Development of real-time PCR and droplet digital PCR based marker for the detection of Tilletia caries inciting common bunt of wheat.

This is the first study reporting droplet digital PCR and quantitative real time PCR for detection of Tilletia caries (syn. T. tritici), which causes common bunt of wheat and leads to yield losses of 80% in many wheat growing areas worldwide. To establish an accurate, rapid and quantifiable detection method, we tested 100 inter simple sequence repeats (ISSR) primers and obtained a species-specific fragment (515 bp) generated by ISSR 827. Then, a specific 266 bp band for the sequence characterized amplified region (SCAR) marker was produced from T. caries. The detection limit reached 50 pg/µL. Based on the SCAR marker, we further developed a higher sensitivity of quantitative real time-polymerase chain reaction (qRT-PCR) with a detection limit of 2.4 fg/µL, and droplet digital PCR (ddPCR) with a detection limit of 0.24 fg/µL. Both methods greatly improved the detection sensitivity of T. caries, which will be contribute a lot for quickly and accurately detection of T. caries, which causes wheat common bunt.

Metabolomics analysis of grains of wheat infected and noninfected with Tilletia controversa Kühn.

Dwarf bunt caused by the pathogen Tilletia controversa Kühn is one of the most serious quarantine diseases of winter wheat. Metabolomics studies provide detailed information about the biochemical changes at the cell and tissue levels of plants. In the present study, a liquid chromatography/mass spectrometry (LC/MS) metabolomics approach was used to investigate the changes in the grain metabolomics of infected and noninfected with T. controversa samples. PCA suggested that T. controversa-infected and noninfected samples were separated during the interaction. LC/MS analysis showed that 62 different metabolites were recorded in the grains, among which a total of 34 metabolites were upregulated and 28 metabolites were downregulated. Prostaglandins (PGs) and 9-hydroxyoctadecadienoic acids (9-HODEs) are fungal toxin-related substances, and their expression significantly increased in T. controversa-infected grains. Additionally, the concentrations of cucurbic acid and octadecatrienoic acid changed significantly after pathogen infection, which play a large role in plant defense. The eight different metabolic pathways activated during T. controversa and wheat plant interactions included phenylalanine metabolism, isoquinoline alkaloid biosynthesis, starch and sucrose metabolism, tyrosine metabolism, sphingolipid metabolism, arginine and proline metabolism, alanine, aspartate, and glutamate metabolism, and tryptophan metabolism. In conclusion, we found differences in the metabolic profiles of wheat grains after T. controversa infection. To our knowledge, this is the first study to evaluate the metabolites in wheat grains after T. controversa infection.