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基于高通量测序技术鉴定青花椒花叶病病原

引用本文：邢飞,夏思丹,戚志恩,金畑宏,淳长品.基于高通量测序技术鉴定青花椒花叶病病原.植物保护学报,2025,52(2):307-315

DOI：10.13802/j.cnki.zwbhxb.2025.2024085

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作者	单位	E-mail
邢飞	西南大学柑桔研究所, 国家柑桔工程技术研究中心, 重庆 400712
夏思丹	西南大学柑桔研究所, 国家柑桔工程技术研究中心, 重庆 400712
戚志恩	西南大学柑桔研究所, 国家柑桔工程技术研究中心, 重庆 400712
金畑宏	西南大学柑桔研究所, 国家柑桔工程技术研究中心, 重庆 400712
淳长品	西南大学柑桔研究所, 国家柑桔工程技术研究中心, 重庆 400712	chuncp@cric.cn

中文摘要:为明确我国青花椒花叶病的病原及其基因组结构和分子特征，在青花椒产区调查花叶病的主要表现症状并采集病样，利用高通量测序技术鉴定病样中的病原种类，采用PCR或反转录PCR技术扩增病原基因组全长序列，并进一步对其结构、序列多样性及其与症状的相关性进行分析。结果表明：青花椒花叶病症状主要表现为叶片出现花叶、不规则的褪绿、黄化斑或带，部分感病叶片还伴随皱缩、叶缘缺刻症状；结合高通量测序结果和PCR或反转录PCR扩增序列，最终确认感花叶病样品中同时存在青花椒脉明伴随病毒（green Sichuan pepper vein clearing-associated virus，GSPV‐CaV）和青花椒耳突花叶病毒（green Sichuan pepper enamovirus，GSPEV），并克隆获得GSPVCaV-DJ分离物基因组，全长为8 099 nt，包含3个开放阅读框，属于杆状DNA病毒属Badnavirus的典型结构特征；序列比对发现GSPVCaV-DJ分离物与其余8条GSPVCaV分离物基因组的核苷酸序列一致性为97.2%~98.8%，相似性较高；在田间采集的感花叶病样品中均检出了GSPVCaV，无明显症状样品中则未检出该病毒，说明GSPVCaV与青花椒花叶病症状有较高的相关性，而GSPEV在这2种症状样品中均有检出，检出率达90%，表明GSPVCaV是引起我国青花椒花叶病的主要病原。

中文关键词:高通量测序青花椒花叶病病毒鉴定基因组特征相关性分析

Identification of viral pathogen associated with mosaic disease in green Sichuan pepper Zanthoxylum armatum with high-throughput sequencing technology

Author Name	Affiliation	E-mail
Xing Fei	National Citrus Engineering Research Center, Citrus Research Institute, Southwest University, Chongqing 400712, China
Xia Sidan	National Citrus Engineering Research Center, Citrus Research Institute, Southwest University, Chongqing 400712, China
Qi Zhi'en	National Citrus Engineering Research Center, Citrus Research Institute, Southwest University, Chongqing 400712, China
Jin Tianhong	National Citrus Engineering Research Center, Citrus Research Institute, Southwest University, Chongqing 400712, China
Chun Changpin	National Citrus Engineering Research Center, Citrus Research Institute, Southwest University, Chongqing 400712, China	chuncp@cric.cn

Abstract:To clarify the causal agent of mosaic disease in green Sichuan pepper Zanthoxylum armatum and to analyze the genome structure and molecular characteristics of the pathogen, field surveys were conducted in major production areas to document the primary symptoms. High-throughput sequencing (HTS) technology was employed to identify possible viruses in mosaic-diseased leaves, and the complete full genome of one virus was subsequently amplified by polymerase chain reaction (PCR) or reverse transcription-PCR (RT-PCR). Additionally, the genome structure, sequence diversity, and symptom correlation were thoroughly analyzed. The results showed that the characteristic symptoms of mosaic disease in green Sichuan pepper leaves include mosaic patterns, irregular chlorosis or yellow spots/bands, and in some cases, leaf wrinkling and notched leaf margins. HTS combined with PCR/RTPCR confirmed the presence of green Sichuan pepper vein clearing-associated virus (GSPVCaV) and green Sichuan pepper enamovirus (GSPEV) in symptomatic samples. The genome of the GSPVCaV-DJ isolate, 8 099 nt in length, containing three open reading frames (ORFs), was cloned and sequenced, revealing typical features of the genus Badnavirus. Multiple sequence alignment indicated that the DJ isolate shares 97.2%-98.8% nucleotide identity with eight other isolates, suggesting a high degree of genetic similarity. Diagnostic testing of field-collected samples showed that GSPVCaV was consistently detected in symptomatic samples but absent in asymptomatic ones, indicating a strong correlation between GSPVCaV and mosaic disease. In contrast, GSPEV was detected in approximately 90% of both symptomatic and asymptomatic leaf samples, suggesting that it is not directly associated with symptom development. These findings demonstrate that GSPVCaV is the primary causal agent of mosaic disease in green Sichuan pepper in China, providing a critical reference for further studies on the epidemiology, pathogenicity, and management of this virus.

keywords:high-throughput sequencing green Sichuan pepper mosaic disease virus identification genome characteristics relationship analysis

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