Nucleotide Sequence and Structural Features of Hop Stunt Viroid and Citrus Bent Leaf Viroid Variants from Blighted Citrus Plants in Kohgiluyeh–Boyerahmad Province of Iran

Authors
M. Amiri Mazhar 1
S. A. A. Bagherian 1
A. Salahi Ardakani 2
K. Izadpanah 3
1 Plant Virology Research Center, College of Agriculture, Shiraz University, Shiraz, Islamic Republic of Iran.
2 Department of Horticulture, Faculty of Agricultural Science, Jahrom University, Islamic Republic of Iran.
3 Agricultural Research Center, Kohgiluyeh-Boyerahmad Province, Islamic Republic of Iran.
Abstract
Hop stunt viroid (HSVd) isolates have been reported as the causal agent of citrus cachexia in Mazandaran Province and recently shown to be associated with yellow corky vein disease of sweet orange and split bark disorder of sweet lime in the Fars Province, Iran. In the present work isolation and partial characterization of viroids from citrus trees affected by gummy stem blight is reported from Kohgiluyeh–Boyerahmad (KB) Province of Iran. Fifteen samples of citrus trees from Dehdasht area (KB Province) showing bark necrosis, gum exudation and die-back as well as seven citrus symptompless trees from the same area were tested for the prevalence of viroids, through Reverse Transcription Polymerase Chain Reaction (RT-PCR) followed by sequencing of PCR products. They were also tested for Citrus tristeza virus through Double-Antibody Sandwich Enzyme Linked Immunosorbent Assay (DAS-ELISA). Two variants of HSVd which differed from GenBank isolates in nucleotide sequence and two variants of Citrus Bent Leaf Viroid (CBLVd) were identified in any of the symptomatic samples. Moreover, a Citrus Exocortis Viroid (CEVd) was found only in symptomatic sweet lime. An HSVd isolate from KB (HSVd-bn1) was selected and used for comparison with a number of HSVd variants from Iran (Fars and Mazandaran Provinces) and the related accessions from GenBank. On the basis of nucleotide sequence and secondary structure analysis, HSVd-bn1 and HSVd-bn2 belong to non-cachexia variants of HSVd and have about 95% similarity to Citrus gummy bark viroid, a sub-species of HSVd. CTV was not detected in the diseased plants. It is yet to be determined whether bark necrosis of sweet lime and of sweet orange plants is caused solely by the associated viroid(s) or other factors are involved as well.

Keywords

1. Alavi, S. M., Ahoonmanesh, A. and Rahimian, H. 2006. Nucleotide Sequence of Hop Stunt Viroid cDNA from Cachexia-infected Citrus Plants in Mazandaran Province. Iran. J. Plant Path., 43: 49-64.
2. Ashulin, L., Lachman, O., Hadas, R. and Bar-Joseph, M. 1991. Nucleotide Sequence of a New Viroid Species, Citrus Bent Leaf Viroid (CBLVd) Isolated from Grapefruit in Israel. Nucleic Acids Res., 19: 4767.
3. Astruc, N., Marcos, J. F., Macquaire, G., Candresse, T. and Pallas, V. 1996. Studies on the Diagnosis of Hhop Stunt Viroid in Fruit Trees: Identification of New Hosts and Application of a Nucleic Acid Extraction Procedure Based on Non-organic Solvents. Eur. J. Plant Pathol., 102: 837-846.
4. Bagherian, S. A. A., Amid Motlagh, M. H. and Izadpanah, K. 2009. Molecular Features of Three Novel Variants of Citrus Exocortis Viroid from Fars Province. The 6th National Biotechnol. Cong. of Iran, Tehran.
5. Bagherian, S. A. A. and Izadpanah, K. 2010. Two Novel Variants of Hop Stunt Viroid Associated with Yellow Corky Vein Disease of Sweet Orange and Split Bark Disorder of Sweet Lime. 21st International Conference on Virus and other Graft Transmissible Diseases of Fruit Crops in Julius-Kühn-Archiv, 427: 105-113.
6. Bagherian, S. A. A. and Izadpanah, K. 2011. A Novel Variant of Citrus Bent Leaf Viroid from Fars Province of Iran. 1st Plant Virologists Symposium, Shiraz, Iran. 1: 8-10.
7. Banihashemian, S. M., Murcia, N., Gomez Trenor, I. and Duran-Vila, N. 2010. Low Performance of Citrus Trees Grafted on Carrizo Citrange is Associated with Viroid Infection. J. Plant Pathol., 92: 511-517.
8. Bar-Joseph, M., Garnsey, S. M. and Gonsalves, D. 1979. The Closteroviruses: A Distinct Group of Elongated Plant Viruses. Adv. Virus Res., 25: 93-168.
9. Bernad, L. and Duran-Vila, N. 2006. A Novel RT-PCR Approach for Detection and Characterization of Citrus Viroids. Mol. Cell. Probe., 20: 105–113.
10. Cañizares, M. C., Marcos, J. F., and Pallás, V. 1999. Molecular Characterization of an Almond Isolate of Hop Stunt Viroid (HSVd) and Conditions for Eliminating Spurious Hybridization in Its Diagnosis in Almond Samples. Eur. J. Plant Pathol., 105: 553-558.
11. Diener, T., Smith, D., Hammond, R., Albanese, G., La Rosa, R. and Davino, M. 1988. Citrus B Viroid Identified as a Strain of Hop Stunt Viroid. Plant Dis., 72: 691-693.
12. Fei, J., Li, Y., Wu, Y., Bai, X., Shi, G., Yu, F. and Kuai, Y. 2009. Identification of a Latent Pathogen on Mulberry Tree with a Disease of Mosaic Dwarf. African J. Biotech., 8: 5358-5361.
13. Gross, H. J., Krupp, G., Domdey, H., Raba, M., Jank, P., Lossow, C., Alberty, H., Ramm, K., and Sanger, H. L. 1982. Nucleotide Sequence and Secondary Structure of Citrus Exocortis and Chrysanthemum Stunt Viroids. Eur. J. Biochem., 121: 249-257.
14. Nour-Eldin, F. 1956. Phloem Discoloration of Sweet Orange. Phytopathol., 46: 238-239.
15. Ohno, T., Takamatsu, N., Meshi, T. and Okada, Y. 1983. Hop Stunt Viroid: Molecular Cloning and Nucleotide Sequence of the Complete cDNA Copy. Nucleic Acids Res., 11: 6184-6196.
16. Onelge, N., Cinar, A., Szychowski, J. A., Vidalakis, G. and Semancik, J. S. 2004. Citrus Viroid II Variants Associated with 'Gummy Bark' Disease. Eur. J. Plant Pathol., 110: 1047-52.
17. Ragozzino, E., Faggioli, F., Amatruda, G. and Barba, M. 2002. Occurrence of Dapple Fruit of Plum in Italy. Phytopathol. Mediterr., 41: 72-75.
18. Reanwarakorn, K. and Semancik, J. S. 1999. Correlation of Hop Stunt Viroid Variants to Cachexia and Xyloporosis Diseases of Citrus. Phytopathol., 89: 568-574.
19. Saitou, N. and Nei, M. 1987. The Neighbor-joining Method: A New Method for Reconstructing Phylogenetic Trees. Mol. Biol. Evol., 4: 406–425.
20. Sano, T., Hataya, T. and Shikata, E. 1988. Complete Nucleotide Sequence of a Viroid Isolated from Etrog Citron, a New Member of Hop Stunt Viroid Group. Nucleic Acids Res., 16: 347.
21. Sano, T., Hataya, T., Terai Y. and Shikata, E. 1989. Hop Stunt Vviroid Strains from Dapple Fruit Disease of Plum and Peach in Japan. J. Gen. Virol., 70: 1311-1319.
22. Semancik, J., Roistacher, C., Rivera-Bustamante, R. and Durán-Vila, N. 1988. Citrus Cachexia Viroid, a New Viroid of Citrus: Relationship to Viroids of the Exocortis Disease Complex. J. Gen. Virol. 69: 3059-3068.
23. Semancik, J. S., Morris, T. J., Weathers, J. G., Rodorf, B. F. and Kearns, D. R. 1975. Physical Properties of a Minimal Infectious RNA (Viroid) Associated with Exocortis Disease. Virol., 63: 160-167.
24. Serra, P., Barbosa, C. J., Daròs, J. A., Flores, R. and Duran-Vila, N. 2007. Citrus Viroid V: Molecular Characterization and Synergistic Interactions with Other Members of the Genus Apscaviroid. Virol., 370: 102-112.
25. Serra, P., Gago, S. and Duran-Vila, N. 2008. A Single Nucleotide Change in Hop Stunt Viroid Modulates Citrus Cachexia Symptoms. Virus Res., 138: 130–134.
26. Shikata, E. 1990. New Viroids from Japan. Semin. Virol., 1: 107-115.
27. Sofy, A. R., Soliman, A. M., Mousa, A. A., Ghazal, S. A. and El-Dougdoug, K. A. 2010. First record of Citrus Viroid II (CVd-II) Associated with Gummy Bark Disease in Sweet Orange (Citrus sinensis) in Egypt. New Disease Rep., 21: 24.
28. Verniere, C., Perrier, X., Dubois, C., Dubois, A., Botella, L., Chabrier, C., Bové, J. M., and Duran-Vila, N. 2006. Interactions between Citrus Viroids Affect Symptom Expression and Field Performance of Clementine Trees Grafted on Trifoliate Orange. Phytopathol., 96: 356–368.
29. Wang, X. F., Zhou, Y., Li, Z. G., Tang, K. Z., Liu, Y. Q., Cao, M. J. and Zhou, C. Y. 2010. Molecular, Biological and Phylogenetic Analysis of Chinese Isolates of Hop Stunt Viroid Associated with Citrus Cachexia Disease. J. Phytopathol., 158(5): 372-377.
Journal of Agricultural Science and Technology
Volume 16, Issue 3
May and June 2014
Pages 657-665