Distribution of chrysanthemum chlorotic mottle viroid in shoot meristem and flower buds of chrysanthemum

  • Mami Ebata
  • Yosuke Matsushita
  • Masayuki Morimoto
  • Tomofumi MochizukiEmail author


Detailed information on viroid invasion of the shoot apical meristem is important for the efficient production of viroid-free plants by meristem tip culture. Additionally, knowledge on viroid distribution in plant reproductive tissues, especially the embryo, is crucial to assess seed transmission of viroids. In this study, we analyzed histochemically the distribution of chrysanthemum chlorotic mottle viroid (CChMVd) in the shoot meristems and flower buds of CChMVd-infected chrysanthemum plants (cv. ‘Okayama-heiwa’). In situ hybridization revealed that CChMVd invaded the shoot apical meristems and leaf primordia in the shoot meristem at a high frequency (ca. 70%). In flower buds, CChMVd was found in the bract, receptacle vascular, petals, ovules, and immature stamens and pistils. Furthermore, we showed that seed transmission of CChMVd occurred at a high frequency when CChMVd-infected plants were used as female parents (♀). Overall, our findings suggest that CChMVd infection of ovules in flower buds is involved in seed transmission of the viroid.


Chrysanthemum Flower bud In situ hybridization: seed transmission Shoot apical meristem Viroid 


Compliance with ethical standards

Conflict of interest

All the authors declare that they have no conflict of interest.

This article does not contain any studies with human participants or animals performed by any of the authors.


  1. Barba, M., Ragozzino, E., & Faggioli, F. (2007). Pollen transmission of Peach latent mosaic viroid. Journal of Plant Pathology, 89(2), 287–289.Google Scholar
  2. Card, S. D., Pearson, M. N., & Clover, G. R. G. (2007). Plant pathogens transmitted by pollen. Australasian Plant Pathology, 36(5), 455–461.Google Scholar
  3. Cho, W. K., Jo, Y., Jo, K. M., & Kim, K. H. (2013). A current overview of two viroids that infect chrysanthemums: Chrysanthemum stunt viroid and Chrysanthemum chlorotic mottle viroid. Viruses, 5(4), 1099–1113.Google Scholar
  4. Chung, B.-N., & Pak, H.-S. (2008). Seed transmission of Chrysanthemum stunt viroid in Chrysanthemum. Plant Pathology Journal, 24(1), 31–35.Google Scholar
  5. Chung, B.-N., Cho, J. D., Cho, I. S., & Choi, G. S. (2009). Transmission of Chrysanthemum stunt viroid in chrysanthemum by contaminated cutting tool. Horticulture, Environment, and Biotechnology, 50(5), 536–538.Google Scholar
  6. de la Peña, M., & Flores, R. (2002). Chrysanthemum chlorotic mottle viroid RNA: dissection of the pathogenicity determinant and comparative fitness of symptomatic and non-symptomatic variants. Journal of Molecular Biology, 321(3), 411–421.Google Scholar
  7. de la Peña, M., Navarro, B., & Flores, R. (1999). Mapping the molecular determinant of pathogenicity in a hammerhead viroid: a tetraloop within the in vivo branched RNA conformation. Proceedings of the National Academy of Sciences of the United States of America, 96(17), 9960–9965.Google Scholar
  8. Di Serio, F., Martínez de Alba, A. E., Navarro, B., Gisel, A., & Flores, R. (2010). RNA-dependent RNA polymerase 6 delays accumulation and precludes meristem invasion of a viroid that replicates in the nucleus. Journal of Virology, 84(5), 2477–2489.Google Scholar
  9. Di Serio, F., Li, S. F., Matoušek, J., Owens, R. A., Pallás, V., Randles, J. W., Sano, T., Verhoeven, J. T. J., Vidalakis, G., & Flores, R. (2018). ICTV report consortium. ICTV virus taxonomy profile: Avsunviroidae. Journal of General Virology, 99(5), 611–612.Google Scholar
  10. Dimock, A.W. (1947). Chrysanthemum stunt. N. Y. State Flower Grower's Bull. 26th October, 2.Google Scholar
  11. Dimock, A. W., Geissinger, C. M., & Horst, R. K. (1971). Chlorotic mottle: a newly recognized disease of chrysanthemum. Phytopathology, 61(4), 415–419.Google Scholar
  12. Ding, B. (2010). Viroids: self-replicating, mobile, and fast-evolving noncoding regulatory RNAs. Wiley Interdisciplinary Reviews: RNA, 1(3), 362–375.Google Scholar
  13. Ding, B., & Itaya, A. (2007). Viroid: a useful model for studying the basic principles of infection and RNA biology. Molecular Plant-Microbe Interactions, 20(1), 7–20.Google Scholar
  14. Flores, R., Hernández, C., Alba, A. E. M. D., Daròs, J. A., & Serio, F. D. (2005). Viroids and viroid-host interactions. Annual Review of Phytopathology, 43, 117–139.Google Scholar
  15. Flores, R., Gas, M. E., Molina-Serrano, D., Nohales, M. Á., Carbonell, A., Gago, S., De la Peña, M., & Daròs, J. A. (2009). Viroid replication: rolling-circles, enzymes and ribozymes. Viruses, 1(2), 317–334.Google Scholar
  16. Gross, H. J., Krupp, G., Domdey, H., Raba, M., Jank, P., Lossow, C., Alberty, H., Ramm, K., & Sänger, H. L. (1982). Nucleotide sequence and secondary structure of citrus exocortis and chrysanthemum stunt viroid. European Journal of Biochemistry, 121(2), 249–257.Google Scholar
  17. Haseloff, J., & Symons, R. H. (1981). Chrysanthemum stunt viroid: primary sequence and secondary structure. Nucleic Acids Research, 9(12), 2741–2752.Google Scholar
  18. Hollings, M., & Stone, O. M. (1970). Attempts to eliminate chrysanthemum stunt from chrysanthemum by meristem-tip culture after heat-treatment. Annals of Applied Biology, 65(2), 311–315.Google Scholar
  19. Horst, R. K. (1975). Detection of a latent infectious agent that protects against infection by chrysanthemum chlorotic mottle viroid. Phytopathology, 65(9), 1000–1003.Google Scholar
  20. Horst, R. K., & Cohen, D. (1980). Amantadine supplemented tissue culture medium: a method for obtaining chrysanthemum free of chrysanthemum stunt viroid. Acta Horticulturae, 110(1), 315–319.Google Scholar
  21. Hosokawa, M. (2008). Leaf primordia-free shoot apical meristem culture: a new method for production of viroid-free plants. Journal of the Japanese Society for Horticultural Science, 77(4), 341–349.Google Scholar
  22. Hosokawa, M., Otake, A., Ohishi, K., Ueda, E., Hayashi, T., & Yazawa, S. (2004). Elimination of chrysanthemum stunt viroid from an infected chrysanthemum cultivar by shoot regeneration from a leaf primordium-free shoot apical meristem dome attached to a root tip. Plant Cell Reports, 22(11), 859–863.Google Scholar
  23. Hosokawa, M., Matsushita, Y., Ohishi, K., & Yazawa, S. (2005). Elimination of chrysanthemum chlorotic mottle viroid (CChMVd) recently detected in Japan by leaf-primordia free shoot apical meristem culture from infected cultivars. Journal of the Japanese Society for Horticultural Science, 74(5), 386–391.Google Scholar
  24. Johansen, E., Edwards, M. C., & Hampton, R. O. (1994). Seed transmission of viruses: current perspectives. Annual Review of Phytopathology, 32(1), 363–386.Google Scholar
  25. Matsushita, Y. (2011). Distribution of viroid variants and their infectivity in horticultural plants in Japan. Bulletin of the National Institute of Floricultural Science, 11, 9–48.Google Scholar
  26. Matsushita, Y., & Shima, Y. (2015). Effect of low temperature on the distribution of Chrysanthemum stunt viroid in Chrysanthemum morifolium. Phytoparasitica, 43(5), 609–614.Google Scholar
  27. Matsushita, Y., & Tsuda, S. (2014). Distribution of Potato spindle tuber viroid in reproductive organs of petunia during its developmental stages. Phytopathology, 104(9), 964–969.Google Scholar
  28. Matsushita, Y., Usugi, T., & Tsuda, S. (2011). Distribution of tomato chlorotic dwarf viroid in floral organs of tomato. European Journal of Plant Pathology, 130(4), 441–447.Google Scholar
  29. Mink, G. I. (1993). Pollen and seed-transmitted viruses and viroids. Annual Review of Phytopathology, 31(1), 375–402.Google Scholar
  30. Navarro, B., & Flores, R. (1997). Chrysanthemum chlorotic mottle viroid: unusual structural properties of a subgroup of self-cleaving viroids with hammerhead ribozymes. Proceedings of the National Academy of Sciences of the United States of America, 94(21), 11262–11267.Google Scholar
  31. Paduch-Cichal, E., & Kryczyński, S. (1987). A low temperature therapy and meristem-tip culture for eliminating four viroids from infected plants. Journal of Phytopathology, 118(4), 341–346.Google Scholar
  32. Rodio, M. E., Delgado, S., De Stradis, A., Gómez, M. D., Flores, R., & Di Serio, F. (2007). A viroid RNA with a specific structural motif inhibits chloroplast development. The Plant Cell, 19(11), 3610–3626.Google Scholar
  33. Singh, D., & Mathur, S. B. (2004). Histopathology of seed-borne infections. Boca Raton: CRC Press.Google Scholar
  34. Yamamoto, H., & Sano, T. (2005). Occurrence of Chrysanthemum chlorotic mottle viroid in Japan. Journal of General Plant Pathology, 71(2), 156–157.Google Scholar
  35. Yamamoto, H., & Sano, T. (2006). An epidemiological survey of Chrysanthemum chlorotic mottle viroid in Akita Prefecture as a model region in Japan. Journal of General Plant Pathology, 72(6), 387–390.Google Scholar
  36. Zhang, Z., Lee, Y., Spetz, C., Clarke, J. L., Wang, Q., & Blystad, D. R. (2015). Invasion of shoot apical meristems by Chrysanthemum stunt viroid differs among Argyranthemum cultivars. Frontiers in Plant Science, 6, 53.Google Scholar
  37. Zhu, Y., Green, L., Woo, Y. M., Owens, R., & Ding, B. (2001). Cellular basis of potato spindle tuber viroid systemic movement. Virology, 279(1), 69–77.Google Scholar

Copyright information

© Koninklijke Nederlandse Planteziektenkundige Vereniging 2019

Authors and Affiliations

  1. 1.Graduate School of Life and Environmental SciencesOsaka Prefecture UniversityOsakaJapan
  2. 2.Institute of Vegetable and Floriculture ScienceNational Agriculture and Food Research OrganizationIbarakiJapan
  3. 3.Japan Agribio Company LimitedShizuokaJapan

Personalised recommendations