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1 w ³wz The Korean Journal of Mycology The Korean Society of Mycology Kor. J. Mycol. 36(2) : (2008) q ³(Botrytis cinerea) PCR w p Primer 1 Á w 1 Áû x 2 Á½y» 1 Á½ 3 1 û w w œ, 2» x, 3 w w Development of PCR Primers for Specific Identification and Detection of Botrytis cinerea on Tomato Jeong Young Song 1, Jin Ha Lim, Myeong Hyeon Nam 2, Hong Gi Kim 1 1 Dept. of Agricultural Biology, Chungnam National University, 2 Nonsan Strawberry Experiment Station, Chungnam ARES, 3 Dept. of Plant Science, Kangnung National University (Received November 24, Accepted December 19, 2008) and Byung-Sup Kim 3 ABSTRACT: Botrytis cinerea, gray mold pathogen, causes serious losses in greenhouse tomato crop. In this study, a primer set was developed for identification and specific PCR detection of B. cinerea from tomato plants. The primer pair (BTF1/BTR1) was designed from polymorphic sequence region in pyruvate carboxylase gene (pyc) of B. cinerea. A PCR product (112 bp) was amplified on genomic DNA of 13 B. cinerea isolates from 10 different host plants, but not on those from 6 other Botrytis spp., 4 Botryotinia spp., 5 Sclerotinia spp. and 16 other genus of phytopathogenic fungi. The sensitivity limit of the primer set was 2 pg of genomic DNA of B. cinerea, approximately. The PCR assay using species-specific primer set was specifically able to detect the pathogen on naturally infected tomato plants and artificially infected plants. These results suggest that the sensitivity and specificity of this primer set can be applied in a rapid and accurate diagnosis of tomato disease caused by B. cinerea. KEYWORDS : Botrytis cinerea, Pyruvate carboxylase gene, Species-specific primer, Tomato m m q ³(Botrytis cinerea, : Botryotinia fuckeliana) m m ƒ, š,»,, y{, 200» w j ³ ü sw ƒw vw k (Coley-Smith et al., 1980; ½, 1997) B. cinerea» ³ ù ³w w ƒ ƒ w m m ƒ w wš w w w yz w» w (Chastagner and Ogawa, 1979; Coley-Smith et al., 1980; Droby and Lichter, 2004; Williamson et al., 2007). w, w ³ ³w l s œ» w ù w e w w p wš ƒ. m m q w» ³ w *Corresponding author < bskim@kangnung.ac.kr> l w yw mw z w. beta-tublin, SCAR(sequence characterized amplified region) marker, IGS(intergenic spacer of the nuclear ribosomal DNA), ITS(internal spacer of the nuclear ribosomal DNA) š cutinase A w p DNA» l p molecular marker w PCR (polymerase chain reaction)» Botrytis ³ Ÿ w y š (Chilvers et al., 2007; Gachon and Saindrenan, 2004; Rigotti, et al., 2002; Suarez, et al., 2005). p molecular marker w» w B. cinerea w Botrytis ü Sclerotinia pyruvate carboxylase(pyc) ü w, B. cinerea p primer PCR» w y w» w 138

2 q ³(Botrytis cinerea) PCR w p Primer 139 Table 1. Fungal species used in this study and their PCR detection using specific primer set (BTF1/BTR1) Isolates Fungal species Host plants Source a PCR test b (BTF1/BTR1) BC-RU Botrytis cinerea rubber tree KN BC-RU + BC-ST B. cinerea strawberry CNU BC-ST + BC-MA B. cinerea magnificus CNU BC-MA + BC-TO B. cinerea tomato CNU BC-TO + BC-YY2 B. cinerea tomato KN BC-YY2 + BC-GR B. cinerea grape KACC BC-IK21 B. cinerea paprika KN BC-IK21 + BC-RRS B. cinerea paprika KN BC-RRS + BC-LI B. cinerea lily KACC BC-RO B. cinerea rose KACC BC-58 B. cinerea geranium CNU BC-58 + BC-447 B. cinerea strawberry CNU BC BC-449 B. cinerea strawberry CNU BC BOAC B. aclada allium KACC BOBY B. byssoidea watermelon KCTC BOCR B. croci crocus KCTC BOEL B. elliptica lily KACC BOFA B. fabae broad bean KACC BOGA B. galanthina snowdrop KCTC BTCA Botryotinia calthae caltha KCTC BTCO Botryotinia convoluta iris KCTC BTDR Botryotinia draytonii gladiolus KCTC BTFI Botryotinia ficariarum plant debris KCTC SLSC Scleotinia sclerotiorum lectucae KACC SLBU Sc. bulborum peziza KACC SLTR Sc. trifoliorum white clover KACC SLSP Sc. spermophila white clover KACC SLMI Sc. minor chinese cabbage KACC SCCE Sc. cepivorum garlic KACC SCDE Sc. delphinii german iris KACC SCDE Sclerotium cepivorum lily KACC SCHY Sclerotium delphinii victoria regia KACC SCPE Sclerotium denigrans tulip KACC SCRO Sclerotium hydrophilum apple KACC SCTU Sclerotium perniciosum tulip KACC SCWA Sclerotium rolfsii tulip KACC SCBA Sclerotium tuliparum bean KACC COAC Colletotrichum acutatum pepper KACC PHIN Phytophthora infestans tomato CNU-PHIN ALSO Alternaria solani carrot CNU-3509 CLCU Cladosporium cucumerinum cucumber KACC STLY Stemphylium lycopersici pepper CNU-STLY MOFR Monilinia fructicola apple KACC ASNI Aspergillus niger pinus CNU-ASNI RHSO Rhizoctonia solani rice KACC PYUL Pythium ultimum cucumber KACC DIBR Didymella bryoniae melon KACC FUFU Fulvia fulvum tomato CNU-FUFU PEDI Penicillium digitatum citrus CNU-PEDI RHOR Rhizopus oryzae sudan grass KACC FOLY Fusarium oxysporum f. sp. lycopersici tomato CNU-FOLY STNA Stromatinia narcissi narcissus KACC a CNU, Chungnam National University; KN, Kangnung National University, KACC, Korean Agricultural Culture Collection and KCTC, Korean Collection for Type Cultures. b, negative; +, positive.

3 140 p w. œ ³ œ ³ B. cinerea»k ³ û (CNU) (KNU), l (KCTC) š w l(kacc) l w (Table 1). Genomic DNA œ ³ PDA w ƒƒ 20~27 C o 5 w z ³ PD broth w z 5 k w ³ ³ 2 ƒ effendorf p š 70 C z o w. ³ w 400 µl extraction buffer[200 mm Tris-HCl(pH 8.0), 200 mm NaCl, 30 mm EDTA, 0.5% SDS] proteinase K(50 µg) ƒw 37 C 1 o w. 400 µl 2ÜCTAB solution[2% CTAB(w/v), 100 mm Tris- HCl(pH 8.0), 20 mm EDTA(pH 8.0), 1.4 M NaCl, 1% PVP(polyvinylpyrrolidone)] ƒw z 600 µl chloroform:isoamylalcohol(24 : 1) wš 12,000 rpm 10 w 1.5 ml tube. 0.7 isopropanol ƒ wš 10 ew z 12,000rpm 10 w. e DNA 70% ethanol w w. p ü ethanol, k z DNA 100 µl TE buffer 2 µl(10 mg/ml) RNase ƒw RNA w z agarose gel» w DNA y w. m m s w q (100 mg) B. cinerea k z m m (100 mg) ³w(20 mg) l genomic DNA w w. Pyruvate carboxylase(pyc) PCR s GenBank( œ Botryotinia fuckeliana(xm_ ), Sclerotinia sclerotiorum (XM_ ), Podospora anserine(xm_ ), Gibberella zeae(xm_387251), Magnaporthe grisea(xm_ ) JGI( œ Mycosphaerella graminicola(scaffold_10: ) Nectria haematococa(scaffold_10: ) pyruvate carboxylase(pyc) DNA» w œm sw x universal primer set PYCUF1(5'-GGACHACNTTCACGA-3')/ PYCUR5(5'-GGNGTYACCTTGAC-3') PYCUF(5'- CACTTCTGTGAGCAAGC-3')/PYCUR(5'-GATGTTGG- AGTGACCTTG-3') w. w pyruvate carboxylase(pyc) DNA» š B. cinerea, B. aclada B. fabae ³ w PCR s w. ƒ s w mixture total volume 50 µl wš template DNA 2~10 ng, ƒ primer set 10 pmole, 250 µm dntps, 10ÜPCR buffer 5 µl, 2 mm MgCl 2, Taq DNA Polymerase 2 unit(solgent Co.) ƒw PCR ww. s initial denaturation 94 C 5 w z o denaturation 94 o C/30, annealing 55 o C/90, extension 72 o C/ cycles wš final extension 72 o C/10 w. MJ Research PTC-100 w PCR s w s PCR 1.5% agarose gel 100 V 30» w ethidium bromide w z w.» PCR s PCR prep kit(intron Biotechnology) wš, BigDye teminator cycle sequencing kit (Applied Biosystems, Forster City, CA, U.S.A), template 40 ng, primer(pycuf1, PYCUR5, PYCUF, PYCUR) ƒƒ 3.2 pmol, teminator ready reaction mixture 8 µl ƒw 20 µl volume w 96 o C/10, 50 o C/5, 60 C/4 o 25 cycle PCR ww z ABI prismtm 3730 Genetic Analyzer(PE Applied Biosystems) w sequencing w. p primer p primer w B. cinerea, B. aclada B. fabae pyc» B. fuckeliana Sc. Sclerotiorum wì EMBL-EBI( clastralw v w, Oligonucleotide Properties Calculator( w B. cinerea p primer BTNF1; 5'-GCT TGA CCC AGG CTT GAA C-3' BTNR1 5'-TGG GTC TGG TCC CGT GTA A-3' w. p primer p primer p wš 10» w B. cinerea 13³, 6 Botrytis, 4 Botryotinia, 7 Sclerotinia 16 ³ 16³ w BTNF1/ BTNR1 primer set w PCR w. w š B. cinerea ³ (BC-TO) genomic DNA 10 k z PCR s w. PCR initial denaturation 94 o C

4 q ³(Botrytis cinerea) PCR w p Primer w z denaturation 94 o C/30, annealing 65 o C/30, extension 72 C/30 o 35 cycles wš final extension 72 C/5 o w. PCR mixture genomic DNA pyc sw w w. l ³ m m s q w ³ w. r 1% ùp (NaOCl) t w z Streptomycin 300 ppm ƒ potato dextrose agar(pda) e w š, r l ù ³ ³ w z 4 o C þ š w œ ³ w. w ³»» DNA w w, p primer p w PCR x ww. š Pyruvate carboxylase(pyc)» mg ü w w w z pyc (Jitrapakdee and Wallace, 1999) w Botrytis ³ p š GenBank œ DNA» w ( š). q ³ B. cinerea Botryotinia fuckeliana(3,630 bp) Sc. sclerotiorum (3,624 bp)ƒ ƒƒ 90%, 94% DNA» ƒ ƒà ùkû. w pyc 70%ƒ w w ³ DNA» w z ³ pyc sw ü sw wš p primer ƒ ƒ w PCR s j universal primer. p primer» w B. cinerea w ³ B. aclada B. fabae ³ w pyc PCR sw z DNA» w. 437 bp j» ü m m ³ B. cinerea(bo-to) PCR s» ƒ œ ³ B. cinerea Botryotinia fuckeliana 2,339 l 2,775 ¾ w j» DNA w» ƒ 2,628 w œ ü» ƒ w p ùkü š (Fig. 1). wr, Sc. sclerotiorum (437 bp) 2,495 l 2,686 DNA 437 bp j Fig. 1. Partial sequence alignment of pyruvate carboxylase gene (pyc). Underlined sequences indicates specific primers for Botrytis cinerea.» B. cinerea DNA» ¼ ùk ù»ü 88% ù kþ. w B. cinerea B. aclada(488 bp, 96%) š B. fabae (494 bp, 90%) w DNA»¼ Sc. sclerotiorum w ƒƒ ùkþ. Botrytis ³ pyc w» ƒ ü w w. p primer p B. cinerea w ³ pyc ü w DNA» mw 112 bp s j p primer(btf1/ BTR1)ƒ (Fig. 2). primer p wš ü m m sww 10ƒ w» w B. cinerea 13³ 6 Botrytis ³, 4 Botryotinia ³, 5 Sclerotinia ³ 16 ³ w BTNF1/ BTNR1 primer set w PCR w B. cinerea ³ p 112 bp s

5 142 송정영 등 Fig. 2. Specific PCR amplification of Botrytis cinerea isolates by species-specific primer set (BTF1/BTR1). Genomic DNAs of Botrytis cinerea isolates (lanes 1~13: BCRU, BC-ST, BC-MA, BC-TO, BC-YY2, BC-GR, BCIK21, BC-RRS, BC-LI, BC-RO, BC-58, BC-447, BC449) from various host plants were used for PCR analysis. M: 100 bp DNA ladder (intron, Korea), lane 14: negative control (addition of sterile water to the PCR mixture). Fig. 3. PCR sensitivity of primer set BTF1/BTR1 for serially diluted genomic DNA of Botrytis cinerea (BC-TO), M, 100 bp DNA ladder (intron, Korea); lane 1, 2 ng; lane 2, 200 pg; lane 3, 20 pg; lane 4, 2 pg; lane 5, 200 fg; lane 6, 20 fg; lane 7, 2 fg; lane 8, negative control (addition of sterile water to the PCR mixture). 들을 얻을 수 있었다(Fig. 2와 3). 또한 이들의 PCR 반응 민감도를 알아보고자 B. cinerea(bc-to) genomic DNA 를 10배씩 연속적으로 희석시킨 후 PCR 증폭을 실시한 결과 반응민감도 한계는 대략 2 pg임을 알 수 있었다(Fig. 4). Real time PCR은 본 연구에서와 같이 전기영동을 통 해 결과를 확인하는 conventional PCR에 비해 PCR 반응 민감도가 약 10~100배 이상 높게 나타나는 것으로 알려 져 있지만 보다 정밀한 결과분석을 위해 작은 크기의 증 폭산물을 만드는 종 특이적 primer가 우선적으로 요구되 며, 증폭산물 내부에 probe의 제작이 가능한 종 특이적 변 이영역을 필요로 하기도 한다(Minerdi et al., 2008; Suarez, et al., 2005). 따라서 본 실험에서 새롭게 개발된 종 특이적 primer는 112 bp의 최적 크기의 단일 증폭산물 을 만들며 probe 제조시 필요한 증폭산물 내부의 종 특이 적 변이영역이 존재해 real time PCR에서도 매우 유용하 게 활용될 수 있을 것이다. Fig. 4. Specificity test for primer set BTF1/BTR1 in different fungal species. No product was amplified on DNAs from negative control (addition of sterile water to the PCR mixture) (lane 1), 6 other Botrytis spp. (lanes 2~6), 4 Botryotinia spp. (lanes 7~10), 5 Sclerotinia spp. (lanes 11~15) and Sclerotium cepivorum (lane 16) isolates. Lane 17: positive control (2 ng/µl of B. cinerea (BC-TO) genomic DNA) and M: 100 bp DNA ladder (intron, Korea). Fig. 5. PCR detection of Botrytis cinerea using DNAs extracted from diseased tomato plants, M: 100 bp DNA ladder (intron, Korea), lane 1, artificially inoculated tomato plant; lane 2, naturally infected tomato fruit; lane 3, not infected tomato fruit; lane 4, negative control (addition of sterile water to the PCR mixture); lane 5, sclerotia of B. cinerea; lane 6, positive control (2 ng/µl of B. cinerea (BC-TO) genomic DNA). 병든 식물체로부터 병원균 검출 자연적으로 B. cinerea에 의해 감염된 토마토 식물체와 인공적으로 접종된 토마토 열매병반과 균핵으로부터 DNA 를 분리 후 종 특이적 primer를 이용한 PCR을 수행한 결 과 병원균의 검출이 이루어졌다(Fig. 5). 또한 이들 이병 식물체로부터 B. cinerea를 배지상에서 모두 순수하게 분 리하였으며 이들 DNA에 대한 PCR 반응에 있어서도 112 bp 크기의 증폭산물들을 모두 확인할 수 있었다. B. cinerea 는 식물체의 이병잔재 및 토양에서 생존하며 식물체를 침 입하여 병징을 나타낼 때까지 발병전 꽃이 진 부위에 부 생적으로 존재하기도하며 식물체에 잠복하다가 수확 후 저장시 피해를 주는 것으로 알려져 있다(Coley-Smith et al., 1981; Droby and Lichter, 2004). 이러한 토마토 잿빛 곰팡이병 발생에 의한 피해를 최소화하기 위해서는 신속

6 q ³(Botrytis cinerea) PCR w p Primer 143 yw mw w w ƒ v w q. primer p ùkü z m m q ³ yw y q. m m q ³(B. cinerea) w w m m» mw ƒ w vw. m m w q ³ w p primer setƒ. p primer(btf1/btr1) B. cinerea w ³ pyruvate carboxylase(pyc) ü l. 10» 13³ B. cinerea 112 bp j» PCR. ù 6 Botrytis ³, 4 Botryotinia ³, 5 Sclerotinia ³ 16 ³ w PCR ùkù. p primer w 2pg. k B. cinerea m m œ l p primer y w ³ PCR. primer p ùk ü z m m q ³ yw y q. w w (KRF C00157) 2008 FTA» m m w. ³ w û w w x w ½ ³ Ì. š x ½,, y, Ÿ q ³(Botrytis cinerea) xkx. w ³wz 25: Chastagner, G. A. and Ogawa, J. M A fungicide-wax treatment to suppress Botrytis cinerea and protect fresh-market tomatoes. Phytopathology 69: Chilvers, M. I., du Toit, L. J., Akamatsu, H. and Peever, T. L A real-time, quantitative PCR seed assay for Botrytis spp. that cause neck rot of onion. Plant Dis. 91: Coley-Smith, J. R., Verhoeff, K. and Jarvis, W. R The Biology of Botrytis, edited by Academic Press, London, New York. Droby, A. and Lichter, A Post-harvest Botrytis infection: etiology, development and management. In: Botrytis: Biology, Pathology and Control. (Elad, Y., Williamson, B., Tudzynski, P. and Delen, N., eds), pp Dordrecht, The Netherlands: Kluwer Academic Press. Gachon, C. and Saindrenan, P Real-time PCR monitoring of fungal development in Arabidopsis thaliana infected by Alternaria brassicicola and Botrytis cinerea. Plant Physiol. Biochem. 42: Jitrapakdee, S. and Wallace, J. C Structure, function and regulation of pyruvate carboxylase. Biochem. J. 340:1-16. Minerdi, D., Moretti, M., Li., Y., Gaggero, L., Garibaldi, A. and Gullino, M. L Conventional PCR and real time quantitative PCR detection of Phytophthora cryptogea on Gerbera jamesonii. Eur. J. Plant Pathol. 122: Rigotti, S., Gindro, K., Richter, H. and Viret, O Characterization for molecular markers for specific and sensitive detection of Botrytis cinerea Pers.: Fr. In strawberry (Fragaria x ananassa) using PCR. FEMS Microbiol. 209: Suarez, M. B., Walsh, K., Boonham, N., O Neill, T., Pearson, S. and Barker, I Development of real-time PCR (Taq- Man ) assays for the detection and quantification of Botrytis cinerea in planta. Plant Physiol. Biochem. 43: Williamson, B., Tudzynski, P., Van, K. and Jan, A. L Botrytis cinerea: the cause of grey mold disease. Mol. Plant Pathol. 8:

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