystal violet 2 ml(sol. 0,1 % ethanol) Nutrient agar 23 g Distilled water 1 l. Adjust pH to 7.0-7.2; 115, 10 min sterilisation by autoclaving 0.025 g 3g 10 ml. 242 g 100 ml 57.1 ml Adjust volume to 1 l 24.2 g 14.6 g 9.3 g 5g 20 g Adjust volume to 1 l

Part 2 (to add to part 1 after sterilisation) Thallium nitrate* 2 ml (1%w/v aqueous solution) Cycloheximide* 0,05 g Sterilise by filtration (0.45 ). Add to 1 l of the sterile first part (at about 45C). *Highly toxic reagents, handle with precautions

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KINGS B medium (King et al., 1954) Proteose peptone N 3 Glycerol K2HPO4 MgSO4 .7H2O Agar Distilled water Adjust pH to 7.0-7.2 120C, 15 min sterilisation by autoclaving 20 g 10 ml 1,5 g 1,5 g 15 g 1l

LEVAN medium

Yeast extract Bactopeptone NaCl Sucrose Agar Distilled water Adjust pH to 7-7.2 120C, 15 min sterilisation by autoclaving

2g 5g 5g 50 g 20 g 1l

AYERS Medium (Ayers et al., 1919) NH4 H2 PO4 1g K Cl 0.2 g Mg SO4 0.2 g Bromothymol blue 75 ml (solution 0.2%) Distilled water 1l Adjust pH to 7 120C, 15 min sterilisation by autoclaving

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Primers: Oligonucleotide primer sequences (Bereswill et al., 1992): Primer A: Primer B: 5 CGG TTT TTA ACG CTG GG 5 GGG CAA ATA CTC GGA TT

Oligonucleotide primer sequences for nested PCR in a single closed tube (Llop et al., 1992): External primers Internal primers AJ75: 5 CGT ATT CAC GGC TTC GCA GAT AJ76: 5 ACC CGC CAG GAT AGT CGC ATA PEANT1: 5 TAT CCC TAA AAA CCT CAG TGC PEANT2: 5 GCA ACC TTG TGC CCT TTA

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APPENDIX 2

Commercially available standardised control material Bacterial isolates: The following E. amylovora isolates are recommended for use as positive controls: NCPPB683 and CFBP 1430. The following collections can provide different E. amylovora reference strains. 1. National Collection of Plant Pathogenic Bacteria (NCPPB), Central Science Laboratory, York, UK. 2. Culture Collection of the Plant Protection Service (PD), Wageningen, The Netherlands. 3. Collection Franaise de Bactries Phytopathognes (CFBP), INRA Station Phytobactriologie, Angers, France. Note: Authenticity of the strains can be guaranteed only if directly obtained from the culture collections. Antibodies to E. amylovora currently recommended for use in detection and identification tests. Antibody E.amylovora1 IVIA EPS 14302 IVIA Mab 7A3 IVIA Mab 8B+5H4 1 Type Polyclonal Polyclonal Monoclonal Monoclonals Source Loewe Biochemica, Germany Plant Print Diagnostics, Spain Plant Print Diagnostics, Spain Plant Print Diagnostics, Spain

Recommended for detection using IF test (validated in ring tests). LOEWE Biochemica GmbH. Mhiweg 2a D-82054 Sauerlach.Germany Recommended for detection using IF test (validated in ring tests). Recommended for detection using IF test (validated in ring tests). Recommended for detection using Enrichment DASI-ELISA test (validated in ring tests). PLANT PRINT Diagnstics, S.L. De la Mar 36, 46512 Faura, Valencia. Spain

2 3 4

Note: After an enrichment step the use of validated specific monoclonal antibodies is recommended to avoid cross reactions.

36 Enrichment DASI-ELISA complete kit based on polyclonal and monoclonal antibodies (8B+5H IVIA), including extraction buffer, semi-selective media, ELISA plates and reagents is commercially available from: PLANT PRINT Diagnstics, S.L. De la Mar 36 46512 Faura, Valencia. Spain Direct Tissue print-ELISA kit based on specific monoclonal antibodies including positive and negative pre-printed controls, nitrocellulose membranes, antibodies alkaline phosphatase conjugated and substrate: PLANT PRINT Diagnstics, S.L. De la Mar 36, 46512 Faura, Valencia. Spain E-mail: plantprint@wanadoo.es

Goat anti-mouse immunoglobulins alkaline phosphatase linked. Sigma (Steinhein), Germany Goat anti-mouse immunoglobulins fluorescein labelled. BioRad (Marnes-laCoquette), France Goat anti-rabbit immunoglobulins fluorescein labelled. BioRad (Marnes-laCoquette), France

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LIST OF PARTICIPANTS IN THE RING TEST The participation of the following laboratories in the ring test and their suggestions are very much acknowledged: Jaap Janse j.d.janse@pd.agro.nl Plant Protection Service Geertjesweg 15, P.O.Box 9102, 6700 HC, Wageningen The Netherlands Marianne Keck mkeck@bfl.at Bundesamt und Forschungszentrum fuer Landwirtschaft Spargelfeldstrasse 191, A-1226 Vienna P.O. BOX 400 Austria Arild Sletten arild.sletten@planteforsk.no Plant Protection Centre Hogskolevein 7, N-9432 AS Norway Miguel Angel Cambra mcambra@aragob.es Centro de Proteccin Vegetal Avda. Montaana 930, 5009 Zaragoza Spain Jose Luis Palomo jlpg@gugu.usal.es Centro Regional de Diagnstico Ctra. De Babilafuente Km 6, 37340 Aldearrubia Salamanca Spain Sean Simpkins s.simpkins@csl.gov.uk Plant Health Group Central Science Laboratory, Sand Hutton York YO41 1LZ United Kingdom Teresa Teixeira Duarte teixeiraduarte@dgpc.min-agricultura.pt Direcao Geral de Protecao das Culturas Tapada de Ajuda Edificio 1 1348-018 Lisboa Portugal

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Franoise Poliakoff Francoise.POLIAKOFF@agriculture.gouv.fr LNPV Unit Bactriologie 10, rue Le Ntre F-49044, ANGERS Cedex 01 France Johan Van Vaerenbergh jvanvaerenbergh@clo.fgov.be Rijkstation voor Plantenziekten 9820 Merelbeke Belgium Mara M. Lpez mlopez@ivia.es Instituto Valenciano de Investigaciones Agrarias Cta. Moncada-Nquera Km 4,5, Moncada, Valencia, 46113 Spain