engleski

Preliminary findings on Coleus blumei viroids in Croatia

Plectranthus scutellarioides (L.) R. Br. is the accepted botanical name of Coleus blumei Benth. It is the main host of six Coleus blumei viroids (CbVds) belonging to the genus Coleviroid (Pospiviroidae) including three CbVd ICTV recognized species (CbVd-1 to -3), an in vitro-generated inverse chimera CbVd-4 and two tentative species (CbVd-5 and CbVd-6) [2, 3, 4]. Whilst CbVd-1 occurs globally, CbVd-2 and -4 have been reported only in Germany, CbVd-3 in Germany and China, and CbVd-5 and -6 only in Southeast Asia [1, 3, 4]. Interestingly, CbVds are efficiently transmissible by seeds [3, 4]. The aim of this study was to investigate the occurrence of coleviroids in three asymptomatic coleus cultivars (cvs labelled 6, 8 and V) grown in the Botanical garden of the Faculty of Science in Zagreb. Leaf and stem tissue from 20 plants were used for obtaining total RNAs. Generic primers covering the central conserved region of all six CbVds were used in RT-PCR based detection procedure [3], as well as specific primers covering either TR region of CbVd-1 or the border V-CCR region in the “lower strand” of CbVd-3 [1]. Two amplicons of approximately 250 and 360 bp were obtained from 7 out of 7 (7/7) plants of cv 6 and 2/11 plants of cv 8. A single band indicating the presence of a smaller viroid was obtained from the remaining cv 8 plants (9/11), whilst both cv V plants (2/2) showed a single amplicon of a larger viroid. An amplicon of each size from two singly infected plants and amplicons of both sizes from one doubly infected plant were AT-cloned. After SSCP-screening, all identified variants of each viroid population structure were sequenced. CbVd-1 was identified in cultivars 6, in mixed infections, and 8, in mixed and single infections. CbVd-1 sequence variants of 249-251 nucleotides were obtained. CbVd-3 variants of 361-362 nucleotides were identified either in mixed infections, in cultivars 6 and 8, or in single infections, in cultivar V. The incidence levels of 45% for CbVd-1 and 10% for CbVd-3 in single infections, as well as 45% in double infections are high for such a small population of investigated plants (20 in total) but not uncommon for these viroids [2, 4]. CbVd-3 presence is reported here only for the second time in Europe. Our findings suggest it could be more widespread than assumed. REFERENCES [1] C. R. Adkar-Purushothama, H. Nagaraja, M. Y., Sreenivasa, and T. Sano (2013) First report of Coleus blumei viroid infecting coleus in India. Plant Disease, 97:149. [2] F. Di Serio, R. Flores, J. Th. J. Verhoeven, S.-F. Li, V. Pallás, J. W. Randles, T. Sano, G. Vidalakis, and R. A. Owens (2014) Current status of viroid taxonomy. Archives of Virology, 159:3467–3478. [3] D. Jiang, Z. Wu, L. Xie, T. Sano, and S. Li (2011) Sap-direct RT-PCR for the rapid detection of coleus blumei viroids of the genus Coleviroid from natural host plants. Journal of Virological Methods, 174:123-127. [4] R. P. Singh, M. E. N. F. Boiteux, K. F. M Ready, and X. Nie (2003) Viroids. CSIRO Publishing.

Plectranthus scutellarioides; CbVd-1; CbVd-3; RT-PCR; sequencing; SSCP

Dio rada realiziran je i obranjen u sklopu diplomskog zadatka Karla Jezernika 2014. godine(diplomski studij molekularne biologije).