A reproducible methodology for absolute viral quantification and viability determination in mechanical inoculations of wheat streak mosaic virus

Authors: RANABHAT, NAR - Kansas State University; BRUCE, MYRON - Kansas State University; Fellers, John; RUPP, JESSICA L.S - Kansas State University

Submitted to: Tropical Plant Pathology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 3/25/2022
Publication Date: 4/12/2022
Citation: Ranabhat, N.B., Bruce, M.A., Fellers, J.P., Rupp, J. 2022. A reproducible methodology for absolute viral quantification and viability determination in mechanical inoculations of wheat streak mosaic virus. Tropical Plant Pathology. https://doi.org/10.1007/s40858-022-00507-y.
DOI: https://doi.org/10.1007/s40858-022-00507-y

Interpretive Summary: Wheat streak mosaic virus (WSMV) is a common wheat virus causing significant wheat production losses in the US. Cultural practices and genetic resistance are the main methods for the management of the virus. Resistant varieties are limited in number and it is difficult to identify and develop new WSMV-resistant wheat varieties. Wheat is screened for virus resistance by inoculation with infected plant sap and resistance level is evaluated based on expressed foliar symptoms. However, information about how long the virus is active in sap and how to accurately measure the number of virus particles in the sap and in infected plants is needed. This manuscript describes methods of measuring active virus in sap and in infected wheat plants over time. We found that the symptoms expressed by infected plants and the number of virus particles are only loosely correlated. Thus, the measurement of virus particle number is the best means of getting accurate results and determining viral resistance when selecting wheat varieties in breeding programs.

Technical Abstract: Wheat streak mosaic virus (WSMV) is a common wheat virus causing economic losses to production in the Great Plains of North America. Reproducible inoculation of WSMV by mechanical methods is essential to evaluate the resistance in breeding lines and relies on successful inoculation and infectivity of the virus particles. We used reverse transcription quantitative PCR (RT-qPCR) for absolute quantification of viral genome copy number in both WSMV inoculum and in infected wheat leaves. A time-course study was designed to determine the viability of WSMV in inoculum over time as well as the examination of the copy number related to the phenotypic rating scale. In the phosphate inoculation buffer, WSMV was stable with average viral genome copy number 1.86 × 10^6 ± 4.85 × 10^5. Plants inoculated with this inoculation buffer using finger rub mechanical inoculation contained WSMV genome copy numbers in the infected leaves ranging between 2.66 × 10^4 and 4.69 × 10^6 at 21 to 28 days’ post-inoculation. Viral copy numbers in wheat leaves inoculated at different times were statistically similar with leaves inoculated at 0 h. There was a weak linear relationship between phenotypic rating score and copy number in infected leaves with the linear model explaining 40% of the variability (R^2 = 0.40). This work describes an accurate methodology to quantify virus concentration in the inoculum and infected plants, as well as empathize the demand of absolute measurement of virus load to validate the subjective assessment for unbiased viral disease assessment