식물병연구 Research Article Open Access Res. Plant Dis. 20(4) : (2014) Efficient Screening Method for Re

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1 식물병연구 Research Article Open Access Res. Plant Dis. 20(4) : (2014) Efficient Screening Method for Resistance of Cucumber Cultivars to Fusarium oxysporum f. sp. cucumerinum * 1, 2 *Corresponding author Tel : Fax: kjchoi@krict.re.kr Ji Hyun Lee 1, Jin-Cheol Kim 2, Kyoung Soo Jang 1, Yong Ho Choi 1 and Gyung Ja Choi 1 * 1 Research Center for Biobased Chemistry, Korea Research Institute of Chemical Technology, Daejeon , Korea 2 Division of Applied Bioscience and Biotechnology, Institute of Environmentally-Friendly Agriculture, Chonnam National University, Gwangju , Korea Received September 3, 2014 Revised September 23, 2014 Accepted October 31, 2014 The study was performed to establish an efficient screening method for resistant cucumber to Fusarium oxysporum f. sp. cucumerinum. The isolate KR5 was identified as F. oxysporum f. sp. cucumerinum based on molecular analyses of ITS and TEF genes and host-specificity test on cucurbits including melon, oriental melon, cucumber, and watermelon. Then four cucumber and two rootstock cultivars showing different resistance degrees to the Fusarium wilt pathogen KR5 were selected. And development of Fusarium wilt of the six cultivars according to several conditions, including incubation temperature after inoculation, inoculum concentration, root wounding, and growth stages of seedlings, was investigated. Disease severity of Fusarium wilt on the resistant cultivars was changed with incubation temperatures after inoculation. The resistant cultivars showed the higher resistance when inoculated plants were kept at 25 or 30 o C than at 20 o C. Among four different growth stages of the seedlings, seven-day-old seedling represented the most difference of resistance and susceptibility to Fusarium wilt. From above results, we suggest that an efficient screening method for resistant cucumber to F. oxysporum f. sp. cucumerinum is to dip the non-cut roots of seven-day-old seedlings in spore suspension of conidia/ml and to transplant the seedling into a non-infected soil, and then to incubate the inoculated plants in a growth room at 25 o C for 3 weeks to develop Fusarium wilt. Keywords : Breeding, Disease resistance, Fusarium wilt, Resistant screening Fusarium oxysporum 100 (Both, 1971). Research in Plant Disease The Korean Society of Plant Pathology pissn , eissn ,, (Lee Lee, 1994). F. oxysporum 80 (formae speciales, f. sp.) (Armstrong Armstrong, 1981; Snyder Hansen, 1940), f. sp. cucumerinum, f. sp. niveum, f. sp. melonis, f. sp. lagenaria, f. sp. luffae (Armstrong Armstrong, 1981; Kawai, 1958; Leach 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.

2 246 Research in Plant Disease Vol. 20 No. 4 Currence, 1938; Matuo Yamamoto, 1957; Owen, 1956). F. oxysporum f. sp. niveum (Owen, 1955; 1956), F. oxysporum f. sp. cucumerinum (Owen, 1955). (Bouhot, 1981; Davis, 1966; Gerlagh Blok, 1988; Martyn McLaughlin, 1983b; McMillan, 1986; Owen, 1955; 1956). F. oxysporum f. sp. cucumerinum (Ahn, 1998; Chen, 2010; Vakalounakis, 1993)., (Freeman, 2002; Sherf MacNab, 1985), (Yang Kim, 1994). (Sherf MacNab, 1985). F. oxysporum f. sp. cucumerinum race(1, 2, 3) (Armstrong, 1978), Fusarium, (Kanno, 1991; Toshimitsh Noguchi, 1975). Ano 2 Aofushinari (Kanno, 1991; Vakalounakis, 1996) WIS-248 F. oxysporum f. sp. cucumerinum race 2 Foc (Netzer, 1977)., SMR-18 WI-2757 F. oxysporum f. sp. cucumerinum race 1 2 Fcu-1 (Vakalounakis, 1993; 1995; Vakalounakis Smardas, 1995).,.,. F. oxysporum f. sp. cucumerinum KR5, 4 2,,. 병원균동정. (KR5). PDA 25 o C 7 InstaGene matrix(bio-rad, USA) ITS(internal transcribed spacer) TEF(translation elongation factor 1α) polymerase chain reaction(pcr). ITS universal primer ITS1(5 -TCC GTA GGT GAA CCT GCG G-3 )/ITS4(5 -TCC TCC GCT TAT TGA TAT GC-3 ) (White, 1990), TEF EF1T(5 -ATG GGT AAG GAG GAC AAG AC-3 )/EF2T(5 -GGA AGT ACC AGT GAT CAT GTT-3 ) (O Donell, 1998). PCR InstaGene matrix 1 µl, 10 mm dntp 1 µl, 10 pmol primer 1 µl, 10 Buffer 3 µl, 2.5 unit/µl EF-Taq DNA polymerase(solgent, Korea) 0.2 µl 30 µl. ITS 95 o C 5 initial denaturation denaturation 95 o C/45, annealing 55 o C/1, extension 70 o C/1 35 cycle final extension 70 o C 10, TEF 95 o C 2 initial denaturation denaturation 95 o C/30, annealing 54 o C/30, extension 72 o C/1 35 cycle final extension 72 o C 10. PCR Big Dye terminator v3.1 cycle sequencing kits(applied Biosystems, USA) primer DNA Engine Tetrad 2 peltier thermal cycler(bio-rad, USA) PCR ABI Prism 3730xl Analyzer 96 capillary type(applied Biosystems, USA). BLAST search GenBank ITS TEF, GenBank CLUSTAL X(Thompson, 1997) PHY- DIT program version 3.0(Chun, 1995). Neighbor-joining tree PHYLIP 3.57c package(felsenstein, 1985) Kimura s 2-parameter distance model(kimura, 1980). 식물재배 (15 ml/pot, ) 5 ( ) 1 7. KR5 ( ) ( ), ( ) ( ),

Research in Plant Disease Vol. 20 No. 4 247 ( ) ( ), ( ) ( ) 8. 4, ( ), ( ), ( ), ( ) 2 ( ), ( ). 6 (25 ± 5 o C) 7, 10, 13, 16. 접종원준비. KR5 potato dextrose broth(pdb; Becton, Dickinson and Co.

3 Research in Plant Disease Vol. 20 No ( ) ( ), ( ) ( ) 8. 4, ( ), ( ), ( ), ( ) 2 ( ), ( ). 6 (25 ± 5 o C) 7, 10, 13, 16. 접종원준비. KR5 potato dextrose broth(pdb; Becton, Dickinson and Co.) 25 o C 7 V8-juice broth[v8 broth; V8 Juice(Campbell Soup Co.) 200 ml, CaCO 3 3 g (Samchun Chem. Co., Ltd.), distilled water 1 l] rpm 25 o C. KR5 4 (4300 g, 10, 4 o C, Beckman Coulter Inc.). hematocytometer ( ) conidia/ml , , , , conidia/ml. 덩굴쪼김병균접종. KR (80 ml/ pot, ) 5 ( ) cm. 발병및병조사. 25 o C o C , o C (disease index) 0 =, 1 = Fig. 1. Disease index of Fusarium wilt on cucumber seedlings., 2 =, 3 =, 4 = 5 (Fig. 1). 1.0, , 2.0, SAS(SAS Institute, Inc., 1989, Cary, NC) ANOVA, Duncan s multiple range test (P = 0.05). 균주동정. KR5 ITS Fusarium TEF, KR5 ITS (White, 1990) F. oxysporum(dq459007) 100% GenBank Fusarirum ITS phylogenic tree F. oxysporum ( ). TEF (O Donell, 1998) F. oxysporum(af160312) 99.2%, TEF Fusarium phylogenic tree KR5 F. oxysporum (Fig. 2). KR5 F. oxysporum. KR5,, 4 2,, (Table 1). F. oxysporum 80 (formae speciales) (Armstrong Armstrong, 1981; Snyder Hansen, 1940).

4 248 Research in Plant Disease Vol. 20 No. 4 Fig. 2. Phylogenetic tree based on sequences of translation elongation factor 1α genes of Fusarium oxysporum isolates and related species. The values above each branch indicate the percentage levels of bootstrap support (>50%) for the branch point based on 1000 resamplings. The bar represents 0.01 substitutions per nucleotide position. ( ) is GenBank accession number. Table 1. Pathogenicity of Fusarium oxysporum f. sp. cucumerinum KR5 to cucumber, melon, watermelon and oriental melon a Crop Cultivar Scientific name Disease index b Cucumber Asiacheongjang Cucumis sativus 4.0 ± 0.0 Beakmibaekdadagi Cucumis sativus 2.8 ± 1.1 Melon Betarich Cucumia melo 2.0 ± 0.0 Asiahwanggeum Cucumia melo 2.6 ± 0.9 Watermelon Seotaja Citrullus lanatus 1.4 ± 1.1 Kokoma Citrullus lanatus 1.0 ± 0.0 Oriental melon Geumje C. melo var. makuwa 2.0 ± 0.0 Joeundae C. melo var. makuwa 3.0 ± 1.0 a Seven-day-old seedlings of each cultivar were inoculated with F. oxysporum f. sp. cucumerinum KR5 by dipping the roots in spore suspension of conidia/ml for 30 min. The inoculated plants were incubated in a dew chamber at 25 o C for 24 hr and then transferred to a growth room at 25 o C with 12-hour light a day. After 3 weeks, disease severity of the plants was investigated. b Each value represents the mean disease severity ± standard deviation of two runs with ten replicates each. F. oxysporum (Bouhot, 1981; Davis, 1966; Gerlagh Blok, 1988; Martyn McLaughlin, 1983b; McMillan, 1986; Owen, 1955; 1956). KR5 F. oxysporum f. sp. cucumerinum.,, Owen(1955). 재배온도에따른오이덩굴쪼김병발생 F. oxysporum f. sp. cucumerinum KR5, ( ).

Research in Plant Disease Vol. 20 No. 4 249 Fig. 3. Development of Fusarium wilt on four cucumber and two rootstock cultivars incubated at three temperatures.

5 Research in Plant Disease Vol. 20 No Fig. 3. Development of Fusarium wilt on four cucumber and two rootstock cultivars incubated at three temperatures. Seven-day-old seedlings of each cultivar were inoculated with Fusarium oxysporum f. sp. cucumerinum KR5 by dipping the roots in spore suspension of conidia/ml for 30 min. The inoculated plants were incubated in a dew chamber at 20, 25, and 30 o C for 24 hr and then transferred to a growth room at 20, 25, and 30 o C with 12-hour light a day, respectively. After 3 weeks, disease severity of the plants was investigated. Each value represents the mean of two runs with ten replicates each. Values in the labeled with the same letter within each incubation temperature of seedlings are not significantly different in Duncan s multiple range test at P = o C, 25 o C 30 o C,., 25 o C 30 o C 0.8, 20 o C 2.2 (Fig. 3). 25 o C 30 o C, 30 o C. F. oxysporum f. sp. cucumerinum 24 o C 26 o C (Vakalounakis, 1993; 1995; 1996; Vakalounakis Smardas, 1995), 25 o C. 접종원농도에따른오이덩굴쪼김병발생. F. oxysporum f. sp. cucumerinum KR5, Fig. 4. Fusarium wilt occurrence of four cucumber and two rootstock cultivars according to inoculum concentration. Seven-day-old seedlings of each cultivar were inoculated with Fusarium oxysporum f. sp. cucumerinum KR5 by dipping the roots in spore suspensions of , , , , and conidia/ml for 30 min. The inoculated plants were incubated in a dew chamber at 25 o C for 24 hr and then transferred to a growth room at 25 o C with 12-hour light a day. After 3 weeks, disease severity of the plants was investigated. Each value represents the mean of two runs with ten replicates each. Values in the labeled with the same letter within each inoculum concentration of seedlings are not significantly different in Duncan s multiple range test at P = , conidia/ml (Fig. 4). Martyn McLaughlin(1983a) F. oxysporum f. sp. niveum conidia/ml Dixielee Smokylee, conidia/ml (Freeman Rodriguez, 1993; Freeman, 2002; Zhou, 2010). ( conidia/ml) (Vakalounakis, 1993; 1995; Vakalounakis Smardas, 1995), conidia/ml conidia/ml. 뿌리상처에따른오이덩굴쪼김병발생.,

6 250 Research in Plant Disease Vol. 20 No. 4 Fig. 5. Development of Fusarium wilt on four cucumber and two rootstock cultivars when cut and non-cut roots were dipped in a spore suspension of Fusarium oxysporum f. sp. cucumerinum. Sevenday-old seedlings of each cultivar were inoculated with F. oxysporum f. sp. cucumerinum KR5 by dipping the cut and non-cut roots in spore suspension of conidia/ml for 30 min. The inoculated plants were incubated in a dew chamber at 25 o C for 24 hr and then transferred to a growth room at 25 o C with 12-hour light a day. After 3 weeks, disease severity of the plants was investigated. Each value represents the mean of two runs with ten replicates each. Values in the labeled with the same letter within each inoculation method of seedlings are not significantly different in Duncan s multiple range test at P = (Fig. 5). F. oxysporum f. sp. cucumerinum (Ahn, 1998; Vakalounakis, 1993; 1995),. 유묘의생육시기에따른오이덩굴쪼김병발생 (Fig. 6). F. oxysporum f. sp. melonis Latin Snell(1986) Fig. 6. Fusarium wilt occurrence on four cucumber and two rootstock cultivars inoculated at four growth stages of seedlings. Seven-, ten-, thirteen- and sixteen-day-old seedlings of each cultivar were inoculated with Fusarium oxysporum f. sp. cucumerinum KR5 by dipping the roots in spore suspension of conidia/ml for 30 min. The inoculated plants were incubated in a dew chamber at 25 o C for 24 hr and then transferred to a growth room at 25 o C with 12-hour light a day. Three weeks after inoculation, disease severity of the plants was investigated. Each value represents the mean of two runs with ten replicates each. Values in the labeled with the same letter within each growth stage of seedlings are not significantly different in Duncan s multiple range test at P = , 13, F. oxysporum f. sp. cucumerinum conidia/ml F. oxysporum f. sp. cucumerinum 25 o C 12 3.,. Fusarium oxysporum f. sp. cucumerinum. ITS TEF,, KR5 F. oxysporum f. sp. cucumerinum. KR5

7 Research in Plant Disease Vol. 20 No ,,., 20 o C 25 o C 30 o C. 7. 7, conidia/ml F. oxysporum f. sp. cucumerinum 25 o C 3. Acknowledgment This research was supported by Golden Seed Project Vegetable Seed Center, Ministry of Agriculture, Food and Rural Affairs (MAFRA), Ministry of Oceans and Fisheries (MOF), Rural Development Administration (RDA) and Korea Forest Service (KFS). References Ahn, I. P., Chung, H. S. and Lee, Y. H Vegetative compatibility groups and pathogenicity among isolates of Fusarium oxysporum f. sp. cucumerinum. Plant Dis. 82: Armstrong, G. M. and Armstrong, J. K Formae speciales and races of Fusarium oxysporum causing wilt disease. In: Fusarium: Disease, Biology, and Taxonomy, ed. by P. E. Nelson, T. A. Toussoun and R. J. Cook, pp Pennsylvania State University Press, University Park. Armstrong, G. M., Armstrong, J. K. and Netzer, D Pathogenic races of the cucumber-wilt Fusarium. Plant Dis. 62: Both, C The genus Fusarium. Commonwealth Mycological Institute, Kew, Surrey, England. Bouhot, D Some aspects of the pathogenic potential in formae speciales and races of Fusarium oxysporum on Cucurbitaceae. In: Fusarium: Disease, Biology, and Taxonomy. ed. by P. E. Nelson, T. A. Toussoun and R. J. Cook, pp Pennsylvania State University Press, University Park. Chen, F., Wang, M., Zheng, Y., Luo, J. M., Yang, X. R. and Wang, X. L Quantitative changes of plant defense enzymes and phytohormone in biocontrol of cucumber Fusarium wilt by Bacillus subtilis B579. World J. Microbiol. Biotechnol. 26: Chun, J Computer-assisted classification and identification of Actinomycetes. Ph. D. thesis, University of Newcastle, Newcastle upon Tyne, UK. Davis, D Cross-infection in Fusarium wilt diseases. Phytopathology 56: Felsenstein, J Confidence limits on phylogenies: An approach using the bootstrap. Evolution 39: Freeman, S. and Rodriquez, R. J A rapid inoculation technique for assessing pathogenicity of Fusarium oxysporum f. sp. niveum and F. o. melonis on cucurbits. Plant Dis. 77: Freeman, S., Zveibil, A., Vintal, H. and Maymon, M Isolation of nonpathogenic mutants of Fusarium oxysporum f. sp. melonis for biological control of Fusarium wilt in cucurbits. Phytopathology 92: Gerlagh, M. and Blok, W. J Fusarium oxysporum f. sp. cucurbitacearum n. f. embracing all formae speciales of F. oxysporum attacking cucurbitaceous crops. Neth. J. Plant Pathol. 94: Kanno, T., Han, X. and Tabei, Y Genetic analysis of resistance in cucumber to Fusarium oxysporum f. sp. cucumerinum. Bull. Natl. Res. Inst. Veg. Ornament. Plants Tea 4: Kawai, I., Suzuki, H. and Kawai, K On the pathogenicity of the wilt Fusarium of the cucurbitaceous plants and their forms. Shizuoka Agric. Exp. Stn. Bull. 3: Kimura, M A simple method for estimating evolutionary rate of base substitution through comparative studies of nucleotide sequence. J. Mol. Evol. 16: Latin, R. X. and Snell, S. J Comparison of methods for inoculation of muskmelon with Fusarium oxysporum f. sp. melonis. Plant Dis. 70: Leach, J. G. and Currence, T. M Fusarium wilt of muskmelon in Minnesota. Minn. Agric. Exp. Stn. Tech. Bull. 129: 32. Lee, S. G. and Lee, W. H Control of Fusarium wilt of watermelon with the root-stock grafting of Sicyos angulatus L. Korean J. Plant Pathol. 10: Martyn, R. D. and McLaughlin, R. J. 1983a. Effects of inoculum concentration on the apparent resistance of watermelons to Fusarium oxysporum f. sp. niveum. Plant Dis. 67: Martyn, R. D. and McLaughlin, R. J. 1983b. Susceptibility of summer squash to the watermelon wilt pathogen (Fusarium oxysporum f. sp. niveum). Plant Dis. 67: Matuo, T. and Yamamoto, I On Fusarium oxysporum f. sp. lagenariae n. f. causing wilt of Lagenaria vulgaris var. hispida. Trans. Mycol. Soc. Jpn. 2: McMillan, R. T Cross pathogenicity studies with isolates of Fusarium oxysporum from either cucumber or watermelon pathogenic to both crop species. Ann. Appl. Biol. 109: Netzer, D., Niego, S. and Galun, E A dominant gene conferring resistance to Fusarium wilt in cucumber. Phytopathology 67: O Donnell, K., Cigelnik, E. and Casper, H. H Molecular phylogenetic, morphological, and mycotoxin data support reidentification of the Quorn mycoprotein fungus as Fusarium venenatum. Fungal Genet. Biol. 23: Owen, J. H Fusarium wilt of cucumber. Phytopathology 45: Owen, J. H Cucumber wilt, caused by Fusarium oxysporum f.

8 252 Research in Plant Disease Vol. 20 No. 4 sp. cucumerinum. Phytopathology 46: Sherf, A. F. and MacNab, A. A Vegetable disease and their control. John Wiley and Sons, New York. 728 pp. Snyder, W. C. and Hansen, H. N The species concept in Fusarium. Am. J. Bot. 27: Thompson, J. D., Gibson, T. J., Plewniak, F., Jeanmougin, F. and Higgins, D. G The Clustal X windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucleic Acids Res. 24: Toshimitsh, Y. and Noguchi, T Inheritance of resistance to Fusarium wilt in cucumber. Proc. Mtg. Jpn. Hort. Society Vakalounakis, D. J Inheritance and genetic linkage of fusarium wilt (Fusarium oxysporum f. sp. cucumerinum race 1) and scab (Cladosporium cucumerinum) resistance genes in cucumber (Cucumis sativus). Ann. Appl. Biol. 123: Vakalounakis, D. J Inheritance and linkage of resistance in cucumber line SMR-18 to races 1 and 2 of Fusarium oxysporum f. sp. cucumerinum. Plant Pathol. 44: Vakalounakis, D. J Allelism of the Fcu-1 and Foc genes conferring resistance to Fusarium wilt in cucumber. Eur. J. Plant Pathol. 102: Vakalounakis, D. J. and Smardas, K Genetics of resistance to Fusarium oxysporum f. sp. cucumerinum races 1 and 2 in cucumber line Wisconsin Ann. Appl. Biol. 127: White, T. J., Bruns, T., Lee, S. and Taylor, J Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics, In: PCR protocols: A guide to methods and applications, ed. by M. A. Innis, D. H. Gelfand, J. J. Sninsky and T. J. White, pp Academic Press, San Diego. Yang, S. S. and Kim, C. H Studies on cross protection of Fusarium wilt of cucumber Ⅲ. Selection of nonpathogenic isolates and their protective effects in the greenhouse. Korean J. Plant Pathol. 10: (In Korean) Zhou, X. G., Everts, K. L. and Bruton, B. D Race 3, a new and highly virulent race of Fusarium oxysporum f. sp. niveum causing Fusarium wilt in watermelon. Plant Dis. 94:

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