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This work reports the identification of Hop latent viroid (HLVd) in commercial hop (Humulus lupulus L.) plants in Belgium. Hop plants without clear symptoms were randomly collected in July 2015 during a survey at nine companies near the city of Poperinge, the prime hop-growing region in Belgium. RNA was extracted from twenty six samples using the Spectrum™ Plant Total RNA Kit (Sigma-Aldrich, St. Louis, MO,USA), and cDNA was prepared using the iScriptTM cDNA synthesis kit (Bio-Rad, Hercules, CA, USA). The samples were tested for the presence of pospiviroids using a set of generic Pospiviroidae primers (pospi1-RE/FW, Verhoeven et al. 2004). Additionally, specific RT-PCR tests were conducted for Hop stunt viroid (HSVd), Hop latent viroid (HLVd), and Citrus bark cracking viroid (CBCVd) using primer sets and protocols described by Matousek et al. (2003) and Bernad & Duran-Vila (2006). Of the 26 hop samples, 13 tested positive for the HLVd. No HSVd or CBCVd was detected in any of the 26 samples. Direct sequencing of the PCR-products (Macrogen, Amsterdam, the Netherlands) generated in the HLVd-specific RT-PCR confirmed the presence of HLVd in these plants. Two HLVd-isolates, originating from hops from two different locations, were further identified and the nearly identical sequences were deposited at GenBank® under the accession numbers KT600317 and KT600318. Hop plants (cv. Goldings) originating from the U.K. and testing negative in the lab for the presence of viroids, including HLVd, were inoculated individually with the two characterized HLVd-isolates (KT600317 and KT600318) in order to fulfil Koch’s postulates. The inoculum was prepared by bringing 100 g of HLVd-infected leaf tissue into extraction bags (Bioreba, Reinach, Switzerland) containing 10 ml of ice-cold inoculation buffer (0.01 M potassium phosphate buffer, pH 7), followed by homogenization using a Homex 6 instrument (Bioreba AG, Reinach, Switzerland). A sterile cotton swab was dusted with carborundum (silicon carbide, size C500, Department Physics & Astronomy, Ghent University), dipped into the filtered inoculum and applied onto three randomly chosen leaves of each hop plant. The inoculated leaves were immediately rinsed with distilled water. Four weeks after inoculation, systemic leaves of the plants tested positive for the presence of HLVd, yet no specific symptoms developed on the plants. It is known that infection by HLVd generally does not produce clear symptoms in hop plants. On the other hand, HLVd infection is reported to be associated with changes in the production of secondary metabolites in the lupulin glands (Patzak et al. 2001), for instance by alterations in the amount of alpha acids that play a role in beer production (Matousek et al. 2003). In that respect, further assessment of the economic impact of HLVd infection in the Belgian commercial hop production is recommended.
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