Application of Next Generation Sequencing for Diagnostic Testing of Tree Fruit Viruses and Viroids

Abstract

Conventional detection of viruses and virus-like diseases of plants is accomplished using a combination of molecular, serological, and biological indexing. These are the primary tools used by plant virologists to monitor and ensure trees are free of known viral pathogens. The biological indexing assay, or bioassay, is considered to be the “gold standard” as it is the only method of the three that can detect new, uncharacterized, or poorly characterized viral disease agents. Unfortunately, this method is also the most labor intensive and can take up to three years to complete. Next generation sequencing (NGS) is a technology with rapidly expanding possibilities including potential applications for the detection of plant viruses. In this study, comparisons are made between tree fruit testing by conventional and NGS methods, to demonstrate the efficacy of NGS. A comparison of 178 infected trees, many infected with several viral pathogens, demonstrated that conventional and NGS were equally capable of detecting known viruses and viroids. Comparable results were obtained for 170 of 178 of the specimens. Of the remaining eight specimens, some discrepancies were observed between viruses detected by the two methods, representing less than 5% of the specimens. NGS was further demonstrated to be equal or superior for the detection of new or poorly characterized viruses when compared with a conventional bioassay. These results validated both the effectiveness of conventional virus testing methods and the use of NGS as an additional or alternative method for plant virus detection.

Updating the Quarantine Status of Prunus Infecting Viruses in Australia

“…Molecular tests should be developed and validated in Australia for detection of these viruses and surveillance should be done to determine their presence within Australian almond and stone fruit production areas. HTS is increasingly being applied for plant pathogen detection to support plant biosecurity and certification programs [77,[92][93][94] and the results of this study further highlight its value as a non-discriminatory diagnostic alternative to traditional virus diagnostic methods such as PCR to support the Australian Prunus industry biosecurity.…”

“…Metagenomics HTS detection of additional virus species, specifically ApLV, AVCaV, APV2, NSPaV and LChV1, and strains of CGRMV, CNRMV, LChV2 and PBNSPaV, which were not detected by species-specific or genus-or family-based RT-PCR tests, highlights the high potential for a false-negative diagnosis by RT-PCR due to virus genetic variability. It also highlights the value of metagenomics HTS as a non-discriminative diagnostic tool for virus detection compared to RT-PCR, because it does not rely on knowledge of the virus genome sequences [77]. Given the high number of viruses that may need to be tested in some Prunus species, which could make RT-PCR a cost-prohibitive diagnostic tool, the reducing cost of HTS [28] makes this technology more attractive for routine virus detection in Prunus species in Australia.…”

The impact of high throughput sequencing on plant health diagnostics

“…Subsequent investigations have shown that MLBVV is the more probable cause of big-vein disease (Lot et al 2002), but that LBVaV may still be associated with other symptoms such as localised necrosis (Verbeek et al 2013). This eight-decade arc of investigation moved on with advances in diagnostic methods, but ultimately progress was hampered by the limitations of these techniques: Electron microscopy can only observe the morphology of viral particles present, but cannot be used to give a conclusive diagnosis at species level; Biological indexing can give an indication of the pathogens present in a sample, but is prone to failure where a virus is labile or is not amenable to mechanical transmission; Targeted methods can only detect the targets the assays have been designed against, at best a broad number of species in a given genus (Adams et al 2013;De Clerck et al 2017;Rott et al 2017).…”

High‐throughput sequencing technologies for plant pest diagnosis: challenges and opportunities

“…HTS technologies open new possibilities and opportunities in routine diagnostics for (a) understanding the status of a pest in a region through surveillance programmes, (b) certifying nuclear stock and plant propagation material, (c) (post-entry) quarantine testing to prevent the introduction of pests into a country or area, and (d) monitoring of imported commodities for new potential risks. HTS offers important benefits for each of these applications (Al Rwahnih et al, 2015;Hadidi et al, 2016;Rott et al, 2017).…”