| Summary: | 博士 === 國立臺灣大學 === 植物病理與微生物學研究所 === 103 === Two previously reported citrus viroids, Citrus exocortis viroid (CEVd) and Hop stunt viroid (HSVd), represent main threats to the citrus industry in Taiwan. However, studies on these citrus viroids remain largely to be explored. To establish study about the current status of the citrus viroids in Taiwan, we focused on development of multiplex detection, disease ecology of the citrus viroids, and relationship between the two citrus viroids. In multiplex detections, a multiplex RT-PCR and a real-time RT-PCR were developed for detecting of CEVd and HSVd. Our field-survey assay of 689 citrus samples in Taiwan revealed that HSVd was slightly more prevalent than CEVd (32.2% vs. 30.4%). Furthermore, CEVd and HSVd commonly co-existed within the citrus cultivars (up to 55%). Results of the multiplex quantitative analysis suggest uneven distributions of both viroids within different plant tissues, of which twig bark appears to be the most appropriate and reliable material for viroid detections, thereby being used for quarantine inspections. The genetic diversity assay appeared the existence of 10 and five major mutation sites in CEVd and HSVd in Taiwan, respectively. A phylogenetic analysis revealed that Taiwanese isolates of CEVd and HSVd were grouped in three and two clusters, respectively. To obtain single isolate of each viroid for disease ecology studies, different forms of infectious clones of CEVd and HSVd were established and successful inoculated to indicator plants, includings ‘Etrog citron’ Arizona 861-S (C. medica), Gynura aurantiaca, Solanum lycopersicum ‘Rutgers’ and Cucumis sativus, along with sensitive citrus rootstock of Rangpur lime (Citrus × limonia Osb.). To facilitate the availability and application of indicator plants for viroid detection, a parallel effort was directed toward developing alternative indicator plants for CEVd detection. Toward this end, 29 tomato commercial cultivars were screened by inoculations with two Taiwanese isolates of CEVd’s infectious RNAs. Only two tomato cultivars, Honey tomato (Xiaomi, ‘HV-206’) and Cherry tomato (‘V-191’), showed leaf distortion, and necrotic or green-island symptoms. Higher titers of both CEVd isolates also indicate that ‘V-191’ is a more susceptible and suitable indicator for CEVd detection. In disease transmission studies, CEVd and HSVd were detected only rarely on seed coats, seed membranes and inner seeds obtained from blood orange, Murcott mandarin, and lemon. The seedlings originating from infected seeds did not present detectable titers for the two viroids, suggesting that these two viroids do not exhibit seed-borne transmission in citrus hosts. In addition, two viroids showed similar patterns when invading shoot apical meristem (SAM) of tomato and citrus as shown by DIG-labeled in situ hybridization. The two viroids displayed the ability to invade into SAM and leaf primordia of citrus but only leaf primordia of tomato. The study provided useful information for improving of the experimental protocols designed for obtaining viroid-free meristem tissues for control of viroid diseases. For interactive studies, titers of the two viroids were examined by real-time RT-PCR in 17 citrus plants (including blood orange and Murcott mandarin) every 3 months (spring, summer, fall and winter) from 2011 to 2013. No correlation was found between temperature and titer of each viroid, except with HSVd-infected blood orange. This result suggests that temperature is likely not a critical environmental factor during the life cycles of the two viroids. Based on the results of real-time RT-PCR, statistical methods, significant positive correlations, the two viroids appeared in specific tissues of both orange cultivars, except for blood orange at high temperatures. At the cellular/subcellular levels, the two viroids showed similar localization patterns in four tissues and cells, as shown by in situ hybridization, fluorescence microscopy, and transmission electron microscopy. Our results demonstrate that the two viroids have a positive relationship and a similar infection pattern, while displaying titer enhancement and localization similarity with no symptom aggravation.
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