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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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Establishment of multiplex real-time RT-PCR and one-step multiplex RT-PCR in detecting of CEVd and HSVd. (A) Standard curves of CEVd and HSVd for absolute quantification obtained by plotting Ct values versus actual pTOPO-CEVd and pTOPO-HSVd copy number. The Ct values for each dilution are the means of three replicates. (B) One-step multiplexe RT-PCR detected CEVd and HSVd transcripts alone and in combination. Samples A and B were collected from the field. Lane 1, viroids co-infection citrus sample; 2,3, unknown samples in the field; 4, Mixed 100 ng/μL RNA transcripts of plasmids of pTOPO-CEVd and pTOPO-HSVd; 5, 100 ng/μL RNA transcript of plasmid pTOPO-CEVd; 6, 100 ng/μL RNA transcript of plasmid pTOPO-HSVd; 7, healthy control; 8, water control; M, 100-bp molecular marker.
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Fig2: Establishment of multiplex real-time RT-PCR and one-step multiplex RT-PCR in detecting of CEVd and HSVd. (A) Standard curves of CEVd and HSVd for absolute quantification obtained by plotting Ct values versus actual pTOPO-CEVd and pTOPO-HSVd copy number. The Ct values for each dilution are the means of three replicates. (B) One-step multiplexe RT-PCR detected CEVd and HSVd transcripts alone and in combination. Samples A and B were collected from the field. Lane 1, viroids co-infection citrus sample; 2,3, unknown samples in the field; 4, Mixed 100 ng/μL RNA transcripts of plasmids of pTOPO-CEVd and pTOPO-HSVd; 5, 100 ng/μL RNA transcript of plasmid pTOPO-CEVd; 6, 100 ng/μL RNA transcript of plasmid pTOPO-HSVd; 7, healthy control; 8, water control; M, 100-bp molecular marker.

Mentions: Typical symptoms of stunting and exocortis (Figure 1A) were first confirmed on citrus trees by bioassays. Three weeks after inoculation or grafting onto indicator plants, the symptoms were obvious on the main indicator plants: Etrog citron Arizona 861-S (Figure 1C) and Gynura aurantiaca (Figure 1F) for CEVd infection and Cucumis sativus for HSVd infection (Figure 1H and I). However, positive results were obtained using molecular methods at 2 weeks after inoculation, when the exterior plants still showed no symptoms (Figure 1D). According to our field observations and bioassay surveys, the two viroids existed throughout the citrus field. The development of a multiplex method that could simultaneously detect the two viroids was therefore necessary. As shown in Figure 2A, at a concentration ratio of 1:1 for the two primer sets, CEVd F194/R18 primers worked perfectly with HSVd F1/R1 primers [16] for the detection of either single or double targets. To further enhance the ability to detect early infection and to quantify viroids in citrus, we developed a real-time RT-PCR protocol for CEVd and HSVd. Samples infected by CEVd and HSVd from Yunlin County, Taiwan as well as indicator plants were analyzed and quantified by real-time RT-PCR. Ten aliquots (Figure 2B) of each viroid corresponding to 10 serial dilution concentrations were used as samples for standard curve quantification. The resulting data, such as a correlation of efficiency of 0.996-0.999 and a high amplification efficiency (92–99%), demonstrated that the standard curves were reliable for the calculation of copy numbers of unknown samples.Figure 1


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)

Establishment of multiplex real-time RT-PCR and one-step multiplex RT-PCR in detecting of CEVd and HSVd. (A) Standard curves of CEVd and HSVd for absolute quantification obtained by plotting Ct values versus actual pTOPO-CEVd and pTOPO-HSVd copy number. The Ct values for each dilution are the means of three replicates. (B) One-step multiplexe RT-PCR detected CEVd and HSVd transcripts alone and in combination. Samples A and B were collected from the field. Lane 1, viroids co-infection citrus sample; 2,3, unknown samples in the field; 4, Mixed 100 ng/μL RNA transcripts of plasmids of pTOPO-CEVd and pTOPO-HSVd; 5, 100 ng/μL RNA transcript of plasmid pTOPO-CEVd; 6, 100 ng/μL RNA transcript of plasmid pTOPO-HSVd; 7, healthy control; 8, water control; M, 100-bp molecular marker.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Fig2: Establishment of multiplex real-time RT-PCR and one-step multiplex RT-PCR in detecting of CEVd and HSVd. (A) Standard curves of CEVd and HSVd for absolute quantification obtained by plotting Ct values versus actual pTOPO-CEVd and pTOPO-HSVd copy number. The Ct values for each dilution are the means of three replicates. (B) One-step multiplexe RT-PCR detected CEVd and HSVd transcripts alone and in combination. Samples A and B were collected from the field. Lane 1, viroids co-infection citrus sample; 2,3, unknown samples in the field; 4, Mixed 100 ng/μL RNA transcripts of plasmids of pTOPO-CEVd and pTOPO-HSVd; 5, 100 ng/μL RNA transcript of plasmid pTOPO-CEVd; 6, 100 ng/μL RNA transcript of plasmid pTOPO-HSVd; 7, healthy control; 8, water control; M, 100-bp molecular marker.
Mentions: Typical symptoms of stunting and exocortis (Figure 1A) were first confirmed on citrus trees by bioassays. Three weeks after inoculation or grafting onto indicator plants, the symptoms were obvious on the main indicator plants: Etrog citron Arizona 861-S (Figure 1C) and Gynura aurantiaca (Figure 1F) for CEVd infection and Cucumis sativus for HSVd infection (Figure 1H and I). However, positive results were obtained using molecular methods at 2 weeks after inoculation, when the exterior plants still showed no symptoms (Figure 1D). According to our field observations and bioassay surveys, the two viroids existed throughout the citrus field. The development of a multiplex method that could simultaneously detect the two viroids was therefore necessary. As shown in Figure 2A, at a concentration ratio of 1:1 for the two primer sets, CEVd F194/R18 primers worked perfectly with HSVd F1/R1 primers [16] for the detection of either single or double targets. To further enhance the ability to detect early infection and to quantify viroids in citrus, we developed a real-time RT-PCR protocol for CEVd and HSVd. Samples infected by CEVd and HSVd from Yunlin County, Taiwan as well as indicator plants were analyzed and quantified by real-time RT-PCR. Ten aliquots (Figure 2B) of each viroid corresponding to 10 serial dilution concentrations were used as samples for standard curve quantification. The resulting data, such as a correlation of efficiency of 0.996-0.999 and a high amplification efficiency (92–99%), demonstrated that the standard curves were reliable for the calculation of copy numbers of unknown samples.Figure 1

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