侵染云南草原龙胆的凤仙花坏死斑病毒鉴定

doi:10.16420/j.issn.0513-353x.2024-1043

摘要/Abstract

摘要：

在云南省昆明市呈贡区斗南镇进行花卉病害调查时，发现叶片表现坏死斑症状的草原龙胆植株。采集典型症状样品进行免疫试纸条检测，初步确定其病原为凤仙花坏死斑病毒（Impatiens necrotic spot virus，INSV）。利用电子显微镜观察发现直径为60 ~ 110 nm的球状病毒粒体，聚集于内质网膜内，叶绿体结构不完整，部分双层膜消失。为进一步明确病原，应用INSV的N、NSm和RdRp基因的特异性引物进行RT-PCR扩增、克隆和测序，分别获得长度为883、712和1 201 bp目的基因片段。序列比对分析发现，INSV-草原龙胆云南分离物与INSV-蜘蛛兰云南分离物的N、NSm基因氨基酸序列相似性达100%，与INSV-紫娇花云南分离物的RdRp基因氨基酸序列相似性达99.7%。基于INSV的N、NSm和RdRp基因序列构建系统进化树，发现INSV云南分离物之间的亲缘关系较近。综合试纸条检测、病毒粒体形态观察与分类相关基因的序列比对分析结果，明确感染云南草原龙胆的病毒为INSV。

关键词: 草原龙胆, 凤仙花坏死斑病毒, N基因, NSm基因, RdRp基因, 系统进化树

Abstract:

During the investigation of flower diseases in Dounan Town，Chenggong District，Kunming City，Yunnan Province，Eustoma grandiflorum plants with necrotic spot symptoms on their leaves were discovered. Samples with typical symptoms were collected and detected using immunostrip test strips，and the pathogen was preliminarily identified as Impatiens necrotic spot virus（INSV）. Observation by electron microscopy revealed spherical virus particles with a diameter of 60-110 nm，which aggregated within the endoplasmic reticulum membrane. The chloroplast structure was incomplete，and part of the double membrane had disappeared. To further confirm the pathogen，the specific primers of the INSV for N，NSm and RdRp genes were used RT-PCR amplification，cloning and sequencing. Target gene fragments with lengths of 883，712，and 1 201 bp were obtained，respectively. Sequence alignment analysis showed that the amino acid sequence similarities of the N and NSm genes between the E. grandiflorum isolate of INSV from Yunnan and the Hymenocallis littoralis isolate of INSV from Yunnan reached 100%，and the amino acid sequence similarity of the RdRp gene between the E. grandiflorum isolate of INSV from Yunnan and the Tulbaghia violacea isolate of INSV from Yunnan reached 99.7%. A phylogenetic tree was constructed based on the INSV for N，NSm and RdRp gene sequences，indicating that the INSV isolates from Yunnan plant materials were closely related. By integrating the results of test strip detection，observation of virus particle morphology，and sequence alignment analysis of classification- related genes，it was confirmed that the virus infecting E. grandiflorum in Yunnan was INSV.

Key words: Eustoma grandiflorum, Impatiens necrosis spot virus, N gene, NSm gene, RdRp gene, phylogenetic tree

杨秀梅, 张艺萍, 彭月, 李金泽, 黄望启, 苏艳, 王丽花. 侵染云南草原龙胆的凤仙花坏死斑病毒鉴定[J]. 园艺学报, 2026, 53(1): 275-284.

YANG Xiumei, ZHANG Yiping, PENG Yue, LI Jinze, HUANG Wangqi, SU Yan, WANG Lihua. Identification of Impatiens Necrosis Spot Virus Infecting Eustoma grandiflorum in Yunnan Province[J]. Acta Horticulturae Sinica, 2026, 53(1): 275-284.

图/表 8

表 1 INSV不同基因的扩增引物

Table 1 Primers of different genes of INSV

基因 Gene	引物名称 Primer name	引物序列（5′-3′） Primer sequence
N	INSV-S2016	ATTTAACACAACACAAAGCAAAC
	INSV-S2873	TTTAGTAATTTGAACACTTTAAGC
NSm	INSV-NsM9	CAGTGCATCAAAATTATATCTAGCCG
	INSV-NsM716	CATCTTTCTTGTAAACTTGATCACAT
RdRp	L1	AGAGCAATCAGGCACAACTA
	L866	ATGGTTTTGTTGTTTCTGGT
	L580	CCCGGCAAAAATAGTCAC AT
	L1198	TGATGCTTCTGGCTTCAAAA

图1 感染INSV的草原龙胆植株症状和免疫试纸条检测结果 A：发病初期叶片上的环状坏死斑；B：叶片上的坏死枯斑；C：茎杆上的坏死枯斑；D：叶片发黄、干枯；E：植株枯萎死亡；F：免疫试纸条检测结果

Fig. 1 Symptoms of lisianthus infected by INSV and immune test results A：Necrotic ringspot at early stage of infection；B：Necrotic spots on leaves；C：Necrotic spots on the stem；D：Leaves yellowing and withered；E：Plants withered and dead；F：Immune test results

图2 侵染草原龙胆的INSV病毒粒体及其形态特征 A：病毒粒体；B、C：细胞病变特征。VP：病毒粒体；CW：细胞壁；Chl：叶绿体

Fig. 2 Morphological characteristics of INSV in lisianthus A：Virus particles；B，C：Cytopathic features. VP：Virus particles；CW：Cell wall；Chl：Chloroplast

图3 指示植物接种测定 A：接种INSV的草原龙胆；B：接种INSV的黄花烟；C：接种INSV的一点红

Fig. 3 Indicator plants inoculation A：Eustoma grandiflorum inoculated with INSV；B：Nicotianal rustica inoculated with INSV；C：Emilia sonchifolia inoculated with INSV

图4 草原龙胆样品INSV的N、NSm和RdRp基因扩增产物 A：N基因；B：NSm基因；C：RdRp基因（引物L580/L1198）；D：RdRp基因（引物L1/L866）。 M：DL1000 marker；1 ~ 4：草原龙胆样品；5：阴性对照

Fig. 4 Amplified products of N，NSm and RdRp genes from INSV of lisianthus A：N gene；B：NSm gene；C：RdRp gene（primer：L580/L1198）；D：RdRp gene（primer: L1/L866）. M：DL1000 marker；1-4：Lisianthus samples；5：Negative control

图5 基于INSV N基因构建的系统进化树

Fig. 5 Phylogenetic tree constructed based on the INSV N gene sequences

图6 基于INSV NSm基因构建的系统进化树

Fig. 6 Phylogenetic tree constructed based on the INSV NSm gene sequences

图7 基于INSV RdRp基因构建的系统进化树

Fig. 7 Phylogenetic tree constructed based on the INSV RdRp gene sequences

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