First report of Cocksfoot mottle virus infecting wheat in Estonia

On 4th July 2022, samples of organically grown winter wheat (Triticum aestivum cv. Edvins), showing symptoms of possible virus infection were collected in Nõmavere, Jõgeva county, Estonia (58°38'21.3“N 25°51'48.4”E). The symptoms included chlorotic mottling, stunting of the plants and a reduction in head size (Figure 1). Diseased plants were seen around the edge of the field. The field has been grassland for the previous three years. Total RNA was extracted from a pool of seven field-collected wheat leaves using Trizol (Thermo Fisher Scientific, USA) according to the manufacturer´s protocol. Library construction and high throughput sequencing of ribo-depleted total RNA was performed on an Illumina Platform by Novogene Inc., USA. Virus identification was achieved through a reference-based assembly of the 150 bp paired-end reads using Geneious Prime software version 2023.1.2 (Biomatters Ltd., New Zealand). Overall, 11.8M out of 38.2M reads mapped to a reference sequence of Cocksfoot mottle virus (CfMV; GenBank Accession No. NC_002618.2). The assembled complete genome of the CfMV-Estonian isolate (OQ974933), consisting of 4,081 nt, shared the highest identity (97.1%) with a CfMV isolate from Japan (AB040447) according to a BLASTn search. The presence of CfMV in the original RNA sample was confirmed by RT-PCR using the primer pair CfMV3010F (5´-AAGCTTGGAGATGGAGCTAA-3´) and CfMV3940R (5´-TGGAGTTAGATTTCCAAGTG-3´) which amplified a product of the expected size (931 bp). The infectivity of the field-collected leaf material was confirmed by mechanical inoculation to spring wheat cv. Zebra plants at the three-leaf stage. Out of 16 inoculated plants, six exhibited foliar mottling two-three weeks post inoculation. The mottling symptoms were most prominent four-five weeks post inoculation and were accompanied by stunted growth (caused by shortened internodes) and reduced head size. These symptoms resembled those observed in field-infected wheat plants. CfMV is a single-stranded, positive-sense RNA virus in the genus Sobemovirus, family Solemoviridae. It was first reported in England causing yellow and necrotic streaking and mottling of cocksfoot (Dactylis glomerata), leading to the decline of infected crops in the third and fourth year. The virus was shown experimentally to be transmittable to oat and barley, resulting in mild mottling, and to wheat, where the infection caused severe mottling, stunting and death within six-eight weeks when infected at the seedling stage (Serjeant, 1967). Under experimental conditions, both adults and larvae of the cereal leaf beetles Oulema melanopa and O. lichenis, acquired CfMV and transmitted the infection to wheat seedlings. CfMV has since been detected in cocksfoot pastures in Europe (France, Germany, Norway, Poland & Russia), Asia/Oceania (Japan, New Zealand) and North America (Canada and USA) (Alderman et al., 2016; Trzmiel & Jeżewska, 2017). The only previous report of a natural CfMV infection in wheat is from Ohio, USA (Hodge et al., 2018). This is the first report of Cocksfoot mottle virus infecting wheat in Estonia. The detection of CfMV infection in wheat provides evidence of the risk of transmission to cereal crops under favourable circumstances. However, CfMV has been detected only once in our cereal virus surveys during the last ten years. This study is part of a research project was funded through the Euphresco network for phytosanitary research coordination and funding 2021-A-374.