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从云南保山采集具典型花叶症状的香料烟病株,用病株汁液接种心叶烟,进行单斑分离纯化。电子显微镜观察到杆状病毒粒体(18nm×300nm),三抗体夹心酶联免疫吸附(TAS-ELISA)检测与烟草花叶病毒(Tobacco mosaic virus,TMV)抗血清呈阳性反应;用TMV外壳蛋白(CP)基因特异性引物进行RT-PCR扩增,克隆,经序列测定分析,侵染云南保山香料烟的病原分离物CP基因含480个核苷酸,编码159个氨基酸,通过核苷酸和氨基酸序列分析,与TMV-152的相似性最高,核苷酸序列的相似性为98.8%,推导编码氨基酸相似性达到100.0%,亲缘关系最近。根据这些特性,引起云南保山香料烟花叶病的病原分离物是烟草花叶病毒的一个分离物。
From the Baoshan in Yunnan Province, the typical tobacco plant with the typical mosaic symptoms was collected and the leaf tobacco was inoculated with the diseased plantlets for single spot separation and purification. The baculoviruses (18 nm × 300 nm) were observed by electron microscopy. The positive antibodies were detected by TAS-ELISA and TMV anti-sera. The TMV coat protein (CP) gene-specific primers were amplified by RT-PCR, cloned, sequenced and analyzed. The CP gene of pathogen isolate that infected Yunnan Baoshan fragrant tobacco contains 480 nucleotides and encodes 159 amino acids. Amino acid sequence analysis showed the highest similarity with TMV-152, the similarity of nucleotide sequence was 98.8%, the deduced amino acid similarity was 100.0%, and the closest genetic relationship was found. According to these characteristics, the isolate of the pathogen causing Tobacco mosaic leaf disease in Baoshan, Yunnan is an isolate of tobacco mosaic virus.