Pseudomonas Cichorii

Plant Pathology Bulletin 15:275-285, 2006

Pseudomonas cichorii

1 1 1, 2

2006 Pseudomonas cichorii 15 275-285

60 (RAPD) Pseudomonas cichorii (Swingle) Stapp 1,100 bp Topo pCR¨II-TOPO Pseudomonas cichorii SfL1/SfR2 (polymerase chain reaction, PCR) 379 bp 6 21 SfL1 / SfR2 P. cichorii DNA 5~10 pg 5.5~9 (P. cichorii) 10 6 cfu/ml 10 5~10 8 cfu/ml SfL1/ SfR2 P. cichorii 3 - 4 hr

SfL1/SfR2 P. cichorii

PCR

Pseudomonas cichorii (3) (Swingle) Stapp P. cichorii (10) (13) (lettuce) (10)

(27) (15) (33) (cabbage) (celery) (tomato) (14) (chrysanthemum) (16) (geranium) (16) (dwarf schefflera) (11) (sunflower) (22) (magnolia) (19)

(5, 10)

(5)

(3) ( polymerase chain reaction PCR) (20) RFLP ( 276 15 4 2006 restriction fragment length polymorphism) AFLP RAPD PCR (amplified fragment length polymorphism) RAPD Table 1. Bacterial isolates used in experiments of RAPD (random amplified polymorphic DNA) (17, 18, 32) and PCR. Bacterium Strain Pseudomonas Burkholderia andropogonis Pan1 Pseudomonas B. caryophylli Tw7, Tw9 syringae pv. cannabina efe gene DNA B. gladioli pv. gladioli Bg ETH1 ETH2 ETH3 P. syringae pv. Erwinia carotovora subsp. carotovora Zan01~15 Ech10~22 cannabina P. syringae pv. glycinea P. syringae pv. Erwinia chrysanthemi Sr53~56 (24) phaseolicola P. Pantoea agglomerans Yx5, Yx7 syringae pv. atropurpurea cfl gene DNA Pseudomonas aeruginosa Pae Primer 1 Primer 2 P. syringae pv. glycinea P. P. cichorii Sf syringae pv. maculicola P. syringae pv. tomato P. fluorescens Pf (8) (30) P. putida Pu P. syringae pv. atropurpurea P. P. syringae pv. glycinea Psg (7) (25) syringae pv. pisi P. syringae pv. phaseolicola P. syringae pv. lachrymans Psl P. syringae pv. phaseolicola Psph P. syringae pv. pisi Pspi P. syringae pv. syringae Pss pel Erwinia P. syringae pv. tabaci Psta (12) carotovora RFLP P. syringae pv. tomato Psto Erwinia (6, Ralstonia solanacearum Ia57, 29 12, 29) DNA Xanthomonas axonopodis pv. dieffenbachiae Ao72 X. axonopodis pv. vesicatoria Xv15 X. campestris pv. mangiferaeindicae X58 PCR Seal (26) RAPD Ralstonia solanacearum PCR 30 min RNase A ( 50 g/ml ) RAPD 37 30 min phenol/chloroform Pseudomonas cichorii (phenol chloroform isoamylalcohol 25 24 1) Pseudomonas 12,000 rpm 4 30 min cichorii PCR 1/10 3 M NaOAc 2 95 % -20 1 hr DNA 70 % 12,000 rpm 4 30 min 50 15 min 100 l 4 DNA -30 (23)

35 Pseudomonas cichorii (Swingle) Stapp (plasmid) Sf Plasmid miniprep purification kit ( ) (Genemark) Kanamycin (50 g/ml) 5 ml LB DNA 37 16 hr 2 ml 5 ml LB broth 30 12,000 rpm 5 min 200 l 16 hr 12,000 rpm 10 min 1 ml STE solution I 200 l solution II 12,000 rpm 10 min 200 l solution III 12,000 0.5 ml STE 50 ml 10 % SDS rpm 4 10 min DNA 65 30 min proteinase K (100 g/ ml ) 700 l washing solution 12,000 rpm Pseudomonas cichorii 277

4 30 sec 12,000 rpm 4 DNA OPY-20 RAPD 3 min DNA 1.5 ml 0.25 N HCl 10 min 60 5~10 min 50 l denature buffer (0.5 M NaOH 1.5 M 12,000 rpm 4 30 sec DNA NaCl) 30 min neutralization buffer (0.1 M Tris-HCl, pH 7.5; 1.5 M RAPD NaCl) 30 min Sf01 Sf02 Sf07 Sf09 10X SSC Sf31 5 DNA OPERON 10- 10X SSC 16 MER KITS OPX 01~20, OPY 01~20 OPZ 01~20 hr DNA 60 RAPD 20 l UVC-515 100 ng DNA 200 M dNTPs 0.25 M Ultraviolet Crosslinker (ULTRA-LUM, Inc. U.S.A.) 0.8 U DNA 1 254 nm GeneAmp PCR System 2400 94 1 2X SSC salmon sperm min 94 1 min, 40 1 min, 72 2 min DNA 200 l ( hybridization 40 72 20 min 8 solution: 5X SSC, 0.5% SDS, 50% Formamide, 5X l 1X TAE 1.5% agarose Denhardts) 3 min 5 min gel 100 V hybridization solution 65 UV box 16 hr 0.1 % SDS 2X SSC 5 min 2 0.1 % DNA SDS 0.2X SSC 65 15 min 2 RAPD buffer I (0.1 M Tris-HCl , pH 7.5 ; 0.15 M NaCl) DNA 5 min buffer (0.5 % blocking reagent in buffer DNA II, 4 ) 1 hr Anti-fluorescein-AP conjugate (NEN¨ Life Science Products, Boston, MA) buffer II (1: 5000) 1 hr buffer I 4 DNA 5 min buffer (0.1 M Tris-HCl, pH RAPD 1X TAE 9.5 0.15 M NaCl) 2 5 min buffer 1.2% agarose CDP-Star 12.5 mM (Tropix, DNA Ultrafree¨-DA (MILLIPORE USA U.S.A.) buffer (1 200) ) KIT Gel Nebulizer Ultrafree-MC Vial 5 min X-ray 10 min 6,000 rpm 10 min DNA -30 DNA Random Primer Fluorescein Labeling Kit DNA with Antifluorescein-AP (NEN) (probe) TOPO TA Cloning¨ kit DNA 19 l DNA 5 4 l DNA min 5 min 5 l Random 1 l salt solution 1 l pCR¨II-TOPO vector primer and reaction buffer mix 5 l Fluorescein 5 min 2~6 l nucleotide mix 1 l Klenow fragment (competent cell) 30 min 42 30 l 37 1 hr 30 sec 2 min 250 l 5 l 0.1 M EDTA (pH8.0) SOC 200 rpm 37 1 hr -30 40 l 100 mM IPTG 40 l 40 g/ml X-gal Kanamycin (50 ppm) LA (Southern hybridization) 37 Southern (28) DNA DNA EcoRI Sf41 Sf80 278 15 4 2006

200 l 50 l 0.5 N NaOH NCBI (National Center for Biotechnology 50 l 50 l 1 M Tris-HCl (pH 8.0) Information) (GenBank) 2 l SfL1/SfR2 PCR Vector-NTI 8 (InforMax) spiral plater G+C hairpin (model D, Spiral systems, Inc. Bethesde, Maryland) duplex structure (forward primer) NA 30 24 hr ( reverse primer)

(dNTP) Wang (31) (primer) Taq polymerase (Dynazyme) 50 l 0.5 N PCR PCR NaOH 25 l PCR 25 l 1 M Tris-HCl (pH 8.0) 2 l SfL1/SfR2 PCR

DNA Sf DNA Pseudomonas putida, P. aeruginosa, P. fluorescens, P. PCR syringae pv. syringae, Agrobacterium tumefaciens, Erwinia carotovota pv. carotovota, E. chysanthemi, (Pseudomonas cichorii) Sf06 Ralstonia solanacearum, Xanthomonas axonopodis pv. Sf09 Sf26 Sf28 Sf30 Sf62 NA dieffenbachiae 5 9 10 24 hr ng SfL1/SfR2 PCR Spectrophotometer A620 nm O.D SfL1/SfR2 P. 0.3 ( 1 108 cfu/ml) cichorii DNA 30 24 Sf NA 24 hr 48 72 96 120 hr 1 10 108 cfu/ml 10 1 30 1 106 cfu/ml Pseudomonas 50 50 l syringae pv. syringae P. putida Agrobacterium 500 l PBS-Tween-20 ( phosphate-buffer saline tumefaciens 0.5% Tween-20) 10,000 rpm 20 104 ~108 cfu/ml min 100 l Sf 106 cfu/ml PBS-Tween-20 (19) 2 l 50 l 200 ml 50 l 0.5 SfL1/SfR2 PCR N NaOH 50 l 50 l 1 M Tris- SfL1/SfR2 HCl (pH 8.0) 2 l SfL1/SfR2 PCR DNA Sf02 Sf76 DNA 100, 10, 1 ng 100, 10, 1, 5 1 0.1 0.5 pg SfL1/SfR2 PCR 20 l PCR RAPD SfL1/SfR2 DNA Sf01 Sf02 Sf07 Sf02 Sf058 NA Sf09 Sf31 5 DNA OPX (nutrient agar) 24 hr 01~20 OPY 01~20 OPZ 01~20 60 8 OD620 0.3 ( 10 cfu/ml) 10 RAPD 101~107 cfu/ml 50 l 10 OPX- Pseudomonas cichorii 279

vector

DNA DNA 99% (identity) NCBI DNA 1,100 bp 8~242 bp 272~304 bp 480~537 bp 765~1138 bp 4 P. syringae pv. tomato DC3000 84~93% ( ) P. syringae pv. tomato 304~765 ( DNA 15%) Vector NTI suite 8 (InforMax) 2 SfL1 SfL2 2 SfR1 SfR2 PCR SfL1/SfR2 (Data not shown) Sf 379 bp P. cichorii

OPX-17 RAPD (A), Sf X17- SfL1/SfR2 PCR Sf1100 (B). Lane 1 lane 2 Sf M Bio 100 marker (-) PCR 20 l Lane 3-20 50 ng DNA 1X Taq buffer 200 M Fig 1. Agarose gel electrophoresis shows RAPD patterns dNTP 0.25 M Primer 0.4 Unit Dynazyme of strains of Pseudomonas cichorii and other bacteria PCR (a) 94 5 min 1 using OPX-17 random primer (A) and southern hybridization of the RAPD products with X17-Sf1100 (b) 94 1 min 65 30 sec 72 30 sec 30 probe cloned from Pseudomonas cichorii (B). Lanes 1 and (c) 72 10 min 1 2, Pseudomonas cichorii Sf isolates; M, Bio100 marker; 2 % agarose lanes 3 - 20, other bacteria; (-), negative control.

17 SfL1/SfR2 1,100 bp ( ) OPX-17 Sf02 Sf10 Sf28 Sf33 Sf41 Sf68 Sf74 DNA ( ) RAPD Sf85 DNA Pseudomonas putida, P. 1,100 bp DNA aeruginosa, P. fluorescens, P. syringae pv. syringae, Sf Agrobacterium tumefaciens, Erwinia carotovota pv. 1,100 bp 1,100 bp carotovota, E. chysanthemi, Ralstonia solanacearum, 1,100 bp Xanthomonas axonopodis pv. dieffenbachiae 5 9 1,100 bp DNA Sf DNA DNA ( ) P. cichorii Sf41 DNA Sf41 106 cfu/ml 2 3 (108~104 cfu/ml) PCR Sf RAPD Sf 379 bp 1,100 bp DNA pCR¨II-TOPO P. cichorii 280 15 4 2006

pX17-Sf1100 bp DNA SfL1 SfL2 SfR1 SfR2 Fig.2. The sequence of the inserted DNA fragment in recombinant plasmid pX17-Sf1100 bp. The sequence of the primers SfL1, SfL2, SfR1 and SfR2 designed are underlined, and the sequence of random primers OPX 17 are indicated red bold-faced.

SfL1/SfR2 SfL1/SfR2 Sf02 Sf76 Sf02 Sf07 Sf09 Sf27 Sf31 Sf35 DNA PCR Sf41 Sf58 Sf68 Sf70 Sf71 Sf75 Sf77 Sf80 Sf76 DNA 5 pg 10 pg ( ) Sf83 Sf85 0.5 N NaOH Sf02 Sf58 DNA SfL1/SfR2 PCR NaOH 379 bp DNA P. cichorii PCR 5.5 ~ 9 ( ) PCR Pseudomonas cichorii 281

Sf41 SfL1/SfR2 DNA SfL1/SfR2 Sf76 DNA 1-8 Pseudomonas M 100 bp marker (-) 1-9 100 putida, P. fluorescens, P. syringae pv. syringae, Erwinia ng 10 ng 1 ng 100 pg 10 pg 5 pg 1 pg 0.5 pg carotovora pv. carotovora, E. chrysanthemi, Xanthomonas 0.1 pg axonopodis pv. dieffenbachiae, Agrobacterium Fig 4. Sensitivity of polymerase chain reaction using trmefaciens, Ralstonia solanacerum 9 primer pair SfL1 / SfR2 to detect total DNA of Pseudomonas aeruginosa DNA; M 100 bp marker (-) Pseudomonas cichorii Sf76. Lanes 1-9, 100 ng 10 ng 1 ng 100 pg 10 pg 5 pg 1 pg 0.5 pg and 0.1 pg of Fig 3. Polymerase chain reaction amplification products of DNA, respectively. M, Bio 100 DNA marker; (-), total DNA from strains of Pseudomonas cichorii Sf41 negative control. mixed with other bacterial with primer pair SfL1/SfR2. Lanes 1-8, Pseudomonas putida, P. fluorescens, P. syringae pv. syringae, Erwinia carotovora pv. carotovora, E. chrysanthemi, Xanthomonas axonopodis pv. dieffenbachine, Agrobacterium trmefaciens, Ralstonia solanacerum, respectively; lane 9, only Pseudomonas aeruginosa DNA; M, 100 bp marker; (-), negative control.

1 10 1 30 1 50 PBST 2 l SfL1/SfR2 PCR 24 SfL1/SfR2 hr 48 hr Sf58 72 hr 1-3 5.5~9.0 103 cfu 4-6 5.5~9.0 102 cfu 1 (1 10 1 30 1 50) 7-9 5.5~9.0 10 cfu 10-12 5.5~9.0 cfu 13-15 5.5~9.0 10-1 cfu 16-18 5.5~9.0 10-2 cfu 19-21 96 hr 120 hr 5.5~9.0 10-3 cfu M 100 bp marker (-) ( ) Fig 5. Sensitivity of PCR for detection of cells of Pseudomonas cichorii Sf58 with primer pair SfL1/SfR2. Lanes 1-3, 5.5~9.0 103 cfu; lanes 4-6, 5.5~9.0 102 cfu; lanes 7-9, 5.5~9.0 101 cfu; lanes 10-12, 5.5~9.0 cfu; Pseudomonas spp. -1 -2 (7, lanes 13-15, 5.5~9.0 10 cfu; lanes 16-18, 5.5~9.0 10 cfu; lanes 19-21, 5.5~9.0 10-3 cfu; M, 100 bp marker; (-), 8, 25, 30) negative control. P. cichorii 282 15 4 2006

SfL1/SfR2 P. cichorii P. cichorii (No. 12682) Agrobacterium, Burkholderia, Erwinia, Xanthomonas, Ralstonia Pseudomonas spp. PCR P. cichorii P. cichorii 379 bp

Sato et al. (24) efe gene ETH1 ETH2 ETH3 P. syringae pv. cannabina, pv. SfL1/SfR2 PCR glycinea, pv. phaseolicola, pv. sesame Bereswill et al. (8) efl gene Pseudomonas cichorii M 100 bp marker (-) Primer 1 Primer 2 1-3 24 hr 4-6 48 hr 7-8 P. syringae pv. atropurpurea, pv. glycinea, pv. 72 hr 9-12 96 hr maculicola, pv. tomato 13-15 120 hr 1-15 DNA 1 10 1 30 P. 50 16 Pseudomonas cichorii DNA cichorii SfL1/SfR2 ( Fig 6. Detection of Pseudomonas cichorii in artificially P. cichorii P. syringae ) infested leaf tissures of sunflower by polymerase chain DNA RAPD reaction using primer pair SfL1/SfR2. Lanes 1-3, 24hr; P. cichorii lanes 4-6, 48 hr; lanes 7-9, 72 hr; lanes 10-12, 96 hr; lanes 13-15, 120 hr; lanes 1-15, symbol 1 10 1 30 and 1 SfL1/SfR2 50 of sample, respectively. 16, Pseudomonas cichorii ; M, 5 pg~10 pg DNA Bio 100 DNA marker; (-), negative control. Bereswill (9) Ewinia amylovora 10 ng (2) SL1/SR1 Acidovorax avenae subsp. citrulli 100 RAPD pg SfL1/SfR2 P. cichorii Sf02 Sf58 RAPD P. (4) cichorii RAPD 5.5 ~ 9 Arnold et al. (7) 5A/5B, Ec1/Ec2 Erwinia carotovora pv. pX17-sf-1100 carotovora E. chrysanthemi Ecc 1.3 2 1 (2) OPX-17 1.1 kb NCBI 10 Ech 5.2 10 8~242 bp 272~304 bp SL1/SR1 Acidovorax avenae subsp. citrulli 2 (1) 480~537 bp 765~1,138 bp 4 P. 1.2 10 nL/nR Burkholderia (21) syringae pv. tomato DC3000 (AE016853) caryophylli 20 Prosen et al. P. 3 84~93% ( ) 813-956 bp P. syringae pv. phaseolicola 10 cfu aeruginosa PAO1 (AE004091) 88% SfL1/SfR2 P. cichorii (21) P. cichorii Prosen et al. P. syringae pv. P. syringae pv. tomato P. phaseolicola (probe aeruginosa PAO1 304~765 bp hybridization) 101 cfu/ml ( DNA SfL1/SfR2 5.5 15%) Vector NTI suite 8 (InforMax) 101 cfu 2 SfL1 SfL2 2 SfR1 SfL1/SfR2 SfR2 PCR P. cichorii SfL1 SfR2 DNA P. cichorii PCR Pseudomonas cichorii 283

379 bp 31 346-357 P. 6. Anna, O. A., Hyman, L. J., Toth, R. L. and Toth, I. K. cichorii P. 2002. Application of amplified fragment length polymorphism fingerprinting for taxonomy and cichorii identification of the soft rot bacteria Erwinia P. cichorii carotovora and Erwinia chysanthemi. Appl. Environ. (31) Wang et al. 0.5 N NaOH Microbiol. 68 : 1499-1508. genomic DNA Tris- 7. Arnold, D. L., Atheypollard, A., Gibbon, M. J., Taylor, HCl buffer genomic DNA 0.5 N NaOH J. D. and Vivian, A. 1996. Specific oligonucleotide PCR primers for the identification of Pseudomonas syringae (2) (1) pv pisi yield one of two possible DNA fragments by DNA PCR amplification : evidence for phylogenetic divergence. Physiol. Mol. Plant Pathol. 49 : 233-245. P. cichorii 8. Bereswill, S., Pshl, A., Bellemann, P., Zeller, W. and SfL1/SfR2 P. Geider, K. 1992. Sensitive and species-specific cichorii detection of Erwinia amylovora by polymerase chain DNA P. detection analysis. Appl. Environ. Microbiol. 58 : cichorii 3522-3526. 9. Bereswill, S., Bugert, P., Volksch, B., Ullrich, M., PCR Bender, C. L., et al. 1994. Identification and 24 hr 48 hr relatedness of coronatine-producing pseudomonas 72 hr 96 hr syringae pathovars by PCR analysis and sequence 120 hr determination of the amplification products. Appl. Environ. Microbiol 60:2924-2930. 10. Chase, A. R. 1988. Compendium of ornamental foliage plant diseases. APS PRESS 92pp. 11. Chase, A. R. and Jones, J. B. 1986. Effects of host SfL1/SfR2 nutrition, leaf age, and Preinoculation light levels on severity of leaf spot of dwarf schefflera caused by Pseudomonas cichorii. Plant Dis. 70 : 561-563. 12. Derrasse, A., Prious, S., Kotoujansky, A. and Bertheau, Y. 1994. PCR and restriction fragment SfL1/SfR2 P. length polymorphism of a pel gene as a tool to identify Erwinia carotovora in relation to potato disease. Appl. cichorii Environ. Microbiol. 60: 1437-1443. P. cichorii 13. Grogan, R. G., Misaghi, I. J., Kimble, K. A., Greathead, A. S., Ririe, D. and Bardin, R. 1977. Varnish spot, destructive disease of lettuce in California caused by Pseudomonas cichorii. 1. 1999 Burkholderia caryophylli Phytopathology. 67 : 957-960. 14. Gulya, T. J., Woods, D. M., Bell, R. and Mancl, M. K. 1991. Disease of sunflower in calfornia. Plant Dis. 75 2. 1999 : 572-574. 15. Jagger, I. C. 1914. Bacterial leaf spot disease of celery. Phytopathology. 4 : 395(Abstr.). 3. 2004 16. Jones, J. B., B. C. Raju., and A. W. Engelhard. 1984. 13 Effects if temperature and leaf wetness on 329-334 development of bacterial spot of geranium and 4. 1998 chrysanthemum incited by Pseudomonas cichorii. Erwinia Plant Dis. 68 : 248-251. 17. Louws, F. J., Rademarker, J. L. W. and de Bruijn, F. J. 5. 1989 1999. The three Ds of PCR-based genomic analysis of 284 15 4 2006

phytobacteria: diversity, detection, and disease N. J. 1989. Identification of Pseudomonas syringae pv. diagnosis. Annu. Rev. Phytopathol. 37: 81-125. phaseolicola by a DNA hybridization probe. 18. Manulis, S., Valinsky, L., Lichter, A. and Gabriel, D. Phytopathology 79 : 903-907. W. 1994. Sensitive and specific detection of 26. Seal, S. E., Jackson, L. A. and Daniels, M. J. 1992. Xanthomonas campestris pv. pelargonii with DNA Isolation of a Pseudomonas solanacearum specific primers and probes identified by random amplified DNA probe by hybridization and construction of polymorphic DNA analysis. Appl. Environ. Microbiol. species-specific oligonucleotide primers for sensitive 60 : 4094-4099. detection by the polymerase chain-reaction. Appl. 19. Mullen, J. M. and G. S. Cobb. 1984. Leaf spot of Environ. Microbiol. 58 : 3759-3761. southern magnolia caused by Pseudomonas cichorii. 27. Smith, M. A. and Ramsey, G. B. 1956. Bacterial Plant Dis. 68 : 1013-1015. zonate spot of cabbage. Phytopathology 46 : 210-213. 20. Mullid, K. B. and Faloona, F. A. 1987. Specific 28. Southern, E. M. 1975. Detection of specific sequences synthesis of DNA in virto via a polymerase-catalysed among DNA fragments separated by gel chain reaction. Methods in Enzymology. 155 : 335- electrophoresis. J. Mol. Biol. 98 : 503-517. 350. 29. Toth, I. K., Hyman, L. J. and Wood, J. R. 1999. A one- 21. Prosen, D., Hatziloukas, E., Schaad, N. W. and step PCR-based method for the detection of Panopoulos, N. J. 1993. Specific detection of economically important soft rot Erwinia species on pseudomonas syringae pv. phaseolicola DNA in bean micropropagated potato plants. J. Appl. Microbiol. 87 : seed by polymerase chain reaction-based amplification 158-166. of a phaseolotoxin gene region. Phytopathology 83 : 30. Tskahashi, Y., Omura, T., Hibino, H. and Sato, M. 965-970. 1996. Detection and identification of Pseudomonas 22. Robbs, C. F. and Almeida, A. M. R. 1982. Bacterial syringae pv atropurpurea by PCR amplification of leaf blight of sunflower caused by Pseudomonas specific fragments from an indigenous plasmid. Plant cichorii (Swingle) Stapp. First record in Brazil. Rev. Dis. 80 : 783-788. Plant Pathol. 61 : 356. 31. Wang, H., Qi., M. and Cutler, A. J. 1993. A simple 23. Sambrook, j., Mantis, T. I. and Fritsch, E. F. 1989. method of preparing plants samples for PCR. Nucleic Molecular cloning: a laboratory manual. 2nd ed. Cold Acids Res. 21: 4153-4154. Spring Harbor Laboratory, Press, N. Y. 32. Williams, J. G. K., Kubelik, A. R., Livak, K. J., 24. Sato, M., Watanabe, K., Yazawa, M., Takikawa, Y. and Rafalski, J. A. and Tingey, S. V. 1990. DNA Nishiyama, K. 1997. Detection of new ethylene- polymorphisms amplified by arbitrary primers are producing bacteria Pseudomonas syringae pvs. useful as genetic markers. Nucleic Acids Res. 18: canncbina and sesame, by PCR amplification of genes 6531-6535. for the ethylene-forming enzyme. Phytopathology 87 : 33. Wilk, J. D. and Dye, D. W. 1974. Pseudomonas 1192-1196. cichorii causing tomato and celery diseases in New 25. Schaad, N. W., Azad, H., Peet, R. C. and Panopoulos, Zealand. N. Z. J. Agr. Res. 17 : 123-130. Pseudomonas cichorii 285

ABSTRACT Hseu, S. H.,1 Shentue, H. 1 and Lin, C. Y.1, 2 2006. Development of specific PCR primers for identification of Pseudomonas cichorii. Plant Pathol. Bull. 15: 275-285 (1 Department of Plant Pathology, Agricultural Research Institute, Council of Agriculture, Wufeng, Taichung, Taiwan, ROC.; 2 Corresponding author, E-mail: [email protected],tw)

A specific PCR (polymerase chain reaction) primer pair have been developed using RAPD ( random amplified polymorphic DNA ) to detect Pseudomonas cichorii. Totally sixty random primers were used to find specific DNA fragments of P. cichorii, and a specific DNA fragment of 1,100 bp amplified by the primer OPX17 was cloned into the pCR¨ II-TOPO vector and further sequenced to design a specific primer pair SfL1 / SfR2. The primer pair could amplify a distinct band of 379 bp that was specific to P. cichorii, and no DNA fragment amplified by the same primer pair from the other tested 21 bacterial species in 6 genera. Sensitivity of PCR for detection of P. cichorii with primer pair SfL1 / SfR2 was between 5 ~ 10 pg for purified DNA and 5.5 ~9 cfu for cultured cells. Non-target bacteria did not affect the efficiency of specific amplification of P. cichorii in PCR assay with primer pair SfL1 / SfR2. PCR technique using primer pair SfL1 / SfR2 identifies the culture of P. cichorii within 3 - 4 hours and detected the bacterium in artificially inoculated sunflower leaf tissue. Based on the data provided, we conclude that the primer pair SfL1 / SfR2 might be a useful tool for rapid identification and diagnosis of P. cichorii.