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Multiplex detection, distribution, and genetic diversity of Hop stunt viroid and Citrus exocortis viroid infecting citrus in Taiwan.

Lin CY, Wu ML, Shen TL, Yeh HH, Hung TH - Virol. J. (2015)

Bottom Line: 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%).Our field survey can help clarify citrus-viroid relationships as well as develop proper prevention strategies.

View Article: PubMed Central - PubMed

Affiliation: Department of Plant Pathology and Microbiology, National Taiwan University, Taipei, 10617, Taiwan. f97633012@ntu.edu.tw.

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.

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Phylogenetic relationships of sequence variants of CEVd and HSVd. The CEVd (A) and HSVd (B) sequence variants were obtained from Taiwan and the representative isolates of the world. Trees were constructed by MEGA 6.0 using the neighbor-joining method with 1000 bootstrap replications. Potato spindle tuber viroid (PSTVd) from the same family Pospiviroidae (KF418767.1) was as outgroup. Bars indicated numbers of nucleotide substitutions per site.
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Fig5: Phylogenetic relationships of sequence variants of CEVd and HSVd. The CEVd (A) and HSVd (B) sequence variants were obtained from Taiwan and the representative isolates of the world. Trees were constructed by MEGA 6.0 using the neighbor-joining method with 1000 bootstrap replications. Potato spindle tuber viroid (PSTVd) from the same family Pospiviroidae (KF418767.1) was as outgroup. Bars indicated numbers of nucleotide substitutions per site.

Mentions: Phylogenetic analyses were conducted on two data sets: nine CEVd sequences generated in this study combined with 17 other worldwide CEVd sequences and eight generated HSVd sequences combined with 18 HSVd sequences from worldwide isolates. As shown in FigureĀ 5, the nine CEVd Taiwanese isolates were distributed among three clusters and the eight HSVd Taiwanese isolates fell into two different groups.Figure 5


Multiplex detection, distribution, and genetic diversity of Hop stunt viroid and Citrus exocortis viroid infecting citrus in Taiwan.

Lin CY, Wu ML, Shen TL, Yeh HH, Hung TH - Virol. J. (2015)

Phylogenetic relationships of sequence variants of CEVd and HSVd. The CEVd (A) and HSVd (B) sequence variants were obtained from Taiwan and the representative isolates of the world. Trees were constructed by MEGA 6.0 using the neighbor-joining method with 1000 bootstrap replications. Potato spindle tuber viroid (PSTVd) from the same family Pospiviroidae (KF418767.1) was as outgroup. Bars indicated numbers of nucleotide substitutions per site.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4340875&req=5

Fig5: Phylogenetic relationships of sequence variants of CEVd and HSVd. The CEVd (A) and HSVd (B) sequence variants were obtained from Taiwan and the representative isolates of the world. Trees were constructed by MEGA 6.0 using the neighbor-joining method with 1000 bootstrap replications. Potato spindle tuber viroid (PSTVd) from the same family Pospiviroidae (KF418767.1) was as outgroup. Bars indicated numbers of nucleotide substitutions per site.
Mentions: Phylogenetic analyses were conducted on two data sets: nine CEVd sequences generated in this study combined with 17 other worldwide CEVd sequences and eight generated HSVd sequences combined with 18 HSVd sequences from worldwide isolates. As shown in FigureĀ 5, the nine CEVd Taiwanese isolates were distributed among three clusters and the eight HSVd Taiwanese isolates fell into two different groups.Figure 5

Bottom Line: 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%).Our field survey can help clarify citrus-viroid relationships as well as develop proper prevention strategies.

View Article: PubMed Central - PubMed

Affiliation: Department of Plant Pathology and Microbiology, National Taiwan University, Taipei, 10617, Taiwan. f97633012@ntu.edu.tw.

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.

Show MeSH
Related in: MedlinePlus