Tobacco Mosaic Virus Infection of Chrysanthemums in Thailand: Development of Colorimetric Reverse-Transcription Loop-Mediated Isothermal Amplification (RT–LAMP) Technique for Sensitive and Rapid Detection

Abstract

We detected tobacco mosaic virus (TMV), a member of the genus Tobamovirus 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 (CP) 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.