Presymptomatic and quantitative detection ofMycosphaerella graminicoladevelopment in wheat using a real-time PCR assay

Abstract

Presymptomatic and accurate diagnosis of Mycosphaerella graminicola leaf blotch is desirable for the disease prediction and the timely application of fungicides. To develop a sensitive PCR assay, four specific primer pairs were designed. They were more specific than three known specific primer pairs. Three of them could detect as little as 0.5 pg M. graminicola DNA in a conventional PCR. A real‐time PCR assay was applied for monitoring the disease progression in both inoculated and naturally infected wheat plants using the primer pair ST‐rRNA F/R. In inoculated plants, M. graminicola DNA could be detected immediately after inoculation and a steady increase was detected before visible symptoms appeared at 8 days. The rapid growth period took place between 6 and 16 days postinoculation. In the field, the disease progression in the top three leaf layers was followed during the epidemic period. The results were significantly correlated to the disease indices (R=0.8986) and also to the number of pycnidia per leaf (R=0.9227). These suggest that the real‐time PCR assay is a reliable approach for the presymptomatic and accurate detection of M. graminicola development in the field.

A Review of Conventional PCR Assays for the Detection of Selected Phytopathogens of Wheat

“…This is mainly caused by the relatively low fungal load in environmental samples and the structure of the fungal cell wall, which makes its disruption for nucleic acid extraction difficult [Leite et al, 2012]. For example, the detection limit for DNA extracted from M. graminicola cultures was 100 pg/μL using conventional PCR, whereas it was only 50 fg/μL by qPCR; thus qPCR assay was 20-fold more sensitive than conventional PCR [Guo et al, 2006]. PCR modification involving probe hybridization with fluorescent dye is known as fluorescent amplificationbased specific hybridization (FLASH-PCR).…”

“…There are many detection systems for F. sporotrichioides , P. striiformis f. sp. tritici , and Z. tritici [Beck and Ligon, 1995;Consolo et al, 2009;Demeke et al, 2005;Fraaije et al, 1999Fraaije et al, , 2001Gao et al, 2016;Guo et al, 2006;Konstantinova and Yli-Mattila, 2004;Lihua et al, 2008;Niessen et al, 2004;Wang et al, 2008Wang et al, , 2009Wilson et al, 2004;Zhao et al, 2007]. Particularly for Z. tritici , the number of known diagnostic tests is justified by the significance of STB, one of the most important foliar diseases of wheat caused by this fungus [Keon et al, 2007;Shetty et al, 2007].…”

“…Similar to the ITS1/JB446 primer pair, both assays introduced by Fraaije et al [1999Fraaije et al [ , 2001 finally proved to give multiple bands from some other wheat pathogens [Guo et al, 2006]. Guo et al [2006] proposed 4 newly designed primer pairs amplifying fragments of rDNA, the act1 gene, and RAPD fragment R5870-2. Primer pairs were tested with isolates of Z. tri tici , other fungal pathogens, and healthy and inoculated plants.…”

“…In justified cases it is necessary to validate the assay against samples from different hosts and various countries. Errors at this stage usually lead to misidentifications reported in some studies [Guo et al, 2006;Parry and Nicholson, 1996]. Every PCR method developed in silico requires testing on healthy plant material, isolates of closely related species, and/or environmental samples containing communities of thousands of fungal species [Beck and Ligon, 1995].…”

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

“…Specific PCR tests have been developed for identification of M. graminicola in wheat [12–15]. A PCR system in a fluorescent amplification-based specific hybridization (FLASH) format was developed for the detection and identification of M. graminicola [16].…”