萵苣細茵性葉腐病及品種抗性篩檢Identification of the causal agent of lettuce bacterial midrib rot and resistance screening of lettuce cultivars
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(2) 102. 18. rot) (10). 2. midrib rot (9). 2009. bacterial rot (3, 6, 15) (27). (20). O/F test). (catalase). (yeast extract-dextrose-CaCO3). (8). (fluorescent pigment. (18). YDC. KB. ). (oxidase). (arginine dihydrolase). 96~97. (gelatin). (levan). (pectolytic). Biolog Identification System Pc1. Pc2. Pc3. Pc9. Pc12. 5. BUGM (Biolog Universal Growth Medium, medium 36 g, Bacto agar 15 g). 16. 24 hr. IF buffer (0.4% NaCl, 0.03% pluronic F-68, 0.01% gellan gum). 63% T. Biolog GN2 75% (NaOCl). 150. 0.1%. 30 sec. (Biolog Inc. Hayward l. 30. CA) 16~24 hr Biolog. 3 GN2. (Biolog 6.1. ). (. ) (nutrient agar, NA, DifcoTM). 30 NA. 3. Pc1~14. NA NA. 14. Pseudomonas cichorii (. 30 NA. 3. NA. 12621. ) ). ). BCRC. P. cichorii Sf 010 (. P. syringae pv. lachrymans (. P. syringae pv. phaseolicola (. syringae pv. pisi ( syringae (. NA. ) ). 30 (spectrophotometer). ). (A600). 0.3. (. P. syringe. pathovars ). pathovars. 2 min. 2%. (PTA. 0.1% Bovine serum) 2 min 7000 (4). V). Pc2. NA. RAPD ) (16). (. TAE buffer). (100. (size marker) g/ml). Pc3. 16S rDNA. 30 /. (26). Gen-100 DNA ladder (GeneMark Technology,. (ethidium bromide 0.5. (12). George Pc1. KOH. DNA. (. 2% agarose (1. Formvar ROC). 5. l. PCR (polymerase chain reaction). pH 7.5 Hitach-. Pc12. 6,000. P. cichorii. 5 sec. Braun-Kiewnick. 24 hr. 2. SfL1 / SfR2. 1. P. syringe. 10 min. rpm NA. DNA. l. 100 30. ). NA 400. Pc. Pc9. P. syringae pv.. P. syrinaeg pv. tabaci (. 10 cfu/ml (Nicotiana tabacum cv. Vam-Hicks.). P.. P. syringae pv. tomato (. 8. Schaad (30). ). Pc1. Pc9. Pc12. 3. NA.
(3) 103 DNeasy plant mini kit (QIAGEN, Hilden, Germany). genomic DNA. rDNA universal primers f8-27/r1510 10. M. 2.5 units/. PCR. 55. 30 sec 72. 5 min. 72. 5 l. 94. 2 min 20 sec. E). l RealTaq DNA 50. 94. D) (. 5 mM. polymerase (Real Biotech Corporation, ROC) 10. (. PCR. PCR. 25 mM Mg +. dNTPs. 16S. (22). (. F). l. PCR 30 sec. 40. 5 min 1. 1.0~1.5% agarose. TEM. (sequencing). NCBI (National Center for. Biotechnology Information ). YDC. SDSC-Biology Workbench ( ). 30 KB. BLAST (Basic Local Alignment. Search Tool). L-Rhamnose Sorbitol. Pc1. Pc9. Pc12. 3. Sucrose. (1) ). (3). (2). (. ). (. (4). ). (. A. A. HV-076. A. 3. Biolog syringe. 0.53. ). P. cichorii. ). 3. (. A). ) P. syringae pv.. P. syrinaeg pv. tabaci (. P. syringae pv. tomato (. ). 16S rDNA 16S rDNA. B) 27/r1510. Pc12. ) ). ). (. Sf 010 (. P. syringae pv. phaseolicola (. syringae (. Pc9. P.. P. syringae pv. lachrymans ( ). 16 ~ 32 hr. 0.85. 379 bp. P. syringae pv. pisi (. Pc1. 0.75. ) DNA. 24 hr. 0.88. BCRC 12621). 30. 10 cfu/ml. P.. 14. cichorii (. 9. (. P.. Pc12. SfL1 / SfR2. PCR. 3. 8. Pc1 Pc9. SfL1/SfR2. 10 cfu/ml 4~6. Pc3. 0.94. A. 8. (. Pc2. cichorii (syringe). #183 HV-075. Mannitol. Biolog Identification System. 21 (. D-Trehalose. f8-. PCR. 1,502 bp. DNA. DNA. C) BLAST database. NCBI. SDSC-Biology Workbench Pc1. Pc9. Pc12. P. cichorii 16S rDNA partial sequences (GenBank accession number EF101976 for P. cichorii strain IP1-05.
(4) 104. A D. 18. B C E F. 2. A D. 2009. B. C 2. ( (. ) ). E ( ) F ( ) Fig. 1. Symptoms of bacterial midrib rot of lettuce. A, B, C : Symptoms of leaf lettuce (var. crispa) in fields. D, E, F : Symptoms of lettuce inoculated with Pseudomonas cichorii. D : 1 day after inoculation of stem lettuce (var. augustana),. irregular brown spot on leaf E : 1 week after inoculation of leaf lettuce (var. crispa), rot on leaf and midrib; F: 1 week after inoculation of leaf lettuce (var. crispa), dry rot on leaf..
(5) 105 and AJ308302 for P. cichorii strain ICMP 5707T) (identity). rDNA Pc1. Pc9. Pc12. 16S. 15. 99%. 4. P. syringae 16S rDNA. ). partial sequences (GenBank accession number DQ318868 for P. syringae strain 17) Pc9. Pc12. PGO25. 98%. Pc1 93%. 11. #183 076. EU196772 for P. syringae strain. 16S rDNA. HV-075. A. ) (. Pc9. (. 4~6. #183. HV-075. (. A). ) (. A. A. 2. D). (. E). 12. ) ). ). ( 4. Table 1. Physiological and biochemical characters of strains isolated from lettuce Character Strains from lettuce 1 Pseudomonas cichorii 2 3 KOH test G( ) G ( )3 3 O/F test O O3 Catalase Colonies yellow on YDC Colonies mucoid on YDC at 30 Diffusible non-fluorescent pigments on KB Fluorescent pigment on KB Flagellar number 1 1 Oxidase Arginine dihydrolase Gelatin hydrolysis Levan Pectolytic activity Tobacco HR Utilization of : Cellobiose Mannitol L-Rhamnose Sorbitol Sucrose D-Trehalose 2 3. ). 9 (. 1. HV3. 2. 7 (. (. A. 16 hr 2. ). 3 4. 7. ). HVA. 4. Pc12 076. (. A. A. ( Pc1. (. The tested strains isolated from lettuce were Pc 1, 2, 3, 9 and 12. Data are combined by referring to Braun-Kiewnick, et al., 2001 (4). George, et al., 2005 (12). and Schaad, 2001 (31). G (-) , Gram negative; O, oxidative; F, formation; , positive reaction; , negative reaction; V, 21~79 positive.. P. syringae 2 G ( )3 O3. 1. V.
(6) 106. 18. 2. 2009. Pseudomonas cichorii Table 3. Resistance of commercial varieties of lettuce to Pseudomonas cichorii ( ) , 0/9 , 6/9 , 5/9 , 0/9 , 0/9 , 0/9 (. ). (. ). , 0/9 , 0/9 , 0/9 , 0/9 183. HV-075. A. HV-076. A. , 0/9 , 1/9 , 2/9 , 0/9 , 0/9 , 1/9 , 2/9 , 1/9 , 0/9 , 2/9 , 1/9. A A 3. 16S rDNA P. cichorii 99% (. 16S rDNA. P. syringe. ). 93 ~ 98% rDNA. (group) Biolog. (12). George. (. ). 84 ~ 87%. 16S rDNA. P. cichorii Biolog. P. 16S. P. syringe. cichorii. Pseudomonas. P. SfL1 / SfR2. Hseu. KB. (16). SfL1 / SfR2. Pseudomonas. P. agarici P. marginalis. P. viridiflava. P. cichorii. P. savastanoi. P. syringe. 15%. DNA P. cichorii. P. cichorii. (4). Biolog. P. syringe 0.74~0.94. P. syringe. NCBI DNA. PCR P. syringe. (16). P. cichorii. P.. (4). P. agarici. cichorii. Bootstrap. P. (12). rDNA P. cichorii. P. tolaasii. 16S. P. syringe. syringe. Pseudomonas. fuscovaginae. Pseudomonas. P. cichorii. syringe. cichorii. 16S rDNA. P. cichorii P. syringe. (16). PCR. P. P.. DNA. 379 bp ) (pathovars). DNA. (. P. syringe pv. lachrymans (. ). pv..
(7) 107 phaseolicola ( ). ). pv. pisi (. pv. syringae ( ). ). pv. tabaci (. pv. tomato (. ). P. cichorii. 6. P.. syringe. P. cichorii P. syringe Biolog. PCR. Su. 16S rDNA. P. cichorii. 30-35. 72 hr (32). P. cichorii P. cichorii. (33). Swingle 1925. (19). P. cichorii. (6, 9, 10, 14). (34). (34). (11). (19). (25). (29) (23). ( 21. bacterial rot. midrib rot. bacterial stem rot (32). P. cichorii. varnish spot. (. P. cichorii. ). (. (17). ). (. (. ). A. ). ) A(. #183 ). HV-075. 12. P. cichorii bacterial rot (3, 6, 15) (bacterial stem rot). P. cichorii. (10). midrib rot. HV-076 (9). A (. (varnish spot) (14). ). 3. (. A ). 9 P. cichorii. PCR SfL1 / SfR2 M 100 bp DNA marker 1 negative control 2 Pseudomonas cichorii ( ) 3 P. syringae pv. lachrymans ( ) 4 P. syringae pv. phaseolicola ( ) 5 P. syringae pv. pisi ( ) 6 P. syringae pv. syringae ( ) 7 P. syrinaeg pv. tabaci ( ) 8 P. syringae pv. tomato ( ) 9 Sf 010 ( P. cichorii) 10~16 Pc 1~7 ( P. cichorii) Fig. 2. Identification of Pseudomonas cichorii from lettuce by PCR with primer pair SfL1 / SfR2. Lane M, 100 bp DNA marker; lane 1, negative control; lane 2, P. cichorii; lane 3, P. syringae pv. lachrymans ; lane 4, P. syringae pv. phaseolicola; lane 5, P. syringae pv. pisi; lane 6, P. syringae pv. syringae; lane 7, P. syrinaeg pv. tabaci; lane 8, P. syringae pv. tomato; lane 9, Sf 010 (bacterial leaf spot of sunflower, P. cichorii); lanes 10~16, Pc 1~7 (bacterial midrib rot of lettuce, P. cichorii)..
(8) 108. 18. 2. 2009 Burkholder. Grogan. P. cichorii bacterial rot. (6). varnish spot (14). Cottyn P. cichorii. Dhanvantari. butterhead lettuce (. (10). ). midrib rot (9). Burkholder P. cichorii. (14). 1977 (10). (3). (6). 1954 (15). 1996. 1990. 2003. (9). 2009. P. cichorii 90 P. cichorii (varnish spot) (14) midrib rot. (9). bacterial rot. (bacterial stem rot) (10) (3, 6, 15). P. cichorii. (LITERATURE CITED) 1. Agriculture and Food Agency. 2002. Crop yields of the order query-Lettuce. Retrieved June 12, 2008, from Agricultural report resources network on the world wide web: http://agr.afa.gov.tw/afa/afa_frame.jsp (in Chinese) 2. Alvarez, J., Datnoff, L. E., and Nagata, R. T. 1992. Crop rotation minimizes losses from corky root in Florida lettuce. Hortscience 27: 66-68. 3. Aysan, Y., Sahin, S., Ulke, G., and Sahin, F. 2003. Bacterial rot of lettuce caused by Pseudomonas cichorii in Turkey. Plant Pathol. 52: 782. (Abstract) 4. Braun-Kiewnick, A., and Sands, D. C. 2001. C. Pseudomonas. Pages 84-107 in: Laboratory guide for identification of plant pathogenic bacteria. 3 rd ed. Schaad, N. W., Jones, J. B., and Chun, W. ed. APS, St. Paul, USA. 373 pp. 5. Brown, N. A. 1918. Some bacterial diseases of lettuce. J. Agric. Res. 13: 367-388. 6. Burkholder, W. H. 1954. Three bacteria pathogenic on head lettuce in New York State. Phytopathology 44: 592-596. 7. Ceponis, M. J., Kaufman, J., and Butterfield, J. E. 1970. Relative importance of gray mold rot and bacterial soft rot of western lettuce on the New York. market. Plant Dis. Rep. 54: 263-265. 8. Chen, Y. K., and Chen, M. J. 1994. Lettuce leafroll mosaic-a new lettuce disease caused by caulimoviruslike agent in Taiwan. Plant Pathol. Bull. 3: 209-215. 9. Cottyn, B., Heylen, K., Heyrman, J., Vanhouteghem, K., Pauwelyn, E., Bleyert, P., Van Vaerenbergh, J., H fte, M., De vos, P., and Maes, M. 2009. Pseudomonas cichorii as the casual agent of midrib rot, an emerging disease of greenhouse-grown butterhead lettuce in Flanders. Syst. Appl. Microbiol. 32: 211-225. 10. Dhanvantari, B. N. 1990. Occurrence of bacterial stem rot caused by Pseudomonas cichorii in greenhousegrown lettuce in Ontario. Plant Dis. 74: 394. (Abstract) 11. Englhard, A. W., Mellinger, H. C., Ploetz, R. C., and Miller, J. W. 1983. A lesf spot of florists' geranium incited by Pseudomonas cichorii. Plant Dis. 67: 541544. 12. George, M. G. Julia, A. B., and Timothy, L. 2005. Family I. Pseudomonadaceae Pages 323-377. in: Bergey's manual of systematic bacteriology. part c. George ed. Springer. N.Y, USA.1388 pp. 13. Grogan, R. G. 1980. Control of lettuce mosaic with virus. Plant Dis. 64: 446-449. 14. 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. Phytopathology 67: 957-960. 15. Hikichi, Y., Saito, A., and Suzuki, K. 1996. Infection sites of Pseudomonas cichorii into head leaf of lettuce. Ann. Phytopathol. Soc. Jpn. 63: 125-129. 16. Hseu, S. H., Shentue, H., and Lin, C. Y. 2006. Development of species specific PCR primers for identification of Pseudomonas cichorii. Plant Pathol. Bull. 15: 275-285.(in Chinese with English abstract) 17. Hseu, S. H., Shih, S. C., Ding, P. F., and Lin C. Y. 2004. Characterization of sunflower bacterial leaf spot and its bacteriocide screening. Plant Pathol. Bull.13: 329-334. (in Chinese with English abstract) 18. Huang, J. H. 2005. Vegetables disease of Asteraceae. (plant protection). Pages 175-176 in: Taiwan agriculture encyclopedia. vol. 3. Harvest Farm Magazine of non-profit organization, Ye ed. Taipei, 520 pp. (in Chinese) 19. Jones, J. B., Raju, B. C., and Engelhard, A. W. 1984. Effects of temperature and leaf wetness on development of bacterial spot of geraniums and chrysanthemums incited by Pseudomonas cichorii. Plant Dis. 68: 248-251. 20. Lin, H. J., and Huang, J. W. 2002. A semiselective medium for detecting Acremonium lactucae, the causal.
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(10) 110. 18. 2. 2009. ABSTRACT Hseu, S. H. 1, Lai, W. C. 1, and Lin, C. Y. 2, 3 2009. Identification of the causal agent of lettuce bacterial midrib rot and resistance screening of lettuce cultivars. Plant Pathol. Bull. 18 101-110. (1 Plant Pathology Division, Taiwan Agricultural Research Institute, Council of Agriculture, 2 Health Science College, Asia University, Wufeng, Taichung, Taiwan, ROC, 3 Corresponding author, E-mail: yihlin@asia.edu.tw) An unknown bacterial disease was first found in lettuce cultivation areas of Changhua during summer of 2007 and 2008. Symptoms mainly occurred in the midrib of leaves and started with greenish, brown or black spots and later coalesced and developed leaf rot symptoms. Under humid condition, symptoms development could be rapid, often in less than 24 hours, and always occured on nearly mature plants before harvest. The causal agent was identified as Pseudomonas cichorii based on physiological and chemical tests, Biolog GN MicroPlate Identification System, 16S rDNA sequence analysis, and pathogenicity tests. The pathogen was further confirmed by PCR with SfL1 / SfR2 specific primers to Pseudomonas cichorii. This is the first report of the disease occurred on lettuce in Taiwan. In a disease resistance screening of the twenty one local lettuce cultivars, the result showed that all tested cultivars were sensitive to the disease but the romaine and stem lettuce showed moderate degree of resistance. Key words. lettuce, bacterial midrib rot, Pseudomonas cichorii, variety resistance.
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