식물병연구 Research Article Open Access Res. Plant Dis. 21(3) : (2015) Development of an Efficient Simpl

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1 식물병연구 Research Article Open Access Res. Plant Dis. 21(3) : (2015) Development of an Efficient Simple Mass-Screening Method for Resistant Melon to Fusarium oxysporum f. sp. melonis 1, * 1, 2, 3 *Corresponding author Tel : Fax: kjchoi@krict.re.kr Won Jeong Lee 1,2, Kyoung Soo Jang 1, Yong Ho Choi 1, Heung Tae Kim 2, Jin-Cheol Kim 3 and Gyung Ja Choi 1 * 1 Center for Eco-friendly New Materials, Korea Research Institute of Chemical Technology, Daejeon , Korea 2 Department of Plant Medicine, Chungbuk National University, Cheongju , Korea 3 Division of Applied Bioscience and Biotechnology, Institute of Environmentally-Friendly Agriculture, Chonnam National University, Gwangju , Korea Received July 8, 2015 Revised July 22, 2015 Accepted August 3, 2015 This study was conducted to establish a simple mass-screening method for resistant melon to Fusarium wilt caused by Fusarium oxysporum f. sp. melonis (FOM). Root-dipping inoculation method has been used to investigate resistance of melon plants to Fusarium wilt. However, the inoculation method requires a lot of labor and time because of complicate procedure. To develop a simple screening method on melon Fusarium wilt, occurrence of Fusarium wilt on susceptible and resistant cultivars of melon according to inoculation method including root-dipping, soil-drenching, tip, and scalpel methods was investigated. Scalpel and tip methods showed more clear resistant and susceptible responses in the melon cultivars than root-dipping inoculation method, but tip method represented slightly variable disease severity. In contrast, in the case of soil-drenching inoculation method, disease severity of the susceptible cultivars was very low. Thus we selected scalpel method as inoculation method of a simple screening method for melon Fusarium wilt. By using the scalpel inoculation method, resistance degrees of the cultivars according to incubation temperature after inoculation (25 and 30 o C) and inoculum concentration ( and conidia/ml) were measured. The resistance or susceptibility of the cultivars was hardly affected by all the tested conditions. To look into the effectiveness of scalpel inoculation methods, resistance of 22 commercial melon cultivars to FOM was compare with root-dipping inoculation method. When the melon cultivars were inoculated by scalpel method, resistance responses of all the tested cultivars were clearly distinguished as by root-dipping method. Taken together, we suggest that an efficient simple mass-screening method for resistant melon plant to Fusarium wilt is to sow the seeds of melon in a pot (70 ml of soil) and to grow the seedlings in a greenhouse (25±5 o C) for 7 days, to cut the root of seedlings with a scalpel and then pour a 10 ml-aliquot of the spore suspension of conidia/ml on soil. The infected plants were cultivated in a growth room at 25 to 30 o C for about 3 weeks with 12-hr light a day. Keywords : Breeding, Disease resistance, Fusarium wilt, Inoculation method, Resistant screening Research in Plant Disease The Korean Society of Plant Pathology pissn , eissn 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.

2 202 (Cucumis melo L.), ha 26,000 MT(Seo, 2006). (Kwon, 1999). (C. melo) (C. melo var. makuwa),,,,,,,,, 21 (KSPP, 2009). (Park, 1996). (Fusarium wilt) Fusarium oxysporum f. sp. melonis(fom), (Beckman, 1987; Katan, 1994). (Gordon Okamoto, 1990; Sherf Macnab, 1986). (Yeo, 2013). (Lee, 1994; Traka-Mavrona, 2000). Fom-1 Fom-2, race 0, Fom-1 race 1, Fom-2 race 2 Fom-1 Fom-2 race 1, 2 (Risser, 1976). race 0, 1, 2, race 1, 2. race 1, 2 (Herman Perl-Treves, 2007).., race (root-dipping method) (Cohen, 1989; Lee, 2015; Matsumoto, 2011; Namiki, 1998; Wellman, 1939). (Latin Snell, 1986). (root-dipping).. 6 (rootdipping), (soil-drenching), scalpel tip scalpel. scalpel (110 6, conidia/ml) (25, 30 o C) 6. scalpel, 22 scalpel. 식물재배., (), ( ), (), (), (), FR () (), ( ), (), ( ), (), (), (), (), ( ), (), (), (), (), VIP( ), (), FR(), JJ(), JJ(), (), (), () (). 816 ( 21 ml, ) 5() 1 (255 o C) 7. scalpel, tip 58 ( 70 ml, ) 5() 1

3 203 (25±5oC)에서 7일 동안 재배하여 실험에 사용하였다. 접종원 준비. Potato dextrose agar(pda; Becton, Dickinson and Co.) 배지 중앙에 F. oxysporum f. sp. melonis GR 균주의 균 사 조각을 접종하고 25oC에서 7일 동안 배양한 균총으로부터 균사 조각을 떼어 V8-juice broth 배지에 접종하였다(Lee 등, 2015). 그리고 이를 25oC 암상태에서 7일 동안 150 rpm으로 진 탕배양 하였다. GR 균주 배양액은 4겹의 거즈로 걸러 균사를 제 거하고 원심분리 한(4,300 g, 10분, 4oC, Beckman Coulter Inc.) 후에 배양여액을 버리고 포자(침전물)를 수확하였다. 여기에 멸 균수를 넣고 잘 현탁하고 hematocytometer를 이용하여 광학 현미경 하에서 포자(소형분생포자)의 농도를 측정하였으며, 포 자 농도가 conidia/ml와 conidia/ml이 되도록 멸 균수로 희석하여 접종원을 준비하였다. 한편, 뿌리 침지와 scalpel 접종 방법에 따른 22개 멜론 품종의 덩굴쪼김병 저항성 실 험을 위해서는 포자 농도를 conidia/ml로 조정하여 실 험에 사용하였다. 덩굴쪼김병균 접종. 뿌리 침지 접종 방법은 온실에서 재 배한 멜론 유묘의 뿌리를 물로 세척하여 흙을 제거한 후에 F. oxysporum f. sp. melonis GR 균주의 포자현탁액에 30분 동안 침지하여 접종하였다. 그리고 원예용상토 5호(부농사)를 넣은 5 8 연결 포트(포트 당 토양 70 ml, 범농사)로 접종한 멜론 유 묘를 이식하였다. Scalpel 접종 방법은 5 8 연결 포트에서 재배한 멜론 유묘에 scalpel을 이용하여 지제부에서 1 cm 떨어진 곳에서 45o 각도 로 3 cm 깊이로 뿌리를 향하여 찔러서 상처를 주고, 여기에 멜 론 덩굴쪼김병균 포자현탁액을 10 ml씩 관주하여 접종하였다 (Park 등, 2013). Tip 접종 방법은 5 8 연결 포트에서 재배한 멜론 유묘에 10 ml tip을 이용하여 scalpel 접종 방법과 마찬가지로 방법으 로 뿌리를 향하여 찔러서 상처를 주고 포자현탁액을 10 ml씩 관 주 하였다. 토양 관주 접종 방법은 멜론 유묘가 있는 5 8 연결 포트 토 양에 멜론 덩굴쪼김병균 포자현탁액을 10 ml씩 관주하여 접종 하였다. 발병 및 병조사. 접종한 멜론 유묘는 25oC 습실상에서 1일 동안 배양한 후에 25oC 항온실로 옮기고 하루에 12시간씩 광을 조사하면서 약 3주 동안 재배한 후에 덩굴쪼김병 발생을 조사 하였다. 접종한 후의 재배 온도에 따른 멜론의 덩굴쪼김병 발생 실험 은 접종한 멜론 유묘를 25oC와 30oC 습실상에서 1일 동안 배양 한 후에 각각 25oC와 30oC 항온실로 이동하고 하루에 12시간씩 광을 조사하면서 약 3주 동안 재배한 후에 병 발생을 조사하 였다 (최경자).indd 203 Fig. 1. Disease index of Fusarium wilt in melon plants. 병조사는 각 식물체의 덩굴쪼김병 발생 정도(disease severity)를 조사하였으며, 발병 정도는 다음과 같은 발병도(disease index)로 조사하였다. 0=건전, 1=도관이 갈변되고 생육이 약간 억제된 것, 2=도관이 갈변되고 생육이 억제된 것, 3=도관이 갈 변되고 생육이 심하게 억제되고 황화된 것, 4=고사 등의 5단계 로 하였다(Fig. 1). 그리고 평균 발병도가 1.0 이하인 경우에는 저항성, 는 중도저항성, 2.6 이상은 감수성으로 판정하 였다. 모든 실험은 10반복으로 2회 실시하였다. 결과 및 고찰 네 가지 접종 방법에 따른 멜론 품종의 덩굴쪼김병 발생. 뿌리 침지, scalpel, tip 및 토양 관주 등 네 가지 접종 방법에 따 른 저항성 및 감수성 멜론 품종들의 덩굴쪼김병 발생을 두 가 지 접종원 농도(1 106, conidia/ml)로 실험한 결과, 실험 한 모든 감수성 멜론 품종들은 뿌리 침지 방법으로 접종하였을 때에는 실험한 두 농도 모두에서 4.0의 높은 발병도를 보였다 (Table 1). 그리고 scalpel 및 tip 방법으로 접종하였을 때에도 뿌 리 침지 접종법 보다는 다소 낮지만 발병도 3.3 이상의 높은 감 수성 반응을 나타냈다. 하지만 토양 관주 접종법에서는 저항성 품종들은 저항성을 잘 나타내었으나, 감수성 품종인 아시아황 금 은 의 발병도를 그리고 얼룩파파야 와 장춘FR파파 이야 는 의 낮은 발병도를 보였다. 네 가지 접종 방법 중 토양 관주 접종법이 시간과 노동력이 가장 적게 들고 이식 후 활착 문제 등이 없어 가장 선호하는 접종 방법이나 감수성 품 종에서 덩굴쪼김병 발생이 충분하지 못하여 멜론 덩굴쪼김병 저항성 검정에는 적합하지 않은 접종 방법으로 생각되었다. 저항성 품종 중 레드퀸 과 슈퍼세지 는 뿌리 침지, scalpel 및 tip 방법으로 접종하였을 때 농도에 상관없이 모두 1.0 이하의 낮은 발병도를 보였다(Table 1). 하지만 저항성 품종인 썸머쿨 은 scalpel 및 tip 방법으로 접종하였을 때에는 두 접종 농도 모 두에서 고도의 저항성 반응을 나타냈으나, 뿌리 침지 방법으로 오전 11:37:41

4 204 Table 1. Development of Fusarium wilt on six melon cultivars according to inoculation method a Inoculation method Inoculum concentration (conidia/ml) Redqueen (R) Summercool (R) Superseji (R) Melon cultivar Asiahwanggeum (S) Eolukpapaya (S) Jangchoon- FRpapaiya (S) Root- dipping b ±0.4 c 0.8± ± ± ± ± ± ± ± ± ± ±0.0 Scalpel d ± ± ± ± ± ± ± ± ± ± ± ±0.1 Tip e ± ± ± ± ± ± ± ± ± ± ± ±0.1 Soil-drenching f ± ± ± ± ± ± ± ± ± ± ± ±1.3 a Seven-day-old seedlings of melon cultivars were inoculated with Fusarium oxysporum f. sp. melonis (FOM) GR. The inoculated plants were incubated in a dew chamber at 25 o C for 24 hours and then transferred to a growth room at 25 o C with 12-hour light a day. After 3 weeks, disease severity of the seedling was investigated on a scale of 0 4. b Seedlings of melon cultivars were uprooted and the roots were washed gently in water. And then the plants were inoculated with FOM GR by dipping the roots in inoculum suspensions of conidia/ml and conidia/ml for 30 minutes and were transplanted into 40-cell plastic trays. c Each value represents the mean disease index±standard deviation of two runs with ten replicates each. d Seedlings of melon cultivars were inoculated with FOM GR by cutting the roots with a scalpel and then pouring a 10 ml-aliquot of spore suspensions of conidia/ml and conidia/ml on soil. e Seedlings of melon cultivars were inoculated with FOM GR by cutting the roots with 10 ml tip and then pouring a 10 ml-aliquot of spore suspensions of conidia/ml and conidia/ml on soil. f Seedlings of melon cultivars were inoculated with FOM GR by pouring a 10 ml-aliquot of spore suspensions of conidia/ml and conidia/ml on soil without cutting of the roots conidia/ml conidia/ml conidia/ml (Freeman, 2002; Iori, 2001; Zhou Everts, 2007; Zink, 1983), Fom-1 Fom-2 (Risser, 1976), scalpel tip, conidia/ml., scalpel , tip scalpel tip. Park (2013) (Fusarium wilt) scalpel tip, scalpel tip. scalpel tip. tip Table 1 scalpel scalpel. Latin Snell(1986) 5 ml pipette,. scalpel scalpel (Table 1). Scalpel 접종방법에서재배온도에따른멜론품종들의덩굴쪼김병발생. scalpel conidia/ml 25 o C 30 o C

5 205 Table 2. Development of Fusarium wilt on melon cultivars according to incubation temperature a Cultivar Trait b 25 o C 30 o C c Redqueen R 0.1±0.3 d R e 0.2±0.4 R 0.3±0.7 R 0.2±0.6 R Summercool R 0.4±0.7 R 0.3±0.5 R 0.3±0.7 R 0.4±0.7 R Superseji R 0.3±0.5 R 0.2±0.4 R 0.1±0.3 R 0.2±0.6 R Eolukpapaya S 4.0±0.0 S 3.9±0.3 S 3.9±0.3 S 3.8±0.6 S Asiahwanggeum S 4.0±0.0 S 4.0±0.0 S 3.9±0.3 S 4.0±0.0 S JangchoonFRpapaiya S 3.8±0.6 S 4.0±0.0 S 3.4±1.1 S 3.7±0.9 S a Seven-day-old seedlings of melon cultivars were inoculated with Fusarium oxysporum f. sp. melonis GR by cutting the roots with a scalpel and then pouring a 10 ml-aliquot of spore suspensions of conidia/ml and conidia/ml on soil. The inoculated plants were incubated in dew chambers at 25 o C and 30 o C for 24 hours and then transferred to growth rooms at 25 o C and 30 o C with 12-hour light a day, respectively. After 3 weeks, disease severity of the seedling was investigated on a scale of 0 4. b R, resistant cultivar to Fusarium wilt provided by each seed company; S, susceptible cultivar to the disease. c Spore concentration, conidia/ml. d Each value represents the mean disease index±standard deviation of two runs with ten replicates each. e Resistance response: R, resistance [disease index (DI)=0 1.0]; MR, moderate resistance (DI= ); S, susceptibility (DI= )., 0.4 (Table 2) o C (Cohen, 1989; Matsumoto, 2011; Namiki, 1998; Zhou Everts, 2007; Zink, 1983). Lee (2015) ( ) 20 o C, 25 o C 30 o C o C. scalpel (Table 2). scalpel conidia/ml o C. Scalpel 접종법에의한 22 개멜론품종의덩굴쪼김병에대한저항성. scalpel, 22 scalpel, 5 (,,,, FR ) FR (Table 3). FR 3.4. GR race 1 Fom-1 (Lee, 2015), FR Fom scalpel scalpel 0.9. scalpel. Fom-1 Fom-2 (Risser, 1976), scalpel. scalpel.

6 206 Table 3. Degrees of resistance of 22 melon cultivars to Fusarium wilt according to inoculation method a Cultivar Trait b Root dip c Scalpel d Asiaseongha R 1.0±0.0 e R f 0.6±0.2 R Redqueen R 0.7±0.7 R 0.2±0.2 R Summercool R 0.7±0.5 R 0.1±0.3 R Asiajochunmanchu R 1.3±0.5 MR 0.6±0.5 R JangchoonFRpapaiya R 4.0±0.0 S 3.4±0.9 S Superseji 0.9±0.3 R 0.0±0.0 R Asiapapaya 2.0±0.0 MR 0.5±0.3 R Betarichi 2.0±0.6 MR 0.1±0.2 R Earlselite 2.0±0.0 MR 0.3±0.4 R Earlselysee 1.3±0.5 MR 0.1±0.3 R Earlsgoldking 1.3±0.7 MR 0.3±0.1 R Earlshappy 1.4±0.5 MR 0.3±0.1 R Earlsking 1.9±0.3 MR 0.1±0.3 R Earlsmounthagye 1.3±0.5 MR 0.1±0.3 R Earlsparty 1.1±0.8 MR 0.1±0.0 R Earlstopone 1.5±0.5 MR 0.9±0.2 R EarlsVIP 1.1±0.3 MR 0.4±0.5 R Jjhagye 1.7±0.5 MR 0.4±0.2 R Jjonetop 2.2±0.4 MR 0.3±0.0 R Sejiokay 2.4±0.5 MR 0.2±0.2 R Asiahwanggeum 4.0±0.0 S 3.9±0.5 S Eolukpapaya 4.0±0.0 S 3.3±0.6 S a Seven-day-old seedlings of melon cultivars were inoculated with Fusarium oxysporum f. sp. melonis (FOM) GR. The inoculated plants were incubated in a dew chamber at 25 o C for 24 hours and then transferred to a growth room at 25 o C with 12-hour light a day. After 3 weeks, disease severity of the seedling was investigated on a scale of 0 4. b Resistant cultivar to Fusarium wilt provided by each seed company. c Seedlings of melon cultivars were uprooted and the roots were washed gently in water. And then the plants were inoculated with FOM GR by dipping the roots in inoculum suspensions of conidia/ml and conidia/ml for 30 minutes and were transplanted into 40-cell plastic trays. d Seedlings were inoculated with FOM GR by cutting the roots with a scalpel and then pouring a 10 ml-aliquot of the spore suspension of conidia/ml on soil. e Each value represents the mean disease index±standard deviation of two runs with ten replicates each. f Resistance response: R, resistance [disease index (DI)=0 1.0]; MR, moderate resistance (DI= ); S, susceptibility (DI= ). (255 o C) 7 scalpel conidia/ml 10 ml o C 3. Fusarium oxysporum f. sp. melonis. (root-dipping),., scalpel, tip F. oxysporum f. sp. melonis. scalpel tip. tip scalpel. scalpel. scalpel (110 6, conidia/ml) (25, 30 o C), scalpel. 22. (255 o C) 7 scalpel conidia/ml 10 ml o C 3. Acknowledgement This research was supported by Golden Seed Project Vegetable Seed Center ( SBc10, SBZ10), Ministry of Agriculture Food and Rural Affairs (MAFRA), Ministry of Oceans and Fisheries (MOF), Rural Development Administration (RDA) and Korea Forest Service (KFS). References Beckman, C. H The nature of wilt diseases of plants. APS Press, St. Paul, MN. Cohen, R., Katan, T., Katan, J. and Cohn, R Occurrence of Fusarium oxysporum f. sp. melonis race 1,2 on muskmelon in Israel. Phytoparasitica 17: Freeman, S., Zveibil, A., Vintal, H. and Maymon, M Isolation of nonpathogenic mutants of Fusarium oxysporum f. sp. melonis

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