In this study, five wheat pathogens (Blumeria graminis, F. graminearum, Puccinia graminis var. tritici, P. striiformis and P. triticina) and six Tilletia-related fungi (Ustilago tritici, U. hordei, U. maydis, T. laevis and T. controversa, T. caries) were used to detect the specificity. Information on the pathogens listed in Table S1 . The DNA of all the strains was extracted by the CTAB method (Xu et al., 2020). Then, a NanoDrop 3300 fluorospectrometer (Biotech, USA) was used to evaluate the purity and quantitation of DNA with an OD₂₆₀/OD₂₈₀ between 1.8 and 2.0. The integrity of the DNA was examined by agarose gel electrophoresis with λDNA/HindIII labeling. Finally, the DNA were stored at −20°C for further use.

To obtain the species-specific DNA fragment, 100 ISSR primers published by the University of British Columbia (https://www.michaelsmith.ubc.ca/services/NAPS/PrimerSets) were used to amplify the DNA of all the tested pathogens. The primers were synthesized by Tsingke Biotechnology Co., Ltd. (Beijing, China), and are listed in Table 1 . ISSR-PCR was performed in a programmable optics module thermocycler (Bio-Rad, USA) with a total reaction volume of 25 μL, which included 2 μL of primer (10 μM), 1 μL of template DNA (100 ng/μL), 12.5 μL of 2 × Pro Taq Master Mix (dye plus) (Vazyme Biotech Co., Ltd., China), and 9.5 μL of ddH₂O. The PCR amplification programs were as follows: predenaturation at 94°C for 30 s; followed by 35 cycles of denaturation at 98°C for 10 s, annealing at 45–65°C (depending on the primers) for 30 s, and extension at 72°C for 1 min; and final extension step at 72°C for 2 min. The PCR products were electrophoresed on 2% agarose gel electrophoresis containing ethidium bromide at 150 V for 20 min and visualized by a gel documentation system (WSE-5200 Printgraph 2 M, ATTO, Korea).

The DNA fragment (515 bp) specific to T. caries produced by the ISSR 827 primer was isolated from the gel and purified with a TIANgel Purification Kit (TianGen Biochemical Technology Co., Ltd., China). Then, a pCloone007 Versatile Simple Vector Kit (Tsingke Biotechnology Co., Ltd., China) was used to ligate the specific DNA fragment with the pUC19 vector and transform it into chemically competent Escherichia coli DH5α cells (Tsingke Biotechnology Co., Ltd.). The cloned fragment was sequenced with primers M13F and M13R by Tsingke Biotechnology Co., Ltd. The E. coli plasmid was then extracted by a TIANprep Mini Plasmid Kit (TianGen Biochemical Technology Co., Ltd., China), and the concentration calculated by the NanoDrop 3300 fluorospectrometer (Biotech, USA) was 240 ng/μL. Based on the sequencing results, SCAR marker primers Erc19F and Erc19R were designed by Primer Premier 6 and synthesized by Tsingke Biotechnology Co., Ltd. (Beijing, China).

Five wheat pathogens and five fungi closely related to T. caries were used as controls in this study. SCAR-based PCR amplification was carried out with a total volume of 25 μL, including 1 μL DNA template (100 ng/μL), 1 μL SCAR primer Erc19F (10 μM), 1 μL SCAR primer Erc19R (10 μM), 12.5 μL of 2 × Pro Taq Master Mix II (dye plus) (Vazyme Biotech Co., Ltd., China) and 9.5 μL ddH₂O. The PCR amplification programs were as follows: predenaturation at 94°C for 30 s; followed by 35 cycles of denaturation at 98°C for 10 s, annealing at 61.5°C for 30 s, and extension at 72°C for 1 min; and final extension step at 72°C for 2 min. The PCR products were electrophoresed as mentioned above.

A series of diluted DNA concentrations (100 ng/μL, 50 ng/μL, 25 ng/μL, 10 ng/μL, 5 ng/μL, 1 ng/μL, 0.5 g/μL, 0.1 ng/μL, 50 pg/μL, 25 pg/μL, 10 pg/μL and 1 pg/μL) were used as templates for sensitivity. The amplification system, programs and agarose gel electrophoresis conditions were consistent with those mentioned above.

According to the specific DNA fragment generated by ISSR 827, qRT-PCR primers Qerc19F and Qerc19R were designed and synthesized by Tsingke Biotechnology Co., Ltd. (Beijing, China). To verify the specificity of the primers, we used 10 sets of T. laevis DNA (100 ng/μL) and 3 sets of T. caries DNA (100 ng/μL) to perform qPCR. The reaction was carried out by using an ABI 7500 real-time PCR system (Applied Biosystems, Carlsbad, CA, USA) machine. The reaction was performed in 20 μL, including 1 μL DNA, 0.4 μL Qerc19F (10 μM), 0.4 μL of Qerc19R (10 μM), 8.2 μL of nuclease-free water (TransGen Biotech, China) and 10 μl of 2 × TransStart Top Green qPCR SuperMix (+DyeI/+DyeII) (TransGen Biotech, China). The program settings were as follows: 95°C for 5 min, followed by 40 cycles of 95°C for 10 s and 58°C for 30 s. Then, a series of 10-fold diluted plasmids (2.4 pg-0.24 fg) were used as templates. Three repeats for every concentration and 2 μL of nuclease-free water (TransGen Biotech, China) were used as controls for each repeat. The reaction system was the same as mentioned above.

Based on the success of the SCAR marker and qRT-PCR for the detection of T. caries, we successfully developed ddPCR detection method. Pairs of the primers WX-F and WX-R and probe primer WX-P were synthesized by Tsingke Biotechnology Co., Ltd. (Beijing, China). The probe primer WX-FRP was generated by mixing WX-F, WX-R and WX-P at a ratio of 1:1:0.5 (10 μL of WX-F, 10 μL of WX-R, 5 μL of probe primer WX-P and 225 μL of ddH₂O). The reaction was performed in 20 µL, containing, 10 μL Prenix EX Taq (Probe qPCR) (Takara, Japan), 4 μL WX-FRP, 4 μL ddH₂O and 2 μL DNA template (240 ng/μL), was mixed with 35 μL of droplet-generating oil (186-3005, Bio-Rad, USA) and moved to a droplet-generating card (186-4007, Bio-Rad, USA) to generate droplets in a droplet generator (QX200, Bio-Rad, USA). The products were transferred to a 96-well PCR plate (Eppendorf, Germany), and ddPCR was performed in a C1000 touch thermal cycler (Bio-Rad, USA) with the following program: predenaturation at 95°C for 5 min, 40 cycles of denaturing at 95°C for 30 s, followed by 60 s of annealing at 58°C, and extension at 98°C for 10 min. The products were diluted 10-fold in 10 gradients and transferred to a droplet reader (QX200, Bio-Rad, Hercules, CA, US). Quanta Soft (Version, 1.7.4, Bio-Rad, provided with the ddPCR system) analysis was used to generate data, and the florescence amplitude threshold was set by the JavaScript program “dedinetherain” (Jones et al., 2014) to improve the accuracy and credibility of the results to follow the previous studies (Kubista et al., 2006; Jones et al., 2014). In this experiment, we tested the T. caries samples with plasmid concentrations ranging from 2.4 pg/μL to 0.24 fg/μL in 3 replicates per sample, 7 repeats of T. controversa and T. laevis, and 2 repeats of ddH₂O as negative controls.

Among the 100 ISSR primers from the University of British Columbia, ISSR 827 amplified a polymorphic profile (515 bp) in T. caries but not in the other investigated pathogens ( Figure 1 ). Primer Premier 6 was used to analyze the specific DNA sequence of T. caries, and pairs of SCAR marker primers named Erc 19F and Erc 19R were designed. The primers amplified a specific 266 bp band from the DNA of T. caries ( Figure 2 ).

Some related pathogens (B. graminis, F. graminearum, P. graminis var. tritici, P. striiformis f. sp. tritici, P. triticina, U. tritici, U. hordei, U. maydis, T. caries, T. laevis and T. controversa) were used to detect the specificity of the designed SCAR primers. The specific band (266 bp) occurred only in T. caries and in none of the other tested pathogens ( Figure 3 ). In addition, we tested the sensitivity of the SCAR marker using a series of diluted DNA template concentrations of T. caries (100 ng/μL, 50 ng/μL, 25 ng/μL, 10 ng/μL, 5 ng/μL, 1 ng/μL, 0.5 ng/μL, 0.1 ng/μL, 50 pg/μL, 25 pg/μL, 10 pg/μL, and 1 pg/μL), resulting in a sensitivity of 50 pg/μL of the Erc19F/Erc19R primer ( Figure 4 ).

Based on the specific sequence, primers Qerc19F and Qerc19R were designed to perform qRT-PCR with SYBR Green I. For the specific of the primers, 10 sets of T. laevis DNA and 3 sets of T. caries DNA were used as templates to carry out qPCR. The results showed that 10 sets of T. laevis DNA failed to amplify the target sequence, while 3 sets of T. caries DNA successfully amplified the target sequence ( Supplementary Figure S1 ). For the sensitivity of the detection, 10-fold serial dilutions of plasmids were used as templates (2.4 pg to 0.24 fg) and the detection limit concentration was 2.4 fg/μL ( Figure 5A ). Furthermore, a standard curve with correlation coefficient of the standard curve reached 0.997, suggesting successful development of the SYBR Green I qRT-PCR detection method ( Figure 5B ).

We used 10,000 droplets to increase the accuracy and reliability of the experiment. The increase in the number of positive droplets (blue points) in the DNA samples indicated a greater copy number in the ddPCR products, which suggested that the concentration of T. caries in the DNA samples was increased. In contrast, the lack of positive droplets indicated that T. caries was not detected ( Supplementary Figure S2 ). The results showed that the detection limit of ddPCR was 0.7 copies/μl (0.24 fg/μL) ( Figure 6 ). Furthermore, we conducted a statistical analysis of the number of positive droplets, and the results showed that ddPCR detection is a precise and effective method for the detection of T. caries ( Figure 7 ).