Abstract
Author(s): Steyn, C; Cook, G; Burger, J T; Maree, H J | Abstract: Viroid species identified in citrus induce a range of symptoms in this host as well as in non-citrus hosts. Currently, 7 citrus viroid species are recognized including Citrus bent leaf viroid (CBLVd), Hop stunt viroid (HSVd), Citrus dwarfing viroid (CDVd), Citrus bark cracking viroid (CBCVd), Citrus viroid V (CVd-V), Citrus viroid VI (CVd-VI) and Citrus exocortis viroid (CEVd). Cachexia-inducing variants of HSVd and CEVd, which causes exocortis, are considered severe pathogens of citrus, whereas other viroid species induce less severe symptoms such as stunting, either singly or in combination. Some viroid species, such as CDVd, have previously been used to deliberately induce stunting for high density planting of citrus, but studies on the effects of commercial orchards are limited. Research on the effect of viroid species requires the isolation of single viroid species. Biological isolation of single species from naturally infected citrus is challenging since viroids seldom occur as single infections and are often found in combination with various other pathogens. The production of single viroid species clones for in vitro transcription enables the generation of a single viroid species inoculum for research applications and circumvents the need for maintenance of sources in plants. Complete genomes of 7 viroid strains including CBLVd, 2 HSVd variants (CVd-IIa and CVd-IIb), CDVd, CBCVd, CVd-V and CEVd were cloned with a leading T7 promoter sequence to facilitate in vitro transcription. Circularized RNA transcripts were successfully used to transfect ‘Etrog’ citron (Citrus medica) by slash-inoculation that developed typical citrus viroid symptoms.
Highlights
Viroids are small, circular, naked, single-stranded RNA molecules ranging from 240-400 nucleotides in length, lacking any protein coding ability
All citrus viroids belong to the Pospiviroidae family due to their secondary structure conformation, and characteristically have a rod-like structure with a central conserved region (CCR) that is mostly preserved among strains of a species (Flores et al 2011; Di Serio et al 2014)
The transcripts produced for Citrus dwarfing viroid (CDVd) and Citrus exocortis viroid (CEVd) were noticeably less than for the other viroids, and bands for Hop stunt viroid (HSVd) variants and Citrus bark cracking viroid (CBCVd) were more prominent
Summary
Circular, naked, single-stranded RNA molecules ranging from 240-400 nucleotides in length, lacking any protein coding ability. The high degree of base pair self-complementarity in their genomes promote stable secondary structures that have been shown to influence functionality (Flores et al 2009). All citrus viroids belong to the Pospiviroidae family due to their secondary structure conformation, and characteristically have a rod-like structure with a central conserved region (CCR) that is mostly preserved among strains of a species (Flores et al 2011; Di Serio et al 2014). Each species consists of more than 1 variant, often varying in pathogenicity. The population structure of viroid variants has been shown to be influenced by the citrus host (Bernad et al 2009)
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