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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

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.

SUBMITTER: Xu T

PROVIDER: S-EPMC7528053 | biostudies-literature | 2020 Jan

REPOSITORIES: biostudies-literature

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Project description:Simple Summary Wheat is the staple food crop for many countries. Therefore, avoiding pathogen infection in the crops is a prerequisite for productive and sustainable agriculture. Exploring the molecular mechanisms of pathogenesis related proteins and thaumatin-like proteins is significant for control of dwarf bunt and common bunt. In the present research, a highly susceptible cultivar was used to measure the defense protein expression differences against dwarf bunt and common bunt pathogens. There were huge differences in expression of defense proteins in pathogen-infected and control libraries. Abstract Dwarf bunt and common bunt diseases of wheat are caused by Tilletia controversa Kühn and Tilletia foetida Kühn, respectively, and losses caused by these diseases can reach 70–80% in favourable conditions. T. controversa and T. foetida are fungal pathogens belonging to the Exobasidiomycetes within the basidiomycetous smut fungi (Ustilaginomycotina). In order to illuminate the proteomics differences of wheat spikes after the infection of T. controversa and T. foetida, the isobaric tags for relative and absolute quantification (iTRAQ) technique was used for better clarification. A total of 4553 proteins were differentially detected after T. controversa infection; 4100 were upregulated, and 453 were downregulated. After T. foetida infection, 804 differentially expressed proteins were detected; 447 were upregulated and 357 were downregulated. In-depth data analysis revealed that 44, 50 and 82 proteins after T. controversa and 9, 6 and 16 proteins after T. foetida were differentially expressed, which are antioxidant, plant-pathogen interaction and glutathione proteins, respectively, and 9 proteins showed results consistent with PRM. The top 20 KEGG enrichment pathways were identified after pathogen infection. On the basis of gene ontology, the upregulated proteins were linked with metabolic process, catalytic activity, transferase activity, photosynthetic membrane, extracellular region and oxidoreductase activity. The results expanded our understanding of the proteome in wheat spikes in response to T. controversa and T. foetida infection and provide a basis for further investigation for improving the defense mechanism of the wheat crops.

Project description:Wheat dwarf bunt leads to the replacement of seeds with fungal galls containing millions of teliospores of the pathogen Tilletia controversa Kühn. As one of the most devastating internationally quarantined wheat diseases, wheat dwarf bunt spreads to cause distant outbreaks by seeds containing teliospores. In this study, based on a combination of amplicon sequencing and isolation approaches, we analyzed the seed microbiome signatures of endophytes between resistant and susceptible cultivars after infection with T. controversa. Among 310 bacterial species obtained only by amplicon sequencing and 51 species obtained only by isolation, we found 14 overlapping species by both methods; we detected 128 fungal species only by amplicon sequencing, 56 only by isolation, and 5 species by both methods. The results indicated that resistant uninfected cultivars hosted endophytic communities that were much more stable and beneficial to plant health than those in susceptible infected cultivars. The susceptible group showed higher diversity than the resistant group, the infected group showed more diversity than the uninfected group, and the microbial communities in seeds were related to infection or resistance to the pathogen. Some antagonistic microbes significantly suppressed the germination rate of the pathogen's teliospores, providing clues for future studies aimed at developing strategies against wheat dwarf bunt. Collectively, this research advances the understanding of the microbial assembly of wheat seeds upon exposure to fungal pathogen (T. controversa) infection. IMPORTANCE This is the first study on the microbiome signature of endophytes in wheat seed response to wheat dwarf bunt caused by Tilletia controversa Kühn. Some antagonistic microbes suppressed the germination of teliospores of the pathogen significantly, which will provide clues for future studies against wheat dwarf bunt. Collectively, this research first advances the understanding of the microbial assembly of wheat seed upon exposure to the fungal pathogen (T. controversa) infection.

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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

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