Rapid detection of Mycosphaerella graminicola in wheat using reverse transcription-PCR assay

Mycosphaerella graminicola is a prevalent and economically important fungal pathogen in wheat. Presymptomatic and accurate detection of viable pathogen structures in infected host plants is desirable for determining latent periods of epidemics and for timely fungicide application. A reverse transcription polymerase chain reaction (RT-PCR) assay was used to detect and identify M. graminicola in wheat with the specific primer set E1/STSP2R. One single fragment was amplified only from total RNA of M. graminicola and infected leaves, but not from those of healthy leaves or five other common fungal wheat pathogens. The sensitivity of one-step RT-PCR was compared with that of two-step RT-PCR. The detection limit of two-step RT-PCR was markedly lower (100 pg total RNA) than that of one-step RT-PCR (5 ng total RNA). RT-PCR was used to monitor M. graminicola disease development in inoculated wheat plants and in naturally infected F-1 leaves (the second leaf from top) during the epidemic period. RT-PCR could detect M. graminicola at least 4 days before lesions appeared in inoculated plants. After symptoms were visible on the leaves, the band intensities of the amplified RT-PCR products were in general agreement with the visual disease assessment.