First Report of Brassica Yellows Virus (BrYV) Naturally Infecting Wheat in China.

Abstract

Brassica yellows virus (BrYV) was first reported in rutabaga as a new species in the genus Polerovirus in China in 2011, which was closely related to turnip yellows virus (TuYV) (Zhang et al. 2014). Later, BrYV was widespread in cruciferous and tobacco plants, causing leaf malformations and yellowing (Wang et al. 2015; Zhang et al. 2022). Here, we report its presence in wheat (Triticum aestivum), identified by high-throughput sequencing. In 2023, we collected wheat plants with virus-like symptoms, such as yellow, dwarf, stripe and mosaic, from the fields in Hebei Province, China. Total RNA were extracted from symptomatic leaves using TransZol reagent (TransGen, Beijing, China). RNAs from the same field and having similar symptoms were mixed into a pooled RNA (9 pooled samples from 4 cities, 105 individual samples) to construct a rRNA-depleted RNA-seq library with the TruSeq total RNA Sample Prep Kit and a sRNA-seq library with the Small RNA Sample Prep Kit (Illumina, San Diego, CA). The Illumina Novaseq 6000 was used for the RNA- and sRNA-seq with PE150 and SE50, respectively. A total of tens of millions of clean reads were obtained from each pooled RNA. After de novo assembly using Trinity and Velvet program, and blast analysis using BLASTx and BLASTn program, contigs of four species of wheat-infecting viruses, including barley yellow dwarf virus-PAV, barley yellow striate mosaic virus, wheat yellow dwarf virus and triticum yellow stripe virus, were identified. Beet western yellows virus was also found in our samples, a polerovirus that infects dicotyledonous plants and was first report in wheat in 2023 (Jin et al. 2023). Additionally, three contigs from RNA-seq with high identity to TuYV-5594 (OQ377541) were also identified. The contig DN88565 (5,611 nt) was from the pooled BD-A, contig DN323 (5,662 nt) and DN16914 (699 nt) were from the pooled BD-C, respectively. For sRNA-seq, many short contigs from BD-A and BD-C with high identities to BrYV-R3b (LC428363) were also found. And the 22- and 21-nt BrYV-derived siRNA had the highest percentages, that consistent with BrYV-derived siRNAs in Nicotiana benthamiana (Zhou et al. 2017), which demonstrated the virus infected the wheat samples rather than contaminating them. A Blastn search online showed that the contigs were more identical to BrYV, thus we proposed the virus named as BrYV-Ta. When using contig DN323 as the reference, a total of 20,037 reads of BD-A and 16,156 reads of BD-C from RNA-seq were mapped with a coverage of 100%. For testing the incidence of BrYV-Ta, the contig-specific primers were designed to amplify a 1,444-bp fragment. The assay revealed that 3 of the 32 samples of BD-A and BD-C, from Baoding City, tested positive and no mixed infection happened. The symptoms of the positive samples were yellowing and striping. The full-length genome of BrYV-Ta was amplified by 4 overlapping pairs of primers and sequenced by Sanger sequencing. Identity analysis showed that the virus had more than 90% identity with some isolates of BrYV and TuYV, and shared the highest identity with BrYV (JN015068) from turnip in China (97.44% for genome, 97.10% for RdRp and 99.01% for CP, respectively). The genomic sequence (5,662 nt) of BrYV-Ta, was deposited in GenBank as accession PV008117. Moreover, the BrYV-Ta clustered with some BrYV isolates in the phylogenetic tree based on RdRp and CP amino acid sequences. To our knowledge, this is the first report of BrYV in wheat worldwide. This finding expends the host range of BrYV and suggests that BrYV could become a new threat to wheat due to its widespread in cruciferous plants.