60 Research in Plant Disease Vol. 24 No. 1 X. euvesicacoria (Kyeon, 2016). (Kim, 2015), copper hydroxide oxadixyl, oxine-copper polyoxin- B, validamyc

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1 식물병연구 Research Article Open Access Res. Plant Dis. 24(1): (2018) Fungicide pyraclostrobin Foliar Application of the Fungicide Pyraclostrobin Reduced Bacterial Spot Disease of Pepper * 1, 2 *Corresponding author Tel: Fax: yckimyc@jnu.ac.kr Beom Ryong Kang 1, Jang Hoon Lee 2, and Young Cheol Kim 1 * 1 Department of Applied Biology, Chonnam National University, Gwangju 61186, Korea 2 BASF Company Ltd., ASK/AP, Seoul 04513, Korea Received February 9, 2018 Revised February 15, 2018 Accepted February 15, 2018 Pyraclostrobin is a broad-spectrum fungicide that inhibits mitochondrial respiration. However, it may also induce systemic resistance effective against bacterial and viral diseases. In this study, we evaluated whether pyraclostrobin enhanced resistance against the bacterial spot pathogen, Xanthomonas euvesicatora on pepper (Capsicum annuum). Although pyraclostrobin alone did not suppressed the in vitro growth of X. euvesicatoria, disease severity in pepper was significantly lower by 69% after treatments with pyraclostrobin alone. A combination of pyraclostrobin with streptomycin reduced disease by over 90% that of the control plants. The preventive control of the pyraclostrobin against bacterial spot was required application 1 3 days before pathogen inoculation. Our findings suggest that the fungicide pyraclostrobin can be used with a chemical pesticide to control bacterial leaf spot diseases in pepper. Keywords: Bacterial spot disease, Induced resistance, Pyraclostrobin, Xanthomonas euvesicatoria (Solanum lycopersicum) 1921 Bacterium vesicatoria (Doidge, 1921) B. exitiosum (Gardner Kendrick, 1921) Xanthomonas vesicatoria X. campestris pv. vesicatoria (Young, 1978) (Capsicum annuum),., amylolytic pectolytic activity X. vesicatoria, X. perforans X. euvesicacoria X. gardneri 4 Research in Plant Disease pissn , eissn (Bouzar, 1994; Jones, 2000, 2004). X. euvesicatoria, X. vesicatoria X. gardneri 3 X. perforans (Jones, 1998), X. perforans (Potnis, 2015) (Kyeon, 2016). X. euvesicatoria (Jones, 2004; Obradovic, 2004). X. euvesicatoria % (Kyeon, 2016; Myung, 2015). X. axonopodis pv. vesicatoria X. vesicatoria (Yoo, 2009), The Korean Society of Plant Pathology This is an open access article distributed under the terms of the Creative Commons Attribution Non-Commercial License ( by-nc/4.0/), which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

2 60 Research in Plant Disease Vol. 24 No. 1 X. euvesicacoria (Kyeon, 2016). (Kim, 2015), copper hydroxide oxadixyl, oxine-copper polyoxin- B, validamycin-a, probenazole, chlorothalonil kasugamycin, oxytetracyclin streptomycin sulfate 19 (Korea Crop Protection Association, 2017)., X. campestris pv. vesicatoria streptomycin (Cooksey, 1990), streptomycin copper 1996 (Lee Cho, 1996).,,..,. acibenzolar-s-methyl, probenazole, b- aminobutyric acid (BABA), 2R,3R-butanediol (Oostendorp, 2001; Schreiber Desveaux, 2008). flavonoid diterpenoid, flavonoid indole,,, sesquiterpene coumarin (Harborne, 1999; Kim, 2005; Koga, 1997).,. (Quinone outside inhibitor, QoI) strobilurin Pyraclostrobin (Min, 2014). Pyraclostrobin,. Pyraclostrobin (Herms, 2002; Skandalis, 2016). (X. perforans) (X. axonopodis pv. phaseoli) Pyraclostrobin metiram acibenzolar-s-methyl (Itako, 2014; Vigo, 2012). Pyraclostrobion PR MAPKs (Herms, 2002; Skandalis, 2016; Udayashankar, 2012). pyraclostrobin. Pyraclostrobin. (X. euvesicatoria 173-1). NB (nutrient broth; Becton Dickinson GmbH, Heidelberg, Germany) 48 (28 o C, 120 rpm) 15% glycerol -80 o C. paper disc. NB agar 28 o C, colony forming units(cfu)/ml 0.7% agar NB. pyraclostrobin, streptomycin, copper hydroxide (Table 1). NB agar paper disc (8 mm, Tokyo filter Co., Utsunomiya-shi, Japan) (pyraclostrobin, 0.25 ml/l; streptomycin, 1.25 g/l; copper hydroxide, 2 g/l; pyraclostrobin/streptomycin, (0.2 ml g)/l; pyraclostrobin/copper hydroxide (0.2 ml + 1 g)/l) 28 o C, 48 APS assess 2.0 imaging software (APS Press, St. Paul, MN, USA). 3. Pyraclostrobin.

3 Research in Plant Disease Vol. 24 No Table 1. List of chemical pesticides used in this study Treatments (active ingredient, %) Volume in 20 l Dilution (fold) Inhibition mechanism Pyraclostrobin EC (22.9) 5 ml 4,000 QoI Streptomycin WP (20) 25 g 800 GA Copper hydroxide WP (77) 40 g 500 Multi-site Pyraclostrobin EC (22.9)+Streptomycin WP (20) 4 ml+10 g 5,000+2,000 QoI+GA Pyraclostrobin EC (22.9)+Copper hydroxide WP (77) 4 ml+20 g 5,000+1,000 QoI+Multi-site EC, Emulsifiable Concentrate; WP, suspension concentrate; QoI, Quinone outside inhibitor; GA, glucopyranosyl antibiotic. 1 2 (Yuan Seed, Seoul, Korea). 1% (NaOCl) 1 3. (Farm Hannong, Jeongup, Korea) (9.5 cm 7 cm 9 cm, Greenday, Bucheon, Korea) (30 o C, 45% ) 6-8. pyraclostrobin, streptomycin, copper hydroxide (Table 1), NB 28 o C, 3 OD 600nm =1.0( CFU/ml), 24 Table o C, 95% ;, 1; 1, 2; 2-5, 3; 6-10, 4; 11. (%) {( ) 100}/( 4), (%) (1- / ) , 5, 7,,. Pyraclostrobin. pyraclostrobin (Hungnong Seed, Seoul, Korea). 1% (NaOCl) 1 (9.5 cm 7 cm 9 cm, Greenday) 6 pyraclostrobin 1, 3, 5, 7 4, (OD 600nm =1.0) 30 o C, 95% IBM SPSS Statistics 23.0 software (IBM Co., Armonk, NY, USA) (ANOVA). F Duncan (Duncan s multiple range test) Pyraclostrobin In vitro. streptomycin 400 X. euvesicatoria, pyraclostrobin (Table 2). copper hydroxide in vitro streptomycin. Pyraclostrobin, streptomycin. Pyraclostrobin. 68.9% (Table 2). 12 pyraclostrobin (Table 3). Pyraclostrobin 21%( 69%), streptomycin

4 62 Research in Plant Disease Vol. 24 No. 1 Table 2. Growth inhibition effect of the pyraclostrobin against Xanthomonas euvesicatoria Treatment Clear zone (mm) Pyraclostrobin 0 Streptomycin Copper hydroxide Pyraclostrobin+Streptomycin Pyraclostrobin+Copper hydroxide 15.3±0.01 a 11.0±0.16 c 12.4±0.35 b 10.9±0.01 c Xanthomonas euvesicatoria was cultured at 28 C on nutrient broth agar for 2 days. Bacterial cells were harvested and suspended with sterile water to 10 4 cfu/ml. The bacterial suspension (5 ml) was added to sterile NB soft-agar medium (0.7% agar) which was transferred to Petri dishes. Sterile paper discs placed at the center of these plate were loaded with defined concentrations of the chemical treatments. After 2 days incubation at 22 C, growth inhibition of X. euvesicatoria was imaged and quantified with APS Assess 2.0 imaging software (APS, St. Paul, MN, USA). The values listed are means of the inhibition zones obtained from three replicated studies. Different letters in the same column indicate the significant difference from the inhibition from streptomycin according to the Duncan s multiple range test at P<0.05. Table 3. Reduction of bacterial spot on pepper by pesticide treatments Treatment Disease incidence (%) Control value (%) Pyraclostrobin 21.2±5.6 b 68.8 Streptomycin 15.1±3.9 ab 78.2 Pyraclostrobin+Streptomycin 06.5±1.4 a 90.4 Control (water) 68.9±8.2 c - Six or eight true leaves developed pepper plants (variety, 1bak-2il) were inoculated with a suspension of 10 8 cfu/ml. Xanthomonas euvesicatoria suspension 24 h after spraying the pesticides or with sterile water as a control. The pathogen was cultured at 28 C by shake culture on nutrient broth for 2 days. Cells were pelleted by centrifugation before suspension in sterile water. After 15 days, disease severity on leaves was assessed using the following scale: 0= no symptoms, 1=one bacterial spot, micro spots, 2=up to 5 bacterial spots, micro spots, 3=5-10 bacterial spots, 4=10>bacterial spots, leaves with burned edge, deformed leaves. Disease incidence % was calculated using the equation: (number of diseased leaves disease severity) 100)+(total number of diseased leaves 4). Control value (%)=(1 percentage of disease incidence in fungicide application/percentage of disease incidence in control) 100. The values rare means of triplicates of three independent experiments. Different letters in the same column indicate significant difference according to Duncan s multiple range tests, P< %( 78%). Streptomycin pyraclostrobin Fig. 1. Preventive effect of pyraclostrobin treatments on disease incidence of bacterial spot in pepper. Inoculation involved methods described in Table 3 and disease severity was judged at 22 days after inoculation. (A) Means with standard errors of three independent experiments each with three plants are shown. Different letters in the same column indicate the significant difference according to Duncan s multiple range tests, P<0.05. (B) Representative images of symptoms in plants without (control) or with pre-treated at the defined days with pyraclostrobin before the pathogen, Xanthomonas euvescatoria, inoculation (dpi). The images were photographed at 22 days after the pathogen inoculation. The 0 dpi pepper plant was without pathogen inoculation. 6.5%( 90%) (Table 3). pyraclostrobin, streptomycin pyraclostrobin. Pyraclostrobin. 86%, pyraclostrobin. 7 1

5 Research in Plant Disease Vol. 24 No %, 3 pyraclostrobin (Fig. 1). pyraclostrobin,.. acibenzolar-s-methyl, probenazole, b-aminobutyric acid (BABA), 2R,3R-butanediol BTH (Anfoka, 2000; Dietrich, 2005; Lugtenberg Kamilova, 2009; Oostendorp, 2001; Schreiber Desveaux, 2008; Skandalis, 2016). Strobilurin pyraclostrobin. in vitro X. euvesicatoria pyraclostrobin in planta. Pyraclostrobin Pseudomonas syringae pv. tabaci strobilurin (Herms, 2002; Skandalis, 2016). pyraclostrobin. pyraclostrobin streptomycin. streptomycin 78% pyraclostrobin 90%. in vitro X. euvesicatoria pyraclostronbin 68%, (local) (systemic) (data not shown). pyraclostron streptomycin. pyraclostrobin (Avenot, 2008; Itako, 2014; Min, 2014). pyraclostrobin in vitro, streptomycin (aminoglycoside).. pyraclostrobin cytochrome bc1 (ubiquinol oxidase) Qo (Quinone outside) (QoI, Quinone outside inhibitor) (Bartlett, 2002; Skandalis, 2016)., pyraclostrobin mitogen-activated protein kinases (Skandalis, 2016). pyraclostrobin salicylic acid (SA) Tobacco mosaic virus (TMV) (P. syringae pv. tabaci) (Herms, 2002), (X. axonopodis pv. phaseoli) (Vigo, 2012). pyraclostrobin peroxidase, polyphenol oxidase, phenylalanine ammonia-lyase, b-1,3-glucanase protease (X. perforans) (Itako, 2015). Strobilurin

6 64 Research in Plant Disease Vol. 24 No. 1., 24 (Karadimos, 2005; Turechek, 2006). pyraclostrobin 24 66%,. pyraclostrobin. pyclostrobin P. syringae pv. tomato (strain DC3000) P. syringae pv. tabac 24-48, pyraclostrobin 48-72, (Herms, 2002). TMV PR-1 protein SA pyraclostrobin 12 (Herms, 2002). pyraclostrobin 24 (Herms, 2002; Itako, 2014, 2015; Vigo, 2012).. pyraclostrobin 3. pyraclostrobin. Pyraclostrobin (Quinone outside inhibitor, QoI). pyraclostrobin. pyraclostrobin (Xanthomonas euvesicatoria). Pyraclostrobin in vitro X. euvesicatoria, pyraclostrobin ( 69%) streptomycin ( 90%),. Pyraclostrobin 1-3. pyraclostrobin. Conflicts of Interest No potential conflict of interest relevant to this article was reported. Acknowledgement This study was financially supported by Chonnam National University (Grant number: ). References Anfoka, G. H Benzo-(1, 2, 3)-thiadiazole-7-carbothioic acid S-methyl ester induces systemic resistance in tomato (Lycopersicon esculentum. Mill cv. Vollendung) to Cucumber mosaic virus. Crop Prot. 19: Avenot, H., Morgan, D. and Michailides, T Resistance to pyraclostrobin, boscalid and multiple resistance to Pristine (pyraclostrobin + boscalid) fungicide in Alternaria alternata causing alternaria late blight of pistachios in California. Plant Pathol. 57: Bartlett, D. W., Clough, J. M., Godwin, J. R., Hall, A. A., Hamer, M. and Parr Dobrzanski, B The strobilurin fungicides. Pest Manage. Sci. 58: Bouzar, H., Jones, J., Minsavage, G., Stall, R. and Scott, J Proteins unique to phenotypically distinct groups of Xanthomonas campestris pv. vesicatoria revealed by silver staining. Phytopathology 84: Cooksey, D. A Genetics of bactericide resistance in plant pathogenic bacteria. Annu. Rev. Phytopathol. 28: Dietrich, R., Ploss, K. and Heil, M Growth responses and fitness costs after induction of pathogen resistance depend on environmental conditions. Plant Cell Environ. 28: Doidge, E. M A tomato canker. Ann. Appl. Biol. 7: Gardner, M. W. and Kendrick, J Bacterial spot of tomato. J. Agric. Res. 21: Harborne, J. B The comparative biochemistry of phytoalexin induction in plants. Biochem. Syst. Ecol. 27: Herms, S., Seehaus, K., Koehle, H. and Conrath, U A strobilurin fungicide enhances the resistance of tobacco against tobacco mosaic virus and Pseudomonas syringae pv tabaci. Plant Physiol. 130: Itako, A. T., Tolentino Júnior, J. B., Demant, L. A. R. and Maringoni, A. C Control of bacterial spot of tomato and activation of enzymes related to resistance by chemicals under field conditions. J. Agric. Sci. 6:

7 Research in Plant Disease Vol. 24 No Itako, A. T., Tolentino Junior, J. B., Silva Junior, T. A., Soman, J. M. and Maringoni, A. C Chemical products induce resistance to Xanthomonas perforans in tomato. Braz. J. Microbiol. 46: Jones, J., Bouzar, H., Stall, R., Almira, E., Roberts, P., Bowen, B. W. et al Systematic analysis of xanthomonads (Xanthomonas spp.) associated with pepper and tomato lesions. Int. J. Syst. Evol. Microbiol. 50: Jones, J., Stall, R. and Bouzar, H Diversity among xanthomonads pathogenic on pepper and tomato. Annu. Rev. Phytopathol. 36: Jones, J. B., Lacy, G. H., Bouzar, H., Stall, R. E. and Schaad, N. W Reclassification of the xanthomonads associated with bacterial spot disease of tomato and pepper. Syst. Appl. Microbiol. 27: Karadimos, D., Karaoglanidis, G. and Tzavella-Klonari, K Biological activity and physical modes of action of the Qo inhibitor fungicides trifloxystrobin and pyraclostrobin against Cercospora beticola. Crop Prot. 24: Kim, J.-B Pathogen, insect and weed control effects of secondary metabolites from plants. J. Korean Soc. Appl. Biol. Chem. 48: Kim, J. H., Cheong, S. S., Lee, K. K., Yim, J. R. and Lee, W. H Determination of economic control thresholds for bacterial spot on red pepper caused by Xanthomonas campestris pv. vesicatoria. Res. Plant Dis. 21: Koga, J., Ogawa, N., Yamauchi, T., Kikuchi, M., Ogasawara, N. and Shimura, M Functional moiety for the antifungal activity of phytocassane E, a diterpene phytoalexin from rice. Phytochemistry 44: Korea Crop Protection Association Agrochemicals Use Guide Book. Korea Crop protection Association. URL Kyeon, M. S., Son, S. H., Noh, Y. H., Kim, Y. E., Lee, H. I. and Cha, J. S Xanthomonas euvesicatoria causes bacterial spot disease on pepper plant in Korea. Plant Pathol. J. 32: Lee, S. D. and Cho, Y. S Copper resistance and race distribution of Xanthomonas campestris pv. vesicatoria on pepper in Korea. Plant Pathol. J. 12: Lugtenberg, B. and Kamilova, F Plant-growth-promoting rhizobacteria. Annu. Rev. Microbiol. 63: Min, K. H., Ryu, J. P., Kim, J. M., Kim, S. H., Yim, S. H., Choi, J. J. et al Control efficacy of the mixture of fluxapyroxad plus pyraclostrobin against Pear scab caused by Venturia nashicola. Korean J. Pestic. Sci. 18: Myung, I. S., Yoon, M. J., Lee, J. Y., Kim, Y., Kwon, J. H., Lee, Y. K. et al Bacterial spot of hot pepper, caused by Xanthomonas euvesicatoria, a new disease in Korea. Plant Dis. 99: Obradovic, A., Jones, J., Momol, M., Balogh, B. and Olson, S Management of tomato bacterial spot in the field by foliar applications of bacteriophages and SAR inducers. Plant Dis. 88: Oostendorp, M., Kunz, W., Dietrich, B. and Staub, T Induced disease resistance in plants by chemicals. Eur. J. Plant Pathol. 107: Schreiber, K. and Desveaux, D Message in a bottle: chemical biology of induced disease resistance in plants. Plant Pathol. J. 24: Skandalis, N., Dimopoulou, A., Beri, D., Tzima, A., Malandraki, I., Theologidis, I. et al Effect of pyraclostrobin application on viral and bacterial diseases of tomato. Plant Dis. 100: Potnis, N., Timilsina, S., Strayer, A., Shantharaj, D., Barak, J. D., Paret, M. L. et al Bacterial spot of tomato and pepper: diverse Xanthomonas species with a wide variety of virulence factors posing a worldwide challenge. Mol. Plant Pathol. 16: Turechek, W. W., Peres, N. A. and Werner, N. A Pre-and postinfection activity of pyraclostrobin for control of anthracnose fruit rot of strawberry caused by Colletotrichum acutatum. Plant Dis. 90: Udayashankar, A. C., Nayaka, C. S., Archana, B., Nayak, U., Niranjana, S. R. and Prakash, H Strobilurins seed treatment enhances resistance of common bean against bean common mosaic virus. J. Phytopathol. 160: Vigo, S. C., Maringoni, A. C., Camara, R. C. and Lima, G. P. P Evaluation of pyraclostrobin and acibenzolar-s-methyl on common bacterial blight of snap bean. Semin. Cienc. Agrar. 33: Yoo, S. H List of Plant Diseases in Korea. 5th ed. The Korean Society of Plant Pathology, Suwon, Korea. 76 pp. Young, J., Dye, D., Bradbury, J., Panagopoulos, C. and Robbs, C A proposed nomenclature and classification for plant pathogenic bacteria. N. Z. J. Agric. Res. 21:

212 Research in Plant Disease Vol. 20 No. 3., 7 (Kim, 2004). Colletotrichum (Park Kim, 1992),.. (Agrios, 2008)... 1,202, 256 92, 147, 11 (RDA, 2014).

212 Research in Plant Disease Vol. 20 No. 3., 7 (Kim, 2004). Colletotrichum (Park Kim, 1992),.. (Agrios, 2008)... 1,202, 256 92, 147, 11 (RDA, 2014). 식물병연구 Research Article Open Access Res. Plant Dis. 20(3) : 211-215(2014) http://dx.doi.org/10.5423/rpd.2014.20.3.211 Protective Activity of the Mixtures of Pine Oil and Copper Hydroxide against Bacterial