Establishment of a Method for Quantitative Detection of Latent Infection of Powdery Mildew in Wine Grape by Real-time PCR

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

Acta Horticulturae Sinica ›› 2023, Vol. 50 ›› Issue (6): 1368-1376.doi: 10.16420/j.issn.0513-353x.2022-0333

• New Technology & New Method • Previous Articles Next Articles

Establishment of a Method for Quantitative Detection of Latent Infection of Powdery Mildew in Wine Grape by Real-time PCR

WANG Wenwen, ZHANG Qiangqiang, LI Ling, JIN Jing, WANG Ruotong, GU Peiwen()

College of Agronomy，Ningxia University，Yinchuan 750021，China

Received:2022-10-08 Revised:2023-03-13 Online:2023-06-25 Published:2023-06-27
Contact: * （E-mail：gupeiwen2019@nxu.edu.cn）

Abstract

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

CLC Number:

S663.1

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.

Figures/Tables 6

References 21

[1]	Bohm J. 1999. Real-time quantitative PCR:DNA determination in isolated spores of the mycorrhizal fungus glomus mosseae and monitoring of Phytophthora infestans and Phytophthora citricola in their respective host plants. Journal of Phytopathology, 147 (7/8):409-416. doi: 10.1111/j.1439-0434.1999.tb03842.x URL
[2]	Du Juan. 2021. Quantitative analysis of plasmopara viticola in latent in the fields of wine grape using real-time PCR[M. D. Dissertation]. Yinchuan: Ningxia University. (in Chinese)
	杜娟. 2021. 基于real-time PCR的酿酒葡萄霜霉菌潜伏期田间宏观定量分析[硕士论文]. 银川: 宁夏大学.
[3]	Du Juan, Li Jin, Zhang Tao, Zhang You, Yao Pei, Gu Pei-wen. 2021. Quantitative detection of latent infection of grape downy mildew in field by real-time PCR. Jiangsu Journal of Agricultural Sciences, 37 (4):861-866. (in Chinese)
	杜娟, 李金, 张涛, 张游, 姚珮, 顾沛雯. 2021. 应用real-time PCR定量检测果园葡萄霜霉病菌潜伏侵染. 江苏农业学报, 37 (4):861-866.
[4]	Gu Pei-wen, Du Juan, Shi Hua-rong, Li Wen-xue, Ren Miao-miao. 2020. Real time PCR method for detecting the infection amount of grape powdery mildew in incubation period. China:CN110964847A. 2020-04-07.
	顾沛雯, 杜娟, 石华荣, 李文学, 任苗苗. 2020. 实时荧光PCR检测葡萄白粉病菌潜育期侵染量的方法. 中国:CN110964847A. 2020-04-07.
[5]	Li Wen-xue, Xiao Rui-gang, Lü Miao-miao, Ding Ning, Shi Hua-rong, Gu Pei-wen. 2019. Establishment and application of Real-Time PCR for quantitatively detecting Plasmopara viticola in Vitis vinifera. Scientia Agricultura Sinica, 52 (9):1529-1540. (in Chinese)
	李文学, 肖瑞刚, 吕苗苗, 丁宁, 石华荣, 顾沛雯. 2019. 葡萄霜霉病菌实时荧光定量PCR检测体系的建立和应用. 中国农业科学, 52 (9):1529-1540. doi: 10.3864/j.issn.0578-1752.2019.09.005
[6]	Liu Hui-ning, Zheng Qi. 2002. Study on the resistance to Uncinula necator of several grape varieties. Journal of Fruit Science, 19 (6):426-428. (in Chinese)
	刘会宁, 郑琦. 2002. 葡萄对白粉病的抗性. 果树学报, 19 (6):426-428.
[7]	Liu Qi, Li Wei, Wang Cui-cui, Gu Yi-lin, Wang Rui, Ma Zhan-hong. 2018. Qualitative identification of canopy spectra in wheat stripe rust based on Logistic,IBk and Randomcommittee methods. Journal of Plant Protection, 45 (1):146-152. (in Chinese)
	刘琦, 李薇, 王翠翠, 谷医林, 王睿, 马占鸿. 2018. 基于Logistic、IBk以及Randomcommittee方法的条锈病潜育期小麦冠层光谱的定性识别. 植物保护学报, 45 (1):146-152.
[8]	Luo Y, Gu S, Felts D, Puckett R D, Morgan D P, Michailides T J. 2017. Development of qPCR systems to quantify shoot infections by canker-causing pathogens in stone fruits and nut crops. Journal of Applied Microbiology, 122 (2):416-428. doi: 10.1111/jam.13350 pmid: 27862716
[9]	Luo Yong. 2009. Brief introduction to molecular epidemiology of plant diseases. Acta Phytopathologica Sinica, 39 (1):1-10. (in Chinese)
	骆勇. 2009. 植物病害分子流行学概述. 植物病理学报, 39 (1):1-10.
[10]	Pan Juan-juan, Luo Yong, Huang Chong, Sun Zhen-yu, Zhao Lei, Yan Jia-hui, Ma Zhan-hong. 2010. Quantification of latent infections of wheat stripe rust by using real-time PCR. Acta Phytopathologica Sinica, 40 (5):504-510. (in Chinese)
	潘娟娟, 骆勇, 黄冲, 孙振宇, 赵磊, 闫佳会, 马占鸿. 2010. 应用real-time PCR定量检测小麦条锈菌潜伏侵染量方法的建立. 植物病理学报, 40 (5):504-510.
[11]	Pan Yang, Gu Yi-lin, Luo Yong, Ma Zhan-hong. 2016. Establishment and application of duplex real-time PCR quantitative determination method on latent infection of wheat stripe rust. Acta Phytopathologica Sinica, 46 (4):485-491. (in Chinese)
	潘阳, 谷医林, 骆勇, 马占鸿. 2016. 双重real-time PCR定量测定小麦条锈菌潜伏侵染方法的建立与应用. 植物病理学报, 46 (4):485-491.
[12]	Schena L, Nigro F, Ippolito A, Gallitelli D. 2004. Real-time quantitative PCR:a new technology to detect and study phytopathogenic and antagonistic fungi. European Journal of Plant Pathology, 110 (9):893-908. doi: 10.1007/s10658-004-4842-9 URL
[13]	Wang Wei. 2010. Invasion procedure and effects on physiological characteristics of Uncinula necator in grape leaves[M. D. Dissertation]. Yangling: Northwest A & F University. (in Chinese)
	王伟. 2010. 葡萄白粉菌侵染过程及其对叶片相关生理生化指标的影响[硕士论文]. 杨凌: 西北农林科技大学.
[14]	Wang Yue-jin, He Pu-chao. 1997. Genetic study on leaf powdery mildew resistance of wild Vitis species in China. Scientia Agricultura Sinica, 30 (1):19-25. (in Chinese)
	王跃进, 贺普超. 1997. 中国葡萄属野生种叶片抗白粉病遗传研究. 中国农业科学, 30 (1):19-25.
[15]	Xu Na-na. 2014. Quantitative dectection and dynamic analysis of Rhizoctonia cerealis of wheat in soil by Real-time PCR[M. D. Dissertation]. Taian: Shandong Agricultural University. (in Chinese)
	徐娜娜. 2014. 实时荧光定量PCR技术用于土壤中小麦纹枯病菌的定量检测及动态分析[硕士论文]. 泰安: 山东农业大学.
[16]	Yan J, Luo Y, Chen T. 2012. Field distribution of wheat stripe rust latent infection using real-time PCR. Plant Disease, 96 (4):544-551. doi: 10.1094/PDIS-08-11-0680 pmid: 30727438
[17]	Yan Jia-hui, Luo Yong, Pan Juan-juan, Wang Hai-guang, Jin She-lin, Cao Shi-qin, Ma Zhan-hong. 2011. Quantification of latent infection of wheat stripe rust in the fields using real-time PCR. Acta Phytopathologica Sinica, 41 (6):618-625. (in Chinese)
	闫佳会, 骆勇, 潘娟娟, 王海光, 金社林, 曹世勤, 马占鸿. 2011. 应用real-time PCR定量检测田间小麦条锈菌潜伏侵染的研究. 植物病理学报, 41 (6):618-625.
[18]	Yan L, Yang Q, Zhou Y, Duan X, Ma Z. 2009. A real-time PCR assay for quantification of the Y136F allele in the CYP51 gene associated with Blumeria graminis f.sp. tritici resistance to sterol demethylase inhibitors. Crop Protection, 28 (5):376-380. doi: 10.1016/j.cropro.2008.12.011 URL
[19]	Zheng Y, Luo Y, Zhou Y, Zeng X, Duan X, Cao X, Song Y, Wang B. 2013. Real-time PCR quantification of latent infection of wheat powdery mildew in the field. European Journal of Plant Pathology, 136 (3):565-575. doi: 10.1007/s10658-013-0188-5 URL
[20]	Zhou Ru-jun, Cui Jian-chao, Fu Jun-fan, Xu Zhe, Xue Cai-yun. 2015. Effect of mixed infection of Cercospora arachidicola and Phoma arachidicola on their infection probability and latent periods. Scientia Agricultura Sinica, 48 (21):4264-4271. (in Chinese) doi: 10.3864/j.issn.0578-1752.2015.21.007
	周如军, 崔建潮, 傅俊范, 徐喆, 薛彩云. 2015. 花生褐斑病菌和网斑病菌混合侵染对侵染概率和潜育期的影响. 中国农业科学, 48 (21):4264-4271. doi: 10.3864/j.issn.0578-1752.2015.21.007
[21]	Zhu Xiao-hua, Ma Jun-yi, Patiguli, Yang An-pei, Amanguli, Shadati. 2011. Occurrence regularity of grape powdery mildew and its control techniques in Xinjiang. Xinjiang Agricultural Sciences, 48 (2):282-286. (in Chinese)
	朱晓华, 马俊义, 帕提古丽, 杨安沛, 阿蔓古丽, 沙达提. 2011. 葡萄白粉病发病规律研究及防治技术. 新疆农业科学, 48 (2):282-286.

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

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

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

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

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

Establishment of a Method for Quantitative Detection of Latent Infection of Powdery Mildew in Wine Grape by Real-time PCR

RichHTML

PDF

Knowledge

Abstract

Cite this article

share this article

Figures/Tables 6

References 21

Related Articles 15

Recommended Articles

Metrics

Comments

[1]	LI Zhenxi, PAN Ruixuan, XU Meirong, ZHENG Zheng, DENG Xiaoling. Development of Duplex Real-time PCR Assay of‘Candidatus Liberibacter asiatics’ [J]. Acta Horticulturae Sinica, 2023, 50(1): 188-196.
[2]	WANG Haibo, WANG Baoliang, JI Xiaohao, WANG Xiaodi, SHI Xiangbin, WANG Zhiqiang, WANG Xiaolong, LIU Fengzhi. A New Mid-ripening Table Grape Cultivar‘Huapu Heifeng’ [J]. Acta Horticulturae Sinica, 2021, 48(2): 401-402.
[3]	YANG Ting, XUE Zhenzhen, LI Na, LANG Xiaoan, LI Lingfei, ZHONG Chunmei. Reference Genes Selection and Validation in Begonia masoniana Leaves of Different Developmental Stages [J]. Acta Horticulturae Sinica, 2021, 48(11): 2251-2261.
[4]	HE Honghong1，LU Zhihao2，MA Zonghuan1，LIANG Guoping1，MA Lijuan1，WAN Peng1，and MAO Juan1,*. Genome-Wide Identification and Expression Analysis of the PP2C Gene Family in Vitis vinifera [J]. ACTA HORTICULTURAE SINICA, 2018, 45(7): 1237-1250.
[5]	LIU Xiaoting1,2,，WANG Shunli2,，XUE Jingqi2，XUE Yuqian2，Lü Yingmin1,，and ZHANG Xiuxin2,. Selection of Reference Genes for Quantitative Real-Time PCR in Different Tissue and Organ of Barbadoslily [J]. ACTA HORTICULTURAE SINICA, 2018, 45(5): 919-930.
[6]	WANG An-jun1,2，LI Yu-dan2,3，HUANG He-xun2，LUO Shao-bo2,4，WU Ting-quan2,4，LI Ren-qiang1,，and ZHONG Yu-juan2,4,. Cloning and Characterization of Two Genes Encoding Myo-inositol Monophosphatase 1（CmIMP1）and 2（CmIMP2）from Pumpkin [J]. ACTA HORTICULTURAE SINICA, 2016, 43(7): 1315-1325.
[7]	XU Lan-zhen，HE Yong-rui，LEI Tian-gang，PENG Ai-hong，YAO Li-xiao，JIANG Guo-jin，LI Qiang，ZOU Xiu-ping，and CHEN Shan-chun. Identification of the Copy Number of Exogenous Gene in Transgenic Citrus by Quantitative Real-time PCR [J]. ACTA HORTICULTURAE SINICA, 2016, 43(6): 1186-1194.
[8]	YU Ying1,，CHEN Dan1,，SUN Jun2，Lü Shi-heng1，CHEN Gui-xin1,，and YE Nai-xing1,. Cloning and Expression Analysis of Terpene Synthase Gene from Jasminum sambac [J]. ACTA HORTICULTURAE SINICA, 2016, 43(2): 356-364.
[9]	SUN Jie，WANG Wan，RAO Xue-qin，and LI Hua-ping. Development of TaqMan Real-time PCR Assay of Banana bunchy top virus [J]. ACTA HORTICULTURAE SINICA, 2016, 43(12): 2473-2480.
[10]	BEN Hai-yan1，SHI Yan-xia2，XIE Xue-wen2，CHAI A-li2，and LI Bao-ju2,*. Studies of Soil Improvement Effect of Calcium Cyanamide and Its Control#br# Efficiency on Soil-borne Diseases of Vegetable Crops [J]. ACTA HORTICULTURAE SINICA, 2016, 43(11): 2173-2181.
[11]	FU Li-li，WU Ju-xun，and YI Hua-lin*. Expression Analysis of Citrate Metabolic Related Genes in Late-ripening Mutant of Navel Orange and Its Wild Type [J]. ACTA HORTICULTURAE SINICA, 2016, 43(1): 38-46.
[12]	LI Jia-wei，LIN Ming，HU Hong，WANG Xiao-xuan，GUO Yan-mei，HUANG Ze-jun，DU Yong-chen，and GAO Jian-chang**. The Combined Effects of Different TYLCV Resistant Genes and Temperatures on TYLCV Duplication [J]. ACTA HORTICULTURAE SINICA, 2016, 43(1): 71-79.
[13]	SHI Juan，LI Fang-fang，MA Hong，LI Zhong-yong，and XU Ji-zhong*. Effects of Different Interstocks on Key Enzymes Activities and the Expression of Genes Related to Malic Acid Metabolism in Apple Fruit [J]. ACTA HORTICULTURAE SINICA, 2016, 43(1): 132-140.
[14]	SHEN Yan-hong*，CHEN Xiao-jing，CAI Xue-ling，YE Yi-jiang，GENG Jiao-jiao，and YANG Fei-ying. Cloning and Expression Analysis of Cysteine Protease Gene CpCP from Papaya [J]. ACTA HORTICULTURAE SINICA, 2015, 42(9): 1789-1797.
[15]	JIANG Ting-Ting, GAO Yan-Hui, TONG Zai-Kang. Selection of Reference Genes for Quantitative Real-Time PCR in Lycoris [J]. ACTA HORTICULTURAE SINICA, 2015, 42(6): 1129-1138.