莴笋感染番茄斑萎病毒(TSWV)--病毒粒子分布与亚细胞病变特征

Asparagus lettuce infected with TSWV--the characteristic of virions distribution and subcellular pathological changes

莴笋是西藏昌都市露地栽培的主要蔬菜种类之一,近年来斑萎病发生普遍,发病率达到了80%以上,引起严重的经济损失,前期研究表明发病植株受到番茄斑萎病毒(TSWV)侵染。为明确该区域莴笋感染TSWV不同时期病毒粒体分布与亚细胞病变的特征,为病毒病的精准诊断与绿色防控提供依据,本研究采集了西藏昌都市卡若区莴笋不同发病时期的样品,应用TSWV-N基因序列设计引物,通过RT-PCR扩增克隆与测序分析,根据N基因序列推导N蛋白氨基酸序列比对,明确采集样品受到TSWV侵染。采集不同发病时期的样品进行负染色和超薄切片制样透射电子显微镜观察,发现早期感病叶片(症状为轻微褪绿)中病毒粒体相对含量较少,TSWV粒体分布于细胞质中,叶绿体噬锇颗粒明显增加,线粒体嵴膜部分降解,少部分细胞的液泡内有较多TSWV粒体聚集于细胞质内的囊泡中;中期感病叶片(褪绿或黄化斑)中,病毒粒体相对含量较高,较多TSWV粒体聚集于细胞质内质网池中,叶绿体膜系统降解,噬锇颗粒增加,线粒体增多;后期感病叶片(坏死斑)中,病毒粒体相对含量高,叶绿体、线粒体等细胞器降解,有大量TSWV病毒粒体聚集于残存的细胞质内囊泡中。TSWV粒体相对含量和亚细胞病变程度与植株症状严重度呈正相关,发病早期和后期TSWV粒体在细胞质内囊泡中的聚集为首次发现。上述结果为莴笋感染TSWV的早期诊断和确诊提供了新的依据,可为高原地区TSWV引起的莴笋病毒病的早期预防提供指导。

Asparagus lettuce(Lactuca sativa L.) is one of the main vegetables cultivated in the open field in Qamdo City, Tibet, the prevalence of spot wilt disease has reached more than 80% in recent years, and caused serious economic losses, previously studies showed that the diseased plants were infected with tomato spotted wilt orthotospovirus(TSWV). To identify the characteristics of virion distribution and subcellular cytopathological changes in different stages of asparagus lettuce plants infected with TSWV in this region, and to provide a basis for accurate diagnosis and green prevention and control of viral diseases, in this study, samples of asparagus lettuce at different stages of diseased plants were collected, and used RT-PCR amplification, cloning and sequencing analysis were performed by designed primers of reported TSWV-N gene, amino acid sequence of N protein alignment was deduced according to N gene sequence, and collected samples was confirmed infected with TSWV. The samples collected at different stages of the disease were observed by transmission electron microscopy with negative staining and ultrathin section preparation. In the early stage of diseased leaves(mild chlorosis), the relative content of virions was less, TSWV virions were distributed in the cytoplasm, the osmium particles increased significantly in the chloroplast, the inner carinulae was partially degraded in the mitochondrial, and a small number of cells had more TSWV virions aggregated in the vesicles of the cytoplasm;In the middle stage of diseased leaves(chlorotic or yellow spots), the relative content of virions was higher, and more TSWV virions were aggregated in the endoplasmic reticulum pool of cytoplasm, chloroplast membrane system was degraded, osmium particles and mitochondria were increased;In the late stage of diseased leaves(necrotic spots), the relative content of virions was high, and organelles(e.g. chloroplasts and mitochondria) were degraded, and a large number of TSWV virions were aggregated in the vesicles of remained cytoplasm. The relative content of TSWV virions and cytopathic effect were positively correlated with the severity of plant symptoms, the aggregation of TSWV virions in the vesicles of cytoplasm at early and late stages was first found. These result provided a new basis for the early diagnosis and accurate diagnosis of asparagus lettuce infection with TSWV, and could provide instruction for the early prevention of asparagus lettuce virus disease caused by TSWV in plateau areas.