Improved diagnostic tools to reliably detect modern wheat mosaic virus isolates

Accurate and timely diagnostics are essential for plant disease control, but one challenge is the emergence of new isolates with genomic variation, which limits the binding capabilities of PCR primers and serological antibodies. Triticum mosaic virus (TriMV) and wheat streak mosaic virus (WSMV) are two economically impactful viruses that infect wheat in the US Great Plains. We found that primers traditionally used to detect these are unreliable for Colorado isolates, with 33 of 105 samples tested between 2021-2025 with false negative discrepancies for one or both viruses. We compared all available genomes and developed new universal WSMV and TriMV primers within higher regions of conservation across these genomes. With qPCR, we found that these diagnostic primer pairs were 16% more efficient than the established primers for WSMV, and 18% more efficient for TriMV. Both were 16% more sensitive in detecting viral loads, had higher specificity for the associated viruses (E-values near zero), low specificity for the wheat genome (E-values = 1.9-7.5), and universally detected Colorado isolates. To improve throughput for multiple samples, we subsequently designed a sensitive and specific multiplex PCR bioassay to detect both viruses in a single reaction. Finally, we developed a colorimetric loop-mediated isothermal amplification (LAMP) bioassay to detect WSMV and/or TriMV, using either RNA or cDNA as the genomic template. The LAMP bioassay was reliable, specific, and highly sensitive, consistent with our PCR diagnostic results. Overall, our modified primers and new LAMP assay will serve as improved tools to reliably detect new virus isolates.