Rapid Detection of Mycosphaerella graminicola in Wheat Using Reverse Transcription‐PCR Assay

Abstract

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.www.blackwell-synergy.com

Detection of Mycosphaerella graminicola in Wheat Leaves by a Microsatellite Dinucleotide Specific-Primer

“…The primers amplified similar-sized fragments from fungus culture grown in vitro and wheat leaves inoculated with M. graminicola . This primer pairs allowed a reliable DNA quantification at concentrations as low as 50 fg, which is very sensitive, as reported by other researchers [12,14,15]. Real-time PCR was found to be more sensitive than the standard amplification procedure, and a successful amplification was obtained with M. graminicola DNA.…”

Mycosphaerella graminicola: from genomics to disease control

“…A well tested toolbox of methods is now in place for studying the M. graminicola –wheat interaction. The fungus can be detected and measured in infected leaves and seeds by quantitative polymerase chain reaction (PCR) (Bearchell et al ., 2005; Consolo et al ., 2009; Guo et al ., 2006; Shetty et al ., 2007), which has been used to great effect in studying historical wheat samples in the Rothamsted Research Broadbank archive from 1844 to 2003 (Bearchell et al ., 2005). The study of the interaction has been aided by the use of young plants, requiring a short growth time and no vernalization, and by the use of both attached and detached leaf assays (Arraiano et al ., 2001a; Keon et al ., 2007).…”

A Real‐time PCR Assay for Quantitative and Accurate Assessment of Fungicide Effects on Mycosphaerella graminicola Leaf Blotch

“…The strain was identified by PCR using the species‐specific primers (Guo et al., 2006) and by morphological characters. Conidia were generated on malt‐yeast agar plates (Guo et al., 2005) at 22°C in light for 5 days. A conidial suspension was prepared and adjusted to 10 5 conidia/ml with sterile deionized water.…”

“…The fungicide effect was assessed for two times on 17 May (GS39) and 14 June (GS65). At each time, 30 leaves were randomly sampled from each leaf layer (from leaf 1 (flag leaf) to 5) and the disease severity was scored on a disease scale of 0–5 (scale 1: 1–10% necrotic area of total leaf area; 2: 11–20%; 3: 21–30%; 4: 31–40%, 5: more than 40%) as described previously (Guo et al., 2005). Meanwhile, the pycnidia on each leaf were counted under a light microscope.…”