Characterization of the wheat cultivars against Tilletia controversa Kühn, causal agent of wheat dwarf bunt

Abstract

Wheat is one of the most important staple crops. Tilletia controversa Kühn is the causal agent of wheat dwarf bunt. In this study, a resistant wheat cultivar displayed significantly higher expression of pathogenesis-related genes than a susceptible cultivar at 7 days post inoculation (DPI) with T. controversa. Similarly, the expression was high in the resistant cultivar after exogenous application of phytohormones, including salicylic acid. The expression of pathogenesis-related genes, especially chitinase 4, was high in the resistant cultivar, while LPT-1 was down regulated after T. controversa infection. Callose deposition was greater in the resistant cultivar than in the susceptible cultivar at 10 DPI. Confocal microscopy was used to track the fungal hyphae in both cultivars in anther and ovary cells. The anthers and ovaries of the susceptible cultivar were infected by T. controversa at 7 and 15 DPI. There were no fungal hyphae in anther and ovary cells in the resistant cultivar until 10 and 23 DPI, respectively. Moreover, anther length and width were negatively influenced by T. controversa at 16 DPI. The plant height was also affected by fungal infection. Ultimately, resistance to T. controversa was achieved in cultivars via the regulation of the expression of defense-related and pathogenesis-related genes.

Figure 1

Quantitative real-time PCR (qRT-PCR) analysis of the transcription levels of pathogenesis-related genes in resistant (Ying18) and susceptible (WJ499) cultivars after T. controversa infection. (a) relative expression of CIPDF2.1 (b) relative expression of CIPDF2.4 (c) relative expression of phenylalanine ammonia lyase (PAL) (d) relative expression of chitinase (e) relative expression of ascorbate peroxidase (APX) (f) relative expression of polyphenoloxidase (PPO). The transcript abundances of the genes in the T. controversa-infected plants were relative to those of the control, and the significant differences were statistically analyzed based on three replications (Tukey’s test: **P < 0.01). Bars indicate the SEs.

Figure 2

Quantitative real-time PCR (qRT-PCR) analysis of the transcription levels of pathogenesis-related genes in resistant (Ying18) and susceptible (WJ499) cultivars after salicylic acid (SA) treatment. (a) relative expression of PR1a (b) relative expression of PR2 (c) relative expression of PR5 (d) relative expression of PR10. The significant differences were statistically analyzed based on three replications (Tukey’s test: **P < 0.01). Bars indicate the SEs.

Figure 3

Quantitative real-time PCR (qRT-PCR) analysis of the transcription levels of pathogenesis-related genes in resistant (Ying18) and susceptible (WJ499) cultivars after T. controversa infection. (a) relative expression of chitinase 4 (b) relative expression of lipase (c) relative expression of PR1.1 (d) relative expression of PR1.2 (e) relative expression of defensins (f) relative expression of glucanase 2 (g) relative expression of LTP-1 (h) relative expression of LTP-2. The transcript abundances of the genes in the T. controversa-infected plants were relative to those of the control, and the significant differences were statistically analyzed based on three replications (Tukey’s test: *P < 0.05; **P < 0.01). ns = non-significant. Bars indicate the SEs.

Figure 4

Callose deposition after T. controversa infection in anthers of resistant and susceptible cultivars at 10 DPI. (a) Callose deposition in the susceptible cultivar and (b) callose deposition in the resistant cultivar. Scale bars = 25 µm.

Figure 5

T. controversa location in anthers in the susceptible cultivar on 7 DPI. Propidium Iodide (PI) (red) stains the anther cell types, while Wheat Germ Agglutinin and Alexa Flour 488 conjugate (WGA-AF 488) (green) stains the fungal hyphae in the anthers. (a–c) Fungi located on EPI cell (d–f) hyphae located on EN cell (g–i) fungi located on ML cell (j–l) fungi located on PMC cell (m–o) fungi located on pollen cell (p–r) teliospores on epidermal cells. Scale bars = 25 µm (a–c, g–r), 50 µm (d–f). EPI, EN, ML, PMC, and pollen indicate the cell types during anther development.

Figure 6

Examination of T. controversa in anthers in the resistant cultivar on 15 DPI. (a–c) Examination of fungi on EPI cells (d–o) Examination of fungi on EN cells (d–f), Examination of fungi on ML cells (g–i), Examination of fungi on PMCs (j–l), Examination of fungal pollen cells. Scale bars = 100 µm (a–c), 50 µm (d–o). EPI, EN, ML, PMC, and pollen indicate the cell types during anther development.

Figure 7

Examination of T. controversa in the ovaries of resistant (Ying18) and susceptible (WJ499) cultivars at 23 and 15 DPI, respectively. PI (red) stains the anther cell types, while WGA-AF 488 (green) stains the fungal hyphae in the ovary. (a–c) No fungal hyphae were seen on the cells of the ovaries in the resistant cultivar. (d) There were no symptoms on the ovaries of the resistant cultivar. (e–g) Location of fungi on ovary cells in the susceptible cultivar. (h) T. controversa converts the grain material into millions of teliospores. Scale bars = 50 µm.

Figure 8

Level of disease incidence in resistant (Ying18) and susceptible (WJ499) cultivars to T. controversa infection. WJ499 showed 64% disease incidence, while Ying18 showed 10% disease incidence.