Wheat virus identification within infected tissue using nanopore sequencing technology

Authors: Fellers, John; WEBB, CHRISTIAN - Kansas State University; FELLERS, MADISON - Kansas State University; SHOUP RUPP, JESSICA - Kansas State University; DE WOLF, ERICK - Kansas State University

Submitted to: Plant Disease
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 2/5/2019
Publication Date: 7/19/2019
Citation: Fellers, J.P., Webb, C., Fellers, M.C., Shoup Rupp, J., De Wolf, E. 2019. Wheat virus identification within infected tissue using nanopore sequencing technology. Plant Disease. https://doi.org/10.1094/PDIS-09-18-1700-RE.
DOI: https://doi.org/10.1094/PDIS-09-18-1700-RE

Interpretive Summary: Viruses cause significant loses in crops, but they are difficult to diagnose because their symptoms are similar to damage caused by nutrient deficiencies or environmental factors. This work describes new techniques for the accurate diagnosis of wheat viruses. Prior to this work, antibodies specific to each virus were used for diagnosis. Kits could be obtained and used by field scouts or plant diagnosticians. Though the kits were easy to use, accurate diagnostics were limited to kit sensitivity, virus titers, or availability of the kit. A new DNA sequencing technology has been developed that is convenient, efficient, accurate, and highly portable and is evaluated in this work for the identification of wheat viruses from suspect field samples. Four wheat samples from western Kansas were collected in 2015 and 2017 expressing symptoms similar wheat streak mosaic. However, the varieties contained a wheat streak mosaic resistance gene. Using the new harmonica sized DNA sequencer and a computer, the samples were found to contain Wheat streak mosaic virus, Triticum mosaic virus and Barley yellow dwarf virus. Also, enough sequence was obtained to suggest that a new WSMV virus strain could be responsible for the symptoms. The technique has great potential to be used in rapid diagnosis of plant viruses.

Technical Abstract: Viral diseases are a limiting factor to wheat production. These diseases are difficult to diagnose in the early stages of development and are often confused with nutrient deficiencies or other abiotic problems. Immunological methods are useful to identify viruses, but specific antibodies may not be available or require high virus titer for detection. In 2015 and 2017, wheat plants containing Wheat streak mosaic virus (WSMV) resistance gene Wsm2 were found to have symptoms characteristic of WSMV. Serologically, WSMV was detected in all four samples and High Plains Wheat mosaic virus (HPWMoV) in one sample. Barley yellow dwarf virus (BYDV) was not detected and a detection kit was not readily available for Triticum mosaic virus (TriMV). Initially, cDNA cloning and Sanger sequencing were used to determine the presence of WSMV; however, the process was time-consuming and expensive. Subsequently, cDNA was made from infected wheat tissue and sequenced with single-strand Oxford nanopore sequencing technology (NST). NST was able to confirm WSMV, but also detected TriMV and BYDV. Full length, single-strand sequence was obtained to a deep enough coverage to reveal that the collected WSMV isolates were different from the WSMV reference strain Sidney-81 and may represent a new strain that can overcome Wsm2 deployed in current varieties. These results demonstrate that NST can more accurately identify causal virus agents and also provide sufficient resolution to identify particular viral strains.