Xanthomonas fragariae
2006; Wiley; Volume: 36; Issue: 1 Linguagem: Espanhol
10.1111/j.1365-2338.2006.00926.x
ISSN1365-2338
Tópico(s)Plant Pathogens and Fungal Diseases
ResumoEPPO BulletinVolume 36, Issue 1 p. 135-144 Diagnostics†Free Access Xanthomonas fragariae First published: 30 June 2006 https://doi.org/10.1111/j.1365-2338.2006.00926.xCitations: 23 European and Mediterranean Plant Protection Organization PM 7/65 (1) Organisation Européenne et Méditerranéenne pour la Protection des Plantes AboutSectionsPDF 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 Xanthomonas fragariae. Specific approval and amendment This Standard was developed under the EU DIAGPRO Project (SMT 4-CT98-2252) by partnership of contractor laboratories and intercomparison laboratories in European countries. Approved as an EPPO Standard in 2005-09. Introduction Xanthomonas fragariae is the causal agent of bacterial angular leaf spot of strawberry. It is an insidious and potentially serious disease which was first reported in USA (Kennedy & King, 1962a). It was later described in New Zealand, Australia, a few Asian and African countries and in most European countries where strawberry is cultivated (EPPO/CABI, 1998). The disease has been reported to cause the loss of 75% of fruit in Wisconsin (US) (Epstein, 1966). It is widespread in nurseries in many countries and has been responsible for important production losses in Europe (Mazzucchi et al., 1973; López et al., 1985; Bosshard & Schwind, 1997). X. fragariae is easily transmitted via asymptomatic plants with latent infections and international movement of latently infected plants is blamed for the introduction of X. fragariae from the USA to many other countries (López et al., 1985). The pathogen spreads from plants harvested in infested nurseries and symptoms appear under favourable conditions as well as after cold storage (Rat, 1993). Identity Name: Xanthomonas fragariae Kennedy & King. Taxonomic position: Bacteria, Gracilicutes. EPPO code: XANTFR. Phytosanitary categorization: EPPO A2 list no. 135, EU Annex II/A2. Detection The natural hosts of X. fragariae are Fragaria x ananassa, its parents Fragaria chiloensis and Fragaria virginiana, and various wild strawberries such as Fragaria vesca. Symptoms Small (1–4 mm) angular water-soaked spots appear initially only on the lower leaf surface surrounded by the veins. In the early stage, the spots are only visible on the lower surface and appear translucent when viewed with transmitted light. The bacteria are disseminated from the spots by irrigation, rain or dew to initiate new infections, frequently along the main veins of the leaf (Kennedy & King, 1962b). With high relative humidity, white, milky, cream-coloured or yellow exudates can appear, accumulating as yellow glistening opaque or brown masses. The leaves are most sensitive when they are two weeks to two months old. Older and younger leaves are resistant to infection. The size of the lesions increases progressively and, subsequently, the spots may coalesce and become apparent in the upper surface of the leaf (Rat, 1993). Dead tissues appear as reddish-brown irregular spots, and tear and break off. Systemic infections of the crown of the plants have been described in the USA by Hildebrand et al. (1967) and the endophytic movement of the bacterium confirmed by Stefani et al. (1989). Symptoms of angular leaf spot caused by X. fragariae may be confused with those caused by fungi like Mycosphaerella fragariae and with the symptoms caused by a new pathovar of X. arboricola, pv. fragariae (Janse et al., 2001). Preliminary studies show that X. a. fragariae may be more widespread in the EPPO region than originally estimated. Definitive diagnosis should always be obtained through laboratory analysis. Identification on plants with symptoms The procedures to be followed are summarized in the flow diagram of Fig. 1. Figure 1Open in figure viewerPowerPoint Flow diagram for the diagnosis of bacterial angular leaf spot (Xanthomonas fragariae) on host plants with symptoms. Sampling and sample preparation The sample for analysis of bacterial angular leaf spot on plants with symptoms should if possible be leaves with young water-soaked spots, or otherwise dry spots with or without exudates. In the case of suspected systemic infections, tissue from the crown of the plants should be tested. The general sample preparation procedure (Appendix 1– symptomatic plants) is valid for isolation, bioassay, serological detection and PCR amplification (before or after biological enrichment) of X. fragariae. Rapid screening tests Rapid screening tests (Appendix 2) facilitate presumptive diagnosis. Positive results should be obtained in two tests – a serological test (ELISA or Immunofluorescence (IF) and PCR/Nested PCR) to confirm presumptive diagnosis of X. fragariae, as the bacterium is very difficult to isolate. Isolation Direct isolation of X. fragariae is difficult, even in the presence of symptoms and exudates, because the bacterium is very slow-growing on artificial nutrient media. Leaves with young lesions are selected from plants with symptoms and disinfected individually with a cotton-wool stick soaked in 70% ethanol. Isolations should be made from young, water-soaked lesions, or from the margins of older lesions by cutting out a small piece of tissue with a sharp sterile scalpel. The tissue is crushed in a few mL of sterile distilled water or PBS (Appendix 7) and held for 10–15 min, and processed as in Appendix 3 (method 1). Isolation is usually more successful on Wilbrink's medium with nitrate (Wilbrink-N) (Appendix 7) than on YPGA or other common media. Use of purified agar (Difco) is recommended in all media because impurities from other commercial agars can inhibit the growth of X. fragariae. An alternative method for isolation is based on surface sterilization of the infected plant material, followed by diffusion of bacteria into an extraction buffer, plating and growth of the bacteria on nutrient media (Appendix 3, method 2). Interpretation of isolation results The isolation is negative if no bacterial colonies with morphology similar to X. fragariae are observed after 7 days on either of two media (provided that no inhibition is suspected due to competition or antagonism) and typical X. fragariae colonies are found in the plates of the positive control. A negative result does not guarantee the absence of the pathogen. The isolation is positive if presumptive X. fragariae colonies are isolated in at least one of the media used. When analysing symptomatic samples, good correlation is not always observed between isolation and the rapid screening tests, because isolation frequently fails. Best isolation results can be expected if freshly prepared samples extracted from young lesions are used. Even if isolation fails, the sample should be considered as presumptively affected by X. fragariae if a serological test, and PCR, are positive. Detached leaf assay Samples prepared in the maceration buffer can be used directly to inoculate detached strawberry leaves, following Civerolo et al. (1997) (Appendix 4). The detached leaf assay is negative if no typical angular leaf spots or chlorotic haloes appears in some of the inoculated sites after 28 days. The negative controls should be negative or at least show totally different type of lesions. The detached leaf assay is positive if typical angular leaf spots or chlorotic haloes appear in the inoculated sites after 28 days. The lesions should differ from those which may sometimes be observed in the negative controls. PCR or dilution plating after detached leaf assay The inoculated detached leaves can be used for enrichment prior to PCR or dilution plating. One leaf per sample is taken from those inoculated in the detached leaf assay, 48 h after inoculation, and 10–12 small discs 0.5 cm in diameter are taken from each inoculated site and crushed in 4.5 mL PBS (Appendix 7). For PCR, the samples can be stored at –20°C at this point. The method of the Rapid screening tests (above) is used (Appendix 2). Each of the soaked enriched samples is streaked on Wilbrink N medium plates and the bacterial growth is washed off after 4 days for PCR analysis. This is a modification of the bio-enrichment PCR described by Schaad et al. (1995). For dilution plating, the samples are used directly, without freezing. The enrichments are plated on Wilbrink N medium, after preparation of 1 : 10, 1 : 100 and 1 : 1000 dilutions in PBS (Appendix 7). The dilutions are plated by triple streaking (Appendix 3) to obtain isolated colonies. After incubation at 25°C for 5–7 days, X. fragariae-like colonies are recorded as for Isolation (Appendix 3). Identification in asymptomatic samples Whereas a flow diagram is provided for the identification of the bacterium in plants with symptoms (Fig. 1), there is not enough information to do the same for identification in asymptomatic samples. Sampling and sample preparation Asymptomatic samples can be processed individually or in groups of up to 50 plants. Where surveys are performed, they should be based on statistically representative samples. Samples taken from stored material can be considered random whereas those from orchards or nurseries may not be. Sampling and sample preparation for analysis of asymptomatic runners can be performed according to a modification of the procedure of OEPP/EPPO (1994) (Appendix 1– asymptomatic plants, method 1) or by testing the crown and petioles of the plants (Randhawa California Seed and Plant Laboratory) (Appendix 1– asymptomatic plants, method 2). Isolation The washing liquid, pellet or macerate of crowns can be used for direct isolation as in the method for symptomatic samples (Appendix 3). Unfortunately, isolation of X. fragariae from asymptomatic samples is normally negative due to low bacterial populations. Screening tests The procedures for ELISA, IF and PCR are as for symptomatic samples (Appendix 2). If two of the screening tests are positive but the isolation is negative, an attempt can be made to isolate the pathogen from new fresh samples. Confirmation Biochemical and physiological identification Conventional tests X. fragariae has the common characteristics of all xanthomonads: Gram-negative, aerobic, rods, with single polar flagellum, nitrates not reduced, catalase positive, asparagine not used as a sole source of carbon and nitrogen and producer of xanthomonadin, weak production of acids from carbohydrates. Colonies are mucoid, convex, and shiny on YPGA and Wilbrink-N media (Dye, 1962; van den Mooter & Swings, 1990; Swings et al., 1993). Xanthomonas spp. are easily differentiated from the other genera of aerobic, Gram-negative rods and other yellow pigmented bacteria by the characters shown in Table 1, following (Schaad & Stall, 1989; Schaad et al., 2001). Table 1. Phenotypic characteristics useful for differentiating Xanthomonas from Pseudomonas and other yellow-pigmented bacteria such as Flavobacterium Test Xanthomonas Pseudomonas Flavobacterium Pantoea Flagella 1, polar > 1, polar none peritrichous Xanthomonadin + – – – Fluorescence – V – – Levan from sucrose + V – – H2S from cysteine + – – – Oxidase – V + – Fermentation – – – + Growth on 0.1% TTC – + + + V: variable reaction. The most relevant or useful characteristics for distinguishing X. fragariae from other Xanthomonas (Schaad & Stall 1989; Goszczynska et al., 2000; Janse et al., 2001) are shown in Table 2. Differences from X. campestris justify the status of X. fragariae as a separate species. Growth is poor on nutrient agar but much better when supplemented with 5% glucose. Purified agar is required for growth of most of the strains. For long-term preservation, cultures should be stored at –80°C, as a suspension of more than 1010 cfu mL−1 in sterile 30% glycerol. Table 2. Diagnostic tests for differentiation of Xanthomonas spp Tests X. campestris X. arboricola pv. fragariae X. fragariae Growth at 35°C + ND – Growth on 2% NaCl + + – Esculin hydrolysis + + – Gelatin liquefaction V + + Protein digestion + ND – Starch hydrolysis V + + Urease production – – – Acid from: Arabinose + ND – Galactose + + – Trehalose + ND – Cellobiose + + – V: variable reaction. ND: Not determined. Biochemical characterization by commercial systems X. fragariae can be identified biochemically by its specific profiles in API 20 NE and API 50 CH strips (BioMérieux, France). For API 20 NE, the manufacturer's instructions are followed to prepare a suspension from a 48-h culture on Wilbrink-N medium (Appendix 7) and inoculate a strip, which is incubated at 25–26°C and read after 48 h for enzymatic activities and 96 h for substrate utilization (Table 3). For API 50 CH, a suspension of OD = 1.0 is prepared in PBS (Appendix 7), of which 1 mL is added to 20 mL of medium C of Dye (1962) (Appendix 7). The manufacturer's instructions are followed for inoculation of the strip, which is incubated at 25°C aerobically and read after 2, 3 and 6 days. Utilization of the different carbohydrates is shown by a yellow colour in the well after the incubation period (Table 4). Table 3. Reaction of X. fragariae in API 20 NE tests Test Reaction (48 or 96 h)1 Glucose fermentation – Arginine – Urease – Esculin + Gelatin weak + PNPG + Assimilation of Glucose + Arabinose – Mannose + Mannitol – N-acetyl-glucosamine + Maltose – Gluconate – Caprate – Adipate – Malate + Citrate – Phenylacetate – 1 Common reactions of 90% strains of X. fragariae analysed (Domínguez et al., pers. comm.). For first 6 tests, read at 48 h. For others, read at 96 h. Table 4. Readings after 6 days for a typical X. fragariae culture in API 50 CH Test1 Reaction d-arabinose Variable Galactose + d-glucose + d-fructose + d-mannose + N-acetylglucosamine + Esculin + Melibiose Variable Sucrose + Trehalose + d-xilose + l-fucose + 1 The remaining sugars are not utilized by X. fragariae (Domínguez, pers. comm.) Serological tests For IF-test, a suspension of approximately 106 cells per mL is prepared in PBS buffer and the IF procedure for symptomatic samples is applied (Appendix 2). Alternatively, indirect ELISA or DAS-ELISA can be used (Appendix 2). Recommended antibodies are also given in Appendix 2. If only two identification tests are performed, only one should be serological. PCR test A suspension of approximately 106 cells per mL in molecular grade sterile water is prepared from a 48 h growing culture on Wilbrink-N medium. The PCR procedure given for symptomatic samples can be used (Appendix 2) or alternatively the protocol of Roberts et al. (1996), without any DNA extraction (Appendix 5). Fatty acid profiling (FAP) Cultures are grown on trypticase soy agar for 48 h at 28°C. Fatty acid analysis is available from MIDI (Newark, US), NCPPB (CSL, York, GB), and PD (Wageningen, NL). A positive test is achieved if the profile of the presumptive culture is identical to that of the X. fragariae positive control. REP-PCR The following protocols have been used by Opgenorth et al. (1996) to identify X. fragariae strains specifically. The PCR reaction mixture and amplification conditions are essentially the same as those described by Louws et al. (1994). The sequences of the primers are as follows: REP1R-I, 5′-IIIICGICGICATCIGGC-3′; REP2-I, 5′-ICGICTTATCIGGCCTAC-3′; ERIC1R, 5′-ATGTAAGCTCCTGGGGATTCAC-3′; ERIC2, 5′-AAGTAAGTGACTGGGGTGAGCG-3′. Amplification conditions were: 95°C for 6 min followed by 35 cycles at 94°C for 1 min, 44°C (REP primers) or 52°C (ERIC primers) for 1 min, 65°C for 8 min. The amplification cycles were followed by a final extension cycle of 68°C for 16 min. Inoculation Suspected X. fragariae colonies from the isolation and enrichment plates should be inoculated onto strawberry plants to prove their pathogenicity, specially those from new findings. The detached leaf assay described for symptomatic samples may be used (Appendix 4) or other procedures (Hazel & Civerolo, 1980) (Appendix 6). Reference material See Appendix 8. Reporting and documentation Guidelines on reporting and documentation are given in EPPO Standard PM7/– (in preparation). Further information Further information on this organism can be obtained from: M.M. López, Departamento de Protección Vegetal y Biotecnología, Instituto Valenciano de Investigaciones Agrarias (IVIA), Carretera de Moncada-Náquera km 5, 46113 Moncada (Valencia) (ES). E-mail: (mlopez@ivia.es) E.L. Civerolo, USDA, ARS, PWA, San Joaquin Valley Agricultural Sciences Center, Exotic and Invasive Diseases and Pests Research, 9611 So, Riverbend Ave., Parlier, CA 93648. E-mail: (eciverolo@fresno.ars.usda.gov). Footnotes 1 The Figures in this Standard marked ‘Web Fig.’ are published on the EPPO website http://www.eppo.org. 2 J. Janse (Plant Protection Service, Wageningen, NL); M. Keck (Bundesamt und Forschungszentrum für Landwirtschaft, Vienna, AT); A. Sletten (Plant Protection Centre, Ås, NO); S. Simpkins (Central Science Laboratory,York GB); L. Cruz (Centro Nacional da Producao Tapada de Ajuda, Lisbon, PT); F. Poliakoff (LNPV Unité Bactériologie, Angers, FR); J. Van Vaerenbergh Rijkstation voor Plantenziekten, Merelbeke (BE); A. Baudry NPV, Villenave d’Ornon (FR); M.M. López (IVIA, Moncada-Valencia, ES). Acknowledgements This protocol was originally drafted by M.M. López, F. Domínguez, C. Morente, C.I. Salcedo and A. Olmos (IVIA, 46113, Moncada (Valencia), Spain), and E. Civerolo (USDA-ARS. PWA Parlier, CA 93648, USA). The main diagnostic tests recommended in this protocol were ring-tested in different European laboratories22 J. Janse (Plant Protection Service, Wageningen, NL); M. Keck (Bundesamt und Forschungszentrum für Landwirtschaft, Vienna, AT); A. Sletten (Plant Protection Centre, Ås, NO); S. Simpkins (Central Science Laboratory,York GB); L. Cruz (Centro Nacional da Producao Tapada de Ajuda, Lisbon, PT); F. Poliakoff (LNPV Unité Bactériologie, Angers, FR); J. Van Vaerenbergh Rijkstation voor Plantenziekten, Merelbeke (BE); A. Baudry NPV, Villenave d’Ornon (FR); M.M. López (IVIA, Moncada-Valencia, ES). . Appendices Appendix 1 Sample preparation Symptomatic plants Select plants with young symptoms (water-soaked spots in the lower part of the leaves). Externally disinfect the parts to be tested with 70% ethanol. Aseptically excise 0.1 g from each leaf containing water-soaked spots. If the plants show vascular symptoms, remove the roots and the leaves, carefully rinse the crown and petioles under tap water and then disinfect them by immersion for 1 min in 70% ethanol and rinse each plant separately three times in sterile distilled water. Take 0.1 g per sample from the leaves or crown and petioles and add 9 mL of PBS (Appendix 7) in a plastic bag with a heavy net. Slightly crush the plant material with a hammer or other appropriate apparatus in the plastic bag. Let all the samples macerate for 15 min at room temperature. Transfer 2 mL, 1 mL and 1 mL from each macerated sample to three sterile Eppendorf tubes. Immediately (on the same day), use the 2 mL for isolation, inoculation of detached leaves and IF. Store the other two tubes (with 1 mL), respectively, at –20°C, and at –80°C with 30% glycerol. The ELISA and PCR analysis are performed as soon as convenient, using aliquots from the Eppendorf tubes stored at –20°C. Asymptomatic plants Method 1 Remove leaves and roots from runners, keeping the petioles. Wash the remaining crowns in running water, drain and cut lengthwise into quarters. Collect a 30 g sample at random from the 200 quarters. Place each sample in 150 mL of PBS (Appendix 7). Shake for 30 min and use the washing liquid directly for detection, or after centrifugation at 10 000 g per 10 min. Suspend the final pellet in sterile distilled water to obtain a final volume of 5 mL. After leaving to settle for 15 min collect the upper clarified part and prepare dilutions (1 : 10 and 1 : 100) in sterile distilled water. Keep 2 mL for analysis and put 1 mL at –20°C and 1 mL at –80°C with 30% glycerol. Method 2 Surface-disinfest runners with 0.5% sodium hypochlorite +0.02% Tween 20 for 2 min, followed by two washes with sterile distilled water. Take small discs of tissue about 0.3–0.5 cm in diameter from the crown and petioles of each plant, wash in running water before crushing and macerating them in 10 mm MOPS buffer pH 7.3 for 15–30 min at room temperature. Appendix 2 Rapid screening tests ELISA Use 2 mL of the sample freshly prepared or stored at –20°C. Two ELISA protocols with commercially available polyclonal antibodies (Appendix 8) have been validated in a ring test performed by 10 laboratories. Use a pure culture of X. fragariae and a non X. fragariae strain as positive and negative controls in each plate. Because of the frequent cross reactions with polyclonal antibodies, healthy plant controls should be used. The titre should preferably be determined for each new batch of antibodies. Indirect ELISA Mix 210 µL of each sample with 210 µL coating buffer pH 9.6 (Appendix 7). Add 200 µL of the mix to two wells of one microtiter plate (Nunc-Polysorp or equivalent). Prepare a 109 cfu mL−1 suspension of the positive control in PBS (Appendix 7). Mix 210 µL of this suspension with 210 µL of coating buffer and fill two wells. Prepare a 109 cfu mL−1 suspension of the negative control bacterium, mix 1 : 1 with coating buffer and fill in other two wells. Crush 0.01 g of strawberry (X. fragariae-free) material (leaf or crown) in 0.9 mL PBS (Appendix 7) and add 0.9 mL of coating buffer (Appendix 7). Use this as plant material negative control and fill two wells. Wrap the plate tightly in cling film or place in a plastic box with some damp paper towels and close the box. Incubate the plate at 4°C overnight. Wash the plate three times with PBS (Appendix 7) diluted 1/2 +0.05% Tween 20. Fill the wells of the plate with PBS 1/2 +0.05% Tween 20 and invert to remove the buffer. Repeat twice. Put the plate to dry on paper towels. Add 200 µL of blocking buffer (PBS +1% bovine serum albumin (BSA, or non fat milk powder) to each of the test wells. Wrap the plate as described above and incubate at 37°C for 1 h. Wash the plate as described above. Prepare the X. fragariae antibody at appropriate dilution in PBS (Appendix 7) and add 200 µL to each test well. Wrap the plate as described above and incubate at 37°C for 2 h. Wash the plate as described above. Prepare the antibody-enzyme conjugate at appropriate dilution in PBS (Appendix 7) +0.2% BSA and add 200 µL to each test well. Wrap the plate as described above and incubate at 37°C for 1 h. Wash the plate four times. Prepare the substrate just before use. Add p-nitrophenylphosphate at 1 mg/mL to substrate buffer pH 9.8 (Appendix 7). Add 200 µL of prepared substrate to each test well. Incubate in the dark at room temperature for 15, 30 and 60 min. Read the absorbance at 405 nm. DAS-ELISA Prepare antibodies at the appropriate dilution in coating buffer pH 9.6 (Appendix 1). Add 200 µL to each well of two plates. Incubate at 37°C for 4 h. Wash the wells three times with PBS (Appendix 7) 1/2 +0.05% Tween 20. Add 200 µL of each sample to two wells of each plate. Add also in two wells 200 µL of positive and negative controls, as described for indirect ELISA, to each plate. Incubate for 16 h at 4°C. Wash the wells three times with PBS 1/2 +0.05% Tween 20. Incubate at 37°C for 1 h, and wash three times as before. Prepare the appropriate dilution of the conjugate in PBS (Appendix 7) containing 0.2% BSA and add 200 µL to each well. Incubate at 37°C for 3 h. Wash the wells four times with PBS 1/2 +0.05% Tween 20. Prepare the substrate just before use. Add p-nitrophenylphosphate at 1 mg/mL to substrate buffer pH 9.8 (Appendix 7). Add 200 µL of prepared substrate to each test well. Incubate in the dark at room temperature for 15, 30 and 60 min. Read the absorbance at 405 nm. Interpretation of ELISA test results The ELISA test is negative if the average absorbance reading from duplicate sample wells is < 2x the average absorbance of that in the negative sample extract control wells. This is provided that the absorbances for the positive controls are all above 1.0 after 60 min incubation and are greater than twice the absorbances obtained for negative sample extracts. The ELISA test is positive if the average absorbance readings from duplicate sample wells is > 2x the average absorbance in the negative sample extract well provided that 2x average absorbance 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 that the reagents were not well prepared. Positive ELISA readings in negative control wells indicate that cross-contamination or non-specific antibody binding has occurred. In either case, the test should be repeated or a second test based on a different biological principle should be performed. Immunofluorescence Follow the standard procedure described in EU (1998), preferably using a validated source of antibodies. Two commercially available polyclonal antibodies (Appendix 8) have been validated using FITC-conjugated antispecies immunoglobulins. Use undiluted preparations of the samples and their 1 : 10, 1 : 100 and 1 : 1000 dilutions in PBS (Appendix 7) to spot windows of the IF slides. Prepare one IF slide for each sample and its dilutions. Prepare positive controls with 106 cfu mL−1 suspensions in PBS of a pure culture of a reference strain of X. fragariae and a negative controls (healthy plant controls and another bacterium). Include negative buffer controls with antibodies and conjugate, and with conjugate only. Air-dry, and fix by flaming or by absolute or 95° ethanol. Store slides at –20°C until required. Use the antibodies at the recommended dilutions in PBS (Appendix 7). Use of two dilutions of the antibodies is advised to detect cross reactions with other bacteria. Spot the appropriate amount per well. Incubate slides in a moist chamber for 30 min at room temperature. Shake droplets off the slide and rinse slides with PBS. Wash 10 min with the same buffer. Carefully remove excess moisture. Dilute the appropriate FITC conjugate in PBS (Appendix 7). Cover the windows of all slides with the corresponding diluted conjugate and incubate in a moist chamber for 30 min at room temperature. Repeat the washing step. Add 0.1 m phosphate-buffered glycerol mountant (with antifading) on each window and apply a cover slip. View slides under oil immersion at 500–1000× magnification by scanning windows across 2 diameters at right angles and around the perimeters. Count the cells that show fluorescence and have a similar size to that of the reference strain of X. fragariae as described in EU (1998). Interpretation of the IF test result The test is negative if green fluorescing cells with morphology typical of X. fragariae 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, the IF test is considered positive for samples with > 103 cells per mL. For samples with < 103 cells per mL, the result of the IF test may be considered doubtful in which case further testing or re-sampling should be performed. Samples with large numbers of incomplete or weakly fluorescing cells compared to the positive control need further testing. The test should be based on a different biological principle, or involve a repeat IF test with different dilutions of antibody or pellet or a second source of antibodies. PCR Use the validated PCR reagents and protocol. A nested PCR in two tubes has been developed but is not yet validated (Zimmerman et al., 2004). Care should be taken with nested PCR since the increased sensitivity gained may increase the possibility for false positive results. Every precaution should be taken to avoid contamination of samples with target DNA. As positive controls, use aliquots of strawberry samples which previously gave a negative test result by several techniques, to which 104 and 106 cells mL−1 of a reference X. fragariae strain has been added, and a suspension of 104 cells mL−1 of the bacterium. Positive controls should be prepared separately. As negative controls, use at least a sample extract which previously gave a negative test result for X. fragariae, and a sample of ultra pure water. Perform the DNA extraction from the positive and negative controls as well as from the samples, and also include negative controls after extraction. One protocol for DNA ext
Referência(s)