식물병연구 Research Article Open Access Res. Plant Dis. 20(4) : 283-288(2014) http://dx.doi.org/10.5423/rpd.2014.20.4.283 Inactivation of Xanthomonas citri subsp. citri and Effect on Infection of Citrus Canker by Gamma Irradiation 1 1 2 3 1,2 * 1, 2, 3 *Corresponding author Tel : +82-64-754-3319 Fax: +82-64-725-2351 E-mail: ycjeun@jejunu.ac.kr Kyung Nam Kim 1, Min A Song 1, Sang Heon Han 2, Seong Joon Song 3 and Yong Chull Jeun 1,2 * 1 Major of Plant Resources & Environment, Jeju National University, Jeju 690-756, Korea 2 Major of Horticultural Science, The Research Institute for Subtropical Agriculture and Biotechnology, Jeju National University, Jeju 690-756, Korea 3 Institute for Nuclear Science and Technology, Jeju National University, Jeju 690-756, Korea Received October 12, 2014 Revised December 2, 2014 Accepted December 4, 2014 Citrus canker caused by Xanthomonas citri subsp. citri (Xcc) has been quarantined by many countries in the world. Recently, the usage of methyl bromide should be limited, application by gamma irradiation on the agricultural production is raised as an alternative method. In this study, the level of gamma irradiation which could decrease of population of Xcc in the suspension or on the surface of citrus fruit was investigated. The D 10 value of Xcc, which is radiation dose required to reduce the number of the microorganism, was 55 and 28 Gy in the suspension and on the surface of citrus fruit, respectively. Furthermore, disease severity was suppressed on the citrus leaves inoculated with Xcc suspension pre-treated with gamma irradiation. Based on this study, it is suggested that Xcc on the citrus fruit could be eradicated by gamma irradiation and the results of this study may be valuable for application of gamma ray in quarantine activity. Keywords : Environment friendly, Ionization, Jeju Island, Radio activity, Sterilization Xanthomanas citri subsp. citri (Parnell, 2009).,, (Schubert, 2001). (Satsuma Research in Plant Disease The Korean Society of Plant Pathology pissn 1598-2262, eissn 2233-9191 mandarin) (Shiotani, 2009).. 2006 (Dewdney Graham, 2014). This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
284 Research in Plant Disease Vol. 20 No. 4. (Shiotani, 2009). (Gottwald, 2009; Hyun, 2012) (Behlau, 2010; Gottwald Timmer, 1995). (Penicillium digitatum) (Eckert Eaks, 1989). imazalil, thiabendazole, sodium o-phenylphenate (Holmes Eckert, 1999; Palou, 2002; Smilanick, 2006). methyl bromide (FAO, 2003) methyl bromide., 50 (Farkas Farkas, 2011).,,, (Andres, 2007; Aquino, 2012; Froelicher, 2007) (Mahmoud, 2010)... bio-test. 감귤궤양병균현탁액에감마선처리. ( ) X. citri subsp. citri N5-1(Yang, 2014)., DNA ITS. tryptic soy broth(tsb; Becton, Dickson and Company, France) 28 o C (HB-201SL, Hanbaek Scientific Co., Korea) 120 rpm 3. (Bright-Line Hemacytometer, Hausser Scientific) 1.0 10 7 cfu/ml. 5 15 ml conical tube 10 ml. 60 Co g-ray irradiator(c-188 irradiator, MDS Nordion, Canada) 100, 200, 300, 400 Gy., 100%, 18 o C. 감귤생과실에감마선처리... 1.0 10 7 cfu/ml. 1.., () Turn table(vmd, Korea) 3/min. 30, 50, 70, 100 Gy. 감마선효과검증 D 10 value 산정. 0, 100, 200, 300, 400 Gy 100 ml semi selective medium(ssm, Cleci, 2009) 28 o C 72. 0, 30, 50, 70, 100 Gy 150 ml 500 ml 25 o C 150 rpm 2 100 ml SSM 28 o C 72.. D 10 value. D 10 value 90%
Research in Plant Disease Vol. 20 No. 4 285 10 (Whitby Gelda, 1979). D 10 value = D t Log 10 N 0 - Log 10 N t D t = (Radiation dose) N 0 = N t = D t 3 3. 감마선을처리한감귤궤양병균의병원성조사. (: ) 1% (NaOCl) 30.. 50, 100, 200 Gy (1730MR, Gyrozen) 3,000 rpm 10 3,, 1.0 10 7 cfu/ml 100ml tween20( 0.01% ) 10 ml. 1.0 10 7 cfu/ml. 28 o C 100% 24 28 ± 1 o C. 3 3. 14. Duncan(P = 0.01), Statistical Analysis System(SAS Institute, version 8.02) program. 감마선에의한감귤궤양병균불활성화.. 100 Gy 100 (Fig. 1). 300 400 Gy (Fig. 1). Fig. 1. Number of Xanthomonas citri subsp. citri in the suspension by treatment of different level of gamma irradiation. The concentration of the bacterial suspension was 1.0 10 7 cfu/ml. The vertical bars indicated the standard deviation of three replications of the bacterial suspensions. 90%, D 10 value 53 58 Gy (Table 1). D 10 value,,, (Aquino, 2012).. (OH - ) (e - ) (H + ). OH -
286 Research in Plant Disease Vol. 20 No. 4 Table 1. The D 10 values of Xanthomonas citri subsp. citri in the suspension or in the surface of citrus fruits X. citri subsp. citri Experiment D 10 value (Gy) R 2 Suspension * 2 55 0.9064 1 58 0.9546 3 53 0.9296 Fruit ** 2 31 0.9137 1 29 0.9327 3 25 0.9618 *The concentration of Xanthomonas citri subsp. citri was 1.0 10 7 cfu/ml. **The fruits were sprayed with the suspension of Xanthomonas citri subsp. citri in the concentration of 1.0 10 7 cfu/ml and dried at the room temperature.. 감마선처리에의한감귤생과실의소독효과.. 30 Gy 10 100 Gy (Fig. 2). Fig. 2. Number of Xanthomonas citri subsp. citri on the surface of citrus fruit by treatment of different level of gamma irradiation. The citrus fruits were sprayed with the bacterial suspension at the concentration with 1.0 10 7 cfu/ml before irradiation with gamma ray. The vertical bars indicated the standard deviation of three replications of the bacterial suspensions.. OH - DNA (hydration layer) DNA (Borrely, 1998).. D 10 value 60 Gy D 10 value 29 31 (Table 1).. (Thomas, 1988)., (Mahrouz, 2002), (Mahmoud, 2010). D 10. D 10 value 30 Gy 100 Gy. 감마선에의한감귤궤양병균의병원성변화.. 50 Gy
Research in Plant Disease Vol. 20 No. 4 287 Fig. 3. Disease severity on Satsuma mandarin leaves non-treated (A), pre-treated with 50 (B), 100 (C) and 200 (D) Gy of gamma irradiation at 14 days after inoculation with citrus canker pathogen Xanthomonas criti subsp. citri. The concentration of the suspension of X. criti subsp. citri was 1.0 10 7 cfu/ml.... Fig. 4. Number of lesions on Satsuma mandarin leaves pre-treated with different level of gamma irradiation at 14 days after inoculation with citrus canker pathogen Xanthomonas criti subsp. citri. The concentration of the suspension of X. criti subsp. citri was 1.0 10 7 cfu/ml. 100 Gy 200 Gy (Fig. 3, 4). 1.0 10 7 cfu/ml (Yang, 2014). Botrytis elliptica (Kim Yun, 2014). 1.5 kgy Nagpur Kagzi Mosambi Nagpur Penicillium (Ladaniya, 2003). Xanthomonas citri subsp. citri. methyl bromide.. 90% D 10 value 55 28 Gy... Acknowledgement This research was supported by 2014 Export Promotion Technology Development Program, Ministry for Agriculture, Food and Rural Affairs. References Andres, V. S., Ortego, F. and Castañera, P. 2007. Effects of gammairradiation on midgut proteolytic activity of the mediterranean
288 Research in Plant Disease Vol. 20 No. 4 fruit fly, Ceratitis capitata (Diptera: Tephritidae). Arch. Insect Biochem. 65: 11-19. Aquino, K. A. S. 2012. Sterilization by gamma irradiation, gamma radiation, Prof. Feriz Adrovic (Ed.), ISBN: 978-953-51-0316-5, In: INTECH, available. URL http://www.intechopen.com/books/ gammaradiation/sterilization-by-gamma irradiation. Behlau, F., Belasque, J., Graham, J. H. and Leite, R. P. 2010. Effect of frequency of copper applications on control of citrus canker and the yield of young bearing sweet orange trees. Crop Prot. 29: 300-305. Borrely, S. I., Cruz, A. C., Del Mastro, N. L., Sampa, M. H. O. and Somessari, E. S. 1998. Radiation processing of sewage and sudge. A review. Prog. Nucl. Energ. 33: 3-21. Cleci, D., Antonio, C. M., Jose, O. M. and Renata, C. C. 2009. Semi- Selective culture medium for Xanthomonas axonopodis pv.malvacearum detection in cotton seeds (Gossypium hirsutum L.). Asian J. Plant Pathol. 3: 39-49. Dewdney, M. M. and Graham, J. H. 2014. 2014 Florida Citrus Pest Management Guide: Citrus Canker. IFAS Extension, U Fla. 1-5. Eckert, J. W. and Eaks, I. L. 1989. Postharvest disorders and diseases of citrus fruits. In: Reuther, W., Calavan, E., Clair, Carman, G. E., Jeppson, L. R. (Eds.), The Citrus Industry, revised ed. University of California, Division of Agriculture and Natural Resources, Oakland, CA, pp. 179-259. FAO. 2003. Guideline for the use of irradiation as a phytosanitary measures. International Plant Protection Convention, ISPM No. 18, Rome, Italy. Farkas, J. and Farkas, C. M. 2011. History and future of food irradiation. Trends Food Sci. Tech. 22: 121-126. Froelicher, Y., Bassene, J. B., Jedidi-Neji, E., Dambier, D., Morillon, R., Bernardini, G., Costantino, G. and Ollitrault, P. 2007. Induced parthenogenesis in mandarin for haploid production: induction procedures and genetic analysis of plantlets. Plant Cell Rep. 26: 937-944. Gottwald, T., Graham, J., Bock, C., Bonn, G., Civerolo, E., Irey, M., Leite, R., McCollum, G., Parker, P., Ramallo, J., Riley, T., Schubert, T., Stein, B. and Taylor, E. 2009. The epidemiological significance of post-packinghouse survival of Xantnomonas citri subsp. citri for dissemination of Asiatic citrus canker via infected fruit. Crop Prot. 28: 508-524. Gottwald, T. R. and Timmer, L.W. 1995. The efficacy of windbreaks in reducing the spread of citrus canker caused by Xanthomonas campestris pv. citri. Trop. Agric. 72: 194-201. Holmes, G. J. and Eckert, J. W. 1999. Sensitivity of Penicillium digitatum and P. italicum to postharvest citrus fungicides in California. Phytopathology 89: 716-721. Hyun, J. W., Kim, H. J., Yi, P. H., Hwang, R. Y. and Park, E. W. 2012. Mode of action of streptomycin resistance in the citrus canker pathogen (Xanthomonas smithii subsp. citri) in Jeju Island. Plant Pathology J. 28: 207-211. Kim, J. H. and Yun, S. C. 2014. Effect of gamma irradiation and its convergent treatments on lily leaf blight pathogen, Botrytis elliptica and the disease development. Res. Plant Dis. 20: 71-78. (In Korean) Ladaniya, M. S., Singh, S. and Wadhawan, A. K. 2003. Response of Nagpur mandarin, Mosambi sweet orange and Kagzi acid lime to gamma radiation. Radia. Phys. Chem. 67: 665-675. Mahmoud, G. A., El-Tobgy, K. M. K. and Abo-El-Seoud, M. 2010. Application of combined biocides and gamma radiation for keeping good quality stored grapefruits. Arch. Phytopathol. Pfl. 43: 712-721. Mahrouz, M., Lacroix, M., D aprano, G., Oufedjikh, H., Boubekri, C. and Gagnon, M. 2002. Effect of ç-irradiation combined with washing and waxing treatment on physicochemical properties, vitamin C, and organoleptic quality of Citrus clementina Hort. Ex. Tanaka. J. Agric. Food Chem. 50: 7271-7276. Palou, L., Usall, J., Muñoz, J. A., Smilanick, J. L. and Viñas, I. 2002. Hot water, sodium carbonate, and sodium bicarbonate for the control of postharvest green and blue molds of clementine mandarins. Postharvest Biol. Tec. 24: 93-96. Parnell, S., Gottwald, T. R., van den Bosch, F. and Gilligan, C. A. 2009. Optimal strategies for the eradication of Asiatic citrus canker in heterogeneous host landscapes. Phytopathology 99: 1370-1376. Schubert, T. S., Rizvi, S. A., Sun, X., Gottwald, T. R., Graham, J. H. and Dixion, W. N. 2001. Meeting the challenge of eradication citrus canker in Florida-Again. Plant Dis. 85: 340-356. Shiotani, H., Uematsu, H., Tsukamoto, T., Shimizu, Y., Ueda, K., Mizuno, A., and Sato, S. 2009. Survival and dispersal of Xanthomonas citri subsp. citri from infected Satsuma mandarin fruit. Crop Prot. 28: 19-23. Smilanick, J. L., Mansour, M. F., Mlikota Gabler, F. and Goodwine, W. R., 2006. The effectiveness of pyrimethanil to inhibit germination of Penicillium digitatum and to control citrus green mold after harvest. Postharvest Biol. Tec. 42: 75-85. Thomas, P. 1988. Radiation preservation of foods of plant origin. Part VI. Mushrooms, tomatoes, minor fruits and vegetables, dried fruits and nuts. CRC Crit. Rev. Food Sci. Nutr. 26: 313-358. Whitby, J. L. and Gelda, A. K. 1979. Use of incremental doses of cobalt 60 radiation as a means to determining radiation sterilization dose. J. Parent. Drug Assoc. 33: 144-155. Yang, J. S., Kang, S. Y. and Jeun, Y. C. 2014. Suppression of citrus canker by pretreatment with rhizobacterial strains showing antifungal activity. Res. Plant Dis. 20: 101-106. (In Korean)