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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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Related in: MedlinePlus

Locations and changes of sequence variations were founded in CEVd and HSVd Taiwanese isolates. CEVd type species (A)(GenBank Acc. No. KC290927) and HSVd type species (B) (GenBank Acc. No. KC290929) were as structure models. 17 Taiwanese CEVd isolates and 13 HSVd isolates on secondary structure were compared to each structure models. Red color nucleotides meant that the changes were found in more than three variants, whereas black color nucleotides were detected in less than two.
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Fig4: Locations and changes of sequence variations were founded in CEVd and HSVd Taiwanese isolates. CEVd type species (A)(GenBank Acc. No. KC290927) and HSVd type species (B) (GenBank Acc. No. KC290929) were as structure models. 17 Taiwanese CEVd isolates and 13 HSVd isolates on secondary structure were compared to each structure models. Red color nucleotides meant that the changes were found in more than three variants, whereas black color nucleotides were detected in less than two.

Mentions: Alignment of the nine CEVd sequences and additional non-submitted sequences against the genome sequence of the CEVd Taiwanese type isolate (KC290927) uncovered 35 variable sites. Similarly, alignment of the nine generated HSVd sequences against the genome sequence of the HSVd type isolate (KC290929) revealed 20 mutation sites. As shown in FigureĀ 4, Taiwanese CEVd populations contained 10 sites at which mutations (relative to the type specimen) were present in most sequences, with high overall sequence homology observed. The mutation sites existed at both ends of terminal and variability domains of the CEVd secondary structure. In addition, Taiwanese HSVd populations possessed five sites frequently undergoing point mutations or sequence insertions/deletions (indels) and exhibited high overall sequence homology. The mutation and indel sites were present on the left terminal and pathogenicity domains of the HSVd secondary structure.Figure 4


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)

Locations and changes of sequence variations were founded in CEVd and HSVd Taiwanese isolates. CEVd type species (A)(GenBank Acc. No. KC290927) and HSVd type species (B) (GenBank Acc. No. KC290929) were as structure models. 17 Taiwanese CEVd isolates and 13 HSVd isolates on secondary structure were compared to each structure models. Red color nucleotides meant that the changes were found in more than three variants, whereas black color nucleotides were detected in less than two.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Fig4: Locations and changes of sequence variations were founded in CEVd and HSVd Taiwanese isolates. CEVd type species (A)(GenBank Acc. No. KC290927) and HSVd type species (B) (GenBank Acc. No. KC290929) were as structure models. 17 Taiwanese CEVd isolates and 13 HSVd isolates on secondary structure were compared to each structure models. Red color nucleotides meant that the changes were found in more than three variants, whereas black color nucleotides were detected in less than two.
Mentions: Alignment of the nine CEVd sequences and additional non-submitted sequences against the genome sequence of the CEVd Taiwanese type isolate (KC290927) uncovered 35 variable sites. Similarly, alignment of the nine generated HSVd sequences against the genome sequence of the HSVd type isolate (KC290929) revealed 20 mutation sites. As shown in FigureĀ 4, Taiwanese CEVd populations contained 10 sites at which mutations (relative to the type specimen) were present in most sequences, with high overall sequence homology observed. The mutation sites existed at both ends of terminal and variability domains of the CEVd secondary structure. In addition, Taiwanese HSVd populations possessed five sites frequently undergoing point mutations or sequence insertions/deletions (indels) and exhibited high overall sequence homology. The mutation and indel sites were present on the left terminal and pathogenicity domains of the HSVd secondary structure.Figure 4

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