Viromes of Ten Alfalfa Plants in Australia Reveal Diverse Known Viruses and a Novel RNA Virus.

Samira Samarfard,Nicolás E Bejerman,Murray Sharman,Ralf G Dietzgen,Alistair R Mctaggart

doi:10.3390/pathogens9030214

Abstract

Alfalfa plants in the field can display a range of virus-like symptoms, especially when grown over many years for seed production. Most known alfalfa viruses have RNA genomes, some of which can be detected using diagnostic assays, but many viruses of alfalfa are not well characterized. This study aims to identify the RNA and DNA virus complexes associated with alfalfa plants in Australia. To maximize the detection of RNA viruses, we purified double-stranded RNA (dsRNA) for high throughput sequencing and characterized the viromes of ten alfalfa samples that showed diverse virus-like symptoms. Using Illumina sequencing of tagged cDNA libraries from immune-captured dsRNA, we identified sequences of the single-stranded RNA viruses, alfalfa mosaic virus (AMV), bean leafroll virus, a new emaravirus tentatively named alfalfa ringspot-associated virus, and persistent dsRNA viruses belonging to the families Amalgaviridae and Partitiviridae. Furthermore, rolling circle amplification and restriction enzyme digestion revealed the complete genome of chickpea chlorosis Australia virus, a mastrevirus (family Geminiviridae) previously reported only from chickpea and French bean that was 97% identical to the chickpea isolate. The sequence data also enabled the assembly of the first complete genome (RNAs 1–3) of an Australian AMV isolate from alfalfa.

Highlights

Alfalfa or lucerne (Medicago sativa L.) is a legume cultivated in more than 80 countries on about45 million ha and used as silage for grazing livestock [1,2]
We identified several known and one novel RNA virus through high throughput sequencing (HTS) of 10 alfalfa samples in
We identified several known and onesymptoms novel RNAincluding virus through of 10ring alfalfa samples in Australia that showed a range of virus-like yellowHTS

Summary

Introduction

Alfalfa or lucerne (Medicago sativa L.) is a legume cultivated in more than 80 countries on about45 million ha and used as silage for grazing livestock [1,2]. Alfalfa or lucerne (Medicago sativa L.) is a legume cultivated in more than 80 countries on about. Virus diseases can affect production of alfalfa hay and seed [3]. A large and increasing number of viruses is known to infect alfalfa globally. Surveys of alfalfa using serological assays showed that alfalfa mosaic virus (AMV; genus Alfamovirus, family Bromoviridae), bean leafroll virus (BLRV; genus Luteovirus, family Luteoviridae), and cucumber mosaic virus (CMV; genus Cucumovirus, family Bromoviridae) are the most frequently detected viruses in Australian alfalfa pastures [3,4,5,6]. Several novel viruses have been identified in alfalfa worldwide, with the help of high throughput sequencing (HTS). In 2010, alfalfa dwarf disease (ADD), which affects

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