Integrating High throughput Sequencing into Survey Design Reveals Turnip Yellows Virus and Soybean Dwarf Virus in Pea (Pisum Sativum) in the United Kingdom.

Aimee R Fowkes,Sam Mcgreig,Roy Macarthur,Adrian Fox,Hollie Pufal,Shona Duffy,Becky Howard,Ian P Adams,Rebecca Weekes

doi:10.3390/v13122530

Abstract

There is only limited knowledge of the presence and incidence of viruses in peas within the United Kingdom, therefore high-throughput sequencing (HTS) in combination with a bulk sampling strategy and targeted testing was used to determine the virome in cultivated pea crops. Bulks of 120 leaves collected from twenty fields from around the UK were initially tested by HTS, and presence and incidence of virus was then determined using specific real-time reverse-transcription PCR assays by testing smaller mixed-bulk size samples. This study presents the first finding of turnip yellows virus (TuYV) in peas in the UK and the first finding of soybean dwarf virus (SbDV) in the UK. While TuYV was not previously known to be present in UK peas, it was found in 13 of the 20 sites tested and was present at incidences up to 100%. Pea enation mosaic virus-1, pea enation mosaic virus-2, pea seed-borne mosaic virus, bean yellow mosaic virus, pea enation mosaic virus satellite RNA and turnip yellows virus associated RNA were also identified by HTS. Additionally, a subset of bulked samples were re-sequenced at greater depth to ascertain whether the relatively low depth of sequencing had missed any infections. In each case the same viruses were identified as had been identified using the lower sequencing depth. Sequencing of an isolate of pea seed-borne mosaic virus from 2007 also revealed the presence of TuYV and SbDV, showing that both viruses have been present in the UK for at least a decade, and represents the earliest whole genome of SbDV from Europe. This study demonstrates the potential of HTS to be used as a surveillance tool, or for crop-specific field survey, using a bulk sampling strategy combined with HTS and targeted diagnostics to indicate both presence and incidence of viruses in a crop.

Highlights

Peas are a key horticultural crop with produce for human consumption as fresh or frozen and combinable peas for the production of pea protein e.g., cattle feed
The aims of this study were to provide a baseline for pea viruses in the United Kingdom (UK), to show the feasibility of integrating high-throughput sequencing (HTS) into a survey programme using a bulked field sample’ (BFS) approach
Six viruses, one satellite RNA and an associated RNA were detected by HTS across all 20 sites, and 28 symptomatic samples

Summary

Introduction

Peas are a key horticultural crop with produce for human consumption as fresh or frozen and combinable peas for the production of pea protein e.g., cattle feed. Conventional approaches to conducting virus surveys tend to focus on collecting multiple symptomatic samples which get tested for a set number of target viruses, using targeted approaches such as ELISA or PCR, the suite of viruses would likely comprise those viruses expected to be present and those viruses known to infect the host which have not yet been detected in a given geographic region. Where there is limited knowledge of the presence of a disease in a crop, a diagnostic focus on the viruses expected to be present, may lead to the exclusion of potentially important pathogens which may not previously have been reported in that region or host, or may be as yet undescribed [3,4]. Determining the infection status of asymptomatic individuals can later inform risk assessment or determination of symptom causation [3]

Objectives

Results

Conclusion