葡萄白粉病菌潜伏侵染量Real-time PCR检测方法建立

doi:10.16420/j.issn.0513-353x.2022-0333

摘要/Abstract

摘要：

根据葡萄EF1-α基因序列设计引物F-g-6/R-g-6，根据葡萄白粉菌细胞色素P450基因序列设计特异性引物F-P450-Un/R-Un，分别建立葡萄叶片和葡萄白粉菌的real-time PCR检测方法，用于计算分子病情指数。结果表明：使用real-time PCR方法对酿酒葡萄‘赤霞珠’叶片DNA和葡萄白粉菌DNA测定的灵敏度分别为10^-2和10^-5 ng · µL^-1，分别是常规PCR的100倍和1 000倍。同时建立葡萄叶片DNA和白粉菌DNA各自的标准曲线，标准曲线循环阈值与模板浓度呈良好的线性关系，相关系数分别为0.99637和0.99564。利用建立的real-time PCR检测方法对葡萄尚未发病的30个田间样品进行检测，共检测到22个样品中含有葡萄白粉菌，其分子病情指数与18 d后的田间病情指数极显著相关（相关系数为0.916）。建立的葡萄白粉菌潜伏侵染量real-time PCR分子检测体系，能够为葡萄白粉病的早期诊断和监测预警提供理论支持。

关键词: 葡萄, 葡萄白粉菌, 早期诊断, real-time PCR, 分子病情指数, 田间病情指数

Abstract:

In order to caculate molecular-detected disease index（MDI）of grape powdery mildew，according to the P450 sequence of Uncinula necator and EF1-α sequence of Vitis vinifera L.，pairs of primers，F-P450-Un/R-Un and F-g-6/R-g-6，were designed to establish the real-time PCR reaction system. The sensitivity of real-time PCR of V. vinifera and U. necator was 1.0 × 10^-2 ng · µL^-1 and 1.0 × 10^-5 ng · µL^-1 genomic DNA respectively，which was 100 and 1 000 times greater than that of the conventional PCR. At the same time，the standard curves of V. vinifera and U. necator were established respectively. There were good linear relationship between the cycling threshold（Ct）and template DNA concentration with the correlation coefficient of 0.99637 and 0.99564 respectively. Application of this technology to detect 30 samples in the field. A total of 22 samples were detected to contain grape powdery mildew, and the MDI was significantly correlated with the field DI after 18 days (correlation coefficient was 0.916). The established real time PCR system of U. necator could provide theoretical basis for early detection and monitoring of powdery mildew.

Key words: Vitis vinifera, Uncinula necator, early diagnosis, real-time PCR, molecular-detected diseases index, field disease index

中图分类号:

S663.1

王雯雯, 张强强, 李玲, 金婧, 王若彤, 顾沛雯. 葡萄白粉病菌潜伏侵染量Real-time PCR检测方法建立[J]. 园艺学报, 2023, 50(6): 1368-1376.

WANG Wenwen, ZHANG Qiangqiang, LI Ling, JIN Jing, WANG Ruotong, GU Peiwen. Establishment of a Method for Quantitative Detection of Latent Infection of Powdery Mildew in Wine Grape by Real-time PCR[J]. Acta Horticulturae Sinica, 2023, 50(6): 1368-1376.

图/表 6

表1 供试菌株

Table 1 Tested strains

菌种编号Number	菌株 Bacterial strain	寄主（品种） Host（Variety）
U	葡萄白粉病菌 Uncinula necator	葡萄（赤霞珠）Vitis vinifera（Cabernet Sauvignon）
1	葡萄灰霉病菌 Botrytis cinerea	葡萄（赤霞珠）V. vinifera（Cabernet Sauvignon）
2	葡萄霜霉病菌 Plasmopara viticola	葡萄（赤霞珠）V. vinifera（Cabernet Sauvignon）
3	黄瓜白粉病菌 Podosphaera xanthii	黄瓜 Cucumis sativus
4	西瓜白粉病菌 Po. xanthii	西瓜 Citrullus lanatus
5	南瓜白粉病菌 Po. xanthii	南瓜 Cucurbita moschata
6	地锦白粉病菌 Sphaerotheca pannosa	地锦 Parthenocissus tricuspidata
7	酸模白粉病菌 Erysiphepolygoni	酸模 Rμmex acetasa
8	委陵菜白粉病菌Po. fusca	委陵菜 Potentilla supina
9	秋海棠锈菌 Pucciniastrum boehmeriae	秋海棠 Begonia palmata
10	玉米大斑病菌 Exserohilum turcicum	玉米 Zea mays

表2 本研究中使用的引物序列

Table 2 Sequences of the primers used in this study

引物Primer	序列（5′-3′）Sequence
F-g-6	CAGCACAATCTGCCTGTGAGGTAC
R-g-6	GTCGACTACCACTGGTCACTTGATC
F-P450-Un	GGTCCGTAAATGGTCGAGTAGTAAT
R-Un	CACCCTGAAAGCTAACGCAA

图1 引物F-P450-Un/R-Un常规PCR（A）和real-time PCR（B）特异性检测结果 M：Marker；U：葡萄白粉菌；1 ~ 10：植物病原菌详见表1；CK：RNase-free ddH2O。橘色：葡萄白粉病菌；红色：葡萄灰霉病菌；黄色：葡萄霜霉病菌；浅绿色：黄瓜白粉病菌；墨绿色：西瓜白粉病菌；深蓝色：南瓜白粉病菌；浅蓝色：地锦白粉病菌；粉色：酸模白粉病菌；紫色：委陵菜白粉病菌；黑色：秋海棠锈菌；灰色：玉米大斑病菌。

Fig. 1 Specificity detection of U. necator primers F-P450-Un/R-Un with conventional PCR（A）and real-time PCR（B） M：Marker；U：Uncinula necator；1-10：The name of plant pathogen in Table 1；CK：RNase-free ddH2O. Orange：Uncinula necator; Red: Botrytis cinerea; Yellow: Plasmopara viticola; Light Green: Podosphaera xanthii; Dark Green: Po. Xanthii; Dark Blue: Po. Xanthii; Light blue: Sphaerotheca pannosa; Pink: Erysiphepolygoni; Purple: Po. Fusca; Black: Pucciniastrum boehmeriae; Gray: Exserohilum turcicum.

图2 葡萄叶片和白粉病菌DNA常规PCR（A1、B1）和real-time PCR灵敏度检测（A2、B2）及熔解曲线（A3、B3） M：Marker；CK：RNase-free ddH2O；A2、A3中红、浅绿、深蓝、橙、深绿、浅蓝和粉色分别表示葡萄叶片DNA浓度为1.0 × 102、1.0 ×101、1.0 × 100、1.0 × 10-1、1.0 × 10-2、1.0 × 10-3和1.0 × 10-4 ng · μL-1；B2、B3中浅绿、深蓝、红、深绿、墨蓝、浅蓝、棕和橙色分别表示葡萄白粉病菌DNA浓度为1.0 × 102、1.0 ×101、1.0 × 100、1.0 × 10-1、1.0 × 10-2、1.0 × 10-3、1.0 × 10-4和1.0 × 10-5 ng · μL-1。

Fig. 2 Conventional PCR（A1，B1），real-time PCR sensitivity（A2，B2）and melting curve（A3，B3）of grape leaves DNA and powdery mildew DNA M：Marker；CK：RNase-free ddH2O；In A2, A3, red, light green, dark blue, orange, dark green, light blue and pink indicate the concentrations of DNA in grape leaves 1.0 × 102, 1.0 ×101, 1.0 × 100, 1.0 × 10-1, 1.0 × 10-2, 1.0 × 10-3 and 1.0 × 10-4 ng · μL-1; Respectively in B2 and B3, light green, dark blue, red, dark green, blue black, light blue, brown, orange indicate the DNA concentration of Uncinula necator 1.0 × 102, 1.0 ×101, 1.0 × 100, 1.0 × 10-1, 1.0 × 10-2, 1.0 × 10-3, 1.0 × 10-4 and 1.0 × 10-5 ng · μL-1.

图3 葡萄叶片（A）和葡萄白粉病菌（B）的real-time PCR标准曲线的建立

Fig. 3 Establishment of real-time PCR standard curve for grape leaves（A）and grape powdery mildew（B）

表3 田间样品的分子病情指数与病情指数

Table 3 MDI and DI of samples from field

样点 Sampling point	分子病情指数 MDI	病情指数 DI	样点 Sampling point	分子病情指数 MDI	病情指数 DI
1	0	0	16	0.0017	0.21
2	0	0	17	0.0021	0.32
3	0	0	18	0	0
4	0.0003	0	19	0.0064	0.97
5	0.0002	0	20	0	0
6	0.0001	0	21	0.0002	0
7	0.0036	0.52	22	0.0023	0.39
8	0	0	23	0.0001	0
9	0	0	24	0.0003	0
10	0.0002	0	25	0.0034	0.49
11	0	0	26	0.0009	0.01
12	0.0021	0.29	27	0.0002	0
13	0.0985	2.93	28	0.0001	0
14	0.0015	0.14	29	0.0050	0.77
15	0.0017	0.19	30	0.0113	1.09

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