First report of Medicago trirhavirus 1 infecting alfalfa in Washington State, USA.

ABSTRACT

The first tri-segmented viruses in the family Rhabdoviridae were recently discovered by exploring publicly available plant datasets in several hosts, including alfalfa (Medicago sativa L.) (Bejerman et al. 2023). They were classified in a novel genus "Trirhavirus" within the family Rhabdoviridae. The trirhavirus identified in alfalfa was named Medicago trirhavirus 1 (MeTRV1). Here we report the first confirmation of MeTRV1 in commercial alfalfa fields in Washington State, USA. Samples were collected in 2019-2021 in Benton and Grant Counties, WA. The alfalfa leaves in which the virus was detected displayed irregular chlorotic spotting (Fig.1). Total RNA extraction, library preparation, high throughput sequencing, and bioinformatics analysis were performed as described in Nemchinov et al (2023). Raw reads were trimmed with Trimmomatic 0.39 (Bolger at al. 2014). SPAdes 3.15.5 (Bankevich et al. 2012) was used for assembly. MeTRV1 was identified in four plants out of 100 tested and three complete RNA segments were recovered from one of them. For clarity, the virus found in the alfalfa field samples was designated MeTRV1-Wa. De novo assembly resulted in three contigs, which, when subjected to BLASTn analyses, aligned to the respective RNA segments of MeTRV1. The first contig was 6,498 nucleotides (nts)-long, 99.4% identical to RNA1 of MeTRV1 (BK064256.1), and 5,922 reads mapped to it (coverage 125x). RNA1 of MeTRV1-Wa encoded a protein 2,040 amino acid (aa)-long that aligned with protein L of MeTRV1 (DBA36559.1, 99.8%). The second contig was 4,014 nts-long and 95.2% identical to the RNA2 of MetRV1 (BK064257.1) with 1,751 reads mapping (coverage 59x). It contained four open reading frames (ORFs) encoding proteins N (445 aa, 99.8%, DBA36560.1); P2 (343 aa, 99.4%, DBA36561.1); P3 (183 aa, 99.4%, DBA36562.1); and P4 (72 aa, 98.6%, DBA36563.1). Altogether, 4,653 reads mapped to the third contig (coverage 131x) that was 4,889 nts-long and 99.1% identical to the RNA 3 segment of MeTRV1 (BK064258.1). RNA3 of MeTRV1-Wa encoded four proteins P6 (274 aa, 100%, DBA36565.1); P7 (189 aa, 99.5%, DBA36566.1); P8 (514 aa, 99 %, DBA36567.1); and P5 (303 aa, 99.7%, DBA36564.1). The 5' trailer of each RNA segment had a nearly identical 24 nts at the end. Genomic organization of the MeTRV1-Wa and the locations of its ORFs are shown in Fig.2. To confirm the virus's presence, two sets of primers were designed based on the predicted sequence of the viral RNA 3 segment. The correct-size products were amplified in RT-PCR assays with RNA extracted from infected plants (Fig.3) and verified by Sanger sequencing. Besides MeTRV1-Wa, sequences of the following viruses known to cause symptoms in alfalfa were identified in the same library alfalfa mosaic virus, bean leafroll virus, lucerne transient streak virus, and pea streak virus. Thus, the observed symptomatology may not be clearly attributed to MeTRV1-Wa due to coinfecting organisms. However, a possible association of the disease symptoms with the virus presence could be suggested based on comparison with both asymptomatic and symptomatic plants negative for MeTRV1-Wa (Fig.1). Since plant rhabdoviruses are recognized as a cause of economic losses in alfalfa and other major crops and are transmitted by insects (Bejerman et al. 2011, 2015; Jackson et al. 2005; Man and Dietzgen 2014), this first experimental confirmation of the occurrence of the new virus in the U.S. alfalfa is important for understanding its origin, distribution, and pathogenic potential.