Abstract
BackgroundCitrus exocortis viroid (CEVd) and Hop stunt viroid (HSVd) are commonly found simultaneously infecting different citrus cultivars in Taiwan. A crucial question to be addressed is how accumulations of these two viroids affect each other in an infected plant. In this study, we investigated the relationship between the two viroids at macroscopic and microscopic levels.MethodsCEVd and HSVd titers were examined by real-time RT-PCR in 17 plants of two citrus cultivars (blood orange and Murcott mandarin) every 3 months (spring, summer, fall and winter) from 2011 to 2013. Three nonparametric tests (Spearman’s rank correlation coefficient, Kendall’s tau rank correlation coefficient and Hoeffding’s inequality) were performed to test the correlation between CEVd and HSVd. Cellular and subcellular localizations of the two viroids were detected by digoxigenin- and colloidal gold-labeled in situ hybridization using light and transmission electron microscopy.ResultsThe two viroids were unevenly distributed in four different types of citrus tissues (rootstock bark, roots, twig bark and leaves). Compared with blood orange, Murcott mandarin was generally more susceptible to CEVd and HSVd infection. Both viroids replicated and preferentially accumulated in the underground tissues of the two citrus cultivars. Except for blood orange at high temperatures, significant positive correlations were observed between the two viroids in specific tissues of both cultivars. Relative to concentrations under single-infection conditions, the CEVd population significantly increased under double infection during half of the 12 monitored seasons; in contrast, the population of HSVd significantly increased under double infection during only one season. At cellular/subcellular levels, the two viroids showed similar localization patterns in four tissues and the cells of these tissues in the two citrus cultivars.ConclusionsOur findings of titer enhancement, localization similarity, and lack of symptom aggravation under CEVd and HSVd double infection suggest that the two viroids have a positive relationship in citrus. The combination of molecular and cellular techniques used in this study provided evidence of titer correlation and localization of co-infecting viroids in the host. These methods may thus be useful tools for exploring viroid–viroid and viroid–host interactions.
Highlights
Citrus exocortis viroid (CEVd) and Hop stunt viroid (HSVd) are commonly found simultaneously infecting different citrus cultivars in Taiwan
Distribution of viroids in four tissues of the two citrus cultivars During our 3-year survey, we observed the following distributions of the two viroids in 15 citrus trees: double infection of five blood oranges and four Murcott mandarins, CEVd single infection of two blood oranges, and HSVd single infection of two blood oranges and two Murcott mandarins
Murcott mandarin had higher HSVd titers in roots, rootstock bark and twig bark (103.4, 103.7 and 102.4 RNA copies/μL, respectively) than did blood orange (101.9, 102 and 101.7 RNA copies/μL, respectively) (Fig. 1). These data indicate that Murcott mandarin is generally more susceptible to CEVd and HSVd infection relative to blood orange
Summary
Citrus exocortis viroid (CEVd) and Hop stunt viroid (HSVd) are commonly found simultaneously infecting different citrus cultivars in Taiwan. Mixed virus/viroid and viroid/viroid infections are common in field-grown plants, but only a few studies have addressed this phenomenon. A possible mechanism for virus/viroid interaction was uncovered in a study using viral-encoded silencing suppressors, where a titer of Citrus dwarfing viroid (CDVd) was enhanced by Citrus tristeza virus (CTV) in Mexican lime but not in Etrog citron. Interactions between these pathogens differed among host plant cultivars, indicating that such interactions are likely dictated by the host [6, 7]. Co-infection by the two viroids does not cause severe symptoms in citrus, their interaction is intriguing because of their high co-infection rate in the field and their identical biological properties in the same host
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