ITRAQ-Based Proteomic Analysis of Wheat (Triticum aestivum) Spikes in Response to Tilletia controversa Kühn and Tilletia foetida Kühn Infection, Causal Organisms of Dwarf Bunt and Common Bunt of Wheat.

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.

Characterization of the microbial communities in wheat tissues and rhizosphere soil caused by dwarf bunt of wheat.

Dwarf bunt of wheat, which is caused by Tilletia controversa J.G. Kühn, is a soil-borne disease which may lead up to an 80% loss of yield together with degradation of the quality of the wheat flour by production of a fishy smell. In this study, high-throughput sequencing technology was employed to characterize the microbial composition of wheat tissues (roots, spikes, first stem under the ear, and stem base) and rhizosphere soil of wheat varieties that are resistant and susceptible to T. controversa. We observed that the soil fungal community abundance and diversity were higher in resistant varieties than in susceptible varieties in both inoculated and uninoculated wheat, and the abundances of Sordariomycetes and Mortierellomycetes increased in the resistant varieties infected with T. controversa, while the abundances of Dothideomycetes and Bacteroidia increased in the susceptible varieties. Regarding the bacteria present in wheat tissues, the abundances of Chloroflexi, Bacteroidetes, Gemmatimonadetes, Verrucomicrobia and Acidobacteria in the ear and the first stem under the ear were higher than those in other tissues. Our results indicated that the abundances of Sordariomycetes, Mortierellomycetes, Leotiomycetes, Chryseobacterium and Massilia were higher in T. controversa-infected resistant varieties than in their controls, that Dothideomycetes, Bacteroidia, Nocardioides and Pseudomonas showed higher abundances in T. controversa-infected susceptible varieties, and that Curtobacterium, Exiguobacterium, Planococcus, and Pantoea may have higher abundances in both T. controversa-infected susceptible and resistant varieties than in their own controls.

Characterization of histological changes at the tillering stage (Z21) in resistant and susceptible wheat plants infected by Tilletia controversa Kühn.

BACKGROUND: Dwarf bunt, which is caused by Tilletia controversa Kühn, is a soilborne and seedborne disease that occurs worldwide and can lead to 70% or even total losses of wheat crops. However, very little information is available about the histological changes that occur in dwarf bunt-resistant and dwarf bunt-susceptible wheat plants at the tillering stage (Z21). In this study, we used scanning electron microscopy and transmission electron microscopy to characterize the histological changes at this stage in resistant and susceptible wheat cultivars infected by T. controversa. RESULTS: Using scanning electron microscopy, the root, stem, and leaf structures of resistant and susceptible cultivars were examined after T. controversa infection. The root epidermal and vascular bundles were more severely damaged in the susceptible T. controversa-infected plants than in the resistant plants. The stem cell and longitudinal sections were much more extensively affected in susceptible plants than in resistant plants after pathogen infection. However, slightly deformed mesophyll cells were observed in the leaves of susceptible plants. With transmission electron microscopy, we found that the cortical bundle cells and the cell contents and nuclei in the roots were more severely affected in the susceptible plants than in the resistant plants; in the stems and leaves, the nuclei, chloroplasts, and mesophyll cells changed significantly in the susceptible plants after fungal infection. Moreover, we found that infected susceptible and resistant plants were affected much more severely at the tillering stage (Z21) than at the seedling growth stage (Z13). CONCLUSION: Histological changes in the wheat roots, stems and leaves were much more severe in T. controversa-infected susceptible plants than in infected resistant plants at the tillering stage (Z21).

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.

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.

First Report of Wheat Common Bunt Caused by Tilletia laevis in Henan Province, China.

Wheat common bunt is a serious disease that may lead to yield losses of 75-80% in many wheat regions of the world (Mathre 1996). The disease may reduce yield and flour quality by producing trimethylamine, a compound that smells like rotting fish (Castlebury et al. 2005; Hoffmann 1981; Mathre 1996). Two closely related basidiomycete species, Tilletia caries (DC.) Tul. & C. Tul. [syn. T. tritici (Bjerk.) Wint.] and T. laevis J. G. Kühn [syn. T. foetida (Wallr.) Liro], cause wheat common bunt. Teliospore morphology is used to differentiate the two species. Teliospores of T. caries have reticulates on the surface while teliospores of T. laevis have a smooth surface (Pieczul et al. 2018). T. laevis was reported in Liaoning, Shaanxi, Shandong, Beijing, Hebei, Shanxi, Jilin, Heilongjiang, Jiangsu, Gansu, Xinjiang, Sichuan, Yunnan, Inner Mongolia, and Tibet (Guo 2011; Wang 1963), but not in Henan, the biggest wheat production province in China, before the present study. In July 2019, we found wheat common bunt in three fields grown with cultivar Zhengmai 618 in Yugong Mountain, Henan province. The diseased wheat heads had bunt balls filled with black powder with fishy smell. The disease incidences in these fields were 20-50%, but no common bunt was found in other nearby fields. About 200 diseased heads were sampled from the three fields. Teliospores from each head were observed under a microscope, and they all had smooth surface. Observations using a scanning electron microscope also showed smooth-surfaced teliospores. Teliospores were measured 13.5 to 18.5 µm in diameter. After surface sterilization of diseased heads using 0.25% NaClO for 5 min, teliospore suspension (1×106/ml) was made using sterilized distilled water and spread on water agar (200 µl per plate), and the plates were kept at 15°C with 24 h light (Goates and Hoffman 1987). On the 6th days, teliospores were germinated. Based on the disease symptoms, teliospore morphology, and germination, the bunt fungus was identified as T. laevis. To fulfill Koch's postulates, 1 ml of germinating teliospore suspension at the concentration of 106 spores/ml was injected into the heads of susceptible wheat cultivar (Dongxuan 3) at the boot stage with a syringe, and the plants injected with sterile ddH2O were used as control. The inoculated plants were grown in a growth chamber at 17°C with 50% humidity and 24 h light (300 µmol/m2/s). After one month at the ripening stage, the kernels of the inoculated plants were filled with black teliospores releasing fishy smell, and the control plants did not have bunt heads. Under a scanning electron microscope, teliospores from the inoculated heads had smooth surface and were measured 13.5 to 18.5 µm in diameter, similar to the teliospores of bunt heads from the fields. The fungus was also confirmed through molecular characterization using sequence characterized amplification region (SCAR) markers specific for T. laevis, and the expected 660 bp (Yao et al., 2019) and 286 bp (Zhang et al. 2012) bands were obtained separately from the teliospore samples from both the fields and growth chamber. The collection named as CGMCC 3.20112 was deposited in China General Microbiological Culture Collection Center. To the best of our knowledge, this is the first report of T. laevis causing wheat common bunt in Henan Province of China. Because the pathogen is seedborne and soilborne, the disease may become a high risk to wheat production in Henan and other provinces of China.