Construction and application of infectious citrus viroids for biological indexing

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.