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.
Chrysanthemum (Chrysanthemum x morifolium) belongs to the family Asteraceae, and it is one of the most popular ornamental plants in the world for its wide range of flower colour and structure (Hadizadeh et al., 2021). Chrysanthemum plants are infected by two viroids, the smallest and simplest plant pathogens, consisting of chrysanthemum chlorotic mottle viroid (CChMVd) and chrysanthemum stunt viroid (CSVd) (Cho et al., 2013). Viroids are small (250-400 nt), circular, highly structured RNAs that do not encode proteins and are replicated by nuclear or chloroplastic DNA-dependent RNA polymerases that accept RNA templates of the host plants (Ding & Itaya, 2007). Recently, more than 30 viroids have been identified and classified into two families based on biological, structural, biochemical features and especially, the present of CCR and hammerhead ribozymes that are uniquely
Chrysanthemum is among the world's most important ornamental plants because of its high economic and cultural value. Our report is the first to describe the detection of chrysanthemum virus B (CVB) in chrysanthemum leaf samples collected from Thailand, which showed yellowing and mild mottling symptoms. The coat protein sequences of CVB isolated in this study share 95.15% identity with previously characterised CVB isolates. Biological indexing found that CVB induced both local and systemic symptoms in tobacco plants, while petunia displayed systemic symptoms. To improve the rapidity and sensitivity of CVB detection, the loop-mediated isothermal amplification (LAMP) technique was developed. LAMP detection was found to be optimal when incubation was conducted at 65 °C for 45 min, wherein the LAMP reaction demonstrated 106 times higher sensitivity than polymerase chain reaction. To simplify the interpretation of results, we designed the method such that a positive result is clearly indicated based on a change of colour (colourimetry), from pink to yellow, as observed visually and via gel electrophoresis. To our best knowledge, this is the first report on the characterisation of molecular, biological and morphological characteristics of CVB infecting chrysanthemum in Thailand, along with the development of colourimetric RT-LAMP for improving detection efficiency.
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