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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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Analysis of the viroids distribution in different citrus tissues by RT-PCR and qPCR. For RT-PCR detection, CEVd-F194/R18 primer was used to detect CEVd from five citrus tissues. (A) Murcott oranges from Hsinchu; (B) Blood oranges from Yunlin; (C) Kumquat from Yilan; (D) Tankan from Taichung; (E) Lemon from Pingtung. Lane 1, exocortis symptom on rootstock; 2, root; 3, twig bark; 4, leaf; 5, healthy control; M, 100-bp molecular marker. (F) The quantification data of qPCR were collected among winter, 2011 to autumn, 2013 and the copy numbers of two viroids were measured by log10. The collected data of fruit were discontinuous in winter, 2011 and autumn, 2012. The collected data of leaf were only from one citrus. The bars represented the standard deviation errors.
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Fig3: Analysis of the viroids distribution in different citrus tissues by RT-PCR and qPCR. For RT-PCR detection, CEVd-F194/R18 primer was used to detect CEVd from five citrus tissues. (A) Murcott oranges from Hsinchu; (B) Blood oranges from Yunlin; (C) Kumquat from Yilan; (D) Tankan from Taichung; (E) Lemon from Pingtung. Lane 1, exocortis symptom on rootstock; 2, root; 3, twig bark; 4, leaf; 5, healthy control; M, 100-bp molecular marker. (F) The quantification data of qPCR were collected among winter, 2011 to autumn, 2013 and the copy numbers of two viroids were measured by log10. The collected data of fruit were discontinuous in winter, 2011 and autumn, 2012. The collected data of leaf were only from one citrus. The bars represented the standard deviation errors.

Mentions: Using multiplex RT-PCR, we analyzed the distributions of the two viroids in four citrus plant tissues. In all five citrus cultivars tested, root tissues had the highest concentrations of viroids, while leaf tissues displayed no viroid detection signals (Figure 3A-E). Quantification of the data revealed that the two viroids were similar in abundance and that both were unevenly distributed across the different citrus tissues (Figure 3F). As inferred from the log10-modified copy-number data, rootstocks and roots were the two regions most heavily infected by CEVd and HSVd. Leaf and fruit tissues were not included in this analysis because most of the obtained data were derived from samples in which viroids were not detected or by discontinuous sampling; consequently, most Ct values for these samples could not be determined by real-time RT-PCR.Figure 3


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)

Analysis of the viroids distribution in different citrus tissues by RT-PCR and qPCR. For RT-PCR detection, CEVd-F194/R18 primer was used to detect CEVd from five citrus tissues. (A) Murcott oranges from Hsinchu; (B) Blood oranges from Yunlin; (C) Kumquat from Yilan; (D) Tankan from Taichung; (E) Lemon from Pingtung. Lane 1, exocortis symptom on rootstock; 2, root; 3, twig bark; 4, leaf; 5, healthy control; M, 100-bp molecular marker. (F) The quantification data of qPCR were collected among winter, 2011 to autumn, 2013 and the copy numbers of two viroids were measured by log10. The collected data of fruit were discontinuous in winter, 2011 and autumn, 2012. The collected data of leaf were only from one citrus. The bars represented the standard deviation errors.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Fig3: Analysis of the viroids distribution in different citrus tissues by RT-PCR and qPCR. For RT-PCR detection, CEVd-F194/R18 primer was used to detect CEVd from five citrus tissues. (A) Murcott oranges from Hsinchu; (B) Blood oranges from Yunlin; (C) Kumquat from Yilan; (D) Tankan from Taichung; (E) Lemon from Pingtung. Lane 1, exocortis symptom on rootstock; 2, root; 3, twig bark; 4, leaf; 5, healthy control; M, 100-bp molecular marker. (F) The quantification data of qPCR were collected among winter, 2011 to autumn, 2013 and the copy numbers of two viroids were measured by log10. The collected data of fruit were discontinuous in winter, 2011 and autumn, 2012. The collected data of leaf were only from one citrus. The bars represented the standard deviation errors.
Mentions: Using multiplex RT-PCR, we analyzed the distributions of the two viroids in four citrus plant tissues. In all five citrus cultivars tested, root tissues had the highest concentrations of viroids, while leaf tissues displayed no viroid detection signals (Figure 3A-E). Quantification of the data revealed that the two viroids were similar in abundance and that both were unevenly distributed across the different citrus tissues (Figure 3F). As inferred from the log10-modified copy-number data, rootstocks and roots were the two regions most heavily infected by CEVd and HSVd. Leaf and fruit tissues were not included in this analysis because most of the obtained data were derived from samples in which viroids were not detected or by discontinuous sampling; consequently, most Ct values for these samples could not be determined by real-time RT-PCR.Figure 3

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