Detection of Citrus exocortis viroid, Iresine viroid, and Tomato chlorotic dwarf viroid in New Ornamental Host Plants in India

Abstract

Columnea latent viroid, originating from ornamental plants, is known to be harmful to crop plants (2). Despite the potential threat to crop plants, the importance of ornamental plants in viroid evolution is not fully appreciated. Availability of a Pospiviroid genus-specific primer pair (1) to detect the most prevalent viroids in ornamental plants and a simplified nucleic acid preparation protocol (3) for use in reverse-transcription polymerase chain reaction (RT-PCR) have facilitated surveys of ornamental plants for pospiviroids. Using the above protocol in India, leaf and shoot samples were collected randomly from roadside beds consisting of ground covers or creepers/trailing plants at the IARI campus, New Delhi. These were extracted in 50 mM NaOH + 2.5 mM EDTA solution, centrifuged to sediment the coarse debris from sap, and 10 μl of the supernatant was spotted on a nitrocellulose membrane. Individual spots were eluted with distilled sterile water (30 μl) and the eluates were used for RT-PCR detection of viroids (3). Amplified fragments or subsequently cloned plasmids were also purified using NaOH-EDTA membrane protocol. Cloning and sequencing of amplicons (195 to 224 bp) revealed a very high sequence identity with specific viroids from the viroid sequence database (NCBI). Among the 19 plant samples assayed, only three plant species were infected by viroids, although none of them exhibited any symptoms. The three plant species included: (i) moss verbena, Glandularia puchella (Verbenaceae, native to Argentina and Chile, now established in several regions of the world), infected with a viroid (Accession No. DQ846884) having 99% sequence identity to Citrus exocortis viroid (CEVd) (Accession No. S67446); (ii) trailing verbena, Verbena × hybrida (Verbenaceae, ornamental plant), doubly infected with a viroid (Accession No. DQ846885) having 95% sequence identity to CEVd (Accession No. DQ094297) and infected with another viroid (Accession No. DQ846883) having 98% sequence identity to Tomato chlorotic dwarf viroid (TCDVd) (Accession No. AF162131); and (iii) red joyweed, Alternanthera sessilis (Amaranthaceae, a perennial weed herb) infected with a viroid (Accession No. DQ846886) having 96% sequence identity to Iresine viroid (IrVd) (Accession No. DQ094293). CEVd and TCDVd were mechanically transferred to tomato seedlings causing reduced growth of plants, smaller leaves, and bunchy-top appearance of plants, symptoms similar to those typically observed with other isolates of these viroids. As expected from previous studies, IrVd was not transmitted to tomato plants. Natural infection of verbena with CEVd has been detected in North America (2) but this was a novel observation in India. Additional novel observations include: A. sessilis as a new host for IrVd; and TCDVd is the first crop viroid to be isolated from a naturally infected ornamental plant. The significance of these viroid findings in ground cover and widely grown ornamental plants may lie in their potential role in spreading the viroids to citrus plants in citrus-growing countries such as India. References: (1) H. Bostan et al. J. Virol. Methods 116:189, 2004, (2) R. P. Singh and J. A. Teixeira da Silva. Floriculture, Ornamental Plant Biotechnol. 3:531, 2006. (3) R. P. Singh et al. J. Virol. Methods 132:204, 2006.

Molecular and biological characterization of a severe isolate of Tomato chlorotic dwarf viroid containing a novel terminal right (TR) domain sequence

“…# EU729744) and the USA (Ling et al 2009). In addition to the tomato crop, TCDVd has also been isolated from several greenhouse grown ornamental plants (e.g., Verbena x hybrida (Singh et al 2006a), Petunia x hybrida (Verhoeven et al 2007;James et al 2008), Brugmansia sanguinea (acc. # EF 626530) and Vinca minor (Singh and Dilworth 2008).…”

Citrus exocortis viroid transmission through commercially-distributed seeds of Impatiens and Verbena plants

“…The finding of CEVd infection in seed-grown seedlings, in moss Verbena (Glandularia puchella; Singh et al 2006a) in India and Impatiens (Impatiens walleriana; unpublished observation) in Canada in 2006 pointed to the possibility of seed transmission of CEVd from Impatiens and Verbena seeds.…”

“…Recent surveys to determine the prevalence of pospiviroids in ornamental plants have shown that CEVd is carried symptomlessly in vegetativelypropagated plants of Impatiens and Verbena species (Bostan et al 2004;Nie et al 2005;Singh et al 2006a). The finding of CEVd infection in seed-grown seedlings, in moss Verbena (Glandularia puchella; Singh et al 2006a) in India and Impatiens (Impatiens walleriana; unpublished observation) in Canada in 2006 pointed to the possibility of seed transmission of CEVd from Impatiens and Verbena seeds.…”

The Detection and Diagnosis Methods of Infectious Viroids caused Plant Diseases

“…바이로이드가 주로 감염되는 숙주로는 사과, 배, 포도, 토마 토, 감자, 시트러스, 국화, 코코넛 팜, 아보카도, 홉 등이 있다 [58]. 주로 야기되는 바이로이드는 Pospiviroidae과의 Apple scar skin viroid (ASSVd) [46], Pear blister canker viroid (PBCVd) [24], Citrus bent leaf viroid (CVd-Ib) [63], Citrus exocortis viroid (CEVd) [68], Hop stunt viroid (HSVd) [36], Coconut cadang-cadang viroid (CCCVd) [79], Potato spindle tuber viroid (PSDVd) [67], Coleus blumei viroid 1 (CbVd) [1], Avsunviroidae과의 Avocado sunblotch Viroid (ASVd) [10], Peach latent mosaic viroid (PLMVd) [29], Eggplant latent viroid (ELVd) [14] 등이 있다( Table 1).…”