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

Abstract

AbstractWheat 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 numbers 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 copy number related to the phenotypic rating scale. In the phosphate inoculation buffer, WSMV was stable with average viral genome copy number 1.86 × 106 ± 4.85 × 105. Plants inoculated with this inoculation buffer using finger rub mechanical inoculation contained WSMV genome copy numbers in the infected leaves ranging between 2.66 × 104and 4.69 × 106at 21 to 28 days post-inoculation. Viral copy numbers were statistically similar between leaves inoculated immediately and those inoculated at later time points. 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 (R2 = 0.40) indicating the difficulty in disease assessment based solely on phenotypic symptoms. This work describes an accurate methodology to quantify virus concentration in the inoculum and infected plants, as well as emphasizes the demand for absolute measurement of virus load to validate the subjective assessment for unbiased viral disease assessment.