Molecular and phylogenetic identification of unique isolates of hammerhead viroid-like RNA from ‘Pacific Gala’ apple (Malus domestica) in Canada

Apple hammerhead viroid-like RNA is a bona fide viroid: Autonomous replication and structural features support its inclusion as a new member in the genus Pelamoviroid

“…An RNA preparation from leaves of an AHVd-infected apple tree of cultivar "Pacific Gala" (Messmer et al, 2017), obtained with the RNeasy Plant Mini Kit (Qiagen), was reverse transcribed and PCR-amplified (see below) using the abutted primers RF-1385 and RF-1386 (Table 1). The full-length product was eluted from nondenaturing PAGE in 5% gels and ligated with T4 DNA ligase (Thermofisher), with the resulting dimer being identified by its size, gel-eluted, cloned in pBlueScript II KS+ (Stratagene) and checked for proper head-to-tail orientation and sequence.…”

“…Since their discovery, GHVd RNA and AHVd RNA have retained the status of viroid-like RNAs because, despite some attempts, no proof of autonomous replication has been provided so far (Wu et al, 2012;Zhang et al, 2014;Messmer et al, 2017). Nevertheless, there is a good reason to consider that they could be true…”

A bushel of viruses: Identification of seventeen novel putative viruses by RNA-seq in six apple trees

“…The frequency of recently discovered AhVd [19], ARWaV1 and 2 [15] and CCGaV [16] in apples in Washington state is not known, however they were all detected in at least one sample from this work; AhVd was present in all sequenced trees (Table 3). While CCGaV has been associated with concave gum disease in citrus [16], it has also been reported in apples [40].…”

“…Additional apple samples not submitted for sequencing were screened for the presence of known apple viruses. RT-PCR assays were performed, as described above, for ARWaV-1 and ARWaV-2 [15], AhVd [19], and CCGaV [16]. qRT-PCR was performed for AGCaV and ASGV [17] and ACLSV, ApMV, and ASPV [Harper, manuscript in review] using the Invitrogen TM Superscript TM III Platinum TM One-Step qRT-PCR kit (Fisher Scientific).…”

“…ARWaV1 and 2 have been associated with apple rubbery wood disease [15], however the symptoms of that disease are distinct from what is observed in apple decline. AhVd has been observed in Pacific Gala trees that exhibit limb swelling and cracking [19], however those symptoms also differ from apple decline. Lastly, AGCaV was detected in both healthy and declining plants ( Table 3).…”