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

Tongshuo Xu,Han Zhang,Sifeng Zhao,Li Gao,Wanquan Chen,Ghulam Muhae Ud Din,Taiguo Liu,Jianjian Liu,Zhaoqun Yao

doi:10.1038/s41598-020-72976-7

Tongshuo Xu, Han Zhang + Show 7 more

Open Access

https://doi.org/10.1038/s41598-020-72976-7

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Abstract

Common bunt of wheat caused by Tilletia laevis and/or T. caries (syn. T. tritici), is a major disease in wheat-growing regions worldwide that could lead to 80% or even total loss of production. Even though T. laevis can be distinguished from T. caries on the bases of morphology of teliospores using microscopy technique. However, molecular methods could serve as an additional method to quantify the pathogen. To develop a rapid diagnostic and quantify method, we employed the ISSR molecular marker for T. laevis in this study. The primer ISSR857 generated a polymorphic pattern displaying a 1385 bp T. laevis-specific DNA fragment. A pair of specific primers (L57F/L57R) was designed to amplify a sequence-characterized amplified region (SCAR) (763 bp) for the PCR detection assay. The primers amplified the DNA fragment in the tested isolates of T. laevis but failed in the related species, including T. caries. The detection limit of the primer set (L57F/L57R) was 5 ng/µl of DNA extracted from T. laevis teliospores. A SYBR Green I real-time PCR method for detecting T. laevis with a 100 fg/µl detection limit and droplet digital PCR with a high sensitivity (30 fg/µl detection limit) were developed; this technique showed the most sensitive detection compared to the SCAR marker and SYBR Green I real-time PCR. Additionally, this is the first study related the detection of T. laevis with the droplet digital PCR method.

Highlights

To date, the major detection methods of the pathogens of T. laevis and T. caries have been based on teliospore morphology[6], triacylglycerol features[7], immunological m ethods[8], polypeptide p rofiles[9] and genetic p roperties[10]
We developed a rapid and accurate method for sequence-characterized amplified region (SCAR) marker detection in T. laevis, and based on the SCAR marker, we reported that real-time PCR and droplet digital PCR with high sensitivity contribute to accurate detection
A species-specific SCAR marker of T. laevis was developed with the inter-simple sequence repeat (ISSR) technique

Summary

Introduction

The major detection methods of the pathogens of T. laevis and T. caries have been based on teliospore morphology[6], triacylglycerol features[7], immunological m ethods[8], polypeptide p rofiles[9] and genetic p roperties[10]. We developed a rapid and accurate method for SCAR marker detection in T. laevis, and based on the SCAR marker, we reported that real-time PCR and droplet digital PCR with high sensitivity contribute to accurate detection. This is the first study related to detecting the teliospore of T. laevis with the high-sensitivity ddPCR method

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Results

Conclusion