S.L. 433.14 Art. 175 µl of the be applied DNA from the validated extract (1:10 in validated PCR control. tubes. 6.2.4. and add the 6.2.5. Place testing and run 6.3. 6.3.1. of amplified (Appendix 6) (Appendix 6) 6.3.2. to 45 min 6.3.3. nm) for 6.3.4. For performing by incubating buffer (see under UV undigested and The

Spin down any white precipitate by centrifugation and use supernatant containing DNA for the PCR.

6.1.(b)Other methods Other DNA extraction methods (e.g. Qiagen DNeasy Plant Kit) could providing that they are proven to be equally as effective in purifying control samples containing 103 to 104 pathogen cells per ml.

6.2. PCR 6.2.1. Prepare test and control templates for PCR according to protocol (Appendix 6). Prepare one decimal dilution of sample DNA UPW).

6.2.2. Prepare the appropriate PCR reaction mix in a contamination-free environment according to the published protocol (Appendix 6). The protocol is a multiplex reaction that also incorporates an internal PCR 6.2.3. Add 5 µl of DNA extract per 25 µl PCR reaction in sterile PCR Incorporate a negative control sample containing only PCR reaction mix same source of UPW as used in the PCR mix in place of sample. tubes in the same thermal cycler which was used in preliminary the appropriately optimized PCR programme (Appendix 6). Analysis of the PCR product

Resolve PCR amplicons by agarose gel electrophoresis. Run at least 12 µl DNA reaction mixture from each sample mixed with 3 µl loading buffer in 2,0% (w/v) agarose gels in tris-acetate-EDTA (TAE) buffer at 5 to 8 V per cm. Use an appropriate DNA marker, e.g. 100 bp ladder. Reveal DNA bands by staining in ethidium bromide (0,5 mg per L) for 30 taking appropriate precautions for handling this mutagen. Observe stained gel under short wave UV transillumination (e.g.  = 302 amplified PCR products of the expected size (Appendix 6) and document. all new findings/cases verify authenticity of the PCR amplicon by restriction enzyme analysis on a sample of the remaining amplified DNA at the optimum temperature and time with an appropriate enzyme and Appendix 6). Resolve the digested fragments by agarose gel electrophoresis as before and observe characteristic restriction fragment pattern transillumination after ethidium bromide staining and compare with the digested positive control. Interpretation of the PCR test result:

is negative if the C. m. subsp. sepedonicus-specific PCR amplicon of is not detected for the sample in question but is detected for all control samples (in case of multiplex PCR with plant specific internal primers: a second PCR-product of expected size must be amplified with the question).

test is positive if the C. m. subsp. sepedonicus-specific PCR amplicon of and restriction pattern (when required) is detected, providing that it is from any of the negative control samples. Reliable confirmation of a can also be obtained by repeating the test with a second set of PCR (section 9.3).

of the PCR may be suspected if the expected amplicon is obtained from control sample containing C. m. subsp. sepedonicus in water but results are obtained from positive controls with subsp. in C. m. sepedonicus In multiplex PCR protocols with internal PCR controls, inhibition of indicated when neither of the two amplicons are obtained. Contamination may be suspected if the expected amplicon is obtained from one or negative controls. BIOASSAY TEST

testing with this method should permit reproducible detection of 103 colony-forming units of subsp. per ml added to sample C. m. sepedonicus previously tested negative (preparation see Appendix 2). sensitivity of detection can be expected when using freshly prepared extract and optimal growth conditions. However, the method can be applied to extracts that have been stored under glycerol at -68 to -86°C. Some varieties of eggplant provide an excellent selective enrichment the growth of subsp. even in the absence of symptoms C. m. Sepedonicus provide an excellent confirmatory host test. Growth conditions should be optimal to reduce the risk of false results. For cultural details, see Appendix 8. 7.1. Distribute the whole of the remaining test aliquot of the resuspended from section 3.1.6 or 3.2.5 between eggplants by one of the methods (7.3 or 7.4). Use only plants at leaf stage two to three up to full expansion third true leaf. In order to ensure complete utilisation of the resuspended well as effective inoculation the procedures outlined below will require eggplants per sample. 7.2. Do not water eggplants for one to two days prior to inoculation turgor pressure. 7.3. Slit inoculation 7.3.1. Holding the plant between two fingers, pipette a drop (approximately 10 µl) of the suspended pellet on the stem between the cotyledons and 7.3.2. Using a sterile scalpel, make a diagonal slit, about 1,0 approximately 2/3 of the stem thickness deep, starting the cut from the 7.3.3. Seal the cut with sterile vaseline from a syringe.

7.4. Syringe inoculation Inoculate the eggplant stems just above the cotyledons using a syringe a hypodermic needle (not less than 23 G). Distribute the sample eggplants.

7.5. As the positive controls, inoculate 5 plants with an aqueous 105 to 106 cells per ml of a known culture of subsp. C. m. sepedonicus possible, with naturally infected tuber tissue (see section 4) by the same method (7.3 or 7.4). 7.6. As the negative control, inoculate 5 plants with sterile pellet same inoculation method (7.3 or 7.4). 7.7. Incubate plants in quarantine facilities for up to four weeks Incubate plants with sufficient light and high humidity (70 to 80%) prevent water logging or wilting through water deficiency. C. m. sepedonicus are killed at temperatures above 30°C and the optimum temperature avoid contamination incubate positive control and negative control plants separated benches in a glasshouse or growth chamber or, in case space ensure strict separation between treatments. If plants for different samples incubated close together, separate them with appropriate screens. When watering, inspecting and any other manipulations take great care to contamination. It is essential to keep glasshouses and growth chambers insect pests since they may transmit the bacterium from sample to sample. 7.8. Examine regularly for symptoms starting after a week. Count plants showing symptoms. C. m. subsp. sepedonicus causes leaf wilting which may commence as a marginal or interveinal flaccidity. Wilted initially appear dark green or mottled but turns paler before becoming Interveinal wilts often have a greasy water-soaked appearance. Necrotic has a bright yellow margin. Plants are not necessarily killed; the longer before symptoms develop, the greater the chance of survival. Plants may Young eggplants are much more susceptible to low populations of C. m. than are older plants, hence the necessity to use plants at or just Sepedonicus stage 3. also be induced by populations of other bacteria or fungi present in the pellet. These include Ralstonia solanacearum, Erwinia carotovora and subsp. carotovora E. carotovora atroseptica, Erwinia chrysanthemi, var. as well as large populations of saprophytic bacteria. In foveata, Erwinia chrysanthemi can cause leaf symptoms and wilt that is very symptoms of The only difference is blackening of the C. m. sepedonicus. in infections. Other wilts can be distinguished Erwinia chrysanthemi caused by C. m. subsp. sepedonicus since whole leaves or whole plants Also a Gram stain can be prepared: this test will differentiate C. m. from spp. sepedonicus Erwinia soon as symptoms in eggplants are observed reisolation should be using sections of wilted leaf tissue or stem tissue from plants (see 3.1.3 maceration). Surface disinfect the eggplant leaves and stems by wiping ethanol. Perform an IF test or PCR on the eggplant sap and isolate on to (selective) media (see section 8). A Gram stain (Appendix 9) may also be Identify purified cultures of presumptive C. m. subsp. sepedonicus and pathogenicity (see section 9 and 10).

certain circumstances, in particular where growing conditions are not be possible for subsp. to exist as a latent infection C. m. sepedonicus eggplants even after incubation periods up to 4 weeks. If no symptoms are after 4 weeks perform IF/PCR on a composite sample of 1 cm stem each test plant taken above the inoculation site. If the test is positive on suitable (selective) media should be performed following the under section 8. Identify purified cultures of presumptive C. m. subsp. and confirm pathogenicity (section 9 and 10).

Interpretation of the bioassay test result. Bioassay test results are obtained when plants of the positive control show symptoms, the bacteria can be reisolated from these plants and no symptoms the negative controls.

bioassay test is negative if test plants are not infected by C. m. subsp. and provided that subsp. is detected in positive C. m. sepedonicus bioassay test is positive if the test plants are infected by subsp. C. m.

ISOLATION OF SUBSP. C. M. SEPEDONICUS

is only completed if subsp. is isolated, subsequently C. m. sepedonicus (see section 9). and confirmed by a pathogenicity test (section 10). subsp. is a fastidious organism, it can be isolated from m. sepedonicus tissue. it may be outgrown by rapidly growing saprophytic bacteria and, isolations directly from the tuber or stem tissue pellet (section 3.1.6 or icult. With selective medium and appropriate dilution of the pellet from the heel end cores or stems of potatoes direct isolation of C. may be possible. sepedonicus shall be made from all symptomatic potato tubers or stem segments and from eggplants were no symptoms are observed but IF/PCR test from sample was positive (see section 7.10). Maceration of eggplants necessary should be carried out as described in section 3.1.3.

As positive controls, prepare decimal dilutions from a suspension ml of C. m. subsp. sepedonicus (e.g. NCPPB 4053 or PD 406). possibility of contamination, prepare positive controls totally separated samples to be tested. For each newly prepared batch of a selective medium its suitability the pathogen should be tested before it is used to test routine samples. Test control material in an identical manner as the sample(s).

8.1. Selective plating 8.1.1. From a 100 µl aliquot from a resuspended potato pellet sample sap make 10-fold dilutions in pellet buffer (Appendix 3). 8.1.2. Isolation from undiluted potato pellet usually fails due to growth habit of Cms and competition by saprophytes. Since the bacterium present in high populations in infected tissues, the saprophytes can usually out, whilst the pathogen remains. It is therefore recommended to spread each of the samples, 1/100 up to 1/10 000 dilutions onto MTNA medium medium (Appendix 5) (if using 90 mm diameter petri dishes- adjust alternative dish sizes), using spreaders (hockey sticks) and the technique. Note: An alternative strategy is to spread out the initial 100 µl potato pellet a first agar plate with a spreader and then remove the spreader to a second streaking out any residue left on the spreader; finally repeat this with thus giving a dilution plating effect via the spreader.

8.1.3. Incubate plates in the dark at 21 to 23°C. 8.1.4. Initial examinations of the plates including, by reference plates, counts of any C. m. subsp. sepedonicus like colonies are made with further counts after 5, 7, eventually 10 days.

8.2. Purification of suspicious colonies Note: Subculturing of subsp. colonies should be carried C. m. sepedonicus-like YGM media for eggplant inoculation and/or subsequent identification; done before the plates become too overgrown i.e. preferably after three 8.2.1. Streak subsp. colonies on to one of C. m. sepedonicus-like media: (formulae are given in Appendix 5): nutrient dextrose agar (for use in subculturing only), yeast peptone glucose agar, yeast extract mineral salts agar. Incubate at 21°C to 24°C for up to 10 days. subsp. is slow-growing, usually producing pin-point, C. m. sepedonicus domed colonies within 10 days. Photos of typical colonies of sepedonicus (see web site http://forum.europa.eu.int/Public/irc/sanco/Home/main). 8.2.2. Re-streak to establish purity. are improved with subculture. Typical colonies are creamy-white or occasionally yellow, rounded, smooth, raised, convex-domed, mucoid-fluidal, edges and usually 1 to 3 mm in diameter. Gram stain (Appendix 9) may help to select colonies for further testing. Identify presumptive cultures (see section 9) and perform a pathogenicity section 10). IDENTIFICATION

pure cultures of presumptive C. m. subsp. sepedonicus isolates using at following tests based on different biological principles.

known reference strains where appropriate for each test performed. Nutritional and enzymatic identification tests the following phenotypic properties which are universally present or subsp. according to the methods of Lelliott and Stead m. sepedonicus, Klement et al. (1990), Schaad (2001), Anonymous (1987). should be incubated at 21°C and examined after six days. If no growth incubate for up to 20 days. must include a known subsp. control. Nutritional and C. m. sepedonicus tests must be made using inocula from nutrient agar subcultures. comparisons must be made from nutrient dextrose agar cultures.

TestsExpected result Oxidation/Fermentation (O/F) testInert or weakly oxidative activity -

37°C -activity -hydrolysis +

hydrolysis - or weak of 7% NaCl -production -

activity + production -

utilization -

liquefaction -glycerol -lactose - or weak rhamnose -salicin -(Appendix 9) +

Prepare a suspension of approximately 106 cells per ml in IF buffer (Appendix 3). Prepare a 2-fold dilution series of an appropriate antiserum.

(c) Apply the IF procedure (section 4).

(d) A positive IF test is achieved if the IF titre of the culture that of the positive control. 9.3. PCR test

(a) Prepare a suspension of approximately 106 cells per ml water (UPW).

(b) Heat 100 µl of the cell suspension in closed tubes in a heating boiling waterbath at 100°C for four minutes. If required, freshly-prepared NaOH to a final concentration of 0,05M lysis. The samples may then be stored at -16 to -24°C until

(c) Apply appropriate PCR procedures to amplify subsp. C. m. specific amplicons (e. g. Pastrik, 2000; see Appendix 4; 1995; Mills et al., 1997; Pastrik and Rainey, 1999; Schaad

(d) A positive identification of C. m. subsp. sepedonicus is PCR amplicons are the same size and have the same restriction length polymorphisms as for the positive control strain. 9.4. FISH test

(a) Prepare a suspension of approximately 106 cells per ml in

(b) Apply the FISH procedure (section 5).

(c) A positive FISH test is achieved if the same reactions are the culture and the positive control.

9.5. Fatty acid profiling (FAP)

(a) Grow the culture on trypticase soy agar (Oxoid) for 72 hours -1°).

(b) Apply an appropriate FAP procedure (Janse, 1991; Stead,

(c) A positive FAP test is achieved if the profile of the presumptive is identical to that of the positive control. The presence of fatty acids are 15:1 Anteiso A, 15:0 Iso, 15:0 Anteiso, and 17:0 Anteiso is highly indicative of C. m. sepedonicus. genera such as and Curtobacterium, Arthrobacter Micrococcus have some of these acids but 15:1 Anteiso A is a rare bacteria but occurs in all Clavibacter spp. at between 1 to the value is usually around 5%. sepedonicus 9.6. BOX-PCR

(a) Prepare a suspension of approximately 106 cells per ml in

(b) Apply the test according the procedure (Smith et al., 2001).