Real-time PCR Detection Method of Panax notoginseng Root Rot Pathogen Fusarium oxysporum

doi:10.7668/hbnxb.20190370

Abstract

Abstract: The aim of this paper is to establish an accurate and rapid Real-time fluorescence quantitative PCR method for the detection of Fusarium oxysporum, the pathogenic fungus of Panax notoginseng root rot. A pair of specific primer, Fo-QF and Fo-QR, was designed based on the fatty acid ω-hydroxylase gene of F.oxysporum in this study. The recombinant plasmid of this gene was prepared, moreover, the SYBR Green Ⅰ fluorescence Real-time quantitative PCR method for detecting F. oxysporum was established. In addition, the 14 samples including P. otoginseng plants with typical symptoms of root rot and black spot as well as the P. notoginseng planting soil were collected. The total DNAs of these samples were extracted as templates and detected. The results showed that the Real-time fluorescence quantitative PCR method established was specific. The fatty acid ω-hydroxylase gene could amplify specific PCR products only when F. oxysporum DNA was used as templates. In addition, the method had high sensitive, and the concentration of detection template could be as low as 0.35 pg/μL. The Ct of the standard curve constructed had a good linear relationship with the centrations of templates. The absorption peak of the dissolution curve was single, and the amplification efficiency was good. With the quantitative detection system of this study, the P. notoginseng root rot plants with F. oxysporum were rapidly detected from several different P. notoginseng disease strains. So it could achieve the purpose of accurate diagnosis. This method can be used to reveal the dynamic changes of F. oxysporum numbers in the complex P. notoginseng planting soil and in the diseased plants. Furthermore, it can provide technical supports for the soil treatment, early diagnosis and dynamic monitoring of root rot, and rapid molecular detection of P. notoginsen g seeds and seedlings.

Key words: Panax notoginseng root rot, Fusarium oxysporum, Molecular detection, Real-time PCR, Rapid detection

CLC Number:

S432.4

LI Xin, CUI Xiuming, LIU Diqiu, SUN Tian, GUO Ailing, CHEN Jun. Real-time PCR Detection Method of Panax notoginseng Root Rot Pathogen Fusarium oxysporum[J]. ACTA AGRICULTURAE BOREALI-SINICA, 2019, 34(S1): 324-330. doi: 10.7668/hbnxb.20190370.

/ / Recommend / Download Citations

URL: http://www.hbnxb.net/EN/10.7668/hbnxb.20190370

http://www.hbnxb.net/EN/Y2019/V34/IS1/324

References

[1] 张静静, 刘桂芹, 王庆鹏, 李兰杰. 植物三七中皂苷类化学成分的研究进展[J]. 聊城大学学报(自然科学版), 2018, 31(2):43-59. doi:10.19728/j.issn1672-6634.2018.02.006.
Zang J J, Liu G Q, Wang Q P, Li L J. Advances in studies on chemical constituents of saponin from Panax notoginseng[J]. Journal of Liaocheng (Natural Science Edition), 2018, 31(2):43-59.
[2] Wang J, Huang Z G, Cao H, Wang Y T, Hui P, Hoo C, Li S P. Screening of anti-platelet aggregation agents from Panax notoginseng using human platelet extraction and HPLC-DAD-ESI-MS/MS[J]. Journal of Separation Science, 2008, 31(6-7):1173-1180. doi:10.1002/jssc.200700507.
[3] Wu Z X, Hao Z P, Sun Y Q, Guo L P, Huang L Q, Zeng Y, Wang Y, Yang L, Chen B D. Comparison on the structure and function of the rhizosphere microbial community between healthy and root-rot Panax notoginsen g[J]. Applied Soil Ecology, 2016, 107:99-107. doi:10.1016/j.apsoil.2016.05.017.
[4] 毛忠顺, 龙月娟, 朱书生, 陈中坚, 魏富刚, 朱有勇, 何霞红. 三七根腐病研究进展[J]. 中药材, 2013, 36(12):2051-2054.doi:10.13863/j.issn1001-4454.2013.12.004.
Mo Z S, Long Y J, Zhu S S, Chen Z J, Wei F G, Zhu Y Y, He X H. Research progress on root rot of Panax notoginseng[J]. Journal of Chinese Medicinal Materials, 2013, 36(12):2051-2054.
[5] 缪作清, 李世东, 刘杏忠, 陈昱君, 李云华, 王勇, 郭荣君, 夏振远, 张克勤.三七根腐病病原研究[J]. 中国农业科学, 2006, 39(7):1371-1378. doi:10.3321/j.issn:0578-1752.2006.07.011.
Mao Z Q, Li S D, Liu X Z, Chen Y J, Li Y H, Wang Y, Guo R J, Xia Z Y, Zhang K Q. The causal microorganisms of Panax notoginseng root rot disease[J]. Scientia Agricultura Sinica,2006, 39(7):1371-1378.
[6] Wang W Y, Zhao C L, Chen Z J, Wen G S, Wei F G, Long T G, Li S W, Wang C D. Studies on the isolation, identification and in vitro growth rates of the three pathogenic fungi from Panax notoginseng cultivated in Wenshan Eparchy[J]. Agricultural Science and Technology, 2015, 16(6):1165-1171.doi:10.16175/j.cnki.1009-4229.2015.06.018.
[7] Mao Z S, Long Y J, Zhu Y Y, Zhu S S, He X H, Chen Z J. First report of Cylindrocarpon destructans var. destructans causing black root rot of Sanqi (Panax notoginseng) in China[J]. Plant Disease, 2014, 98(1):162. doi:10.1094/PDIS-11-12-1104-PDN.
[8] 蒋妮, 覃柳燕, 叶云峰. 三七病害研究进展[J]. 南方农业学报, 2011, 42(9):1070-1074. doi:10.3969/j.issn.2095-1191.2011.09.010.
Jang N,Qin L Y,Ye Y F. Research advances in diseases of Panax notoginseng[J]. Journal of Southern Sciences, 2011, 42(9):1070-1074.
[9] 龙月娟, 毛忠顺, 朱书生, 陈中坚, 魏富刚, 尹兆波, 朱有勇, 何霞红. 三七锈斑病的病原菌[J]. 菌物学报, 2015, 34(2):177-185. doi:10.13346/j.mycosystema.140013.
Lng Y J, Mao Z S, Zhu S S, Chen Z J, Wei F G, Yin Z B, Zhu Y Y, He X H.The pathogens of Panax notoginseng root rust spot[J]. Mycosystema, 2015, 34(2):177-185.
[10] Miao C P, Qiao X G, Zheng Y K, Chen Y W, Xu L H, Guan H L, Zhao L X. First report ofFusarium flocciferum causing root rot of Sanqi (Panax notoginseng) in Yunnan, China[J]. Plant Disease, 2015, 99(11):1650.doi:10.1094/PDIS-11-14-1168-PDN.
[11] 汪静, 梁宗锁, 康冰, 罗美佳.文山三七根腐病病原真菌的鉴定与药剂防治[J]. 西北林学院学报, 2015, 30(1):158-163.doi:10.3969/j.issn.1001-7461.2015.01.26.
Wng J, Liang Z S, Kang B, Luo M J. Identification of root rot pathogen of Panax notoginseng from Wenshan[J]. Journal of Northwest Forestry University, 2015, 30(1):158-163.
[12] Zheng Y K, Miao C P, Chen H H, Huang F F, Xia Y M, Chen Y W, Zhao L X. Endophytic fungi harbored in Panax notoginseng:diversity and potential as biological control agents against host plant pathogens of root-rot disease[J]. Journal of Ginseng Research, 2017, 41(3):353-360. doi:10.1016/j.jgr.2016.07.005.
[13] 宋丽敏. 三七病毒病害病原的初步鉴定[D]. 北京:中国农业大学, 2005. doi:10.7666/d.y774579.
Sng L M. Detection and identification of the viruses infecting Panax notoginseng[D]. Beijing:China Agricultural University College of Agronomy and Biotechnology, 2005.
[14] 龚美蓉, 陈凤丽, 曹晨, 孙亦农, 王明艳. 实时荧光定量PCR技术及其在中医药研究中的应用[J]. 中国实验方剂学杂志, 2014, 20(22):238-241. doi:10.13422/j.cnki.syfjx.2014220238.
Gng M R,Chen F L,Cao C,Sun Y N,Wang M Y. Application of Real-time fluorescence quantitative PCR in Chinese medicine research[J]. Chinese Journal of Experimental Traditional Medical Formulae, 2014, 20(22):238-241.
[15] Zheng S, Shan L Y, Zhuang Y L, Shang Y. Identification ofpyrG used as an endogenous reference gene in qualitative and Real-time quantitative PCR detection ofPleurotus ostreatus[J]. Journal of Food Science, 2018, 83(3):750-755. doi:10.1111/1750-3841.14072.
[16] Lin Y H, Su C C, Chao C P, Chen C Y, Chang C J, Huang J W, Chang P F. A molecular diagnosis method using Real-time PCR for quantification and detection of Fusarium oxysporum f. sp.cubense race 4[J]. European Journal of Plant Pathology, 2013, 135(2):395-405. doi:10.1007/s10658-012-0096-0.
[17] Kędrak-Jabłońska A, Budniak S, Krupa M, Szczawińska A, Reksa M, Szulowski K, Iwaniak W. Detection of Listeria spp. and Listeria Monocytogenes in biological samples by SYBR Green I and TaqMan probe-based Real-time PCRs[J]. Journal of Veterinary Research, 2017, 61(4):427-432. doi:10.1515/jvetres-2017-0069.
[18] 董鲜, 马晓惠, 陈传娇, 耿连用, 杨志慧, 鲁荣昌, 徐福荣. 三七根腐病尖孢镰刀菌分离鉴定及致病作用研究[J]. 中药材, 2018, 41(1):8-12. doi:10.13863/j.issn1001-4454.2018.01.002.
Dng X,Ma X H,Chen C J,Geng L Y,Yang Z H,Lu R C,Xu F R. The isolation and identification of Fusarium oxysporum and the study about its pathogenesis effect on Panax notoginseng[J].Journal of Chinese Medicinal Materials, 2018, 41(1):8-12.
[19] 高晓敏, 王琚钢, 马立国, 云兴福. 尖孢镰刀菌致病机理和化感作用研究进展[J]. 微生物学通报, 2014, 41(10):2143-2148. doi:10.13344/j.microbiol.china.140406.
Go X M, Wang J G, Ma L G, Yun X F. Research advances on the mechanism of pathogenesis and allelopathy of Fusarium oxysporium[J]. Microbiology China, 2014, 41(10):2143-2148.
[20] Kashiwa T, Inami K, Teraoka T, Komats K, Arie T. Detection of cabbage yellows fungus Fusarium oxysporum f. sp. conglutinans in soil by PCR and Real-time PCR[J]. Journal of General Plant Pathology, 2016, 82(5):240-247. doi:10.1007/s10327-016-0668-5.
[21] 汲珊珊, 李君文, 安娜, 谌志强, 陈照立, 邱志刚, 刘先宁, 王新为, 郭长江, 金敏. 尖孢镰刀菌TaqMan探针荧光定量PCR快速检测方法的建立及应用[J]. 现代检验医学杂志, 2012, 27(6):30-33. doi:10.3969/j.issn.1671-7414.2012.06.010.
J S S, Li J W, An N, Shen Z Q, Chen Z L, Qiu Z G, Liu X N, Wang X W, Guo C J, Jin M. Establishing and applying of TaqMan probe Real-tirne PCR detection of Fusarium oxysporum[J]. Journal of Modern Laboratory Medicine, 2012, 27(6):30-33.

Please choose a citation manager

Content to export