Dataset Information

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

ABSTRACT: Background and Aims:The dwarf bunt disease of wheat is caused by Tilletia controversa Kühn. This pathogen is primarily involved in the stunted growth of wheat and affects seed quality. Many countries in the world have therefore imposed quarantine bans to prevent the spread of T. controversa. Morphological observations are the main method of detecting teliospores in soil. However, this is a lengthy and laborious process; this method is thus unable to quickly meet the demand for detection of teliospores in the soil. Methods:We compared PCR, real-time PCR and droplet digital PCR (ddPCR) for the qualitative and quantitative measurement of the teliospores of T. controversa in soil. Results:We suggest the use of ddPCR for detection of the soil samples, which was demonstrated to have the most sensitive detection at 2.1 copies/?L. In contract, SYBR Green I real-time PCR could detect 7.97 copies/?L of T. controversa in soil, and this sensitivity was 100 times more sensitive than that of simple PCR. Conclusion:This study was the first report using ddPCR techniques to detect T. controversa teliospores in soil with greatly enhanced sensitivity.

SUBMITTER: Liu J

PROVIDER: S-EPMC7002547 | biostudies-literature | 2020

REPOSITORIES: biostudies-literature

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Development of the Droplet Digital PCR to Detect the Teliospores of <i>Tilletia controversa</i> Kühn in the Soil With Greatly Enhanced Sensitivity.

Liu Jianjian J Li Chao C Muhae-Ud-Din Ghulam G Liu Taiguo T Chen Wanquan W Zhang Jianmin J Gao Li L

Frontiers in microbiology 20200130

<h4>Background and aims</h4>The dwarf bunt disease of wheat is caused by <i>Tilletia controversa</i> Kühn. This pathogen is primarily involved in the stunted growth of wheat and affects seed quality. Many countries in the world have therefore imposed quarantine bans to prevent the spread of <i>T. controversa</i>. Morphological observations are the main method of detecting teliospores in soil. However, this is a lengthy and laborious process; this method is thus unable to quickly meet the demand ...[more]

PMID: 32082275

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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.

Project description:Prevention of canine heartworm disease, caused by Dirofilaria immitis, relies on macrocyclic lactones for which drug resistance is now a concern. Although genetic polymorphisms have been associated with resistance in D. immitis populations, the mechanism is still not well understood. The lack of reliable in vitro assays to detect resistance is a limitation for confirming resistance. Ten single nucleotide polymorphisms (SNPs) were previously clinically validated in D. immitis resistant isolates, using the MiSeq platform. This technique although useful for research studies is expensive and does not facilitate rapid detection of these markers in small numbers of clinical samples. We developed a droplet digital PCR protocol for detecting SNPs correlating with ML resistance. Specific primers and hydrolysis probes encompassing the wildtype and mutant alleles were designed to amplify the SNP targets from genomic DNA of different D. immitis isolates. Allele frequencies were determined and the suitability of the ddPCR assay was assessed and compared with MiSeq data. The ddPCR assay accurately detected and quantified alternate nucleotides in two isolates of reference, the ML-susceptible Missouri (MO) and ML-resistant JYD-34, at the previously identified SNP positions. The presence of the SNPs was also determined in additional isolates with known or putative susceptible or resistant phenotypes. We observed SNP1 and SNP2 are more predictive markers and appear suitable for rapid detection and monitoring of drug resistance. Our results suggested that ddPCR could be employed to distinguish infection due to actual genetic resistance from infection with susceptible parasites and also for rapid detection of isolates not only with ML susceptible and resistant genotypes but also mixed genotypes that correspond to heterogeneous isolates containing a mixed population of ML susceptible and resistant parasites. DdPCR may be a useful tool for conducting surveys, or assessments of individual isolates, for genetic evidence of resistance or developing resistance.

Dataset Information

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

Publications

Development of the Droplet Digital PCR to Detect the Teliospores of <i>Tilletia controversa</i> Kühn in the Soil With Greatly Enhanced Sensitivity.

Similar Datasets

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