A mutual titer-enhancing relationship and similar localization patterns between Citrus exocortis viroid and Hop stunt viroid co-infecting two citrus cultivars

Abstract

Background: Citrus 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.

Methods: CEVd 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.

Results: The 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.

Conclusions: Our 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.

Fig. 1

Titers of Citrus exocortis viroid (CEVd) and Hop stunt viroid (HSVd) in two citrus cultivars. Comparison of CEVd and HSVd quantities in four tissues of 15 blood oranges and Murcott mandarins by real-time RT-PCR. Copy numbers per microliter of (a) CEVd and (b) HSVd were analyzed in four tissues (rootstock bark, roots, twig bark and leaves) of two citrus cultivars. Data was averaged for nine blood oranges and six Murcott mandarins over 12 spring, summer, fall and winter 3-month periods, except for the data for the last two 3-month periods during which only two blood orange and five Murcott mandarin plants were available. Viroid copy numbers per microliter determined by real-time RT-PCR are displayed as log₁₀ values. Bars represent standard deviation errors

Fig. 2

Seasonal dynamics analysis of Citrus exocortis viroid (CEVd) and Hop stunt viroid (HSVd) in citrus plants. Overview of 3-year population dynamics of CEVd and HSVd in 17 citrus plants from an infected field over 12 spring, summer, fall and winter 3-month periods. The statistical analysis incorporated 0.1 substitutions for under-determined values. The blue and red curves represent log₁₀-transformed copy numbers of CEVd and HSVd, respectively. The blue and red bars indicate standard deviation errors. The green bars correspond to monthly average temperatures (MAT) during each 3-month period

Fig. 3

Comparison of populations of Citrus exocortis viroid (CEVd) and Hop stunt viroid (HSVd) under double- or single-infection conditions over 12 seasons. The doubly infected citrus trees consisted of five blood oranges and four Murcott mandarins. CEVd singly infected citrus trees comprised two blood oranges, and HSVd singly infected citrus trees were represented by two blood oranges and two Murcott mandarins. a CEVd populations under double- and single-infection conditions. b HSVd populations under double- and single-infection conditions. The y-axis corresponds to log₁₀-transformed copy numbers of each viroid and the bars represent standard deviation errors. Statistically significant differences between values were determined by Student’s t-test (*P < 0.05; **P < 0.01; ***P < 0.001)

Fig. 4

Tissue distributions of Citrus exocortis viroid (CEVd) and Hop stunt viroid (HSVd) revealed by digoxigenin-labeled in situ hybridization (DIG-ISH) under a transmitted light microscope. a-d) Viroid-negative citrus tissues hybridized with viroid DIG-labeled probes as controls. e-h) Co-infected citrus tissues hybridized with CEVd DIG-labeled probes. i-l) Co-infected citrus tissues hybridized with HSVd DIG-labeled probes. a, e and i Transverse sections of viroid-negative (a) and co-infected (e and i) citrus roots. b, f and j Transverse sections of viroid-negative (b) and co-infected (f and j) citrus rootstock bark. c, g and k) Transverse sections of viroid-negative (c) and co-infected (g and k) citrus twig bark. d, h and l) Transverse sections of viroid-negative (d) and co-infected (h and l) citrus leaves. Bars in original photographs (a-l) = 5 μM; bars in small photographs (e-l) = 15 μM. PT = pith; P = phloem; X = xylem; E = endodermis; C = cortex; CC = cork cambium; PF = phloem fiber; OG = oil gland; PST = palisade tissue

Fig. 5

Transmission electron micrographs showing subcellular localization of Citrus exocortis viroid (CEVd) and Hop stunt viroid (HSVd) in two citrus cultivars. The locations of CEVd and HSVd in three tissues of blood oranges and Murcott mandarins were detected by in situ hybridization with digoxigenin (DIG)-labeled and biotinylated anti-sense riboprobes, respectively. The locations of CEVd DIG-labeled probes were detected by anti-DIG monoclonal antibody as a primary antibody and a 10-nm diameter colloidal gold conjugate of Alexa Fluor 488 goat anti-rabbit IgG as a secondary antibody. HSVd biotin-labeled probes were detected with 20-nm colloidal streptavidin-gold from Streptomyces avidinii. Ultrathin sections of viroid-negative roots (a), rootstock bark (d) and twig bark (g) hybridized with the same probes revealed neither CEVd nor HSVd signals in the nucleus nor in any other subcellular structures. b Ultrathin sections of mature blood orange roots infected by the two viroids. Most of the probe signals were associated with the nucleus and present near the plasma membrane and cytoplasm. c Ultrathin sections of mature roots of co-infected Murcott mandarin. The viroid signals were associated with the vacuole and cell wall. e Ultrathin sections of rootstock bark of co-infected blood orange. The probes were associated with the nucleoplasm or cytoplasm. f Ultrathin sections of rootstock bark of co-infected Murcott mandarins. The viroid signals were associated with cell walls, cytoplasm and the nucleoplasm. h Ultrathin sections of twig bark of co-infected blood orange. The probes were associated with the cytoplasm and cell walls. i Ultrathin sections of twig bark of co-infected Murcott mandarins. The viroid signals were associated with the cytoplasm. NP = nucleoplasm; V = vacuole; PM = plasma membrane; CP = cytoplasm; CW = cell wall