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

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.

High-Throughput Sequencing for Deciphering the Virome of Alfalfa (Medicago sativa L.)

“…Viral genomes are assembled using Spades ( François et al, 2018 ), CLC Genomics Workbench ( Candresse et al, 2014 ), IDBA-UD ( Peng et al, 2012 ; Ma et al, 2019 ), CAP3 ( Huang and Madan, 1999 ; Bernardo et al, 2018 ), and other assemblers. Another approach allowing the enrichment of viral sequences was carried out by Samarfard et al (2020) , who applied the dsRNA immunocapture technique ( Blouin et al, 2016 ) to characterize the virome of alfalfa plants in Australia. dsRNA was captured with specific monoclonal antibodies prior to HTS on an Illumina platform.…”

“…dsRNA was captured with specific monoclonal antibodies prior to HTS on an Illumina platform. This work led to the identification of several known alfalfa viruses and the discovery of a new emaravirus tentatively named alfalfa ringspot-associated virus ( Samarfard et al, 2020 ).…”

“…A novel putative emaravirus, provisionally named alfalfa ringspot-associated virus (ARaV), was recently discovered in Australia ( Samarfard et al, 2020 ) using a dsRNA immunocapture technique ( Blouin et al, 2016 ). Emaraviruses (family Fimoviridae , order Bunyavirales ) have negative-sense, linear, segmented RNA genomes.…”

“…Alfalfa ( Medicago sativa L.), also known as lucerne, is a major forage crop worldwide cultivated in more than 80 countries, where it is mainly used as silage for grazing livestock ( Samarfard et al, 2020 ). In the United States, it has recently become the third most valuable field crop planted on 22 million acres, with an estimated value of over $9.3 billion ( National Alfalfa and Forage Alliance, 2018 ; Miller, 2019 ).…”

“…Many of the viruses infecting alfalfa have long been known ( Babadoost, 1990 ; Samac et al, 2015 ). In recent years, however, a number of new viral pathogens or pathogens that were not previously described in the crop have been discovered ( Bejerman et al, 2011 , 2015 , 2016 , 2019 ; Roumagnac et al, 2015 ; Nemchinov et al, 2017a , b , 2018a ; Kim et al, 2018 ; Gaafar et al, 2019 ; Jiang et al, 2019a , b ; Samarfard et al, 2020 ). This became possible due to revolutionary advances in nucleic acid sequencing that have been leading to the replacement of traditional detection methods in plant virology with comprehensive, large-scale, unbiased, reliable high-throughput sequencing (HTS) technologies ( Wren et al, 2006 ; Roossinck, 2012b , 2017 ; Nagano et al, 2015 ; Roossinck et al, 2015 ; Jones et al, 2017 ; Villamor et al, 2019 ).…”