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.

Athelia rolfsii is one of the most destructive and aggressive fungal pathogens worldwide and causes southern blight disease of lettuce. A nonthermal atmospheric-pressure dielectric barrier discharge (DBD) plasma has attracted interest as an alternative control method to chemical usage because of its antimicrobial activity. Exposure of A. rolfsii to DBD plasma for 5, 10, 15, and 20 min resulted in in vitro fungal inhibition of mycelial discs and sclerotia. The results showed that DBD plasma exposure for 10 min completely inhibited fungal growth of mycelial discs, whereas exposure for over 20 min was required to inhibit the hyphal growth of sclerotia. Scanning electron microscopy (SEM) observations of mycelia and sclerotia abnormalities revealed laceration and damage of both mycelia and sclerotia. In addition, disease incidence and severity were reduced in mycelial and sclerotia inoculation following DBD plasma exposure for 15 and 20 min, respectively, compared with the positive control. In conclusion, the DBD plasma demonstrates antifungal activity against A. rolfsii via inhibition of fungal growth and reduction in disease incidence and severity. Therefore, DBD plasma has the potential to be applied in controlling southern blight disease of lettuce.

Cercospora leaf spot disease is a serious problem for lettuce cultivation worldwide. Cercospora lactucae-sativa, the causative agent of leaf spot disease on lettuce, was treated with non-thermal atmospheric pressure gliding arc (GA), tesla coil (TC) and dielectric barrier discharge (DBD) plasmas for the in vitro fungal inactivation of both mycelial growth and conidial germination. The fungus was exposed to the three plasmas individually for 5, 10, 15 and 20 min. The results showed that DBD plasma inactivated fungal growth during all exposure periods, and the highest inhibitory effect was caused by exposure to DBD plasma for 20 min, at 93.33% inhibition. The germination of fungal conidia was completely inactivated after exposure to all three non-thermal plasmas for 5 min, as observed 4 and 24 h after incubation. The pathogenesis of C. lactucae-sativa on lettuce after plasma treatments for 5 min was examined by spraying an inoculation of the treated conidia on lettuce. The results showed that all three plasmas reduced the disease incidence and severity compared to the non-treated control. Therefore, the non-thermal atmospheric pressure GA, TC and DBD plasmas have antifungal potential for the inactivation of C. lactucae-sativa, making them an interesting novel technology for plant disease control.

Chrysanthemum stunt viroid (CSVd) is a serious pathogen infecting chrysanthemum worldwide. To improve and enhance the detection procedure, a colorimetric loop-mediated isothermal amplification (LAMP) technique was developed. Six LAMP primers were newly designed and tested to determine the optimal conditions using a recombinant plasmid of CSVd as a DNA template. The optimal conditions for colorimetric LAMP were incubation at 65°C for 45 min. Under these conditions, a ladder-like pattern of LAMP products was detected along with a change of color from pink to yellow in the positive reactions. Limits of the detection (LOD) of colorimetric LAMP were up to 1 fg • µl-1 of plasmid DNA concentration which was 10 4 times greater than that of polymerase chain reaction (PCR). The developed colorimetric LAMP was not cross reacted to other viruses and viroids. From detection of actual samples and chrysanthemum plantlets which were obtained from meristem tip culture, the colorimetric LAMP showed effective potential in detecting CSVd. Therefore, the colorimetric LAMP can be used as a main technique to detect CSVd and ensure CSVd-free chrysanthemum plantlet production due to its accuracy, rapidness and sensitivity.

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 10 ⁶ 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.