Real-time RT-PCR detection of Citrus bark cracking viroid (CBCVd) in hops including an mRNA-based internal positive cont
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ORIGINAL ARTICLE
Real‑time RT‑PCR detection of Citrus bark cracking viroid (CBCVd) in hops including an mRNA‑based internal positive control Luitgardis Seigner1 · Marion Liebrecht1 · Linda Keckel1 · Katharina Einberger1 · Carolin Absmeier1 Received: 10 January 2020 / Accepted: 27 March 2020 © The Author(s) 2020
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. Keywords Cbcvd · Citrus bark cracking viroid · Duplex RT-PCR · Internal control · Hops · Real-time RT-PCR · Validation
Background 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 highly infectious and can cause significant economic losses in important agricultural and horticultural crops. During cultivation operations, 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 also hop production is threatened by viroid infections which may lead to detrimental reduction in yield and quality and thus affect hop growers and brewers as well. Eastwell and Nelson (2007) reported of serious damage * Luitgardis Seigner [email protected] 1
Bavarian State Research Center for Agriculture, Institute for Plant Protection, Lange Point 10, 85354 Freising, Germany
in hops (Humulus lupulus L., Cannabaceae) caused by the Hop stunt viroid (HpSVd). In 2007, a similar disease was observed in Slovenian hop gardens which resulted in severely stunted hop plants. Symptoms were comparable to those known from HpSVd infections, but disease progress was much faster and in contrast to HpSVd stunting plants died off within 3–5 years (Jakse et al. 2015). Using highthroughput sequencing deep sequ
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