Abstract
Citrus bark cracking viroid (CBCVd), formerly known as pathogen in the genus Citrus and first detected in Slovenian hops in 2014, threatens hop production as it leads to important economic losses. Reduction in yield and quality and even death of the infected plants within a few years are typical observations due to CBCVd infections of hops. The viroid is easily transmitted and spreads rapidly. As it cannot be controlled by plant protection measures, avoiding its introduction into hop gardens and eradicating first centres of infection are of utmost importance. An indispensable prerequisite is a reliable detection method suitable for large-scale routine testing. In this study, the development of primers and probe for real-time RT-PCR for sensitive CBCVd detection is described. To exclude “false negative” results, a nad5 mRNA-based internal positive control was included. To our knowledge, this is the first time such a duplex real-time RT-PCR detection method for CBCVd at least in hops is described. In addition, first method validation data are presented.
Highlights
Viroids are the smallest plant pathogenic particles known
For Citrus bark cracking viroid (CBCVd) originating from hops, only a single sequence was available which was obtained from sequencing an RT-PCR amplicon obtained with primers published by Ito et al (2002)
The CBCVd plasmid standard revealed that the newly developed primers and probe are well suited for a sensitive real-time PCR detection of CBCVd with PCR efficiencies between 93 and 110%
Summary
Viroids are the smallest plant pathogenic particles known. They only consist of an un-encapsidated, circular, singlestranded RNA which forms rods due to intra-molecular base paring. Viroids are readily transmitted, spread within a field and are disseminated from one place of production to another. Viroidspecific small RNAs (sRNAs) emerging after infection are assumed to affect the host metabolism and lead to symptom expression by post transcriptional gene silencing (PTGS) or RNA silencing of host mRNAs (Qi and Ding 2003; Gas et al 2007; Voinnet 2008). At present hop production is threatened by viroid infections which may lead to detrimental reduction in yield and quality and affect hop growers and brewers as well.
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