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.

Figure 1

Sensitivity of bioassay and RT-PCR methods for indicator plants infected by CEVd and HSVd. Indicator plants as Etrog citron Arizona 861-S and Gynura aurantiaca were infected by CEVd; Cucumis sativus was infected by HSVd. Viroids resources were collected from infected field and were inoculated on indicator plants. (A) Viroids infected citrus tree in the field which showed typical symptoms such as exocortis and stunting compared to (B) Healthy citrus tree as control in the field. (C) Infected leaves showed symptoms such as epinasty and curling at 3 months after grafting with CEVd-infected buds. (D) No symptoms were shown on infected leaves at 1 month after grafting with CEVd-infected buds. (E) Healthy citron was a negative control. (F) Infected leaves showed epinasty at 3 weeks after mechanical inoculation with CEVd-infected sap. (G) Healthy G. aurantiaca was a negative control. (H, I) Infected C. sativus showed stunting after mechanical inoculation with HSVd-infected sap. (J) Healthy C. sativus was a negative control. (K) RT-PCR detection of CEVd and HSVd RNAs in infected indicator plants. M, 100-bp molecular marker; each lane represents the plants which described above.

Figure 2

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.

Figure 3

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.

Figure 4

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.

Figure 5

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.

Figure 6

The map showing different citrus cultivars regions used for field survey in Taiwan. Samples were collected from Hsinchu, Taichung, Yunlin, Chiayi, Tainan, Pingtung, Yilan and Hualien counties, which contains seven citrus cultivars of citrus production.