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Development of multiplex viroid rapid detection system for Solanaceae plants and seeds

Fuh-Jyh Jan: Department of Plant Pathology, National Chung Hsing University

<div><em>Solanaceae </em>plants including tomato, potato, eggplant, and tobacco are commercially important crops, widely planting around the world. Viruses and viroids are the obligatory parasitic pathogens which could infect and cause severe yield loss on<em> Solanaceae </em>plants. Viroids are single strand circular RNA which could be transmitted by mechanical operation or seeds. Several viroids which can be transmitted by seeds are important quarantine pathogens in many countries including Taiwan. Six quarantined viroids including <em>Columnea latent viroid </em>(CLVd), <em>Pepper chat fruit viroid </em>(PCFVd), <em>Potato spindle tuber viroid </em>(PSTVd), <em>Tomato apical stunt viroid </em>(TASVd), <em>Tomato chlorotic dwarf viroid </em>(TCDVd), and <em>Tomato planta macho viroid </em>(TPMVd) are the major concerns. The objective of this study was to develop the rapid one step reverse transcription polymerase chain reaction (RT-PCR) and RT- Loop-mediated Isothermal Amplification (LAMP) system for detecting these six viroids simultaneously in seeds and infected plants. The infectious DNA clones of the above-mentioned six viroids were successfully constructed and inoculated into plants. The infectious clones after being inoculated into tomato seedlings showed stunting, stem necrosis, and chlorosis symptoms which were very similar to those inoculated with mechanical means. Degenerate primer sets for one-step RT-PCR and specific primer sets used for RT-LAMP were designed and tested. The primers could simultaneously detect six target viroids. This multiplex system conjunction with modified methods for rapid total RNA extraction can be used for detection of viroids from seeds and field-collected samples.</div>