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1 I Pythium Pythium Pythium 1 2 Qualitative and Quantitative Detection of Plant Pathogens. Pythium By Koji KAGEYAMA PCR Pythium Pythium 2006 II PCR PCRPolymerase Chain ReactionDNA PCR 2005PCR PCR DNA Pythium 1 PCR 1 1 PCR 1 Singleplex PCR2 Multiplex PCR ASANO et al., 2010 PCR Real Time PCRLI et al., 2010 Pythium Singleplex PCRMutiplex PCRReal Time PCR 1 rdna ITS 52

2 691 1 Pythium PCR Singleplex PCR P. oligandrum P. helicoides P. porphyrae P. intermedium P. sulcatum P. sylvaticum P. violate Multiplex PCR P. torulosum P. vanterpoolii Real time PCR P. abappressorium P. attarantheridium P. heterohtallicum P. irregulare group I P. irregulare group IV P. paroecandrum P. rostratifingens P. sylvaticum P. irregulare P. intermedium KAGEYAMA et al., 1997 GODFREY et al., 2003, 2007 PARK, 2006 KLEMSDAL et al., 2008 ASANO et al., 2010 SCHROEDER et al., 2006 KERNAGHAN et al., 2008 LI et al., 2010 III Mutliplex PCR 5 Pythium Multiplex PCR ASANO et al., 2010Pythium Multiplex PCR Multiplex PCR rdna ITS 2 1 DNA Tm PCR PCR Mutliplex PCR rdna 18S 18S 69F/18S 1118R ; The Helicosporidia Project Online, http//helicosporidia.ifta.ufl.edu/ Pythium DNA Pythium helicoides Multiplex PCR 2Multiplex PCR HENEGARIU et al IV Real Time PCR LAMP Real Time PCR Pythium Real Time PCR 3 P. intermedium LI et al., 2010 rdna ITS Pythium rdna P. sylvaticum MARTIN, 1995 rdna ITS P. intermedium Real Time PCR TaqMan SYBR Green I PCR PCR 300 bp P. intermedium Real Time PCR DNA Real Time PCR 53

3 Pythium graminicola PCR 5 3 Tm bp APH1 APH2 CAT GTT CTG TGC TCT CTT TC AGT TGT TCA CAA TAA ATT GC ARRF ARR5 AAT TCT GTA CGC GTG GTC TCC AAG AGC AAT AAC CAC TCT CA GRA6 GRA7 TGG GCT GCA TGT ATG TGT AGT CT CTC CTT TAC CCT CGA GAG GGC P. torlosum TOR3 TOR4 TAG AGC TGC ATG TAA AAG TGC GGT T CCT TTA CCC TAC GAG TAG GG P. vernterpoolii VAN1 VAN2 GGT GGA TAG CGG CGT ATT TA GTT TAC AAG CAG CAA CTA GA All microorganisms 18S 69F a 18S 1118R a CTG CGA ATG GCT CAT TAA ATC AGT GGT GGT GCC CTT CCG TCA A b Pythium group P. torlosum PyF APH2B ARRR TOR6 CTG TTC TTT CCT TGA GGT G GCG CGT TGT TCA CAA TAA ATT GC CGT CCA AGA GCA ATA ACC ACT C CGC CTG CCG AAA CAG ACT AG Pythium PyR ATT CTG CAA TTC GCA TTA C 49.8 GRAF GGG CTG CAT GTA TGT GTA GT 55.6 P. vernterpoolii VANF GGT GGA TAG CGG CGT ATT T 55.8 a 18S 69F/18S 1118R The Helicosporidia Project Onlinehttp//helicosporidia.ifta.ufl.edu/. b 18S69 F/18S 1118R S 18S ITS1 5.8S ITS2 28S 18SF 1,000 bp 18SR GRAF VANF 181 bp P. vanterpoolii 208 bp PyR PyF 163 bp 89 bp APH2B ARRR P. torlosum 150 bp TOR6 1 Pythium graminicola PCR rdna ITS 18S F 18S R DNA Real Time PCR 3 V PCR P. helicoides PCR 1996 KAGEYAMA et al.,

693 100 bp ladder 1 2 3 4 5 6 D N A 1g pg 3 2.5 2 1.5 1 0.5 y1.2803x1.3595 R 2 0.9655 0 1 gcfu P.

4 bp ladder D N A 1g pg y1.2803x R gcfu P. helicoides 3 Pythium intermedium Real Time PCR DNA 2 Pythium helicoides rdna 18S Mutliplex PCR No P. helicoides 3 PCR Pythium helicoides a /5 2/5 5/6 b 2/6 2/6 2/6 1/1 1/1 1/2 1/2 1/3 2/3 0/3 2/3 1/1 1/1 2/2 2/2 a b 55

5 PCR P. helicoides PCR DNA KAGEYAMA et al. 2003LI et al., 2010 DNA DNA g 0.2 g P. intermedium Real Time PCR P. helicoides 1ASANO, T. et al.2010: J. Phytopath. 158 : GODFREY, S. A. C. et al.2003: Mycol. Res. 107 : HENEGARIU, O. et al.1997: BioTechniques 23 : KAGEYAMA K. et al.1997: Plant Dis. 81 : et al.2002: J. Gen. Plant Path. 68 : et al.2003: 69 : : 59 : : 60 : KERNAGHAN, G. et al.2008: Appl. Soil Ecol. 40 : KLEMSDAL, S. S. et al.2008: Plant Path. 57 : LI, M. et al.2010: Microbiol. Res.in press, doi : /j. micres MARTIN, F. N.1995: Mycologia 87 : PARK, C. S.2006: J. Appl. Phycol. 18 : SCHROEDER, K. I. et al.2006: Phytopathology 96 : : 73 : UPL10/08/

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2 3 4 TTT TCT TAT TGT TTC TCC TAC TGC TTA TCA TAA TGA TTG TCG TAG TGG CTT CCT CAT CGT CTC CCC CAC CGC CTA CCA CAA CGA CTG CCG CAG CGG ATT ACT AAT AGT ATC ACC AAC AGC ATA ACA AAA AGA ATG ACG AAG AGG GTT