title: Multiplex detection, distribution, and genetic diversity of Hop stunt viroid and Citrus exocortis viroid infecting citrus in Taiwan.
abstract: BACKGROUND
  Two citrus viroids, Citrus exocortis viroid (CEVd) and Hop stunt viroid (HSVd), have been reported and become potential threats to the citrus industry in Taiwan. The distributions and infection rates of two viroids have not been investigated since the two diseases were presented decades ago. The genetic diversities and evolutionary relationships of two viroids also remain unclear in the mix citrus planted region.
  
  
  METHODS
  Multiplex RT-PCR was used to detect the two viroids for the first time in seven main cultivars of citrus. Multiplex real-time RT-PCR quantified the distributions of two viroids in four citrus tissues. Sequence alignment and phylogenetic analysis were performed using the ClustalW and MEGA6 (neighbor-joining with p-distance model), respectively.
  
  
  RESULTS
  HSVd was found more prevalent than CEVd (32.2% vs. 30.4%). Both CEVd and HSVd were commonly found simultaneously in the different citrus cultivars (up to 55%). Results of the multiplex quantitative analysis suggested that uneven distributions of both viroids with twig bark as the most appropriate material for studies involving viroid sampling such as quarantine inspection. Sequence alignment against Taiwanese isolates, along with analysis of secondary structure, revealed the existence of 10 and 5 major mutation sites in CEVd and HSVd, respectively. The mutation sites in CEVd were located at both ends of terminal and variability domains, whereas those in HSVd were situated in left terminal and pathogenicity domains. A phylogenetic analysis incorporating worldwide viroid isolates indicated three and two clusters for the Taiwanese isolates of CEVd and HSVd, respectively.
  
  
  CONCLUSIONS
  Moderately high infection and co-infection rates of two viroids in certain citrus cultivars suggest that different citrus cultivars may play important roles in viroid infection and evolution. These data also demonstrate that two multiplex molecular detection methods developed in the present study provide powerful tools to understand the genetic diversities among viroid isolates and quantify viroids in citrus host. Our field survey can help clarify citrus-viroid relationships as well as develop proper prevention strategies.
authors: Department of Plant Pathology and Microbiology, National Taiwan University, Taipei, 10617, Taiwan. f97633012@ntu.edu.tw.
authors: Chun-Yi
authors: CY
authors: Lin
authors: Division of Forest Protection, Taiwan Forestry Research Institute, Taipei, 10066, Taiwan. mlw@tfri.gov.tw.
authors: Meng-Ling
authors: ML
authors: Wu
authors: Department of Plant Pathology and Microbiology, National Taiwan University, Taipei, 10617, Taiwan. shentl@ntu.edu.tw.
authors: Tang-Long
authors: TL
authors: Shen
authors: Agricultural Biotechnology Research Center, Academia Sinica, Taipei, 11529, Taiwan. hyeh@sinica.edu.tw.
authors: Hsin-Hung
authors: HH
authors: Yeh
authors: Department of Plant Pathology and Microbiology, National Taiwan University, Taipei, 10617, Taiwan. thhung@ntu.edu.tw.
authors: Ting-Hsuan
authors: TH
authors: Hung
mesh_terms: D002957:Citrus / Q000821:virology*; D014644:Genetic Variation; D005838:Genotype; D008969:Molecular Sequence Data; D060885:Multiplex Polymerase Chain Reaction; D010802:Phylogeny; D010935:Plant Diseases / Q000821:virology*; D060888:Real-Time Polymerase Chain Reaction; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017422:Sequence Analysis, DNA; D017385:Sequence Homology; D013624:Taiwan; D014772:Viroids / Q000145:classification* / Q000235:genetics / Q000302:isolation & purification*
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