Southern Hybridization and PCR for Specific Detection of Phytopathogenic Clavibacter michiganensis subsp. michiganensis Jens Dreier, Andreas Bermpohl, and Rudolf Eichenlaub Universitit Bielefeld, Fakultat fr Biologie, Gentechnologie/ Mikrobiologie, 33501 Bielefeld, Germany. This work was supported by the Deutsche Forschungsgemeinschalt SFB 223/C08, We thank Dr. Rabenstein for providing the monoclonal C. m. michiganensis antiserum; R. Bahro and all the members of our laboratory for helpful discussions. ‘Accepted for publication 20 December 1994. ‘ABSTRACT Bermpohl, A. and Eichenlaub, R. 1995, Southern hybridization 1nd PCR for specific detection of phytopathogenic Claibacter mic _ganenss subsp. michigonenss, Phytopathology #5462468, Clavibacter michiganenss subsp. michiganonss isthe causal agent of bacterial wilt and canker in tomato (Lpeopesicon esculentum), Spciic ‘detection of his important bacterial pathogen was possible sing Southera hybridization with DNA-probes derived from plasmid-borne genes, ce/A ‘encoding an endocellulae and pa-t involved in pathogenicity. The cel probe diferentated the C.m. subsp. michiganentis,insdionus, nebrask- ‘enss,vessellarlus, and sepedonicu, moreover, the pat-l probe distin. {uished virulent from avirulent strains of C.m. michiganenss. A poy merase chain reaction based on primers derived from the pat-l region ‘was developed. Vrulent strain were detectable in homogenates prepared from infected tomato plants, naturally contaminated seds, and healthy plant Homogenates containing a few as 2% 10" bacteria pr aililter. Bacterial wilt and canker of tomato (Lycopersicon esculentum Mill) is caused by the coryneform bacterium Clavibacter michi- ganensis subsp. michiganensis (8). In this vascular disease, the bacteria spread throughout the plant via the xylem vessels (32) C. m. michiganensis is the cause of crop failures in all main cultivation areas (35,40) since neither resistant tomato cultivars nor effective chemical controls of this pathogen are available (42) C. m. michiganensis generally is transmitted by contaminated seeds (14, 43) or transplants (18, 19), and therefore, there is great demand for reliable diagnostic methods to detect this important pathogen, Diagnostic tests useful for a general application have to be highly specific, reliable, and rapid. Although C. michiganensis subspecies are very host specific, the presence of saprophytes on plant material and low bacterial titers during early stages of infec- tion impede detection ofthese bacteria. Chemotaxonomic markers ‘employed for classification of coryneform bacteria-like fatty acids (6,21), menaguinons (5), polar lipids (5), cellular proteins (4), allozyme patterns (31), and enzymatic relatedness (10) are useless for arapid diagnosis because they require time-consuming entich- ‘ment and complicated detection procedures. Phage typing is hampered due to the limited number of phage available (12,36,48) Currently, serological techniques such as enzyme-linked immuno- ‘©1005 The American Phytopathoiogical Society 462 PHYTOPATHOLOGY sorbent assay (ELISA) and immunofluoreseence assays with poly- clonal antisera are in use. However, they are not always reliable due to strong cross-reactions with’ other subspecies (17,28,30), although the introduction of monoclonal antibodies has improved specificity (9). The best procedure, the isolation of the micro- ‘organism on semiselective media (4) followed by a direct test for virulence on the host plant (44) or induction ofa hypersensitive reaction on nonhost plants (18), is very time-consuming and un- suitable for large-scale sereening. ‘Nucleic acid hybridization and polymerase chain reaction (PCR) offer alternatives for rapid, highly sensitive, and specific identification of pathogenic bacteria (16,22,34). In revent reports, DNA-probes have been used to identify genes for virulence factors (33), ribosomal RNA (11,27), and eryptie chromosomal fragments (41) unique to pathogens. The extensive chromosomal homology among C. michiganensis subspecies has complicated the develop- ment of specific DNA-probes (38). However, we recently found that in strain NCPPB382 pathogenicity genes and a gene for an cendocellulase are carried on two plasmids, pCMI and pCM2 (13,25). The discovery of the genes ce/A, encoding the endocellu- lase, and pai-l, with unknown function, suggested they might be useful targets in specific DNA-based detection methods. We report DNA-probes derived from celA and pat- that allow specific identification of C. michiganensis subspecies. We also describe DNA primers for PCR that distinguish between virulent and avirulent strains of C. m, michiganensis. The PCR assay provides 4 rapid test for identification of virulent C. m. michiganensis strains in infected plant tissueMATERIALS AND METHODS Bacterial strains and growth conditions. Bacteria (Table 1) were obtained either from the National Collection of Plant Pathogenic Bacteria (NCPPB; Hatching Green, Harpenden, England) or the ‘American Type Culture Collection (ATCC; Rockville, MD). Excherichiacolland Bacillus subtilis were grown in TBY medium (10 g of tryptone, 5 g of yeast extract, and 5 g of NaCl per liter, pH 7.2) at 37 C, whereas Rhizobium melilot! was cultured in TBY at 25 C. Clavibacter and other coryneform bacterial strains ‘were grown in TBY medium supplemented with 5 g of glucose per liter (C medium) at 24-26 C. A modified semiselective SCM Phage assay. The lytic phage CMPI has been useful for detecting C. m. michiganensis (12). Lysates containing 10" plaque-forming units per milliliter of CMPi were prepared from confluent lysed agar lawns of C. m. michiganensis strain NCPPB382 as described by Shirako etal (36) Serial dilutions of the phage lysates were made in SM bufler (58 g of NaCl, 24 g of Tris-HCl, 2.5 g fof MgSO,, and 0.1 g of gelatin per liter, pH 7.5); 10-ul drops fof each dilution were spotted on agar pistes seeded with cells ff the indicator bacteria as described by Echandi and Sun (12). ‘After three days of incubation at 20 C, the plates were scored for the occurrence of plaques. ‘Preparation of antisera. Cultures of C.m. michiganensis NCPPB382 Imedium was used (2,14) to determine bacterial titers of plant were grown in TBY, and cells were harvested by centrifugation homogenates, for 10 min at 3,000 g, Pelleted cells were washed twice in PBS ‘TABLE 1, Bacterial strains used inthis study Species Source ‘Orisin Host plant (Clavibacier michiganensis subsp. ‘michiganensis™ NePPBSR? UK, 1986 Lycopersicon esculentum NCPPI aly, 1961 Leesculentum NCPPBIS6S UK, Channel Is, 1962 esculentum NCPPBIS96 CM, Moskovets, 1963, TE. excalentum NCPPBIS72 Hungary, 1983 . excdentum NCPPBIS73 Hungary, 1983 Loesculentum NCPPB2979 Hungary, 1957 esculentum NCPPBSI20 UK.1975, esculentum NCPPBSI2I UK, 1979 Eeselentuon NCPPB3223, UK, 1981 esculentum NCPPB3224 UK, 1981 L esculentum NCPPBI226 UK, Jersey, 1981 exelent NCPPBI227 UK, Jersey, 1981 esculentum NCPPBI264 UK, 1983 esculentum NCPPBI256 UK, 1983 L esculentum NEPPBISS Hungary, 1985 L.esculentiom Com michiganensi™ NCPPBSe ‘Australia, 1933, esculentum NCPPB3S9 HLL Jensen, 1957 1 esadentum NEPPBSIS| Tals, 1986 1 excadenture NCPPBR6I USA, 1939 Cyphomandra betacea NCPPB3I23 USA, 1972 Lesclentume NCPPBI2A5 UK, i983, esculentum [NCPPBIZ86 UK, 1985, 1 esculentum Com inidosus NeppBss, USA, 1934 Medicago sativa NCPPBIIO9 USA, 1935 Mesaiva NCPPBILIO USA, 1983 Msaiva NCPPBIGGO Msaiva NCPPBIG86 Msatva NCPPBIGS? Mosaiva Com nebraskensis NCPPB2SIE Zeamays NCPPB2SI9| Zmays NCPPB2SE0 Z mays NCPPBOSSI Z mass NCPPHOSE2 Z mass ATCC27794 USA, 1971 Z mays Com sepedonicus NCPPBI09 Canad, 1951 Solanum euberosum NCPPBITE Sweden, 1986 ‘tuberosum NCPPBI40 USA, 1942 'S tuberosum NCPPB2S3, ‘Sweden, 1977 'S tuberosum NCPPBII23 J-van Vaerenburgh, 1983, 'S tuberosum NCPPBMs Poland, 1985, ‘tuberosum Com. tessllaris ATCC33566, ALK. Vidaver, USA Trcum aestivum iranicus NePPB2283 lean, 1966 Triticum aesthrum Cates, NCPPRISS T-aestbum NCPPBIES7 Epp! T. aestivum toxious NCPPB2980 New Zealand, 1969 Dacipsglomerata Gurtobacierium flaccumfaciens voor NePPB2II3 ‘Netherlands, 1967 Tulipa gesneriana Rhodococcus fscians NCPPBIAES UK, 1963, Lashyrasodoratus dnthrobacer iis NCPPBI22% USA, 1960, Tex opaca Pacis subtilis ATCCHOSI H.J..Conn, Marburg strain Excherchia col ECI94 Hanahan, 1983 Rhizobium meliot 2011 A. Pabler, Bielefeld Medicago sativa fruleat on Lycopersicon esculentum ev. Moneymaker. “avirulent on ev. Moneymaker Vol. 85, No. 4, 1985 469,buffer (68.5 mM NaCl, 20 mM KH;PO,/K,HPO, pH 7.2), and the bacterial titer was’ adjusted to approximately 1X 10* cells ‘per milliliter with PBS bulfer. The cell suspension (0.5 ml) was injected subcutaneously into rabbits, followed by a second injec- tion after 3-4 wk. Serum was prepared from blood 10 wk after the second injection. The serum was tested at different dilutions with strain NCPPB382 in an ELISA, as described below, and then generally used diluted 1:200, Specificity was tested in com- parison to commercial antisera with the strains listed in Figure 1 ELISA. C.m. michiganensis antisera, used for indirect sandwich ELISA, were obtained from several sources. A mouse monoclonal antibody against. m. michiganensis was provided by Dr. Rabenstein (Bundesanstalt lr Zuchtungsforschung, Aschersleben, Germany): polyclonal rabbit antisera against C. m. michiganensis were ob- tained ether from Loewe Biochemica GmbH! (kit07063K, Otterfing, Germany) or were prepared as described above. ELISA plates (Corning Ine., Corning, NY) were coated with antiserum diluted 1:500 in coating buffer (1.5 g of Na;COy, 2.91 g of NaHCOs, and 0.20 g of NaN; per liter, pH 9.6) by incubation for 4h at 37 C, and then washed five times with washing buffer (8g of NaCl, 29 g of NayHPO,X12 HO, 0.2 g of KH.PO, 0.2'g of KCI, 02 g of NaN}, and 0.5 ml of Tween 20 per lit pH 7.2-7.4), Bacteria were colleced from agar plates and resus” pended in PBS buffer, inactivated by heating (10 min, 95 ©), land washed three times with PBS buffer. The inactivated cells Were adjusted to a concentration of 10" cells per milliliter in wash- ing buffer containing 20 mg of polyvinylpyrrolidone (Kyy-Ka) per milliter and 2 mg of bovine serum albumin per milliliter fand were transferred to the wells of the ELISA plate (0.2 ml per well). The plates were incubated overnight at 4 C and washed five times with washing buffer. Alkaline-phosphatase-labeled rabbit antibodies (diluted 1:500, 0.2 mi) directed against C. m. mmichiganensis (Loewe Biochemica) or 0.2 ml of alkaline- phosphatase-labeled_ goat antibodies diluted 1:7,500 directed Against the F. domain of mouse immunoglobulin (Ig) G (Serva, Heidelberg, Germany) were added, and the plates incubated for 4 hat 37°C. Plates were then washed five times with washing buffer followed by the addition of 0.2 ml of substrate solution (1mg of p-aitrophenyiphosphate per slits of subsate buffer containing 73 ml of glycerin, 0.2 of MgCl, 0.014 g of ZnCl, per liter adjusted to pH 10.5 with KOH). The ODas was deter: fined 30 min after the addition of the substrate using an ELISA plate reader. B. suri strain ATCCOOSI served asthe negative onto in a assays, Virulence assay. Infection of tomato plants was carried out as described previously (48) The petiole of the frst wue leaf of 4-wkeold tomato plants (L. esculentum ev. Moneymaker) was cut off near the stem with 4 scalpel that had been dipped into the bacterial suspension (10 efu per milter) to be texted. A virulent strain wil ease wilting of eaves 6-20 days aftr infection. Plants were cultivated in a growth chamber with a T6-h photo- period at 25 C and 805 relative humidity Seeds contaminated with Cm. michiganensis were obtained from infected tomato plants (cx. Moneymaker), The peduncles of -wk-od plants were inoculated with approximately 50 al of ‘bacterial suspension containing IO" cfu per milter of NCPPB382, using a syringe with a 27-gauge needle, Ripe fruits were harvested, land the seeds were removed, washed in tap wate, placed onts Paper towels, and dried overnight at room temperature DNA preparation and Southern hybridization. Preparation of plasmid DNA from E. coll or Cm. michiganensis was carted ‘out as deseribed previously (26). Plasmids pCMI and pCM2 were prepared {rom the two partially cured Cm michiganensis strains, CMMIOL with pCMI and CMMI02 with pCM2, respectively (26). Total DNA was prepared from 5 ml of lat log-phase cultures 15 described by Hopwood etal 23). ‘Senaiviy to] Strain [ Bandwien ELIGA wth polyclonal Gnmeantinarum phage cM er a ee = [ermnerranee cae ©. michiganensis [on nePres7e var Capeecioe + |smnncronneo ne 7 F [eeancroener “ michiganensis : a(t on tomate) + nase 3 Saher ase tempera ao C. michiganensis Te Neorene sex Subspeci Z| oem neresre| a michiganensi (werent on omit) other . michiganensis subspecies other phytopathogenic corynebacteria Fig. 1 Detection of Cavibater michganensis subsp. michiganenss by enayme-linked immunosorbent arsay (ELISA) and sensitivity to phage CMPL ELISA was performed withthe polyclonal C.m. michiganenss antiserum (Loewe Biochemica GmbH). Phage typing was perormed with the C. m Imichiganents phage CMPI. Strain abbreviations: Com (C. m. michiganensis), Cms (C. m. sepedanicus), Cm (C. mnebraskensis), Cm! Insiiesus), Cot (Cm. escilrius), Ci (C. tranieus), Ct (C. aie, Cto(C. toxcus), Clo (Curtobacterium flaccumfaciens pv oar), RI (Rho fascians), hi Arthrobacter Hic) 48 PHYTOPATHOLOGY (Bacillus subs), Rra (Rhizobium melo), and Ee (Escherichia cl,For extraction of Cm. miehiganensis, DNA from tomato plants was harvested 3 wk after inoculation by cutting above the root At this time, plants inoculated with virulent strains had developed discase symptoms, Infected and noninfected control plants were homogenized by grinding in a mortar with the addition of | ml of TE buffer A (1 mM EDTA, 10 mM Tris-HCl, pH 8.0) per fram of plant material, Serial dilutions of this homogenat plated on semiselective SCM-medium (2,14). When scoring the plates after incubation at 24-26 C for 5 days, the yellow-orange Colonies formed by C. m. michiganensis could be distinguished tasily from the few colonies produced by saprophytes. To remove free DNA, 400-ul aliquots of the plant homogenate were centri- Faged (10 min at 3,000), the pellet was resuspended in TE buffer A, and the process was repeated with TE buffer B (100 mM EDTA, 200 mM Tris-HCl, pH 8.0). The final pellet, consisting of plant tmaterial and bacteria, was resuspended in 400 yl of extraction bulfer (TE buffer B plus 75 yl of NaCl, 25 yl of 10% sodium dodecyl sulfate[ SDS}, and 60 of 10% cetyltri-methylammonium bromide [CTAB]) and incubated at 65 C with shaking at 1,300 rpm for 10 min on a heating str plate. The mixture was extracted twice with phenol-chloroform, and the DNA in the aqueous phase was precipitated with isopropanol, The resulting pellet was washed with 705 ethanol, and the DNA’ was dissolved in 100 yl of TE buffer A. Fifty tomato sceds from infected or noninfected plants ‘ere homogenized by grinding in a mortar after addition of 0.5 ml A | ccmerpannse owen mengaronss (cm sueap | ote opspahagee ‘evet) va ne pac Pte B | _cmcazenmnae sate menbanws Com cep| ota jet eae (wee] vn [cs ne) “yma Fig. 2, Southern hybridization of genomic DNA with probes derived from A, refA and B, the pat! region. Lanes 1% avirulent Clavibacter inichiganensi suns, michitanents sins (Crm) NCPPBIS8, NCPPBSIS, find NCPPB3I23, respectively, Lanes 4-13. vitulent Cm. michiganenss ‘rains NCPPR3R2, NCPPHD68, NCPPBLA5, NCPPBIST2, NCPPBII79. NCPPB320, NCPPH3223, NCPPB3264, NCPPBS284, and NCPPBMSS, respectively, Lanes 14-17: €. michiganensis subspecies: lane 14, C. Insdforas NCPPBUGO (Cri); Lane 15, C. m. sepedonicus NCPPBDI4O (Crs, ane 16, Cm. nebraskensis NCPPB2STS (Crm and lane 17, C.'m.terlarins ATCC33866 (Co). Lanes. 18-22: phytopathogenic ‘corynebacteria: lane 18, C. irnicus NCPPB2253, lane 19, C. toxicus NEPPBIOHO, lane 20, C.irine! NCPPB2SS; lane 21, Curtohacterium Alaccumfacens ps. corti NCPPB2I13; and lane 22, Rhodococcus fascians NCPPMLaES of TE buffer A, and DNA was prepared as described above. To determine the deteetion limit of C. m. michiganensis in plant tissue, bacteria from a freshly prepared overnight culture (about $ * 10" ctu per uililiter) were added at various concentra- tions to homogenate from noninfected plants. After removing ‘a sample for verification ofthe bacterial titer, DNA was extracted as described above. “The DNA. probes used for Southern hybridization were derived from two plasmid-encoded pathogenicity regions, cefA and pat-1, ‘of strain NCPPB382 (25). The ce/A probe was an internal 1-1-kb DNA PCR fragment isolated from pCMI (D. Meletzus, unpub- lished dara). The pat-t probe represents a 0.2-kb fragment of the repetitive sequence motif found on the 3.75-kb Bell fragment of plasmid pCM2 (13), which was obtained by PCR. Both DNA Fragments were cloned with the vector pSVB30 (1) and labeled with digoxigenin-IL-dUTP by nick-translation as deseribed by Maniatis et al (24) For Southern hybridization, DNA fragments were generated using appropriate restriction enzymes, resolved by agarose gel electrophoresis (37), and transferred to Hybond N nylon mem- branes (Amersham ine., Arlington Heights, IL) with a LKB 2016 ‘VactGene blotting apparatus. Prehybridizations were performed at 42 C for 2 h in a solution of 3x SSC (IX SSC fs 0.15 M [NaCl and 0.015 M sodium citrate), 50% formamide, 2% blocking reagent (Boehringer Mannheim GmbH, Mannheim, Germany), 0.02% SDS, and 0.1% N-lauroylsareosine. The denatured probe ‘was added to the prebybridization solution followed by gentle agitation at 42 C for 16 h. The nylon membranes were washed twice for 10 min at room temperature in a 2X SSC and 0.1% SSDS solution and twice for 15 min at 65 C ina solution consisting fof 0.1X SSC and 0.1% SDS. The hybridization results were visualized by using the DIG nucleic acid detection kit supplied by Boehringer. PCR. Polymerase chain reactions were carried out using the GeneAmp DNA amplification kit and a DNA thermal eycler, both from Perkin-Elmer Cetus (Norwalk, CT). In general, approx: imately 50 ng of template DNA and 30 pmol af each primer DNA were used per reaction. The set of synthetic oligonucleotide primers used had the following nucleotide sequence: 5'-GCG- AATAAGCCCATATCAA-3” (CMM-5) and. 5-CGTCAGG- AGGTCGCTAATA-3'(CMM.6), They were derived from partial ‘mucleotide sequence information of the par-| region and generated 4 614-bp PCR amplification product. Generally, 30 PCR cycles ‘were run with denaturation at 94 C for I min, annealing at 55 C for 1.5 min, and extension at 72 C for I min, RESULTS Virulence assay. The virulence of all the C. m. michiganensis strains used in our study was determined by inoculation of @ susceptible tomato cultivar. Only those strains inducing typical wilt symptoms were classified as virulent (Table 1). Our tests ‘confirmed the classification by NCPPB, with the exception of strains NCPPB3285 and NCPPB3286, which were described as ‘weakly aggressive but were avirulent under our test conditions. ‘Serological specificity. To evaluate whether a serotogical pro cedure could specifically detect C. m. mickiganensis, we tested ‘wo polyclonal antisera and a monoclonal antibody in an ELISA. Allthree antibodies gave similar results, and representative ELISA data obtained with the antibody from Loewe Biochemica are shown in Figure |, All C.m. michiganensis strains gave a positive reaction (ODas > 0.4) in indirect ELISA, with the exception of two of the avirulent strains, NCPPB2S4 and NCPPBS6I. A Strong teaction also was observed with all subspecies of C. ‘michiganensis and some phytopathogenic corynebactera, ‘Sensitivity 10 phage CMPI. Reliable identification of C. m. ‘michiganensis was not possible with the lytic phage CMPL. This bacteriophage is related to two other phage, MiP, and MiP), whieh are described as specific for C. m. michiganensis (43). Although C. m:.insidiosus, sepedonicus, nebraskenss, and tssel- Trius were not lysed by CMPI, the phage also did not lyse six strains of C. m. michiganensis (Fig. 1), For example, strains Vol.85, No.4, 1995. 465Fig, 3. Gel electrophoresis and Southern analysis of plasmid DNA isolated from virulent strains of Clvibacier michiganensis subsp. muchige om & 0.85 agarose gel, and tained with ethdiu r {rom strains NCPPH3HD (anes 2 and 3, pCML and pCM2, respectively), NCPPBIA9® (lane 4), NCPPHII79 (ane 3), NCPPBS120 (lie 6), NCPPBSIOL (lane 9) Lanes Tand 10, ‘nolecular marker A-DNA di and C, part A, Plasmid DNA was digested with Bef, subjected t electropho (Gane 7), NCPPB3223 (ane 8), NCPR 5 probed with digoxigenin-1-aUTP- labeled probes By Fig. 4, Electrophoretic analysis of DNA from Clawbacter michiganensis subsp. michizanensis stains amplified by polymerase chin reaction. The ppoutton ofthe predicted product (0.6 Xb) is indicated. Lanes 1 und 12, Pal marker, lane 2-6, avirulent strains of Cm. mlehiganensts NCPPB2S4, NCPPBEGI, NCBBPS99, NCPPBSIS, and NCPPBSI25, lanes 7-11 virulent strains of Cm. michiganensis, NCPPB3S2, NCPPBIA96 NCPPH2979, NCPPBSI20, and NCPPDS2ES NCPPBI496 and NCPPB3223, which were virulent on tomato plants, were resistant in this phage assay Detection of C. michiganensis subspecies by Southern hybrid zation, When total bacterial DNA was cleaved with Bell and subjected to Southern hybridizations with the celA probe, a distinct restriction fragment length polymorphism (RFLP) was characteristic of C. m. michiganensis (Fig. 2A). All C. m. michi- ‘anensis strains listed in Table | gave a trong hybridization signal wwith a 3.2-kb BigMll DNA fragment from which the I.I-kb eelA subfragment was derived, Additional hybridizing bands, especially an 8.6-Kb fragment, indicated other homologies. Other C. michi- ‘ganensis subspecies exhibited different hybridization patterns, Which were characteristic for these bacteria, vhat always lacked the 3.2-kb band. Figure 2A (lanes 14-17) shows the characteristic RFLP common toall strains of C. michiganensis subspecies tested (Table I). Strains NCPPB2S4 and NCPPBS6I did not have the 32-kb hybridizing band (data not shown). The probe for the pat-l region hybridized with a3.75-kb Befll DNA fragment found only in virulent strains of C. m. michiganensis, whereas no signal was obtained with avirulent strains or other phytopathogenic bacteria (Fig. 2B). Both DNA-probes used were derived from plasmid-encoded pathogenicity genes of strain NCPPB382. Since most Clavibacter 485 PHYTOPATHOLOGY ‘wromide. DNA was sd with Pal A, Southern blot of the a strains harbor cryptic plasmids (15,20), we examined the poss bility that these genes are carried on the plasmids present in these Clavibacter strains. Southern blots probed withthe celA region (Fig. 3B) revealed that this gene was not always located. on plasmids; for example, in strains NCPPB2979 and NCPPB3223 i was located on the chromosome because hybridization signals ‘were only obtained with total DNA preparations (Fig. 2A). In contrast, the pal region was always plasmid-borne (Fig. 3C). In summary, the results from Southern hybriization withthe two DNA-probes demonstrated that the cefA probe could be used the detection of Cm. michiganensi, whereas the pall probe allowed further differentiation between virulent and avirulent C. Detection of C: m. michiganensis by PCR. The speciticiy of PCR withthe CMM and CMN-6 primers was tested by sereen- ing for an amplification product with the predicted size (614 6p) using total DNA isolated from 23 C. m. michiganensis strains (Table 1). The proper amplification product was observed only with DNA extracted from virulent C.m. michiganensis strains, ‘Whereas no amplification products were observed with DNA from avirulent strains (Fig 4); result that agrees with the hybeidization data. Abo, DNA prepared from other bacterial species and related phytopathogenic corynebacteia listed in Table 1 did not yield A PCR amplification product (data not shown), ‘The PCR assay was evaluated for detection of C. m. michi= {ganensis in infected plant tissue and seeds as would be eequiced {or routine diagnosis. Amplification of target DNA with imac untreated cells of C. m. michiganensis using the method deseribed for Com. sepedonicus (16) was unsuecesfal. However, the CTAB protocol for extraction of Cm. michiganensis DNA from plant Fomogenste gave satisfactory results. A specific amplification of target DNA ‘was observed with extracts prepared feom plants Infected with strain NCPPB3®2, which induced typical wilt symp- {om and colonized the plat eifectvely (1-3 X 10" fu per gram of plant material) (Fig. 5). Amplification products were not ob Served when extracts from healthy, noninfected control plants ‘were assayed nor when extrats from plants infected ‘with the avirulent strain NCPPB3123 were tested (1-2 X 10" ef per mil Iter of plant homogenate) PCR also was tested on seeds harvested fiom tomato plans infeted with Cm. michiganenss sain NCPPD382. It was possible 10 detect the pathogen in DNA extract from $0 seeds containing 1% 10" bacteria (Fig. 6). Sensitivity of the PCR assay. To estimate the detection lit of PCR assay, DNA preparations from plant homogenates con- taining 2 % 10" to 2% 10" fu per milliliter of strain NCPPB382 were tested in three independent experiments, It was possible to detect the 614-bp amplification product by Southern hybridiza. tion against a 3.75:kb Bell patel probe down to concentrationFig 5. Detection of Cavibacer mihiganensi subsp. michiganentis strain NCDPIMD after cerytr-methylammonivm bromide extraction of infected tomato plants by polymerase chain reaction asay- Lanes | and 10, 4 Parl mater. lane 2, negative control (ides 1:0} lane 3, positive contra (XCPPHIS? DNA}; lanes 4-9: DNA extracted from tomato plants anes ‘Pand S: noninfected plants: lanes 6 and 7, NCPPB3I2S infected plans, Sand lanes 8 and 9, NCPPB3S2 infected plants of 2 X 10° eels of € homogenate (Fig. 6). im. michiganensis per milliliter of plant DISCUSSION Since the main source for transmission of C. m. michiganensis, {contaminated seeds and transplants, the control of this plant disease requites reliable and sensitive methods of detecting the ppathogen in these tissues, Immunofluorescence and ELISA are hhampered by the fact that available antibodies lack specificity to allow differentiation at the subspecies level (17), Strong cross- reactions observed in our work indicate that similar antigenic epitopes are common to all subspecies of C. michiganensis. A prominent antigen is the exopolysaccharide with an identical Carbohydrate composition in C: m. michiganensis and the alfalfa pathogen C. m. insidiosus (3). The detection limit for ELISA with pure cultures was approximately 10" cfu per milliliter (39) sensitivity sufficient to detect the pathogen in symptomless plants (9), Phage typing is not practicable for rapid diagnosis, because bacterial cultures are required, and thus, phage typing is mo useful for taxonomic classification, provided a number of phage swith different host specificity are available cently a DNA-probedirected against the 16S ribosomal RNA. genes of C. m, sepedonicus was described (27), However, due to high conservation of 168 eRNA genes among C. michiganensis subspecies, detection of C. m.michiganenss depends on an appro- priate hybridization stringency. Thompson et al (41) described DNA-probes derived from a chromosomal library of C. m. michi- sanensis to detect C. m. michiganensis by dot blot that suggested sufficient specificity; however, very few strains were tested ‘As reported here, plasmid-borne genes (25) proved to be useful for identification of C. m. michiganensis. Plasmids frequently play a role in virulence of phytopathogenic bacteria (7), and this twas the ease in C. m. michiganensis strain NCPPB382 (25,26) The probing of plasmids from various strains for the presence of the pathogenicity gene par-l (Fig. 3) suggests that in C. m. Imichiganensis virulence genes generally seem to be located on plasmids. This contradicts earlier reports by Grosset al (20) who saw no correlation between plasmids and virulence in C. m. ‘michiganensis. Therefore, as discussed by Finnen et al (15), it ‘would be interesting to study the molecular properties of plasmids from C. m. michiganensis isolates to gain mote information on their relationship and to obtain data on the epidemiology of this important pathogen. Southern hybridization with the ce/A probe allowed differen- tiation between C. michiganensts subspecies and other phyto- Fig. 6, Sensitivity of polymerase cain reaction (PCR) batter michicanenss subsp. michianensis in tomato homogenates snd ‘cade, Homogenates were inoculated with various cell densities of sain NCPPHSR2. Seeds were obtained rom tomato plant infected with strain NCPPB3E2. A, Alter ge! electrophoretic separation ofthe amplifiation predts, th twas blotted on nylon membrane and B, hybridized against the marl probe (2.75 kb) labeled with digonigenin-tL-dUTP. Lanes I fand’ 4, 0" Pall marker lane 2, negative contol (homogenate from foninfected plants); lanes 3-8, i0-old dilations (2% 10" to 2% 10" efu per mililte) of NCPPHMD; lanes 10 and 11, homogenate from nonin fected tomato seeds: and lanes 12 and 13, homogenate from 30 seeds collected from tomato plants infested with NCPPB3E2 containing 1 10" pathogenic coryneform bacteria. In addition, an analysis of RFLP land the development of specific PCR primers for the ce/A region ‘may be helpful for identification of other subspecies. ‘The avirulent C. m. michiganensis strains NCPPBIS4 and NCPPBBG1 were negative in Southern hybridizations with the ee1A probe (data not shawn), and since these strains also gave zo positive signal in ELISA (Fig. 1), they seem to be incorrectly classified, In fact, strain NCPPB86i was isolated from Cypho- ‘mandra betacea and, thus, may represent a different bacterial species ‘An important finding of this work is the specificity of the pat-1 probe and its correlation with virulence, This region can be ampli- Fied by PCR and provides a sensitive detection assay for virulent strains of C. m. michiganensis. Our results indicate that PCR. based analysis without prior isolation and enrichment of the pathogen from plant tissue has a detection limit of 2 X 10" cells Per milliliter of plant homogenate. This sensitivity should even be sulficient for the detection of the pathogen in latently infected plants, provided that the titer is in the range of the detection limit. Disease symptoms are observed only when bacteria reach a titer of 10° 10" efu per gram of plant tissue (25). We demon Strated that ths technique also is appropriate for rapid sereening of heavily contaminated seeds. The application of “booster” PCR ‘may further enhance the sensitivity, as has been shown recently for Agrobacterium tumefaciens (29). Asis the case for serological assays, DNA methods do not provide information about the Viability of the pathogen but rapidly identify potentially contami- ‘nated plant and seed sample fr suceessive confirmation by micro- biological tests, Therefore, PCR assay should be a useful addition to standard detection methods because it combines speed, sensi- tivity, and specificity, which are crtictl parameters for any detec- tion assay, Vol. 86,.No. 4, 1995 467LITERATURE CITED. ‘Arnold, W., and Publer, A. 1988. A family of high-copy-number plasmid vectors with single end-label sites for rapid nucleotide se ‘quencing. Gene 70171179, Beimen, A., Bermpobl, A, Melevus, D., Eichenlaub, Rand Bara, W. 1992, Accumulation of phenolic compounds in leaver of tomato plants after infection with Clavibacter michiganence subsp. Imichiganense stains difering in virulence. Naturforsch 4:89:90. Bermpohl, A. 1994. 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