2018 ASHS Annual Conference

Over the past several decades Rose Rosette Disease has become very serious and threatens to decimate the US rose industry. The causal agent, Rose rosette virus (RRV, Emaravirus), is transmitted by wind-blown eriophyid mites (Phyllocoptes fructiphilus) and can kill a rose within 2-3 years of infection. A recently-funded USDA, NIFA, Specialty Crops Research Initiative grant involves 17 scientists in 6 states working on a range of approaches to study this disease and determine how best to manage it. The only strategy currently available for disease management is early identification and eradication of the infected plants, thereby limiting its potential spread. Key to this effort is the development of efficient and affordable diagnostic tools to enable accurate detection of the virus. Molecular- and serological-based assays with potential for technology transfer and/or on-site implementation should be easy to use, offering visual detection, reliability and sensitivity to the end user. RRV-specific primers and probes (for nucleic acid-based tests) and monoclonal and polyclonal antibodies (for serology-based tests) have been developed. The rabbit polyclonal and five mouse monoclonal RRV-specific antibodies can detect viral nucleoprotein (NP) in various ELISA and western-blot formats. Several unique NP-specific epitopes have been identified. Validation of virus detection from field-collected samples in is progress. An additional goal is to produce a serological lateral flow device for in-field diagnosis. Loop-mediated isothermal amplification (LAMP) and thermophilic helicase dependent amplification (tHDA) assays have been developed and are isothermal DNA amplifications which combine several desirable criteria and do not require a thermocycler. A rapid 15 minute probe-based isothermal Reverse Transcription-recombinase polymerase amplification (RT-exoRPA) assay was also developed. Broad detection of Emaravirus and species discrimination was targeted with Reverse Transcription polymerase chain reaction (RT-PCR) coupled to High Resolution Melting (HRM) analysis. A single primer-set suitable for use with three different chemistries [Endpoint RT-PCR, TaqMan-quantitative RT-PCR (RT-qPCR) and SYBR Green RT-qPCR with High Resolution Melting] was also developed. An immune-capture RT-PCR based protocol is also under evaluation. The most consistent assay(s) will be tested and validated by several diagnostic labs and then moved via outreach to other plant diagnostic labs. The contribution of these methods within a holistic perspective of RRV diagnostics will be presented.