中国农业科学 ›› 2019, Vol. 52 ›› Issue (9): 1529-1540.doi: 10.3864/j.issn.0578-1752.2019.09.005
李文学1,肖瑞刚2,吕苗苗1,丁宁1,石华荣2,顾沛雯1()
收稿日期:
2018-12-04
接受日期:
2019-01-17
出版日期:
2019-05-01
发布日期:
2019-05-16
通讯作者:
顾沛雯
作者简介:
李文学,E-mail: 基金资助:
LI WenXue1,XIAO RuiGang2,LÜ MiaoMiao1,DING Ning1,SHI HuaRong2,GU PeiWen1()
Received:
2018-12-04
Accepted:
2019-01-17
Online:
2019-05-01
Published:
2019-05-16
Contact:
PeiWen GU
摘要:
【目的】葡萄霜霉病是葡萄生产上重要的单年流行病害,研究旨在构建葡萄霜霉病菌(Plasmopara viticola)的实时荧光定量PCR(real-time PCR)检测体系,为葡萄霜霉病的早期诊断和预测预报提供依据。【方法】依据GenBank中葡萄霜霉病菌cox2基因序列设计1对特异性引物(F-cox-Pv/R-Pv),建立并优化常规PCR和real-time PCR反应体系,利用葡萄霜霉病菌、葡萄炭疽病菌(Colletotrichum gloeosporioides)、葡萄白粉病菌(Uncinula necator)、葡萄灰霉病菌(Botrytis cinerea)、葡萄白腐病菌(Coniella diplodiella)、葡萄溃疡病菌(Botryosphaeria dothidea)、白菜黑斑病菌(Alternaria brassicae)、辣椒炭疽病菌(C. capsica)、甘草根腐病菌(Fusarium solani)、西葫芦白粉病菌(Sphaerotheca fuliginea)、番茄菌核病菌(Sclerotinia sclerotiorum)、马铃薯干腐病菌(F. equiseti)、西瓜枯萎病菌(F. oxysporum)、哈茨木霉(Trichoderma harzianum)等14种葡萄及其他作物病原菌和拮抗菌的菌丝DNA进行常规PCR和real-time PCR特异性检测,并对灵敏度和可重复性进行评价。运用已构建的real-time PCR体系对人工接种葡萄霜霉病菌的潜育期叶片内病原菌DNA进行定量检测,利用SPSS 19.0软件分析接种时间与叶片内葡萄霜霉病菌潜伏侵染量的关系。【结果】研究设计的引物特异性高,常规PCR仅对葡萄霜霉病菌DNA有扩增条带,为139 bp;real-time PCR检测结果表明该对引物对葡萄霜霉病菌有唯一的产物吸收峰,对其他供试菌株均未检测到产物吸收峰。常规PCR检测的灵敏度为10 pg·μL -1,real-time PCR的灵敏度可达到0.1 pg·μL -1,是常规PCR检测灵敏度的100倍。以携带目的基因片段的重组质粒为标准品,构建real-time PCR循环阈值(Ct)与模板浓度的线性关系,标准曲线y=42.27-3.36x,相关系数R 2=0.997,扩增效率为98.50%,线性范围达7个数量级,在2.4×10 3—2.4×10 9 copies/μL呈现良好的线性关系。对人工接种葡萄霜霉病菌的潜育期叶片内病原菌DNA进行real-time PCR检测,结果表明叶片内病原菌潜伏侵染量随接种时间的变化呈指数关系增长,y=6.34×10 4·e 0.084 x,相关系数R 2=0.936。该real-time PCR检测体系在接种6 h后就可以检测到葡萄霜霉病菌DNA,检测量为5.68×10 4 copies/μL病原菌DNA。 【结论】构建的葡萄霜霉病菌real-time PCR检测体系的灵敏度远高于常规PCR,且特异性强、重复性好,其Ct值与模板浓度呈较好的线性关系,扩增效率高,可用于定量检测葡萄霜霉病菌的潜伏侵染量。
李文学, 肖瑞刚, 吕苗苗, 丁宁, 石华荣, 顾沛雯. 葡萄霜霉病菌实时荧光定量PCR检测体系的建立和应用[J]. 中国农业科学, 2019, 52(9): 1529-1540.
LI WenXue, XIAO RuiGang, LÜ MiaoMiao, DING Ning, SHI HuaRong, GU PeiWen. Establishment and Application of Real-Time PCR for Quantitatively Detecting Plasmopara viticola in Vitis vinifera[J]. Scientia Agricultura Sinica, 2019, 52(9): 1529-1540.
表1
供试菌株"
菌株编号 Number | 拉丁学名 Latin name | 寄主(品种) Host (Variety) | 菌株编号 Number | 拉丁学名 Latin name | 寄主(品种) Host (Variety) | |
---|---|---|---|---|---|---|
P1 | P. viticola | 葡萄(黑比诺) Vitis vinifera (Pinot Noir) | 6 | S. fuliginea | 西葫芦 Cucurbita pepo | |
P2 | P. viticola | 葡萄(霞多丽) V. vinifera (Chardonnay) | 7 | C. diplodiella | 葡萄(威代尔) V. vinifera (Vidal) | |
P3 | P. viticola | 葡萄(赤霞珠) V. vinifera (Cabernet Sauvignon) | 8 | B. cinerea | 葡萄(红地球) V. vinifera (Red Globe) | |
1 | A. brassicae | 白菜 Brassica pekinensis | 9 | S. sclerotiorum | 番茄 Lycopersicon esculentum | |
2 | C. gloeosporioides | 葡萄(红地球) V. vinifera (Red Globe) | 10 | F. equiseti | 马铃薯 Solanum tuberosum | |
3 | C. capsici | 辣椒 Capsicum annuum | 11 | F. oxysporum | 西瓜 Citrullus lanatus | |
4 | F. solani | 甘草 Glycyrrhiza uralensis | 12 | B. dothidea | 葡萄(红地球) V. vinifera (Red Globe) | |
5 | U. necator | 葡萄(赤霞珠) V. vinifera (Cabernet Sauvignon) | 13 | T. harzianum | 马铃薯 S. tuberosum |
表2
潜伏侵染叶片中葡萄霜霉病菌的real-time PCR定量测定"
接种后时间 Time after inoculation (h) | Ct值 Ct value | 检测结果 Detection result (copies/μL) |
---|---|---|
6 | 32.36±0.57 | (5.68±2.76) ×104c |
12 | 31.04±0.81 | (2.12±1.17) ×105c |
24 | 28.89±1.06 | (1.26±0.72) ×106c |
48 | 26.33±0.40 | (5.95±1.52) ×106c |
72 | 23.77±0.21 | (2.62±0.37) ×107b |
96 | 20.15±0.29 | (1.30±0.20) ×108a |
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