柑橘黄化花叶病毒的实时定量PCR检测及其在寄主植株中的时空分布规律

doi:10.3864/j.issn.0578-1752.2023.18.007

摘要/Abstract

摘要：

【背景】柑橘黄化花叶病毒（citrus yellow mosaic virus，CYMV）是首先发现于印度并对其柑橘产业造成严重危害的一种杆状DNA病毒。目前，CYMV已被美国、日本、新西兰、欧洲和地中海地区列入检疫性有害生物名单，具有传入我国的潜在风险。【目的】建立CYMV的实时荧光定量PCR（real-time quantitative PCR，qPCR）检测体系；筛选CYMV敏感柑橘品种；明确CYMV在寄主植株中的时空分布规律，为该病毒的监控提供技术支持。【方法】根据NCBI中CYMV外壳蛋白（coat protein，CP）基因保守序列，利用软件Primer Premier 5设计qPCR检测引物8对，通过常规PCR筛选扩增效果好、特异性强的引物。通过对引物浓度、退火温度等反应条件优化，建立CYMV的qPCR检测体系，并进一步通过对不同柑橘病原的检测评价所建体系的特异性；以梯度稀释的1.98×10⁹—1.98 copies/μL的质粒标准品平行进行常规PCR及qPCR检测，评价所建方法的灵敏度；随机采集田间柑橘样品，平行进行常规PCR及qPCR检测，评价所建方法的适用性。将CYMV接种到玉环柚（Citrus grandis）、强德勒柚（C. grandis）、22号枳（Poncirus trifoliata）等15个柑橘品种，进行症状观察和分子检测以筛选敏感指示植物。在接种后不同时间，分别自MV甜橙（C. sinensis）植株不同组织部位取样，以柑橘生长因子1α基因为内参基因，利用所建体系进行qPCR检测，从而明确CYMV在寄主植株中的时空分布规律。【结果】建立了CYMV的qPCR检测体系，其最佳引物为CYMV-qF7/R7，最佳引物浓度为200 nmol·L^-1，最佳退火温度为63 ℃。该检测体系的特异性强，检测灵敏度是常规PCR的1 000倍。对来自不同地区的660个田间柑橘样品的检测结果表明，qPCR检测与常规PCR检测结果一致，除阳性对照外均未检测到CYMV阳性植株。不同柑橘品种接种试验结果表明，15个柑橘品种中，玉环柚和强德勒柚最早表现出强烈的黄化花叶典型侵染症状，可以作为敏感指示植物。qPCR检测结果表明，老叶中的CYMV滴度最高，老皮和根病毒滴度较高，嫩皮中的滴度较低。CYMV在植株中的相对含量，7—9月最高，此后逐月下降，并在次年1月达到最低值，从次年2月开始，CYMV的相对含量开始上升，与环境温度变化趋势一致。【结论】建立了特异性强、灵敏度高的CYMV实时荧光定量PCR检测方法，利用该方法明确了CYMV在寄主植株中的时空分布规律。此外，玉环柚和强德勒柚可作为CYMV敏感指示植物。

关键词: 柑橘黄化花叶病毒, 实时荧光定量PCR, 时空分布

Abstract:

【Background】Citrus yellow mosaic virus (CYMV) is a member of badnaviruses that was first discovered in India and caused serious damage to its citrus industry. At present, CYMV has been listed as a quarantine pest in the United States, Japan, New Zealand, Europe and the Mediterranean region, which has the potential risk of being introduced into China.【Objective】The objective of this study is to establish a real-time quantitative PCR (qPCR) detection system for CYMV, screen sensitive citrus varieties to CYMV, elucidate the spatial and temporal distribution of CYMV in host plant, and to provide technical support for monitoring of the virus.【Method】According to the conserved sequences of CYMV coat protein (CP) gene in NCBI, eight pairs of qPCR primers were designed using software Primer Premier 5, and were screened by conventional PCR for primer pairs with good amplification effect and high specificity. The concentration and annealing temperature of the primers were optimized to establish the CYMV qPCR detection system. The specificity of the system was evaluated by detecting different citrus pathogens. Conventional PCR and qPCR were performed in parallel with 1.98×10⁹-1.98 copies/μL of plasmid standards diluted in a gradient as template to evaluate the sensitivity of the established system. The field citrus samples were collected randomly and detected using conventional PCR and qPCR in parallel to evaluate the applicability of the method. Fifteen citrus varieties including Yuhuanyou (Citrus grandis), Chandler pomelo (C. grandis), No.22 karatachi (Poncirus trifoliata), etc., were inoculated with CYMV for symptom observation and molecular detection to screen sensitive indicator plants. Samples from different tissues of Madam Vinous orange (C. sinensis) plants were collected at different times post inoculation, and qPCR assays were performed with C. sinensis elongation factor 1 alpha as an internal reference gene to clarify the spatial and temporal distribution patterns of CYMV in the host plants.【Result】The qPCR detection system for CYMV was established with the optimal primer of CYMV-qF7/R7, the optimal concentration 200 nmol·L^-1 and the optimal annealing temperature 63 ℃. The results of the evaluation experiments showed that the specificity of the established qPCR system is strong, and the detection sensitivity is 1 000 times that of the conventional PCR. The detection results of 660 field citrus samples from different regions showed that the qPCR assay was consistent with the conventional PCR assay, and no CYMV-positive plants were detected except for the positive control. Among the 15 citrus varieties in the inoculation experiment, Yuhuanyou and Chandler pomelo showed strong and typical yellowing mosaic symptoms of CYMV infection at the earliest, suggesting that both could be used as sensitive indicator plants. The detection results of qPCR showed that the titer of CYMV in old leaves was the highest, the titer of virus in old cortex and root was higher, and the titer in tender cortex was lower. The relative content of CYMV in plants was highest from July to September, then decreased month by month, and reached the lowest value in January of the next year. From February of the next year, the relative content of CYMV began to rise, which was consistent with the change trend of the environmental temperature.【Conclusion】A qPCR assay for CYMV detection with high specificity and sensitivity was established, and the spatial and temporal distribution pattern of CYMV in the host plants was determined. In addition, Yuhuanyou and Chandler pomelo can be used as CYMV-sensitive indicator plants.

Key words: citrus yellow mosaic virus (CYMV), real-time quantitative PCR (qPCR), spatial and temporal distribution

曹鹏, 许建建, 李楚欣, 王新亮, 王春庆, 宋晨虎, 宋震. 柑橘黄化花叶病毒的实时定量PCR检测及其在寄主植株中的时空分布规律[J]. 中国农业科学, 2023, 56(18): 3574-3584.

CAO Peng, XU JianJian, LI ChuXin, WANG XinLiang, WANG ChunQing, SONG ChenHu, SONG Zhen. Real-Time Quantitative PCR Detection of Citrus Yellow Mosaic Virus and Its Spatial and Temporal Distribution in Host Plants[J]. Scientia Agricultura Sinica, 2023, 56(18): 3574-3584.

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引物名称 Primer name	引物序列 Primer sequence (5′-3′)	扩增产物长度 Product length (bp)	用途 Usage
CYMV-CP-F	GATGAATATGAGAACCTTGTTACAACT	642	CP序列扩增 CP sequence amplification
CYMV-CP-R	TCGTTTCACATTTCGCCG	642	CP序列扩增 CP sequence amplification
CYMV-JC-F	ACACCGGAAACGGTGAAGTT	342	CYMV检测 CYMV detection
CYMV-JC-R	ACCCTTCGAACATGGGCATT	342	CYMV检测 CYMV detection
EF-1α F	CTCAAGCCTGGTATGGTGGT	185	内参基因 Reference gene
EF-1α R	GGATCATCCTTCGAGTTGGA	185	内参基因 Reference gene
CYMV-qF1	TTGGTGTTTGCCCTCGGATT	119	qPCR检测 qPCR detection
CYMV-qR1	GCCTGGGATAGGGATTGAGC	119
CYMV-qF2	TGGGCATAGTTCAGGTTCGG	93
CYMV-qR2	CTCCTTGCCACCGATTGTCT	93
CYMV-qF3	CCAGTCGTGCTCGTACAGTT	119
CYMV-qR3	CCGCTGCCACTGCCTATAAT	119
CYMV-qF4	CAGGAGTACATCGCCTGGTG	149
CYMV-qR4	TTTCGCCTGCGAGAGTCTAC	149
CYMV-qF5	CAAAGACTGGGCGCATGTTC	142
CYMV-qR5	AGAATCCGAGGGCAAACACC	142
CYMV-qF6	AACAGTTAAGCAGGAGCGGT	128
CYMV-qR6	TCCTTGCCACCGATTGTCTC	128
CYMV-qF7	GCTCAATCCCTATCCCAGGC	208
CYMV-qR7	ACATTTCGCCGGTCATTTGG	208
CYMV-qF8	TGCAAAGATGCAGCGTTCA	59
CYMV-qR8	GGGATTGAGCTGCAAAAGGA	59

地区 <BOLD>R</BOLD>egion	采集品种 Variety	常规PCR（阳性数/总数） Conventional PCR (Positive/Total)	qPCR（阳性数/总数） qPCR (Positive/Total)
广西Guangxi	砂糖橘C. reticulata	0/60	0/60
湖南Hunan	宫川温州蜜柑C. unshiu	0/150	0/150
湖南Hunan	纽荷尔脐橙C. sinensis	0/150	0/150
重庆Chongqing	柠檬C. limon	0/200	0/200
重庆Chongqing	沙田柚C. maxima ‘Shatian Yu’	0/100	0/100
阳性对照Positive control	MV甜橙C. sinensis	25/30	30/30