Development of SCAR Markers and an SYBR Green Assay to Detect Puccinia striiformis f. sp. tritici in Infected Wheat Leaves

Abstract

Stripe rust, caused by the pathogenic fungus Puccinia striiformis f. sp. tritici, is an important disease of wheat worldwide. A rapid and reliable detection of the pathogen in latent infected wheat leaves is useful for accurate and early forecast of outbreaks and timely application of fungicides for managing the disease. Using the previously reported primer pair Bt2a/Bt2b, a 362-bp amplicon was obtained from P. striiformis f. sp. tritici and a 486-bp amplicon was obtained from both P. triticina (the leaf rust pathogen) and P. graminis f. sp. tritici (the stem rust pathogen). Based on the sequence of the 362-bp fragment, two pairs of sequence characterized amplified region (SCAR) primers were designed. PSTF117/PSTR363 produced a 274-bp amplicon and TF114/TR323 produced a 180-bp amplicon from P. striiformis f. sp. tritici, whereas they did not produce any amplicon from P. triticina, P. graminis f. sp. tritici, or any other wheat-infecting fungi. The detection limit of PSTF117/PSTR363 was 1 pg/µl and TF114/TR323 was 100 fg/µl. Both SCAR markers could be detected in wheat leaves 9 h post inoculation. An SYBR Green RT-PCR method was also developed to detect P. striiformis f. sp. tritici in infected leaves with the detection limit of 1.0 fg DNA from asymptomatic leaf samples of 6 h after inoculation. These methods should be useful for rapid diagnosis and accurate detection of P. striiformis f. sp. tritici in infected wheat leaves for timely control of the disease.

Identification of a Novel Alternaria alternata Strain Able to Hyperparasitize Puccinia striiformis f. sp. tritici, the Causal Agent of Wheat Stripe Rust

“…Because the Pst urediniospores could be dispersed over long distances by the wind, the fungal pathogen is able to cause large-scale epidemics and severe yield losses under conducive environmental conditions ( Brown and Hovmøller, 2002 ; Wan et al, 2007 ; Zhao et al, 2008 ). In China, the devastating epidemics occurring in 1950, 1964, 1990, and 2002, has caused up to 6.0 × 10 9 , 3.0 × 10 9 , 2.6 × 10 9 , and 1.0 × 10 9 kg of yield losses, respectively ( Wan et al, 2004 ; Chen et al, 2009 ; Gao et al, 2015 ). So far, cultivation of resistant varieties is the most effective way to control wheat stripe rust.…”

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

“…This primer set (PST2 f/r) was further examined in the experiment of Wang et al [2009] as outer primers in nested-PCR with inner Nesta/Nests primers. Gao et al [2016] developed 2 pairs of SCAR primers, i.e., PSTF117/ PSTR363 and TF114/TR323. These primers produced 274-and 180-bp amplicons, respectively, which could detect the pathogen on wheat leaves even a few hours after inoculation.…”

“…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].…”

Establishment of a PCR Assay for the Detection and Discrimination of Authentic Cordyceps and Adulterant Species in Food and Herbal Medicines

“…However, the authors designed the SCAR marker only for O. sinensis , which does not address the issue of the market distribution of Cordyceps and related species in Korea. Real-time PCR is used in conjunction with SCAR markers in diverse fields for the identification of the taxonomic origins of pathogens, food ingredients and products, and herbal medicines, because it is fast, easy, and quantitative [ 24 , 25 ]. Real-time PCR is widely employed, and it is considered to be very useful for the detection of adulterants in food products and herbal medicines, such as pork, fish, ginseng, etc.…”