A new disease symptomology observed in wheat is associated with high levels of Triticum mosaic virus transcripts
Authors: Roberts Robyn , West Matthew , Fulladolsa Ana Cristina , Migliano Lukas , Mason Esten 
Published: January 2025

Wheat is an important cereal crop globally and in the United States, and is the largest crop grown by acreage in Colorado. In June 2023, we observed wheat fields displaying severe yellowing and virus-like disease symptoms in plants across seven eastern Colorado counties (Yuma, Prowers, Kit Carson, Washington, Sedgewick, Morgan, and Weld). Symptomatic plants were prominent in fields and appeared bright yellow, with ringspots, mosaic patterning, and streaking on leaves. Incidence across fields was sporadic and appeared random, with a typical cluster of two to four plants exhibiting symptoms in a particular area. To investigate the causal agent(s) of the disease, plants were first tested for six viruses common to Colorado using ELISA, including wheat streak mosaic virus (WSMV), high plains wheat mosaic virus, barley yellow dwarf virus-PAV, barley yellow dwarf virus-MAV, cereal yellow dwarf virus, and brome mosaic virus. Of the thirteen samples tested, including seven different commercial varieties and breeding lines, only one sample from Prowers county tested positive for any of these viruses (WSMV). We next tested for triticum mosaic virus (TriMV) using RT-PCR, and found that the remaining twelve samples were all positive for TriMV. The symptomatic sample testing positive for WSMV was also TriMV-positive (co-infected). Total RNA was extracted from leaves of four plants from each of three varieties: Snowmass 2.0, Amplify, and Guardian (Thermo GeneJet Plant RNA Mini Kit, K0801), and cDNA was synthesized (Promega GoScript Reverse Transcriptase, A5001). RT-PCR was performed using primers specific to the TriMV coat protein (5’- TCTGGTGCTGATCAATCTGG and 5’- CTAACGGGTACCAAACATGG). The 880bp amplified band was gel extracted (Zymoclean Gel DNA Recovery, ZD4002), and Sanger sequencing of the band showed 100% nt identity with TriMV (sequence submitted to NCBI: PQ333013). Next, TriMV transcripts were quantified in RT-qPCR (Thermo SYBR Green RT-qPCR, A25741) using published primers (Tatineni et al., 2010). Relative gene expression was compared against a positive control isolated in 2022, which was co-infected with TriMV and WSMV, which have relatively higher levels of TriMV than a single TriMV infection (Tatineni et al., 2010). All three varieties tested had higher TriMV qPCR levels compared to the positive control. At dilution zero, copies of TriMV were as follows: Amplify = 3.61 x 107, Snowmass 2.0 = 1.09 x 107, Guardian = 8.07 x 106, and Control = 1.66 x 106. This corresponds to changes of 21.7-fold, 6.5-fold, and 4.9-fold respectively, compared to the positive control. To confirm the role of TriMV in symptom development, we inoculated 10-day-old wheat seedlings (varieties Guardian and Amplify) with crude sap prepared from field samples. Inoculum was produced by grinding 1 g of field-symptomatic leaf tissue in 10 mL of inoculation buffer (20mM sodium phosphate, 1% carborundum (w/v), pH 7.2), then applied to one leaf of 16 plants using a cotton swab. Symptoms developed 21-30 days post inoculation and mimicked symptoms observed in the field. We confirmed the presence of TriMV in the symptomatic, inoculated greenhouse plants by extracting total RNA, synthesizing cDNA, and amplifying the entire coat protein region as before. This is the first report of a new disease symptomology associated with TriMV, specifically ringspots and whole-plant severe yellowing, which may indicate a shift in virus population and/or infectivity in Colorado wheat.