荷花腐败病菌的荧光定量PCR检测

doi:10.11924/j.issn.1000-6850.casb2020-0836

中国农学通报 ›› 2021, Vol. 37 ›› Issue (34): 127-132.doi: 10.11924/j.issn.1000-6850.casb2020-0836

所属专题：植物保护；园艺

荷花腐败病菌的荧光定量PCR检测

温华强¹(), 舒灿伟¹, 曾莉莎²(), 周而勋¹()

¹华南农业大学植物保护学院/广东省微生物信号与作物病害防控重点实验室,广州 510642
²东莞市农业科学研究中心,广东东莞 523086

收稿日期:2020-12-25 修回日期:2021-07-13 出版日期:2021-12-05 发布日期:2022-01-06
通讯作者: 曾莉莎,周而勋
作者简介:温华强,男,1996年出生,广东揭阳人,硕士在读,研究方向：植物病原真菌学。通信地址：510642 广东省广州市天河区五山路483号华南农业大学燕山区研究生宿舍,E-mail： 757472743@qq.com。
基金资助:
广东省科技计划项目“荷花腐败病抗病品种资源筛选及创新利用”(2015A020209002);东莞市社会科技发展项目“荷花腐败病发病机理与生态防控技术研究”(2014108101044)

Fluorescence Quantitative PCR Detection of Fusarium commune from Lotus

Wen Huaqiang¹(), Shu Canwei¹, Zeng Lisha²(), Zhou Erxun¹()

¹College of Plant Protection, South China Agricultural University/ Guangdong Province Key Laboratory of Microbial Signals and Disease Control, Guangzhou 510642
²Dongguan Agricultural Science Research Centre, Dongguan Guangdong 523086

Received:2020-12-25 Revised:2021-07-13 Online:2021-12-05 Published:2022-01-06
Contact: Zeng Lisha,Zhou Erxun

摘要/Abstract

摘要：

建立一种快速、特异性强的定量检测荷花腐败病菌(Fusarium commune)的技术,评估该技术用于荷花腐败病菌定量检测的可行性,并对东莞莲湖的土壤、莲藕等样品的荷花腐败病菌数量进行动态监测,为防治该病害提供依据。采用以SYBR Green I为荧光染料的实时荧光定量PCR(qPCR)技术,对不同发病级别的荷花植株及其地下土壤的荷花腐败病菌的含量进行检测,并分析病菌含量与病害级别的相关性。结果表明,利用qPCR技术检测的最低检测限为1 fg/µL,添加健康荷花组织的基因组DNA后,该qPCR技术的最低检测限为10 pg/µL。对该qPCR反应的溶解曲线进行分析,扩增的产物仅有1个峰值,表明采用该qPCR技术扩增的DNA产物具有高度的特异性。染病植株中荷花腐败病菌的DNA含量与其发病程度呈正相关,而莲湖土壤荷花腐败病菌含量与病害级别的相关性低,但土壤中荷花腐败病菌的数量随着病害级别提高而增大。该qPCR检测体系能够对莲湖的土壤、发病莲藕等进行荷花腐败病菌的动态监测,可为该病的有效防治提供依据。

关键词: 荷花腐败病, 荷花腐败病菌, 定量检测, 实时荧光定量PCR, 动态监测

Abstract:

To establish a rapid and specific technique for quantitative detection of lotus rhizome rot pathogen Fusarium commune, evaluate the feasibility of this technique for quantitative detection of F. commune, the dynamic monitoring on the number of F. commune in soil and lotus root samples from lotus lake in Dongguan was carried out, so as to provide a basis for the control of the disease. Real-time fluorescence quantitative PCR (qPCR) technology with SYBR Green I as the fluorescent dye was used to detect the contents of F. commune in lotus plants and underground soil with different disease levels, and the correlation between the content of F. commune and disease level was analyzed. The results showed that the minimal detection limit of qPCR technique was 1 fg/µL, and the minimal detection limit of qPCR technique was 10 pg/µL after the addition of genomic DNA from healthy lotus tissues. The dissolution curve of the qPCR reaction was analyzed, and there was only one peak value of the amplified product, indicating that the DNA product amplified by the qPCR technique was highly specific. There was a positive correlation between the content of F. commune DNA and the degree of disease in infected plants, whereas the content of F. commune in lotus lake soil had a low correlation with the disease levels, however, the number of F. commune in soil increased with the increase of disease level. This qPCR detection system could dynamically mornitor the content of F. commune in the lotus lake soil and infected lotus roots, and thus provides a basis for the effective control of this disease.

Key words: lotus rhizome rot disease, Fusarium commune, quantitative detection, real-time fluorescence quantitative PCR (qPCR), dynamic monitoring

中图分类号:

S432.1

温华强, 舒灿伟, 曾莉莎, 周而勋. 荷花腐败病菌的荧光定量PCR检测[J]. 中国农学通报, 2021, 37(34): 127-132.

Wen Huaqiang, Shu Canwei, Zeng Lisha, Zhou Erxun. Fluorescence Quantitative PCR Detection of Fusarium commune from Lotus[J]. Chinese Agricultural Science Bulletin, 2021, 37(34): 127-132.

图/表 6

图1. 10倍浓度梯度的荷花腐败病菌基因组DNA的qPCR扩增曲线 1为10 ng/μL,2为1 ng/μL,3为100 pg/μL,4为10 pg/μL,5为1 pg/μL,6为100 fg/μL,7为10 fg/μL,8为1fg/μL

图2. SYBR Green I PCR扩增基因组标准曲线

图3. SYBR Green I PCR熔解曲线

图4. 接种土壤的SYBR Green I PCR扩增曲线 1为4×107个分生孢子/(g·土),2为4×106个分生孢子/(g·土),3为4×105个分生孢子/(g·土),4为4×104个分生孢子/(g·土),5为4×103个分生孢子/(g·土),6为4×102个分生孢子/(g·土),7为4×101个分生孢子/(g·土),8为4个分生孢子/(g·土),9为0个分生孢子/(g·土)

图5. 接种荷花腐败病菌的土壤中的荷花腐败病菌基因组DNA的标准曲线

病害级别	植物组织		土壤样品
病害级别	Ct值	DNA含量/ng	Ct值	病菌含量/[×10³个分生孢子/(g·土)]
0	33.190±0.361	0.010±0.003b	35.025±0.115	3.618±2.284b
1	29.937±0.705	0.0827±0.053b	28.526±0.321	34.40±12.891a
2	25.397±0.297	1.432±0.439a	26.217±0.3143	164.733±54.270a

表1. 不同病害级别荷花植株中荷花腐败病菌DNA的含量及土壤中荷花腐败病菌的含量

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荷花腐败病菌的荧光定量PCR检测

Fluorescence Quantitative PCR Detection of Fusarium commune from Lotus

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