Discovery of Negative-Sense RNA Viruses in Trees Infected with Apple Rubbery Wood Disease by Next-Generation Sequencing

Abstract

Apple rubbery wood is a disease of apple found around the world, often associated with Apple flat limb disease, and regulated in many countries. Despite its long history in apple cultivation, the disease’s causal agent has remained elusive. In this study, next-generation sequencing (NGS) was used to identify and characterize several related novel viral agents from apple rubbery wood-infected plants, which have been named Apple rubbery wood virus (ARWV) 1 and 2. Additional specimens with apple rubbery wood disease tested positive by polymerase chain reaction with primers designed to ARWV 1 and 2 genomic RNA segments. In an NGS-based screening of over 100 Malus and 100 Prunus specimens from a collection of virus-infected trees, only one Malus specimen was found to be infected with ARWV not known to be infected with the disease, which strongly suggests that ARWV is not commonly found in Malus spp. or other fruit trees. The two viruses are most closely related to members of the order Bunyavirales. Three RNA segments (large, medium, and small) were characterized and the viruses likely represent a new genus under the family Phenuiviridae, with a suggested name of Rubodvirus (Rubbery wood virus).

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).…”

“…Apple green crinkle associated virus (AGCaV) was detected in trees 1, 2, 5, and 6 and ASGV in trees 1, 5, and 6. The newly reported phleboviruses [15,16] were also present in some of the samples with Apple rubbery wood associated virus 1 (ARWaV1) detected in tree 5, Apple rubbery wood associated virus 2 (ARWaV2) in trees 1, 3, and 6, and Citrus concave gum associated virus (CCGaV) in tree 6. An additional 48 trees were screened by qRT-PCR for the presence of these viruses.…”

“…Currently, the virus has not been associated with any symptoms or diseases in apple. 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.…”

“…NGS is particularly useful when investigating diseases where the pathogen has yet to be identified. Recently, NGS has been used to identify two viruses associated with apple rubbery wood disease [15] and one associated with citrus concave gum disease [16]. ALV1 was also discovered by NGS [2].…”