Development of ISSR-derived SCAR Marker and SYBR Green I Real-time PCR Method for Detection of Teliospores of Tilletia laevis Kühn

Abstract

Common bunt, caused by Tilletia laevis Kühn [syn. T. foetida (Wallr) Liro] and Tilletia tritici (Bjerk.) Wint. [syn. T. caries (DC) Tul.], is an important wheat disease worldwide. To quickly differentiate the closely related fungi T. laevis, T. tritici and Tilletia controversa (a pathogen that causes dwarf bunt of wheat and has been requested as a quarantined pathogen in many countries), a rapid diagnostic and detection method for an ISSR molecular marker was developed for the first time in this study. Based on the T. laevis-specific band (1300 bp) amplified by the primer ISSR860, a pair of SCAR primers (L60F/L60R) was designed to amplify a specific 660-bp DNA fragment from the isolates of T. laevis but not other related pathogens. The detection limit of the SCAR marker was 0.4 ng/μl of DNA from T. laevis; moreover, a SYBR Green I real-time PCR method was also successfully developed based on the SCAR marker with the detection limit of 10 fg/μl T. laevis DNA. This is the first report of a rapid, specific and highly sensitive SCAR marker and SYBR Green I real-time PCR method for detection of the teliospores of T. laevis based on ISSR technology. This method allows highly efficient, rapid and accurate differentiation of the pathogen from related pathogens, especially from the very similar pathogens T. tritici and T. controversa.

Development of the Droplet Digital PCR to Detect the Teliospores of Tilletia controversa Kühn in the Soil With Greatly Enhanced Sensitivity

“…Currently, molecular techniques to differentiate T. controversa from other similar Tilletia species are mainly focused on genetic diversity (Pimentel et al, 1998;Pimentel, 2000), PCR (Kochanová et al, 2004), repetitive extragenic palindromic PCR (Rep-PCR) (McDonald et al, 2000;Zupunski et al, 2011), primer-mediated asymmetric-PCR (RM-PCR) together with SYBR Green I and Taqman real-time PCR was also constructed with the detection limit of 0.1 fg and 1.0 fg, respectively (Yuan et al, 2009). Pieczul et al (2018) reported to identify Tilletia spp.…”

Development of droplet digital PCR for the detection of Tilletia laevis, which causes common bunt of wheat, based on the SCAR marker derived from ISSR and real-time PCR

“…Recent advances in molecular detection and quantification have showed that standard PCR, SCAR markers and real-time PCR are highly efficient for pathogen detection 16 . Yao et al developed a SCAR marker for T. laevis with a detection limit of 0.4 ng/μl of DNA from T. laevis, and a SYBR Green I real-time PCR method was also successfully developed based on the SCAR marker with a detection limit of 10 fg/μl T. laevis DNA 20 . In this study, the sensitivity of real-time PCR was 100 fg/µl, which was much more sensitive than that of traditional PCR detection methods (5 ng/µl).…”

“…The specificity of the SCAR marker was determined with genomic DNA based on three factors 20 , excluding the genomic DNA of T. laevis . First, DNA from the fungal species that shared similarity with T. laevis, including T. controversa and T. caries , was used.…”

“…Compared to common PCR, real-time PCR is better with a high degree of sensitivity, specificity, repeatability, and reliability [19][20][21] , and it does not need to run gels after the reaction, save and time and eliminate the possibility of contamination. Real-time PCR is very popular in high throughput detection and quantification 22 .…”

“…Until now, Zhang et al developed an AFLP-derived SCAR marker (286 bp) for T. laevis, but they only tested a limited number of similar strains and did not mention the detection limit of the SCAR marker 19 . Yao et al developed an ISSR-derived SCAR marker (660 bp) for T. laevis with a detection limit of 0.4 ng/μl of DNA from T. laevis, and they also developed a SYBR Green I real-time PCR method based on the SCAR marker with a detection limit of 10 fg/μl of T. laevis DNA 20 . In this study, we developed a rapid and accurate method for SCAR marker detection in T. laevis, and based on the SCAR marker, we also reported that real-time PCR and droplet digital PCR with high sensitivity contribute to accurate detection.…”