Identification of complete genome sequencing of alfalfa virus S diagnosed in alfalfa plants (Medicago sativa L.) from Washington State, USA

Authors: Nemchinov, Lev; Grinstead, Sam; Irish, Brian; Shao, Jonathan

Submitted to: Plant Disease
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 7/20/2020
Publication Date: 7/20/2020
Citation: Nemchinov, L.G., Grinstead, S.C., Irish, B.M., Shao, J.Y. 2020. Identification of complete genome sequencing of alfalfa virus S diagnosed in alfalfa plants (Medicago sativa L.) from Washington State, USA. Plant Disease. https://doi.org/10.1094/PDIS-06-20-1374-PDN.
DOI: https://doi.org/10.1094/PDIS-06-20-1374-PDN

Interpretive Summary: Alfalfa virus S has been previously identified in alfalfa samples originating from Sudan, Northern Africa and more recently in China. Here we report on the first detection and the complete nucleotide sequence of the virus isolate originating from alfalfa plants in the United States. Although pinpointing the source of the virus in alfalfa is difficult, U.S. commercial seed sources or cultivars were used in both Sudan and China. It is expected that this report will be of interest to plant pathologists and extension specialists working in the field of alfalfa improvement and to plant virologists and molecular biologists in general.

Technical Abstract: Here we report the first detection of AVS in the United States as well as the complete nucleotide sequence of the virus. The alfalfa samples were received from the USDA/ARS Plant Germplasm Introduction and Testing Research Unit, Prosser WA worksite, and originated from a commercial production field in the Prosser, WA area. Alfalfa plants displayed chlorotic symptoms with mosaic pattern of light green and dark green coloration on the leaves. Total RNA was extracted from symptomatic leaves using TRIzol Reagent. Library construction and high throughput sequencing (HTS) were outsourced and performed on an Illumina Platform by Novogene Inc. A complete nucleotide sequence of the AVS-US isolate was obtained by a reference-based assembly of the HTS reads using Qiagen CLC Genomics Workbench software version 9.5.2. and 5'/3' RACE SMARTer kit. The accuracy of the nucleotide sequence was confirmed by reverse transcription-polymerase chain reaction assays using the same RNA preparations as were submitted for HTS. At the nucleotide level, the genome of AVS-US consisting of 8,367 nt was 95.2% and 92.3% identical to AVS and AVS isolate from China (AVS-C), respectively. Amino acid identities of the six putative proteins encoded by the genome of the virus with the corresponding proteins of AVS and AVS-C varied from 96.6 to 98.9% (AVS) and from 92.8 to 96.8% (AVS-C). Analogously to AVS and AVS-C, the AVS-US isolate does not appear to have a clear 3' proximal open reading frame encoding a cysteine-rich nucleic acid-binding protein that is typical for allexiviruses. Phylogenetic analysis performed using Qiagen CLC Main Workbench 20.0 software with the predicted amino acid sequence of AVS-USA RdRp and that of other known allexiviruses, placed the three AVS isolates together in the same sub-cluster within a larger group also including Arachis pitnoi virus and Blackberry virus E. As revealed by HTS, alfalfa samples in which AVS was identified, were coinfected with other viruses including alfalfa mosaic virus, bean leafroll virus, pea streak virus, medicago sativa alphapartitivirus, medicago sativa amalgavirus along with others. This once again demonstrated that viral coinfections are common in alfalfa and may constitute a substantial part of the disease complexes. Thus far AVS has been found in three global geographic locations: Sudan, China and USA. Pinpointing the source of AVS in alfalfa is difficult. U.S. commercial seed sources or cultivars were used in both Sudan and China. However, virus genome diversity could be indicative of independent geographic origins for all three isolates.