甘薯茎腐病菌TaqMan荧光定量PCR检测方法的建立与应用

doi:10.3969/j.issn.1674-7968.2019.03.020

Abstract
Figure/Table
References
Related Citation (15)

Download: PDF (4600 KB) HTML (1 KB)
Export: BibTeX | EndNote (RIS)

Abstract Bacterial stem and root rot caused by Dickeya dadantii is proved to be a serious disease, which is harmful to sweet potato (Ipomoea batatas) production. It can damage the stems, leaves, petioles and seed tubers, infecting and forming brown water-stained lesions. Now, the disease occurs in Zhejiang, Guangdong, Heibei, Henan and other provinces. Long-distance transportation of the seed tuber and germchit are the main way to spread the disease to the disease-free zone. Therefore, strict quarantine measures are the key to ensure healthy production in disease-free areas.An accurate, sensitive and rapid detection method is a powerful tool to guarantee strictly quarantining and insurance for researching on the occurrence of disease epidemics. Compared with closely related species, the flagellin protein gene (fliC) sequence of D. dadantii encoding the flagellin with high specificity which makes it to be a suitable target for molecular detection. With the fliC gene as the target, specific primers and TaqMan probes were designed, thus the method for fluorescence quantitative PCR detection (TaqMan qRT-PCR) of D. dadantii was established in this study. The method showed good specificity against D. dadantii based on the test results, and it could be effectively distinguished from bacteria of different species in the same genus and different genera. The method had high sensitivity and the detection sensitivity of the bacterial DNA was up to 280 fg/μL, which was 1 000 times of that of routine PCR. The detection sensitivity of bacterial liquid was up to 2 CFU/μL (CFU: colony forming units), the sensitivity was 100 times higher than the routine PCR detection method. Using NGM (nematode growth medium) to isolate suspected sweet potato stem and root rot samples, it was found that D. dadantii could form dark brown to blue colonies on the medium, which could significantly improve the efficiency of separation and purification of pathogenic bacteria. The actual samples were tested on 70 sweet potato and soil samples using the constructed TaqMan qRT-PCR method, routine PCR method and separation culture method based on NGM medium. Results showed that the positive detection rates of TaqMan qRT-PCR, routine PCR, and isolation culture method were 91.4%, 77.1%, and 71.4%, respectively. TaqMan qRT-PCR detection was more accurate, and the detection time was shortened from 96 h to 1.5 h. It could be seen that the quantitative PCR detection system based on TaqMan probe established in this study coul d be effectively used for rapid detection of suspected samples of sweet potato stem and root rot, diseased bodies, field soil, seed tuber and seedlings, and provided a fast and accurate molecular detection method of sweet potato stem and root rot for inspection and quarantine department, which was of great significance for effectively controlling the spread of the disease. This method can also be used for the quantitative detection of the pathogenic bacteria of sweet potato stem and root rot in suspect samples, providing technical support for studying the relationship between the population of pathogenic bacteria and disease severity in soil.

Key words： Dickeya dadantii Flagellin protein gene (fliC) TaqMan probe qRT-PCR

Received: 30 July 2018

ZTFLH:

S412

Corresponding Authors: * bglou@zju.edu.cn

	Service

	E-mail this article
	Add to my bookshelf
	Add to citation manager
	E-mail Alert
	RSS
	Articles by authors
	SHEN Xiao-Ling
	YI Jian-Ping
	WANG Rong-Zhou
	QIAN Jun-Ting
	LI Yan-Min
	YAO Hai-Feng
	LOU Bing-Gan

Cite this article:

SHEN Xiao-Ling,YI Jian-Ping,WANG Rong-Zhou, et al. Establishment and Application of TaqMan Fluorescence Quantitative PCR Detection Method for Dickeya dadantii[J]. 农业生物技术学报, 2019, 27(3): 551-563.

URL:

http://journal05.magtech.org.cn/Jwk_ny/EN/10.3969/j.issn.1674-7968.2019.03.020 OR http://journal05.magtech.org.cn/Jwk_ny/EN/Y2019/V27/I3/551

[1] 刘鹏, 黄国明, 刘勇,等. 2007. 菊欧文氏菌分子检测技术的研究[J]. 植物病理学报, 37(6): 584-587.
(Liu P, Huang G M, Liu Y, et al.2007. Molecular detection of Erwinia chrysanthemi[J]. Acta Phytopathologica Sinica, 37(6): 584-587.)
[2] 方中达 .1998. 植病研究方法[M]. 北京: 中国农业出版社, pp. 179-180.
(Fang Z D.1998. Plant Disease Research Methods[M]. China Agriculture Press, Beijing, China, pp. 179-180.)
[3] 沈肖玲, 林钗, 钱俊婷, 等. 2018. 甘薯茎腐病症状及其病原鉴定[J]. 植物病理学报, 48(1): 25-34.
(Shen X L, Lin C, Qian J T, et al.2018. Characterization of stem and root rot symptoms of sweet potato and the causal pathogen of the disease[J]. Acta Phytopathologica Sinica, 48(1): 25-34.)
[4] Chen Y J, Lin Y S, Chung W H.2012. Bacterial wilt of sweet potato caused by Ralstonia solanacearum in Taiwan[J]. Journal of General Plant Pathology, 78(1): 80-84.
[5] Clark C A, Hoy M W, Bond J P, et al.1998. First report of Erwinia carotovora subsp. Carotovora causing bacterial root rot of sweet potato (Ipomoea batatas) in Louisiana[J]. Plant Disease, 82(1): 129-129.
[6] John M, Stephane G, Shimpei M, et al.2012. Top 10 plant pathogenic bacteria in molecular plant pathology[J]. Molecular Plant Pathology, 13(6): 614-629.
[7] Lee Y, Yu C.2006. A differential medium for the isolation and rapid identification of a plant soft rot pathogen, Erwinia chrysanthemi[J]. Journal of Microbiological Methods, 64(2): 200-206.
[8] Marrero G, Schneider K L, Jenkins D M, et al.2013. Phylogeny and classification of Dickeya based on multilocus sequence analysis[J]. International Journal of Systems and Evolutionary Microbiology, 63(Pt 9): 3524-3539.
[9] Schena L, Nigro F, Ippolito A, et al.2004. Real-time quantitative PCR: A new technology to detect and study phytopathogenic and antagonistic fungi[J]. European Journal of Plant Pathology, 110(9): 893-908.
[10] Smid E J, Jansen A J, Gorris L M.1995. Detection of Erwinia carotovora subsp. atroseptica and Erwinia chrysanthemi in potato tubers using polymerase chain reaction[J]. Plant Pathology, 44(6): 1058-1069.
[11] Tian Y L, Zhao Y Q, Yuan X L, et al.2016. Dickeya fangzhongdai sp. nov., a plant-pathogenic bacterium isolated from pear tree (Pyrus pyrifolia)[J]. International Journal of Systematic and Evolutionary Microbiology, 66(8): 2831-2835.
[12] Toth I K, van der Wolf J M, Saddler G, et al.2011. Dickeya species: An emerging problem for potato production in Europe[J]. Plant Pathology, 60(3): 385-399.
[13] van der Wolf J M, Nijhuis E H, Kowalewska, et al.2014. Dickeya solani sp. nov., a pectinolytic plant-pathogenic bacterium isolated from potato (Solanum tuberosum)[J]. International Journal of Systematic and Evolutionary Microbiology, 64(Pt3): 768-774.