식물병연구 Research Article Open Access Res. Plant Dis. 21(2) : 74-81(2015) http://dx.doi.org/10.5423/rpd.2015.21.2.074 Acidovorax citrulli nested-pcr Development of Nested-PCR Assay to Detect Acidovorax citrulli, a Causal Agent of Bacterial Fruit Blotch at Cucurbitaceae 1 1 1 2 3 * 1 (), 2, 3 *Corresponding author Tel : +82-43-261-2554 Fax: +82-43-271-4414 E-mail: jscha@cbnu.ac.kr Young-Tak Kim 1, Kyoung-Soo Park 1, Hye-Seong Kim 1, Hyok-In Lee 2 and Jae-Soon Cha 3 * 1 Seed Certification Team, Quality Assurance Division, Nongwoo Bio Co., LTD., Yeoju 469-885, Korea 2 Animal and Plant Quarantine Agency, Anyang 430-757, Korea 3 Department of Plant Medicine, Chungbuk National University, Cheongju 361-763, Korea Received September 11, 2014 Revised March 12, 2015 Accepted March 25, 2015 The specific and sensitive nested-pcr method to detect Acidovorax citrulli, a causal agent of bacterial fruit blotch on cucurbitaceae, was developed. PCR primers were designed from the draft genome sequence which was obtained with the Next Generation Sequencing of A. citrulli KACC10651, and the nested-pcr primer set (Ac-ORF 21F/Ac-ORF 21R) were selected by checking of specificity to A. citrulli with PCR assays. The selected nested-pcr primer amplified the 140 bp DNA only from A. citrulli strains, and detection sensitivity of the nested PCR increased 10,000 times of 1 st PCR detection limit (10 ng genomic DNA/PCR). The nested PCR detected A. citrulli from the all samples of seed surface wash (external seed detection) of the artificially inoculated watermelon seeds with 10 1 cfu/ml and above population of A. citrulli while the nested PCR could not detected A. citrulli from the mashed seed suspension (internal seed detection) of the all artificially inoculated watermelon seeds. When the naturally infested watermelon seeds (10% seed infested rate with grow-out test) used, the nested PCR detected A. citrulli from 2 seed samples out of 10 replication samples externally and 5 seed samples out of 10 replication samples internally. We believe that the nested-pcr developed in this study will be useful method to detect A. citrulli from the Cucurbitaceae seeds. Keywords : Acidovorax citrulli, Bacterial fruit blotch, Cucurbitaceae seeds, Nested-PCR, Next Generation Sequencing Acidovorax citrulli (bacterial fruit blotch, BFB) (Hopkins Thompsin, 2002; Walcott, 2004). 1965 Research in Plant Disease The Korean Society of Plant Pathology pissn 1598-2262, eissn 2233-9191 (Sowel, 1978), 1980 (Latin, 1990)., (Isakeit, 1997; Langston, 1999; Matin, 1999). (Song, 1991), (Seo, 2006). This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Research in Plant Disease Vol. 21 No. 2 75 A. citrulli Pseudomonas,, 16S rrna Acidovorax (Willems, 1990). A. citrulli (Latin, 1995; Schaad, 2003). A. citrulli, 1 (Hopkins, 2002; Rane, 1992).. A. citrulli grow-out,,,. PCR(Polymerase chain reaction), IMS-PCR(Immunomagnetic separation and polymerase chain reaction), Bio-PCR Real-time PCR A. citrulli (Ha, 2009; Latin, 1995; Randhawa, 2001; Schaad, 2002; Walcott, 2000). A. citrulli, 1 (Bahar, 2008; Ha, 2009). A. citrulli. nested-pcr A. citrulli genome Next Generation Sequencing(NGS) A. citrulli ORFs(open reading frames),. 사용균주및 DNA 분리. Table 1. Bacterial strains used in this study and results of 1 st PCR and nested-pcr No. Strains a Host 1 st PCR b nested-pcr 1 Acidovorax citrulli KACC10651 Unknown + + 2 Acidovorax citrulli CNUPBL AC37 Unknown + + 3 Acidovorax citrulli CNUPBL AC42 Unknown + + 4 Acidovorax citrulli CNUPBL LB09-308 Unknown + + 5 Acidovorax citrulli CNUPBL LB09-309 Unknown + + 6 Acidovorax citrulli CNUPBL LB10-233 Unknown + + 7 Acidovorax citrulli CNUPBL LB10-234 Unknown + + 8 Acidovorax citrulli NWB SC016 Watermelon + + 9 Acidovorax citrulli NWB SC024 Watermelon + + 10 Acidovorax citrulli NWB SC058 Watermelon + + 11 Acidovorax citrulli NWB SC074 Watermelon + + 12 Acidovorax citrulli NWB SC076 Melon + + 13 Acidovorax citrulli NWB SC107 Rootstock + + 14 Acidovorax citrulli NWB SC108 Watermelon + + 15 Acidovorax citrulli NWB SC109 Watermelon + + 16 Acidovorax citrulli NWB SC110 Watermelon + + 17 Acidovorax citrulli NWB SC111 Watermelon + + 18 Acidovorax citrulli NWB SC172 Watermelon + + 19 Acidovorax citrulli NWB SC175 Cucumber + + 20 Acidovorax valerianellae NWB SC185 Watermelon - - 21 Acidovorax valerianellae NWB SC186 Watermelon - - 22 Acidovorax avenae CNUPBL294 Unknown - - 23 Acidovorax avenae subsp. avenae KACC10162 Zea mays - - 24 Acidovorax sp. KACC13277 Soil - - 25 Acidovorax facilis LMG2193 lawn soil - - 26 Acidovorax cattleyae LMG2364 Unknown - - 27 Acidovorax konjaci KACC10652 Konjac - - 28 Pseudomonas syringae pv. lachrymans KACC12856 Melon - - 29 Pseudomonas syringae pv. lachrymans KACC12857 Cucumber - - 30 Pseudomonas syringae pv. lachrymans KACC13264 Cucumber - -
76 Research in Plant Disease Vol. 21 No. 2 Table 1. Continued No. Strains a Host 1 st PCR b nested-pcr 31 Pseudomonas syringae pv. lachrymans LMG5070 Cucumber - - 32 Pseudomonas syringae pv. lachrymans LMG5172 Cucumber - - 33 Pseudomonas syringae pv. lachrymans LMG5458 Cucumber - - 34 Pseudomonas syringae pv. lachrymans LMG5459 Cucumber - - 35 Pseudomonas syringae pv. lachrymans LMG5662 Melon - - 36 Pseudomonas syringae pv. syringae LMG2230 Rice - - 37 Pseudomonas syringae pv. syringae NWB SC125 Squash - - 38 Pseudomonas syringae pv. syringae NWB SC132 Squash - - 39 Pseudomonas syringae pv. syringae NWB SC137 Watermelon - - 40 Pseudomonas syringae pv. tomato Y1 Tomato - - 41 Pseudomonas syringae pv. tomato DC3000 Tomato - - 42 Pseudomonas syringae pv. tomato LMG5093 Tomato - - 43 Pseudomonas syringae pv. tomato LMG5509 Tomato - - 44 Pseudomonas syringae pv. pisi LMG5079 Pea - - 45 Pseudomonas syringae pv. pisi LMG5478 Pea - - 46 Pseudomonas syringae pv. pisi LMG5479 Pea - - 47 Pseudomonas syringae pv. pisi LMG5480 Pea - - 48 Pseudomonas syringae pv. pisi LMG5679 Pea - - 49 Pseudomonas syringae pv. pisi LMG8612 Pea - - 50 Pseudomonas syringae pv. pisi KACC11620 Unknown - - 51 Pseudomonas syringae pv. phaseolicola KACC10575 Kidney bean - - 52 Pseudomonas syringae pv. phaseplicola NWB SC126 Squash - - 53 Pseudomonas syringae pv. tabaci NWB SC127 Squash - - 54 Pseudomonas syringae pv. putida NWB SC136 Watermelon - - 55 Pseudomonas syringae pv. garcae KACC10398 Coffee - - 56 Pseudomonas syringae pv. helianthi KACC11618 Unknown - - 57 Pseudomonas syringae pv. japonica KACC11638 Unknown - - 58 Pseudomonas viridiflava LMG6480 Chicory - - 59 Pseudomonas syringae pv. lapsa KACC12216 Unknown - - 60 Pseudomonas syringae pv. mellea KACC12844 Unknown - - 61 Pseudomonas syringae pv. mori KACC10390 Unknown - - 62 Pseudomonas syringae pv. morsprunorum KACC10397 Unknown - - 63 Pseudomonas syringae pv. myricae KACC12845 Unknown - - 64 Pseudomonas syringae pv. panici KACC11619 Unknown - - 65 Pseudomonas syringae pv. papulans LMG5077 Apple tree - - 66 Pseudomonas syringae pv. persicae KACC12852 Unknown - - 67 Pseudomonas syringae pv. ribicola KACC11622 Unknown - - 68 Pseudomonas syringae pv. sesami KACC10649 Unknown - - 69 Pseudomonas syringae pv. tabaci KACC10388 Tobacco - - 70 Pseudomonas syringae pv. tagetis KACC10389 Unknown - - 71 Pseudomonas syringae pv. ulmi KACC11633 Unknown - - 72 Pseudomonas syringae pv. glycinea LMG5554 Unknown - - 73 Pseudomonas syringae pv. savastanoi KACC10751 Olive - - 74 Pseudomonas syringae pv. passiflorae KACC12846 Unknown - - 75 Pseudomonas syringae pv. maculicola LMG5560 Cabbage - - 76 Xanthomonas campestris pv. campestris LMG7460 Rape - - 77 Xanthomonas campestris pv. vesicatoria KACC11153 Pepper - - 78 Clavibacter michiganensis subsp. michiganensis NWB SC191 Tomato - - a KACC: Korean Agricultural Culture Collection, CNUPBL: Chungbuk National University Plant Bacteriology Lab, LMG: Belgian Coordinated Collections of Micro-organisms, NWB: Nongwoobio Co., LTD. b +: PCR product of the expected size was amplified, -: no PCR product was amplified.
Research in Plant Disease Vol. 21 No. 2 77 A. citrulli 19 Acidovorax Pseudomonas 78. BCCM/LMG(The Belgian Co-ordinated Collections of Micro-organisms/Collection of the Laboratiorium voor Microbiologie en Microbiele Genetica, Belgium) (Korean Agricultural Culture Collection, KACC) (CNUPBL) (Table 1). DNA, (Nongwoobio. Co., LTD.). glycerol 15% Kings B -75 o C. 28 o C 3 Kings B(peptone 20.0 g, MgSO 4 7H 2 O 1.5 g, K 2 HPO 4 1.5 g, glycerol 15.0 ml, agar 18.0 g/l) (ROCHE, Basel, Switzerland) DNA. genomic DNA (Mecasys, Deajeon, Korea) 10 ng/ml -20 o C. Next Generation Sequencing. NCBI A. citrulli Acidovorax A. citrulli (Chunlab, Seoul, Korea) A. citrulli draft genome sequence. A. citrulli draft genome A. citrulli KACC10651 genomic DNA Next Generation Sequencing(NGS), NGS Illumina (BMSKorea, Seoul, Korea). 프라이머제작. NGS A. citrulli KACC10651 contig ORFs(open reading frames) NCBI BLASTN Genbank, 500 bp ORF. ORF Primer3(v. 0.4.0) PCR, A. citrulli 19 Acidovorax 8 27 PCR, PCR A. citrulli DNA 1. 1 2 Pseudomonas 51 PCR. Nested PCR A. citrulli PCR Primer3(v. 0.4.0) 100-200 bp DNA. PCR 조건. PCR 95 o C 5 initial denaturation 95 o C 30 denaturation, 60 o C 30 annealing, 72 o C 45 extension 30 72 o C 10 final extension. PCR (Daejeon, Korea) Ex Taq PCR kit (10 pm/ml), genomic DNA 1 ml(10 ng) 20 ml. Nested-PCR 1 PCR 50 1 ml PCR. nested- PCR, PCR 1.2% Agarose gel 120 V, 60. 자연감염종자와인공접종종자조제. PCR., grow-out 10%. A. citrulli KACC10651 28 o C 48 (Mecasys, Deajeon, Korea) OD 600 = 0.1. 10 8 cfu/ml 10 10 8 cfu/ml 10 0 cfu/ml. 10 ml 3 g 24 28 o C, 200 rpm. 종자처리.. 1 g, 1 g 10. 5 ml 1 g 1 200 rpm DNA. ( 1% 10 3 ). DNA. DNA (ROCHE, Basel, Switzerland). ( ) DNA PCR A. citrulli.
78 Research in Plant Disease Vol. 21 No. 2 Fig. 1. PCR result which was carried out with primer set, Ac-ORF 12F/ Ac-ORF 13R and bacterial DNA. Lanes 1-78 is corresponding strain number 1-78 in Table 1. NE: water as negative control. PCR 프라이머의특이성. NGS A. citrulli KACC10651 DNA 510 contig, ORF 244. 244 A. citrulli 19 Acidovorax 8 27 PCR 1 A. citrulli DNA 26. 26 Pseudomonas 51 PCR 5 2. 2 5 A. citrulli 19 DNA 620 bp DNA Ac-ORF 12F(5 -GCATCTTGTTCAGC- CACGAC-3 ) Ac-ORF 13R(5 -ATTGGCAATCACCAAGACGC-3 ) (Fig. 1). PCR Ac- ORF 12F Ac-ORF 13R nested-pcr Ac-ORF 21F(5 -AGCCCACCTA- ATCCTCCCTT-3 ) Ac-ORF 21R(5 -TTATTCTGGGCGTCACC- GTC-3 ). nested-pcr PCR A. citrulli 19 DNA 140 bp (Fig. 2). PCR Ac-ORF 12F Ac-ORF 13R, nested-pcr Ac-ORF 21F Ac-ORF 21R A. citrulli DNA A. citrulli. Fig. 2. Nested-PCR result which was carried out with primer set (Ac-ORF 21F/Ac-ORF 21R) and bacterial DNA. Lanes 1-78 is corresponding strain number 1-78 in Table 1. NE: water as negative control. PCR 프라이머의민감도. PCR 10 ng genomic DNA 10 PCR. PCR Ac-ORF 12F Ac- ORF 13R PCR, 10 ng 1,000 Fig. 3. PCR and nested-pcr results for determination of detection sensitivity of target bacterial genomic DNA. PCR was carried out with primer set, Ac-ORF 12F/Ac-ORF 13R and 10 ng of genomic DNA of Acidovorax citrulli (lane 1) and its ten-fold serial dilution (lane 2-9). Lane 10 was water as negative control (A). Nested-PCR was carried out with primer set, Ac-ORF 21F/Ac-ORF 21R and 1 ml of 50 times dilution of 1 st PCR product (B).
Research in Plant Disease Vol. 21 No. 2 79 Fig. 4. Nested-PCR results for detection of Acidovorax citrulli from external seed samples (A) and internal seed samples of the artificially inoculated watermelon seeds. Nested-PCR was carried out with primer set, Ac-ORF 21F/Ac-ORF 21R and DNA from external and internal seed preparation of the artificially inoculated seeds in 10 8 cfu/ml-10 0 cfu/ml (lane 1-9). N is negative control and P is positive control. Fig. 5. Nested-PCR results for detection of Acidovorax citrulli from external seed samples (A) and internal seed samples of the naturally infected watermelon seeds. Nested-PCR was carried out with primer set, Ac-ORF 21F/Ac-ORF 21R and DNA from external and internal seed preparation of the naturally infected seeds. Lane 1-10 are 10 replication seed samples. N is negative control and P is positive control. 10 pg DNA (Fig. 3). PCR 50 1 ml nested-pcr 1 PCR 10,000 DNA 140 bp (Fig. 3). 인공접종종자로부터검출. A. citrulli 10 nested-pcr 10 8-10 1 cfu/ml (140 bp) DNA. 10 8-10 0 cfu/ml 140 bp (Fig. 4). A. citrulli nested-pcr A. citrulli, A. citrulli. 자연감염종자로부터검출. 1 g 10 nested-pcr 10 1 7 A. citrulli (Fig. 5). 1 7 2, 3, 6 A. citrulli (Fig. 5). 1 7 A. citrulli, 2, 3 6 A. citrulli. 10 A. citrulli A. citrulli grow-out 10% A. citrulli, 1 g 18-20 A. citrulli, A. citrulli. A. citrulli PCR. PCR target. pathovar(pv.) PCR whole-genome draft genome (An, 2015; Lang, 2010). A. citrulli A. citrulli KACC10651 draft genome
80 Research in Plant Disease Vol. 21 No. 2 sequence Next Generation Sequencing(NGS), Ac-ORF 12F/Ac-ORF 13R PCR Ac-ORF 21F/Ac-ORF 21R nested PCR,, 19 A. citrulli 620 bp 140 bp DNA. PCR PCR A. citrulli genomic DNA 10 pg, nested PCR 1 PCR 10,000. LAMP(Loopmediated Isothermal Amplification) genomic DNA 10 fg(rigano, 2010), nested-pcr. nested- PCR. 10 1 cfu/ml. 24. 10 2 A. citrulli, 10 5 A. citrulli. grow-out 10% 10 5 A. citrulli, nested-pcr. F 1. BFB, A. citrulli A. citrulli. A. citrulli, nested-pcr A. citrulli. (bacterial fruit blotch) Acidovorax citrulli nested-pcr. Next Generation Sequencing draft genome sequencing PCR, A. citrulli Ac-ORF 21F/ Ac-ORF 21R nested PCR. Ac- ORF 21F/Ac-ORF 21R A. citrulli 140 bp DNA, 1 PCR (10 ng genomic DNA/PCR) 10,000. nested-pcr 10 1 cfu/ml,. 10 2, 10 5 A. citrulli. nested-pcr A. citrulli. Acknowledgments This study was financially supported by research fund of Nongwoo Bio CO., LTD in 2014. References An, J. H., Noh, Y. H., Kim, Y. E., Lee, H. I. and Cha, J. S. 2015. Development of PCR and TaqMan PCR assays to detect Pseudomonas corona-faciens, a causal agent of halo blight of oats. Plant Pathol. J. 31: 25-32. Bahar, O., Efrat, M., Hadar, E., Dutta, B., Walcott, R. R. and Burdman, S. 2008. New subspecies-specific polymerase chain reactionbased assay for the detection of Acidovorax avenae subsp. citrulli. Plant Pathol. 57: 754-763. Burdman, S., Kots, N., Kritzman, G. and Kopelowitz, J. 2005. Molecular, physiological, and host-range characterization of Acidovorax avenae subsp. citrulli Isolates from watermelon and melon in Israel. Plant Dis. 89: 1339-1347. Dreier, J., Bermpohl, A. and Eichenlaub, R. 1995. Southern hybridization and PCR for specific detection of phytopathogenic Clavibacter michiganensis subsp. michiganensis. Phytopathology 85: 462-468. Ha, Y., Fessehaie, A., Ling, K. S., Wechter, W. P., Keinath, A. P. and Walcott, R. R. 2009. Simultaneous detection of Acidovorax avenae subsp. citrulli and Didymella bryoniae in cucurbit seedlots using magnetic capture hybridization and real-time polymerase chain reaction. Phytopathology 99: 666-678. Himananto, O., Thummabenjapone, P., Luxananil, P., Kumpoosiri, M., Hongprayoon, R., Kositratana, W. and Gajanandana, O. 2011. Novel and highly specific monoclonal antibody to Acidovorax citrulli and development of ELISA-based detection in cucurbit leaves and seed. Plant Dis. 95: 1172-1178.
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