Detection and Discrimination of Wheat Spindle Streak Mosaic Virus and Wheat Yellow Mosaic Virus Using Multiplex RT-PCR

Abstract

Wheat spindle streak mosaic virus (WSSMV) and wheat yellow mosaic virus (WYMV) are two closely related bymoviruses which cause significant yield losses in wheat. There is no molecular diagnostic protocol available for either virus nor are serological methods able to discriminate them. A multiplex reverse transcription-polymerase chain reaction (RT-PCR) protocol was developed for their detection and discrimination. Twenty-three isolates of WSSMV and WYMV were collected from a range of countries and the sequence of 834–837 nucleotides of the coat protein gene of three representative WSSMV isolates and one WYMV isolate was determined. This sequence data provided further evidence that the two viruses are distinct species and was used together with previously published sequence data to design specific oligonucleotide primers to discriminate the two viruses. Using total RNA isolated from eighteen WSSMV and five WYMV isolates in either one-step or two-step RT-PCR, these primers were shown to detect and discriminate the two viruses reliably. The primers were specific to either WSSMV or WYMV and no PCR product was obtained with either soil-borne wheat mosaic virus, which is frequently associated with both diseases, or with the closely related viruses, barley yellow mosaic virus, barley mild mosaic virus or oat mosaic virus. This new diagnostic protocol will enable more effective management of the diseases caused by these viruses by enabling correct identification of the causal pathogen and earlier detection than is possible by serological methods.