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Clavibacter michiganensis subsp. michiganensis

2005; Wiley; Volume: 35; Issue: 2 Linguagem: Catalão

10.1111/j.1365-2338.2005.00822.x

1365-2338

Plant-Microbe Interactions and Immunity

EPPO BulletinVolume 35, Issue 2 p. 275-283 Free Access Clavibacter michiganensis subsp. michiganensis First published: 03 October 2005 https://doi.org/10.1111/j.1365-2338.2005.00822.xCitations: 28 European and Mediterranean Plant Protection Organization PM 7/42(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 Clavibacter michiganensis subsp. michiganensis. Specific approval and amendment Approved in 2004-09. Introduction Clavibacter michiganensis subsp. michiganensis was originally described in 1910 as the cause of bacterial canker of tomato in North America. The pathogen is now present in all main production areas of tomato and is quite widely distributed in the EPPO region (EPPO/CABI, 1998). Occurrence is usually erratic: years of absence or limited appearance may be followed by an epidemic. Tomato is the most important host but in some cases natural infections have also been determined on capsicum, aubergine and several Solanum weeds (e.g. S. nigrum, S. douglasii, S. trifolium). Other solanaceous plants are susceptible on artificial inoculation (Thyr et al., 1975). Several solanaceous and nonsolanaceous plants, e.g. Datura stramonium, Chenopodium album and Amaranthus retroflexus have been identified as reservoirs for epiphytical survival and spread (Chang et al., 1992). The significance of these epiphytic populations is yet not fully understood although they seem to contribute to infections through pruning wounds (Carlton et al., 1994). The pathogen can survive for several months in contaminated debris, although less in buried debris than in debris on the soil surface where decomposition is slower and interaction of other microorganisms is less (Gleason et al., 1991). Also persistence in dry conditions on equipment, boxes and glasshouse constructions is important for survival. The pathogen can be present in commercial seed lots but generally as a contaminant, deposited on the seed surface from the pulp and not as an internal infection. Seed transmission appears to be very low. Latently infected young plants are considered important for spread. The organism is very contagious under protected cultivation (glasshouses). Direct spread from a few primary infections occurs during culture manipulations, e.g. trimming, defoliation, harvesting. Once the disease has appeared, infected plants (ant adjacent plants) should be destroyed and infected rows isolated. Further information can be found in the EPPO data sheet on C. m. michiganensis (EPPO/CABI, 1997). Identity Name: Clavibacter michiganensis subsp. michiganensis (Smith, 1910; Davis et al., 1984) Synonyms: Corynebacterium michiganense subsp. michiganense (Smith, 1910; Carlson & Vidaver, 1982), Corynebacterium michiganense pv. michiganense (Smith, 1910; Dye & Kemp, 1977), Corynebacterium michiganense (Smith, 1910; Jensen, 1934) Taxonomic position: Procaryotae Kingdom, Division II Firmicutes Gibbons & Murray 1978, Class I Firmibacteria. The genus Clavibacter was designed to accommodate the plant pathogenic coryneform bacteria of which the cell wall peptidoglycan contains 2,4-diaminobutyric acid as dibasic amino acid (Davis et al., 1984). Strictly aerobic, Gram-positive rods which do not produce endospores. V, Y and palisade arrangements of cells are usually present. Infections of most Clavibacter species are systemic or become so when the pathogen reaches the vascular tissues EPPO computer code: CORBMI Phytosanitary categorization: EPPO A2 list n° 50, EU Annex designation II/A2 Detection The diagnostic procedure (Fig. 1) comprises isolation from infected tissue, presumptive diagnosis with a rapid test, identification of presumptive isolates and determination of pathogenicity. A seed test is specifically performed by selective plating followed by identification of presumptive isolates and determination of pathogenicity (Fig. 2). Immunofluorescence and PCR after selective enrichment, combined with a bioassay in tomato plantlets, can be used as accessory tests. Detection of C. m. michiganensis in young plants is erratic since the location of the pathogen (roots, stem or leaves) depends on the culture conditions of the plantlets. Therefore, a method for young plants is not given in detail here. Some useful methodology in this respect can be obtained from the experts mentioned in the chapter ‘Further Information’ of this standard. Figure 1Open in figure viewerPowerPoint Scheme for detection and identification of Clavibacter michiganensis subsp. michiganensis in samples from symptomatic or symptomless tomato plants. Figure 2Open in figure viewerPowerPoint Scheme for detection and identification of Clavibacter michiganensis subsp. michiganensis in samples of tomato seeds. Disease symptoms Generally, C. m. michiganensis causes systemic infection of tomato plants. The pathogen can also cause spots on leaves and fruits as a result of a local infection, usually under overhead irrigation. There is a wide range of symptoms which depend on place of production (glasshouse or field), age of the plant at the time of infection, cultural practices, cultivar, etc. In addition, there is evidence of strains which produce less severe symptoms. In a glasshouse crop, the disease can often be recognized at an early stage by dull green, oily areas between the leaf veins which rapidly desiccate to white and subsequently pale brown necrosis giving the plant a scorched appearance, as if sunburned or overdosed with a chemical. Small affected areas may coalesce and produce larger necrotic zones [Web Fig. 3]. Downward turning of one or a few of the lower leaves occurs as the systemic infection progresses and often the leaflets along one side of a leaf become flaccid [Web Fig. 4] , at least during periods of enhanced evapo-transpiration. Under favourable conditions for bacterial development (25°− 30°C and evapo-transpiration stress), entire leaves wilt and shrivel within a few days [Web Fig. 5]. Finally, the whole plant desiccates. Under less favourable conditions, irreversible wilting will be delayed and the plant may not show any wilting when defoliation is done for crop management. Fruits of systemically infected plants may fail to develop and fall or ripen unevenly. They can appear marbled with longitudinal chlorotic streaks and internal bleaching of vascular and surrounding tissues. Typical leaf or fruit spots, arising from local infection, are not found in glasshouse crops unless overhead irrigation is used. In field crops, the leaflets of the oldest leaves curl, their margins yellow and become necrotic by ingress of the pathogen through hydathodes (Carlton et al., 1998). Plants grow poorly and gradually entire plants desiccate. Yellow streaks may develop along the stem and occasionally these split open at the nodes forming cankers. The pith may collapse completely. Symptoms on affected fruits may begin as small, slightly raised lesions with a white margin or halo. These lesions may expand to a few mm with brown, roughened centres and are then called ‘bird's eye’ spots. Several lesions may be formed near the calyx where fruits touch in a cluster. The vascular tissues under the calyx scar and those leading to the seeds may be dark yellow to brown. The vascular tissues of transversely cut stems of wilted plants usually appear dark yellow to brown, in particular at the nodes. The vascular parenchyma in particular has a mealy appearance resulting from bacterial degradation and ooze production [Web Fig. 6]. Systemic infection of C. m. michiganensis can be detected by suspending a stem section with the cut surface in a glass tube with tap water. Bacterial exudate will spontaneously make a milky suspension after a few minutes. Disease symptoms are unlikely to appear before the setting of the fruits in the third or fourth cluster. Generally, infection by C. m. michiganensis is not expressed on young plants. However, wilting may occur early on grafted plants, possibly even in the nursery, and leaf spots may develop on young plants as a result of local infections under saturated humidity [Web Fig. 5]. Wilt symptoms caused by C. m. michiganensis may be confused with other systemic diseases caused by Ralstonia solanacearum, Fusarium spp. and Verticillium spp. Other disorders of tomato, e.g. tomato pith necrosis (Pseudomonas corrugata) or wilting caused by Erwinia carotovora or Botryotinia fuckeliana are more readily differentiated from C. m. michiganensis. Detection on symptomatic plants Wilting plants are cut at the basis of the stem and the wilted leaves removed. Discoloration of vascular tissues at the basis of the broken petioles should be looked for. The stem at each node is sectioned and dark yellow to brown discoloration of the vascular tissues on the cut surfaces are looked for. The epidermis should be carefully separated from the vessels. In advanced stages of wilting, the presence of bacterial exudate in the vascular parenchyma tissues facilitates the separation of the outer layers of stem tissue. By use of a scalpel disinfected by dipping in ethanol and flaming, small sections of affected vascular tissue are removed and transferred into a small volume of sterile distilled water or phosphate buffer (see Appendix I) and 5–10 min is allowed for diffusion of bacteria. Usually, the suspension rapidly becomes milky. For leaf or fruit spots, the leaf or fruit surface is lightly surface-disinfected by wiping with a tissue paper drenched in 70% ethanol. A few young spots are removed with a disinfected scalpel blade and transferred to a small volume of sterile distilled water or phosphate buffer (see Appendix I). 15–20 min are needed for diffusion of bacteria. Rapid tests for presumptive diagnosis A rapid screening test is provided by specific antibodies absorbed on cells of Staphylococcus aureus which are used as agglutination reagent (Express kit, Adgen, Ayr, GB). The test reagent is coloured to enhance the readability of the test result. A positive and negative reaction control is supplied. 15 µL of tissue suspension from the isolation procedure and 5 µL of test reagent is mixed on a window of the multispot test support. A positive and negative control are prepared accordingly. The support is gently rocked for 15–30 s and observed for any agglutination that may occur. Agglutination should be formed in the positive control. Agglutination in the sample window then indicates the presence of C. m. michiganensis. Suspensions of diseased tomato tissue can also be tested by IF or PCR. Details on these tests are provided elsewhere in this diagnostic protocol. Dilution plating In order to dilute out the cells of C. m. michiganensis onto separate plates of Nutrient Glucose Agar (NGA) or Yeast Peptone Glucose Agar (YPGA) (Appendix I), a plating technique is performed. A minimum dilution of 1 : 1000 should be achieved. Usually, a semiselective medium is not required for isolation of C. m. michiganensis from diseased vascular tissues but it may be useful for isolation of the pathogen from leaf or fruit spots. A thin spreader is dipped in ethanol, flamed and then cooled. The volumes are spread over the surface of the medium. If considered useful, separate isolation plates with a diluted cell suspension of the C. m. michiganensis reference strain are prepared. The plates are incubated at 26°C ( 2°C). On the general nutrient media, 2–3-mm colonies of C. m. michiganensis develop within 3–4 days and are light yellow, flat and semifluidal, round or irregular [Web Fig. 6]. Colonies become deeper yellow, opaque and glistening with longer incubation. Presumptive colonies are purified by subculturing on NGA or YPGA. Detection in symptomless plants The plants are cut at the base and the base of the stem (a few cm) is removed. The epidermis is separated with a disinfected scalpel (as above), and discarded. Sections of vascular tissue are transferred into sterile distilled water or soaking buffer and left soaking for 15–20 min. Dilution plating on the semiselective medium is performed as described for the seed test. Detection on seeds General recommendations on detection of C. m. michiganensis in tomato seeds22 The methods described have not been evaluated on pelleted seed and should therefore not be used for this commodity. can be consulted in the ISHI-VEG Seed Health Testing methods Reference Manual [electronic version at http://www.seedtest.org/]. The standard sample size recommended by ISTA is 10.000 seeds. This implies a statistical 95% probability of detecting a 0.03% level of contamination in the seed lot. The minimum number of seeds in a sample is 2000. This can be accepted for protected crops or high-class hybrid seed. Two methods are available to obtain a seed extract. The first uses a Stomacher Laboratory Blender. If this device is not available, then extended soaking can be used. However, there is evidence that blending increases recovery of C. m. michiganensis (Franken et al., 1993), specifically for seeds with residues of antibacterial products used in the extraction process. The first step is to determine the weight of the sample. The sample is divided into equal subsamples so that a subsample does not contain more than 2000 seeds. Standard size samples are thus tested as 5 subsamples of 2000 seeds each. An aliquot of each seed extract should be kept as reference and for eventual additional testing. 1 mL is transferred into a sterile 1.5 mL microvial and centrifuged for 10 min at 6000 g. The supernatant is discarded and the pellet is resuspended in 1 mL of sterile 25% glycerol, and frozen at −18–24°C. For the Stomacher blending procedure, a (sub)sample of seeds is transferred into a Stomacher bag (e.g. 105 mm × 50 mm – radiation sterile) and 20 mL of sterile sample buffer (see Appendix I) added. The bag is placed in a suitable holder and the seeds are soaked overnight at 4°C (± 1°C). The bag is then transferred into the Stomacher, blended for 20 min, then removed from the Stomacher and the sediment is allowed to settle for 30 min at room temperature. For the extended soaking procedure, a (sub)sample of seeds is transferred in a sterilized flask with screw cap. 20 mL of sterile sample buffer (see Appendix I) is added. Each flask is vigorously shaken for 20–30 s. The flasks are placed on a rotary shaker and the seeds soaked for 36–48 h at 150 rev min−1 at 4°C (± 1°C). Tests of seed extracts Dilution plating on semiselective medium 1 mL of the seed macerate is transferred into a sterile microvial. Three decimal dilutions (1 : 10, 1 : 100 and 1 : 1000) are prepared in sterile 50 mm phosphate buffer. 50 µL of the 1 : 1000, 1 : 100, 1 : 10 diluted and the undiluted seed extract is pipetted onto each of two plates of semiselective medium33 Other semiselective media from the ISHI-VEG manual can be used. There is evidence that at least two isolation media should be used to test tomato seeds which were not produced by an acid fermentation extraction process. (Appendix I). With a thin spreader dipped in ethanol, flamed and then cooled, the pipetted volumes are distributed over the whole surface of the media. Positive controls are prepared on separate test plates of the isolation medium by dilution plating of a cell suspension of C. m. michiganensis reference strain. The plates are incubated at 26°C (± 2°C). The plates are examined after 4 days and then daily for the next 3–4 days. Growth rates of colonies on test plates are compared with the positive control. Suspect colonies are light yellow, round with a tendency to become fluidal or irregular, convex and shining [Web Fig. 7]. They become deeper yellow with longer incubation. If suspect colonies have not developed after 8 days, then the seed test should be considered negative, providing that colonies from the seed microflora have not interfered in the isolation of C. m. michiganensis. If this is the case, then a confirmatory screening test should be done (IF test or enrichment PCR test). When suspect colonies have developed, at least five should be purified by subculturing on NGA or YPGA (see Appendix I) for further identification. The seed test is positive when presumptive colonies are identified as C. m. michiganensis. In this selective plating test, 1 contaminated seed added to a sample of 10.000 seeds from EU commercial seed lots was consistently detected at a minimum contamination level of 8 × 102 c.f.u. Detection was less reliable below this level, and in seed lots with > 105 c.f.u. background population of seed microflora (ISO 17025 validation tests). Seed test by immunofluorescence44 Seed extraction by stomacher blending is not appropriate for the immunofluorescence test. The affinity, sensitivity and, in particular, cross reactivity of the polyclonal antiserum used should have been assessed and documented. The working dilution is determined by titration (usually half the titre) on a cell suspension of a C. m. michiganensis reference strain of 105–106 c.f.u. per mL55 As a guideline: the density of a cell suspension in 50 mm phosphate buffer from a 48 h culture produced on a standard nutrient medium with a A600 value of 0.2 corresponds to 1.0–2.0 × 108 c.f.u. per mL as determined in a spectrophotometer and by dilution plating. . A complementary FITC conjugate is used, at the dilution specified by the manufacturer. It is recommended to include a positive control slide prepared from a tomato seed extract which has been artificially contaminated with C. m. michiganensis at the level of the antiserum titration. An appropriate volume (e.g. 20 µL for 8-mm diameter windows) of the 1 : 1000, 1 : 100, 1 : 10 diluted and the undiluted seed extract is transferred onto windows of a multiwell microscope slide. The slide is air-dried and fixed by gentle heating or by ethanol. Each window is covered with antiserum at the appropriate dilution and incubated for 30 min on moist tissue paper under a cover. A volume is used to cover the windows completely, preferably the same volume of the sample. The droplets are shaken off and the slide is rinsed once with 10 mm phosphate buffer, then washed for 5 min in two changes of 10 mm phosphate buffer Tween and finally air-dried. Each window is covered with FITC conjugate, incubated, rinsed, washed and air-dried as before. Mounting buffer is applied and covered with a cover glass. The windows are examined under oil immersion with a fluorescence microscope suitable for working with FITC. A minimum magnification of 800× is required. A minimum of 20 microscope fields should be analysed. The seed test is considered negative when positive cells have not been detected. When positive cells have been detected, in particular cells with typical claviform morphology (Franken et al., 1993), then the seed test is considered potentially positive. The selective plating test and/or the bioassay test should then be carried out to confirm or refute the result of the IF test. Seed test by enrichment-PCR 500 µL of seed extract is transferred into a test tube containing 5 mL of sterile liquid semiselective medium (Appendix I). The tube should be appropriately labelled. Test tubes are inoculated with a cell suspensions of C. m. michiganensis, at contamination levels of 102–103 c.f.u. The enrichment tubes are incubated for 72 h at 26°C (± 2°C). 100 µL from each enrichment tube is then transferred into a sterile 1.5-mL microvial. Samples in closed microvials are heated for 15 min at 95°C66 Alkaline treatment may assist cell lysis (Zhang & Goodwin, 1997). Add 50 µL of 0.25 N NaOH to 100 µL of suspended bacteria. Vortex. Close vials and heat for 7.5 min at 95°C. After cooling, add 50 µL of 0.25 N HCl. Vortex. Add 25 µL of 0.5 m Tris-HCl (pH 8.0) – Tween 20 (1% v/v). Close vials and heat for another 7.5 min at 95°C. Pulse centrifuge after cooling on ice. . Microvials with heated suspensions are transferred into ice and, after cooling, pulse-centrifuged. The appropriate PCR protocol is then applied to generate species-specific amplicons77 The seed test by enrichment PCR has only been validated with the protocol of Pastrik & Rainey (1999). . The vials are removed from the thermal cycler. PCR products are analysed by agarose gel electrophoresis and staining of DNA. The PCR result of the sample(s) is compared with those of the positive controls and the DNA marker. The test result is only reliable when the water control is negative and the specific DNA has been produced for the enriched dilutions of the C. m. michiganensis strain. The seed test is considered negative when the specific DNA amplicon is not detected from the sample. When the specific DNA amplicon is detected from the sample, then the seed test is considered potentially positive. The bioassay should then be carried out to confirm or refute the result of the enrichment-PCR test.88 Selective plating is not advised in the enrichment protocol. In the enrichment-PCR test, 1 contaminated seed added to a sample of 10.000 seeds from EU commercial seed lots was consistently detected at a minimum contamination level of 4 × 102 c.f.u. Detection was less reliable below this level, but remained reliable in seed lots with > 105 c.f.u. background population of the seed microflora (ISO 17025 validation tests). Bioassay in tomato plantlets This test is performed specifically for seed extracts with a positive IF or a positive enrichment-PCR result. It is carried out in tomato plantlets, preferably of cv. ‘Moneymaker’, at the two true-leaf stage as described by van Vaerenbergh & Chauveau (1987). The reference aliquot of extract is used for this test. Identification If no typical colonies can be isolated from seeds by selective plating or in the tomato bioassay, the diagnosis is considered to be negative. For other types of plant material, if at least one of the rapid tests for presumptive diagnosis is positive but typical colonies are not obtained by isolation, then re-isolation from the plant material should be attempted. A culture is identified as C. m. michiganensis when a positive result is obtained for at least two tests related to two different characteristics of the pathogen (biochemical characteristics, SA-agglutination test, IF test, ELISA, PCR or FAP). Biochemical characteristics The following set of phenotypic properties which are universally present or absent in C. m. michiganensis should be determined: Gram positive; oxidative metabolism of glucose; catalase positive; oxidase negative; aesculin hydrolysis positive; acid produced aerobically from mannose, but not from mannitol; sodium acetate and sodium succinate used as carbon sources; growth in presence of 6% NaCl; potato starch hydrolysed; H2S produced from peptone. The methods of Dye & Kemp (1977) may be used. Serological tests SA agglutination test99 For example: Express Kit, Adgen, Ayr, GB. From each presumptive isolate and from a culture of the reference strain, a single colony is suspended in 100 µL of sterile distilled water in a microvial. 15 µL of the suspended bacteria and 5 µL of test reagent are mixed on a window of a multispot test support. The support is gently rocked for 15–30 s and observed for any agglutination that may occur. Agglutination should be formed in the positive control. Agglutination of a presumptive isolate identifies it as C. m. michiganensis. IF test From each presumptive culture and from a culture of the reference strain, a single colony is suspended in 100 µL of sterile distilled water in a microvial, and a 1 : 100 dilution in IF-buffer (Appendix I) is prepared. When IF is used as a presumptive diagnostic test, the tomato tissue suspension is prepared as described in the section on isolation. A fixed volume of this dilution is applied on each window of a multispot test slide (e.g. 20-µL volumes on 8-mm windows). The droplets are air-dried and the bacterial cells are fixed to the slide by flaming or with ethanol (96%). A validated antiserum is used, as stated before. The antibodies should be used in two-fold dilutions to the endpoint titre. The procedure described for the seed test should be followed. A positive identification of a suspect culture as C. m. michiganensis or a positive presumptive diagnostic test is achieved if the IF titre of the suspect culture is equivalent to that of the positive control strain. Appropriate antibodies are available, for example, from Loewe (DE) (http://www.loewe-info.com); Adgen (GB) (http://www.adgen.co.uk) and Plant Research International (NL) (http://www.plant.wageningen-ur.nl). ELISA test The AGDIA reagent and protocol can be used. PCR test From each presumptive isolate and from a culture of the reference strain, a single colony is suspended in 100 µL of sterile distilled water in a microvial 101 The Figures in this Standard marked ‘Web Fig.’ are published on the EPPO website http://www.eppo.org. . Closed vials are heated at 95°C for 15 min. Microvials with heated suspensions are transferred into ice and, after cooling, pulse-centrifuged. When PCR is used as a presumptive diagnostic test, 100 µL of tomato tissue macerate is prepared as described in the section on isolation. The PCR mix is prepared in a dedicated environment to minimize the possibility of contamination. Filter-plugged pipette tips are used for all PCR manipulations. PCR vials are sterilized by autoclaving. Sterile, molecular grade, ultra-pure water (MG UPW) should be used and 10×PCR buffer, dNTP, each primer and Taq DNA polymerase according to published specifications. The required quantity of each component is calculated for the total number of 25- or 50-µL reaction volumes to be performed. 2.5 µL or 5.0 µL of sample is transferred to PCR vials (and of MG UPW as a negative control). 22.5 µL or 45 µL of PCR mix is added to the samples and mixed by gentle aspiration in the pipette tip. According to the PCR protocol of Dreier et al. (1995), the oligonucleotide primers are derived from the plasmid-borne pathogenicity gene pat-1: Forward primer CMM-5: 5′- GCG AAT AAG CCC ATA TCA A -3′ Reverse primer CMM-6: 5′- CGT CAG GAG GTC GCT AAT A -3′ The amplicon size from C. m. michiganensis DNA is 614 bp [Web Fig. 8(a)]. The following programme was optimized for a Perkin Elmer 9600 thermal cycler: 1 cycle of 2 min at 96°C; 30 cycles of 60 s at 96°C (denaturation of DNA); 90 s at 55°C (annealing of primers); 60 s at 72°C (polymerization of DNA); 1 cycle of 10 min at 72°C (final polymeriztion of DNA); storage at 4°C. Modification of the duration in the amplification cycles may be required for use with other thermal cyclers. According to the PCR protocol of Pastrik & Rainey (1999), the oligonucleotide primers are derived from the 16S–23S rRNA intergenic spacer region: Forward primer PSA-4: 5′-TCA TTG GTC AAT TCT GTC TCC C -3′ Reverse primer PSA-R: 5′-TAC TGA GAT GTT TCA CTT CCC C -3′ The amplicon size from C. michiganensis ssp. michiganensis DNA is 270 bp [Web Fig. 8(b)]. The following programme was optimized for a Perkin Elmer 9600 thermal cycler: 1 cycle of 2.5 min at 94°C; 30 cycles of 30 s at 94°C (denaturation of DNA); 20 s at 63°C (annealing of primers) – ramp of 0.5°C s−1; 45 s at 72°C (polymerization of DNA); 1 cycle of 10 min at 72°C (final polymerization of DNA); storage at 4°C. The duration of the steps in the amplification cycles may be modified if other thermal cyclers are used. The PCR amplicons are resolved by agarose gel electrophoresis. 12 µL of each PCR reaction is mixed with 1 µL of loading buffer on a stretch of parafilm by repeated aspiration in the pipette tip and the mixtures is applied to the wells of a 2% (w/v) agarose gel in Tris-acetate-EDTA (TAE) buffer. DNA fragments are revealed by staining in ethidium bromide solution (0.5 µg mL−1, i.e. 50 µL of a 10 mg mL−1 stock solution in 1 L TAE) for 30-45 min and transillumination under UV. Appropriate precautions should be taken when using ethidium bromide. The water control should be negative and the positive control should have the amplicon of the expected size. Pathogenicity tests Two types of pathogenicity test are described, a seedling test and a cotyledon test. Seedling test From an isolate identified as C. m. michiganensis and from a culture of the reference strain, a single colony is suspended in 100 µL of sterile distilled water in a microvial. Five tomato seedlings, e.g. cv. ‘Moneymaker’, are inoculated at the 2nd true leaf stage for each isolate or strain by injection into the stem at the cotyledons. Test plants are grown at 26°C (± 2°C) and > 70% relative humidity. They should not be watered one day before inoculation. From the fifth day on, plants are observed for wilting. Wil