Development of A Real-Time PCR Assay for Plasmodiophora brassicae and Its Detection in Soil Samples

doi:10.1016/S1671-2927(00)9145

Journal of Integrative Agriculture ›› 2013, Vol. 12 ›› Issue (10): 1799-1806.DOI: 10.1016/S1671-2927(00)9145

Development of A Real-Time PCR Assay for Plasmodiophora brassicae and Its Detection in Soil Samples

LI Jin-ping, LI Yan, SHI Yan-xia, XIE Xue-wen, Chai A-li , LI Bao-ju

Institute of Vegetables and Flowers, Chinese Academy of Agricultural Sciences, Beijing 100081, P.R.China

收稿日期:2012-11-12 出版日期:2013-10-01 发布日期:2013-10-01
通讯作者: Correspondence LI Bao-ju, Tel: +86-10-62197975, E-mail: libaoju@caas.cn
作者简介:LI Jin-ping, E-mail: lijinping0011@163.com
基金资助:
This research was supported by the emarked fund for Moden Agro-Industry Technology Research System, China (CARS- 25), the National Natural Science Foundation of China (31201473) and the Key Laboratory of Biology and Genetic Improvement of Horticulture Crops, Ministry of Agriculture, China.

Development of A Real-Time PCR Assay for Plasmodiophora brassicae and Its Detection in Soil Samples

LI Jin-ping, LI Yan, SHI Yan-xia, XIE Xue-wen, Chai A-li , LI Bao-ju

Institute of Vegetables and Flowers, Chinese Academy of Agricultural Sciences, Beijing 100081, P.R.China

Received:2012-11-12 Online:2013-10-01 Published:2013-10-01
Contact: Correspondence LI Bao-ju, Tel: +86-10-62197975, E-mail: libaoju@caas.cn
About author:LI Jin-ping, E-mail: lijinping0011@163.com
Supported by:
This research was supported by the emarked fund for Moden Agro-Industry Technology Research System, China (CARS- 25), the National Natural Science Foundation of China (31201473) and the Key Laboratory of Biology and Genetic Improvement of Horticulture Crops, Ministry of Agriculture, China.

摘要/Abstract

摘要： A SYBR Green I real-time PCR assay was developed to detect and quantify Plasmodiophora brassicae ribosomal DNA (rDNA) and internal transcribed spacer (ITS). A pair of primers PBF1/PBR1 was designed based on the conservative region of rDNA-ITS of P. brassicae. The positive plasmid pB12 was obtained and used as the template to create standard curve. The specificity, sensitivity, and reproducibility of real-time PCR were evaluated respectively. Naturally and artificially infested soil samples containing different concentrations of P. brassicae were detected. The results demonstrated that standard curve established by recombinant plasmid was shown a fine linear relationship between threshold cycle and template concentration. The melting curve was specific with the correlation coefficient of 0.995 and that the amplification efficiency was 93.8%. The detection limit of P. brassicae genomic DNA was approximately 40 copies per 25 μL. The sensitivity of the assay was at least 100-fold higher than conventional PCR. Only DNA from P. brassicae could be amplified and detected using this assay, suggesting the highly specific of this assay. The coefficient of variation was less than 3%, indicating the PCR method revealed high reproducibility. The detection limit in soil samples corresponded to 1 000 resting spores g-1 soil. Bait plants were used to validate the real-time PCR assay. This developed real-time PCR assay allows for fast and sensitive detection of P. brassicae in soil and should be useful in disease management and pest interception so as to prevent further spread of P. brassicae.

关键词: species-specific , rDNA-ITS gene , Plasmodiophora brassicae , real-time quantitative PCR , SYBR Green I dye

Abstract: A SYBR Green I real-time PCR assay was developed to detect and quantify Plasmodiophora brassicae ribosomal DNA (rDNA) and internal transcribed spacer (ITS). A pair of primers PBF1/PBR1 was designed based on the conservative region of rDNA-ITS of P. brassicae. The positive plasmid pB12 was obtained and used as the template to create standard curve. The specificity, sensitivity, and reproducibility of real-time PCR were evaluated respectively. Naturally and artificially infested soil samples containing different concentrations of P. brassicae were detected. The results demonstrated that standard curve established by recombinant plasmid was shown a fine linear relationship between threshold cycle and template concentration. The melting curve was specific with the correlation coefficient of 0.995 and that the amplification efficiency was 93.8%. The detection limit of P. brassicae genomic DNA was approximately 40 copies per 25 μL. The sensitivity of the assay was at least 100-fold higher than conventional PCR. Only DNA from P. brassicae could be amplified and detected using this assay, suggesting the highly specific of this assay. The coefficient of variation was less than 3%, indicating the PCR method revealed high reproducibility. The detection limit in soil samples corresponded to 1 000 resting spores g-1 soil. Bait plants were used to validate the real-time PCR assay. This developed real-time PCR assay allows for fast and sensitive detection of P. brassicae in soil and should be useful in disease management and pest interception so as to prevent further spread of P. brassicae.

Key words: species-specific , rDNA-ITS gene , Plasmodiophora brassicae , real-time quantitative PCR , SYBR Green I dye

LI Jin-ping, LI Yan, SHI Yan-xia, XIE Xue-wen, Chai A-li , LI Bao-ju. Development of A Real-Time PCR Assay for Plasmodiophora brassicae and Its Detection in Soil Samples[J]. Journal of Integrative Agriculture, 2013, 12(10): 1799-1806.

参考文献

[1]Bilodeau G, Uribe P, Koike S T, Martin F. 2010. Quantification and rapid detection of Verticillum dahliae in soil. Phytopathology, 100(Suppl.), S14.

[2]Buczacki S T. 1983. Plasmodiophora. An inter-relationship between biological and practical problems. In: Buczacki S T, ed., Zoosporic Plant Pathogens: A Modern Perspective. Academic Press, London, UK. pp. 161-191

[3]Cao T, Tewari J, Strelkov S E. 2007. Molecular detection of Plasmodiophora brassicae, causal agent of clubroot of crucifers, in plant and soil. Plant Disease, 91, 80-87

[4]Diederichsen E, Frauen M, Linders E G A, Hatakeyama K, Hirari M. 2009. Status and perspectives of clubroot resistance breeding in crucifer crops. Journal of Plant Growth Regulation, 28, 265-268

[5]Dixon G R. 2009. The occurrence and economic impact of Plasmodiophora brassicae and clubroot disease. Journal of Plant Growth Regulation, 28, 194-202

[6]Donald E C, Porter I J. 2009. Integrated control of clubroot. Journal of Plant Growth Regulation, 28, 289-303

[7]Faggian R, Bulman S R, Lawrie A C, Porter I J. 1999. Specific polymerase chain reaction primers for the detection of Plasmodiophora brassicae in soil and water. Phytopathology, 89, 392-397

[8]Faggian R, Strelkov S E. 2009. Detection and measurement of Plasmodiophora brassicae. Journal of Plant Growth Regulation, 28, 282-288

[9]Friberg H. 2005. Persistence of plasmodiophora brassicae. Influence of non-host plants, soil faua and organic material. PhD thesis, Swedish University of Agricultural Science, Uppsala, Swedeen. Heyman F. 2008. Root rot of pea caused by aphanomyces euteiches; calcium dependent soil suppressiveness, molecular detection and population structure. PhD thesis, Swedish University of Agricultural Sciences, Uppsala, Sweden. Ito S, Maechara T, Tanaka S, Kameya-Iwaki M,Yano S, Kishi F. 1997. Cloning of single-copy DNA sequence unique to Plasmodiophora brassicae. Physiological and Molecular Plant Pathology, 50, 289-300

[10]Lange L, Heide M, Hobolth L. 1989. Serological detection of Plasmodiophora brassicae by dot immunobinding and visualization of the serological reaction by scanning electron microscopy. Phytopathology, 79, 1066-1071

[11]Lees A K, Cullen D W, Sullivan L. 2002. Development of conventional and quantitative real-time PCR assays for the detection and identification of Rhizoctonia solani AG-3 in potato and soil Plant Pathology, 51, 293-302

[12]Li Y. 2010. Diagnose and control technology of plasmodiophora brassicae. MSc thesis, Northeast Agricultural University, Harbin, China. (in Chinese)

[13]Lian H X, Lu D X, Gao M. 2009. Establishment of real-time TaqMan-fluorescence quantitative RT-PCR assay for detection and quantification of porcine lipoprotein lipase mRNA. Agricultural Sciences in China, 10, 1256-1262

[14]Luo Y J, Zhang G H, Xu X Q, Chen J H, Liao M. 2008. Molecular characterization and SYBR Ggreen I-based quantitative PCR for duck hepatitis virus type 1. Agricultural Sciences in China, 9, 1140-1146

[15]Shang X G, Guo W, Yang L R. 2010. Development and preliminary application of real-time fluorescent quantitative PCR method for detection of porcine astrovirus. Acta Veterinaria et Zootechnica Sinica, 41, 1049-1053(in Chinese) Sundelin

[16]T, Christensen C B, Larsen J, Møller K, Lübeck M, Bødker L, Jensen B. 2010. In planta quantification of Plasmodiophora brassicae using signature fatty acids and real-time PCR. Plant Disease, 94, 432-438

[17]Takahashi K, Yamaguchi T. 1989. Assessment of pathogenicity of resting spores of Plasmodiophora brassicae in soil by fluorescence microscopy. Annual Phytopathology Society Japan, 55, 621-628

[18]Tewari J P, Strelkov S E, Orchard D, Hartman M, Lange R M, Turkington T. 2005. Identification of clubroot of crucifers on canola (Brassica napus), in Alberta. Canadian Journal of Plant Pathology, 27, 143-144

[19]Voorrips R E. 1995. Plasmodiophora brassicae: aspects of pathogenesis and resistance in Brassica oleracea. Euphytica, 83, 139-146

[20]Wallenhammar A C. 1996. Prevalence of Plasmodiophora brassicae in a spring oilseed rape growing area in central Sweden and factors influencing soil infestation levels. Plant Pathology, 45, 710-719

[21]Wallenhammar A C. 1998. Observations on yield loss from Plasmodiophora brassicae infections in spring oilseed rape. Zeitschriftfur Pflanzenkrankheit und Pflantzenschutz, 105, 1-7

[22]Wallenhammar A C, Almquist C, Soderstroma M, Jonsson A. 2012. In-field distribution of Plasmodiophora brassicae measured using quantitative real-time PCR. Plant Pathology, 6, 16-28

[23]Wallenhammar A C, Arwidsson O. 2001. Detection of Plasmodiophora brassicae by PCR in naturally infested soils. European Journal of Plant Pathology, 107, 313- 321.

[24]Wallenhammar A C, Johnson L, Gerhardson B. 2000. Agronomic properties of partly clubroot-resistant spring oilseed turnip rape lines. Journal of Phytopatholog, 148, 495-499

[25]Zhou Y L, Wei F L, Li J, Zhang X H, Jiang H X. 2011. Development of real-time fluorescent quantitative PCR method for detection of Mycoplasma gallisepticum. Scientia Agricultura Sinica, 44, 2371-2378. (in Chinese)

Development of A Real-Time PCR Assay for Plasmodiophora brassicae and Its Detection in Soil Samples

Development of A Real-Time PCR Assay for Plasmodiophora brassicae and Its Detection in Soil Samples

PDF

可视化

摘要/Abstract

引用本文

使用本文

参考文献

相关文章 2

编辑推荐

Metrics

[1]	XU Cheng-nan, ZHANG Hong-jun, CHI Fu-mei, JI Zhi-rui, DONG Qing-long, CAO Ke-qiang, ZHOU Zong-shan. Species-specific PCR-based assays for identification and detection of Botryosphaeriaceae species causing stem blight on blueberry in China[J]. Journal of Integrative Agriculture, 2016, 15(3): 573-579.
[2]	LIU Yan, YIN You-ping, WANG Zhong-kang , LUO Yuan-li. Expression of Nitrilases in Brassica juncea var. tumida Tsen in Root Galls Caused by Plasmodiophora brassicae[J]. Journal of Integrative Agriculture, 2012, 12(1): 100-108.