2.1. Preparation of Dimeric CBCVd Construct and Inoculation of the Hop Plants

The full-length monomeric cDNA of CBCVd was amplified from total RNAs isolated from CBCVd-infected leaves of the Slovenian hop cultivar “Celeia” by reverse transcription-polymerase chain reaction (RT-PCR) using specific primers (Table S1). The purified RT-PCR product was cloned into T-Vectors (Takara Bio Inc., Shiga, Japan) and it was confirmed by sequencing. The dimeric construct was generated by digesting SacI termini of monomeric positive cDNA strand of CBCVd and cloning into the SacI site of pBlueScript KS (+) vector (Stratagene, San Diego, CA, USA). For 35S::CBCVd construct, the dimeric cDNA of CBCVd was subcloned into the pLV07 binary vector via intermediate vector pLV68 harboring a 35S expression cassette as described previously [28]. The CBCVd cDNA was immobilized on microcarrier gold particles (1 μm) using a calcium-mediated precipitation protocol [29] and biolistically inoculated on three-month-old hop leaves (cv. Osvald’s 72) grown in a container (10 cm height with at least three shoots-stage) under natural light condition. The individual hop plant was inoculated five times with 250 ng DNA per viroid species and was placed in darkness for 24 h after covering with plastic bags. Subsequently, the mock and CBCVd-inoculated plants were grown under natural condition and inspected visually for symptoms development.