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1 식물병연구 Research Article Open Access Res. Plant Dis. 20(2) : (2014) Suppression of Citrus Canker by Pretreatment with Rhizobacterial Strains Showing Antibacterial Activity 1 1 1,2 * 1, 2 *Corresponding author Tel : Fax: ycjeun@jejunu.ac.kr Ji Seun Yang 1, So Young Kang 1 and Yong Chull Jeun 1,2 * 1 Faculty of Bioscience and Industry, Jeju National University, Jeju , Korea 2 The Research Institute for Subtropical Agriculture and Biotechnology, Jeju National University, Jeju , Korea Received April 30, 2014 Revised May 20, 2014 Accepted May 21, 2014 Citrus canker caused by Xanthomonas citri subsp. citri (Xcc) is one of the most important diseases on citrus. Although Satsuma mandarin cultivating mostly in Korea is moderately resistance to canker, occurrence of the disease were more frequently reported since last decade. Like other diseases in citrus, citrus canker was mainly protected by chemical fungicide in the field. Due to the side effect of the chemicals, alternative method of disease control is recently required. In this study four rhizobacterial strains TRH423-3, MRL408-3, THJ609-3 and TRH415-2 are selected by testing its antifungal activity against Xcc. Pre-inoculation with the selected rhizobacterial strains caused disease suppression on the citrus leaves after inoculation with the citrus canker pathogen. Similarly, in the field test symptoms of citrus canker were less developed in the citrus trees applied several times with the selected rhizobacterial strains compared with those of untreated trees. Therefore, it is suggested that the selected rhizobacterial strains may be valuable as an alternative method in the environmentfriendly citrus farm. Keywords : Antibiotics, Bacterial disease, Biological control, Jeju Island mm.,., 6.5 m/s (Timmer, 1991). Research in Plant Disease The Korean Society of Plant Pathology pissn , eissn DNA Xanthomonas citri, X. fuscans, X. alfalfae. X. citri X. citri subsp. citri X. citri subsp. malvacearum, X. fuscans X. fuscans subsp. fuscans X. fuscans subsp. aurantifolii, X. alfalfae X. alfalfa subsp. alfalfae X. alfalfa subsp. citrumelonis (Schaad, 2006). X. citri subsp. citri (Shiotani, 2009).. This is an open access article distributed under the terms of the Creative Commons Attribution License ( which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

2 102 Research in Plant Disease Vol. 20 No. 2...,., (Hynes Boyetchko, 2006; Timmer, 1991).,,.,.,,,. 감귤궤양병균분리와동정. (: ). 55 mm 1% sodium hypochlorite solution(naocl) % ethanol % peptone 1 ml ml tryptic soy agar(tsa; Becton, Dickson and Company, France) 281 o C 3, colony. colony loop tryptic soy broth(tsb) 28 o C rpm. total DNA NucleoSpin kits(macherey-nagel, Germany),. X. citri subsp. citri primer primer-2(5-cacgggtgcaaaaaatct-3) primer-3(5-tggt- GTCGTCGCTTGTAT-3) (Shiotani, 2009). PCR 2 ml DNA(5-10 ng/ml), 2.5mM dntp 1 ml, 10pmol primer 1 ml, 10Buffer 4 ml, 5 unit/ml Taq DNA polymerase(intron Co., Korea) 1 ml, 40 ml. PCR PCR Thermal Cycler Dice TP600(TaKaRa, Japan) 95 o C 10, 95 o C 70, 58 o C 60, 72.0 o C 60 30, 72.0 o C 2. DNA 8 ml ethidium bromide(etbr) 1% agrose gel UV. DNA elution NucleoSpin kits(macherey-nagel, Germany). DNA sequence Genetic Analyzer(Applied Biosystems 3130xl, USA) Applied Biosystems(AB, version 1.0) National Center for Biotechnology Information(NCBI) Basic Local Alignment Search Tool (Blast; nih.gov/blast.cgi). 항균활성에의한식물근권세균선발 Lee (2003) 200 (Kim Jeun, 2006; 2007), (Ko, 2012), (Kim, 2011), (An, 2010) 12. in vitro. TSA cfu/ml 50 ml 1. TSB rpm, paper disc(8 mm) 70 ml. Streptomycin( 20%,, ) 10 g/l. 28 o C 2 (inhibition zone) 1. 실내에서의식물근권세균처리에의한감귤궤양병진전억제. (: ) 20 6

Research in Plant Disease Vol. 20 No. 2 103. 4 MRL408-3, TRH423-3, TRH415-2, THJ609-3 3.010 7 cfu/ml 0.01% Tween 20. streptomycin 1 g/l. 3 3.010 7 cfu/ ml. 28 o C dew chamber 24 281 o C 14. 3, 14.

3 Research in Plant Disease Vol. 20 No MRL408-3, TRH423-3, TRH415-2, THJ cfu/ml 0.01% Tween 20. streptomycin 1 g/l cfu/ ml. 28 o C dew chamber o C 14. 3, 14. Duncan (P = 0.001), Statistical Analysis System(SAS Institute, version 8.02) program. [ /]. 감귤궤양병균분리. TSA 2 colony, 3 colony. DNA primer-2 primer bp (Fig. 1). NCBI Gene Bank database DNA 100% X. axonopodis pv. citri str (Fig. 1). X. citri subsp. citri (Gottwald, 2009) cfu/ml cfu/ml ( ). 포장에서식물근권세균처리에의한감귤궤양병진전억제., streptomycin, TRH423-3, MRL408-3, THJ609-3 TRH m 3, cfu/ml cfu/ml (%) = [( /)100]. Duncan (P = 0.001), Statistical Analysis System(SAS Institute, version 8.02) program. 항균활성에의한식물근권세균선발. TSA. 12 MRL408-3, TRH423-3, THJ609-3 TRH , streptomycin 0.1 g/l (Fig. 2).., Fusasrium oxysporum. 2,4-diacetyphloroglucinol, pyoluteorin, pyrrolnitrin (Raaijmakers, 1995). Fig. 1. Gel electrophoresis of PCR amplified 16S/23S internal transcript spacer regions (ITS) from Xanthomonas citri pv. citri (left) and ITS sequencing based on rdna of X. citri pv. citri and blast results on the web-site (right). The presented gel electrophoresis of PCR product bands was using primer-2(5 -CACGGGTGCAAAAAATCT-3 )and primer-3(5 -TGGTGTCGTCGCTTGTAT-3 ) (lane 1 and 2). M: 1kb molecular size marker.

104 Research in Plant Disease Vol. 20 No. 2 Fig. 2. Growth inhibition of Xanthomonas criti subsp. citri by the selected bacterial 4 isolates on TSA medium.

The concentration of anti-bacterial isolates and Streptomycin were 1.0 10 7 cfu/ml and 10 g/l, respectively. Fig. 3.

4 104 Research in Plant Disease Vol. 20 No. 2 Fig. 2. Growth inhibition of Xanthomonas criti subsp. citri by the selected bacterial 4 isolates on TSA medium. The presented TSA plates were untreated control (H 2O) (A), treated with MRL408-3 (B), TRH423-3 (C), TRH415-2 (D), THJ609-3 (E) and commercial fungicide Streptomycin (F). The concentration of anti-bacterial isolates and Streptomycin were cfu/ml and 10 g/l, respectively. Fig. 3. Disease severity on Satsuma mandarin leaves pre-treated with H 2O (A), rhizobacterial isolate MRL408-3 (B), TRH423-3 (C), and commercial fungicide Streptomycin (D) at 14 days after inoculation with citrus canker pathogen Xanthomonas criti subsp. citri. The concentration of the bacterial isolates and X. criti subsp. citri were cfu/ml and the fungicide was 1 g/l, respectively. MRL408-3, TRH 423-3, TRH415-2 THJ609-3 ITS 38F 72R primer rdna, MRL408-3 TRH423-3 Burkholderia gladioli, TRH415-2 Pseudomons fluorescens, THJ609-3 Pseudomonas pudia (Ko, 2012). 실내에서의식물근권세균처리에의한감귤궤양병진전억제. X. citri subsp. citri (Fig. 3). (Fig. 3). TRH % THJ609-3 (Table 1). MRL408-3 TRH % (Table 1). MRL408-3, TRH423-3 THJ609-3, (Fig. 2). THJ609-3 Table 1. Lesion numbers on Satsuma mandarin leaves pre-treated with H 2O, the selected rhizobacterial strains and commercial fungicide Streptomycin after inoculation with Xanthomonas citri subsp. citri Treatment a Lesion number Duncan s-test Control ± c a d MRL ± bc TRH ± 55.7 bc TRH ± bc THJ ± b Streptomycin b 0.0 ± 0.0 c a The concentration of the bacterial isolates suspension and citrus canker pathogen X. citri subsp. citri were cuf/ml. The control was pre-treated with sterilized distilled water instead of the bacterial isolates. b The concentration of the fungicide was 1 g/l. c Values represent means ± standard error of three separated experiments, each containing three plants of six leaves per treatment. d The different letters are significantly (P = 0.001) different according to Duncan s multiple test.., THJ609-3 (Table 1).

Research in Plant Disease Vol. 20 No. 2 105 Fig. 4. Disease severity on citrus leaves at 7 days after inoculated with Xanthomonas citri subsp. citri (1.0 10 8 cfu/ml) in the field experiment.

5 Research in Plant Disease Vol. 20 No Fig. 4. Disease severity on citrus leaves at 7 days after inoculated with Xanthomonas citri subsp. citri ( cfu/ml) in the field experiment. Bacterial suspension of the isolates was pre-inoculated on citrus leaves 7 times before inoculation with X. citri subsp. citri. The presented leaves were untreated control (A), pre-sprayed with MRL408-3 (B), TRH423-3 (C), TRH415-2 (D), THJ609-3 (E) and pre-treated with commercial fungicide Streptomycin (F). The concentration of bacterial isolates and Streptomycin were cfu/ml and 1 g/l, respectively.. B. cereus TRL2-3.,,. callose (Jeun, 2004), siderophore, exopolysaccharide, lipopolysaccaride(lps) (Jeun, 2004; Jung, 2007) (Joo, 2002). 포장에서식물근권세균처리에의한감귤궤양병진전억제. 4 MRL408-3, TRH423-3, THJ609-3 TRH415-2(Ko, 2012) (: ) 8, (%). 68.7% (%), 3-4, , ( ). Table 2. Suppression of infected leaves on citrus trees by treatment with the selected bacterial isolates in the citrus orchard Treatment a (Table 2). THJ609-3 TRH %, MRL408-3 TRH % (Table 2). 1, ( ). Rate of infected leaves (%) Duncan s multiple test Untreated 68.7 ± 4.3 c a d MRL ± 9.7 bc TRH ± 12.3 b TRH ± 2.8 c THJ ± 2.7 c Streptomycin b 1.3 ± 0.3 c a The bacterial isolates were pre-inoculated 7 times before pathogen inoculation with Xanthomonas citri subsp. citri ( cfu/ml). The untreated control was sprays-inoculated with an only X. citri subsp. citri. The concentration of the bacterial isolates suspension was cfu/ml. The bacterial isolates suspension and the commercial fungicide Streptomycin were treated four times. b The concentration of the fungicide was 1 g/l. c Values represent, means and standard error calculated using two hundred leaves per tree and three trees per treatment. d The different letters are significantly (P = 0.001) different according to Duncan s multiple test. B. gladioli Colletotrichum orbiculare Pythium ultimum (Bae, 2007; Raupach Kloepper, 1998).

6 106 Research in Plant Disease Vol. 20 No. 2 P. putida WCS358 (Meziane, 2005). B. gladioli MRL408-3, TRH Pseudomons TRH415-2, THJ609-3 in vivo,. Xanthomonas citri subsp. citri (Xcc) Xcc TRH423-3, MRL408-3, THJ609-3, TRH ,.. Acknowledgement This research was supported by 2013 Grant for Export of Fresh Agricultural Products of Animal, Plant and Fisheries Quarantine and Inspection Agency/Ministry for Food, Agriculture, Forestry and Fisheries, Republic of Korea. References An, Y. J., Kang, S. Y., Kim, K. D., Hwang, B. K. and Jeun, Y. C Enhanced defense responses of tomato plants against late blight pathogen Phytophthora infestans by pre-inoculation with rhizobacteria. Crop Prot. 29: Bae, Y. S., Park, K. S. and Choi, O. H Laboratory culture mediadependent biocontrol ability of Burkholderia gladioli strain B543. Plant Pathology J. 23: 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 The epidemiological significance of post-packinghouse survival of Xantnomonas citri subsp. citri for dissemination of Asiatic citrus canker via infected fruit. Crop Prot. 28: Hynes, R. K. and Boyetchko, S. M Research initiatives in the art and science of biopesticide formulations. Soil Biol. Biochem. 38: Jeun, Y. C., Park, K. S. Kim, C. H. Fowler, W. D. and Kloepper, J. W Cytological observations of cucumber plants during induced resistance elicited by rhizobacteria. Biol. Control 29: Joo, G. J., Kim, J-H. and Kang, S. J Isolation of antagonistic Bacillus ehimensis YJ-37 and its antifungal activity against vegetables damping-off fungi. Korean J. Life Sci. 12: Jung, H. K., Kim, J. R., Woo, S. M. and Kim, S. D Selection of the auxin, siderophore, and cellulase-producing PGPR, Bacillus licheniformis K11 and its plant growth promoting mechanisms. J. Korean Soc. Appl. Biol. Chem. 50: Kim, H. J. and Jeun, Y. C Resistance induction and enhanced tuber production by pre-inoculation with bacterial strains in potato plants against Phytophthora infestans. Mycobiology 34: Kim, H. J. and Jeun, Y. C Infection structures on the infected leaves of potato pre-inoculated with bacterial strains and DL- 3-amino butyric acid after challenge inoculation with Phytophthora infestans. Plant Pathology J. 23: Kim, S. Y., Hyun, J. W. and Jeun, Y. C Suppression effect and mechanism of citrus scap in the citrus pre-inoculated with rhizobacterial strains. Res. Plant Dis. 17: (In Korean) Ko, Y. J., Kang, S. Y. and Jeun, Y. C Suppression of citrus melanose on the leaves treated with rhizobacteria strains after inoculation with Diaporthe citri. Res. Plant Dis. 18: (In Korean) Lee, C. S., Kim, K. D., Hyun, J. W. and Jeun, Y. C Isolation of Rhizobacteria in Jeju Island showing anti-fungal effect against various plant pathogens. Mycobiology 31: Meziane, H., van der Sluis, I., van Loon, L. C., Hoefte, M. and Bakker, P. A. H. M Determinants of Pseudomonas putida WCS358 involved in inducing systemic resistance in plants. Mol. Plant Pathol. 6: Raaijmakers, J. M., Leeman, M., van Osrschot, M. M. P., van der Sluis, I., Schippers, B. and Bakker, P. A. H. M Dose-response relationships in biological control of Fusarium wilt of radish by Pseudomonas spp. Phytopathology 85: Raupach, G. S. and Kloepper, J. W Mixtures of plant growth promoting rhizobacteria enhance biological control of multiple cucumber pathogens. Phytopathology 88: Schaad, N. W., Postnikova, E., Lacy, G., Sechler, A., Agarkova, I., Stromberg, P. E., Stromberg, V. K. and Vidaver, A. K Emended classification of xanthomonad pathogens on citrus. Syst. Appl. Microbiol. 29: Shiotani, H., Uematsu, H., Tsukamoto, T., Shimizu, Y., Ueda, K., Mizuno, A. and Sato, S Survival and dispersal of Xanthomonas citri subsp. citri from infected Satsuma mandarin fruit. Crop Prot. 28: Timmer, L. W., Gottwald, T. R. and Zikto, S. E Bacterial exudation from lesions of Asiatic citrus canker and citrus bacterial spot. Plant Dis. 75:

284 Research in Plant Disease Vol. 20 No. 4. (Shiotani, 2009). (Gottwald, 2009; Hyun, 2012) (Behlau, 2010; Gottwald Timmer, 1995). (Penicillium digita

284 Research in Plant Disease Vol. 20 No. 4. (Shiotani, 2009). (Gottwald, 2009; Hyun, 2012) (Behlau, 2010; Gottwald Timmer, 1995). (Penicillium digita 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