Supakitthanakorn Salit, Vichittragoontavorn Kanjana, Sunpapao Anurag, Kunasakdakul Kaewalin, Thapanapongworakul Pilunthana, Ruangwong On-Uma
Division of Plant Pathology, Department of Entomology and Plant Pathology, Faculty of Agriculture, Chiang Mai University, Chiang Mai 50200, Thailand.
Plant Protection Center, Royal Project Foundation, Chiang Mai 50200, Thailand.
Plants (Basel). 2022 Jul 6;11(14):1788. doi: 10.3390/plants11141788.
We detected tobacco mosaic virus (TMV), a member of the genus and one of the most significant plant-infecting viruses, for the first time in a chrysanthemum in Thailand using reverse-transcription polymerase chain reaction (RT-PCR). The TMV-infected chrysanthemum leaves exhibited mosaic symptoms. We conducted a sequence analysis of the coat protein () gene and found that the TMV detected in the chrysanthemum had 98% identity with other TMV isolates in GenBank. We carried out bioassays and showed that TMV induced mosaic and stunting symptoms in inoculated chrysanthemums. We observed the rigid rod structure of TMV under a transmission electron microscope (TEM). To enhance the speed and sensitivity of detection, we developed a colorimetric RT loop-mediated isothermal amplification (LAMP) technique. We achieved LAMP detection after 30 min incubation in isothermal conditions at 65 °C, and distinguished the positive results according to the color change from pink to yellow. The sensitivity of the LAMP technique was 1000-fold greater than that of RT-PCR, and we found no cross-reactivity with other viruses or viroids. This is the first reported case of a TMV-infected chrysanthemum in Thailand, and our colorimetric RT-LAMP TMV detection method is the first of its kind.
我们首次在泰国的一株菊花中，使用逆转录聚合酶链反应（RT-PCR）检测到烟草花叶病毒（TMV），它是烟草花叶病毒属的成员之一，也是最重要的植物感染病毒之一。被TMV感染的菊花叶片呈现出花叶症状。我们对衣壳蛋白（CP）基因进行了序列分析，发现从菊花中检测到的TMV与GenBank中其他TMV分离株有98%的同源性。我们进行了生物测定，结果表明TMV对接种的菊花会引发花叶和发育迟缓症状。我们在透射电子显微镜（TEM）下观察到了TMV的刚性杆状结构。为提高检测速度和灵敏度，我们开发了一种比色逆转录环介导等温扩增（LAMP）技术。在65℃等温条件下孵育30分钟后实现了LAMP检测，并根据颜色从粉红色变为黄色来区分阳性结果。LAMP技术的灵敏度比RT-PCR高1000倍，且我们未发现其与其他病毒或类病毒有交叉反应。这是泰国首次报道的菊花感染TMV病例，我们的比色逆转录LAMP TMV检测方法也是首例此类方法。
