Methyljasmonate and salicylic acid contribute to the control of Tilletia controversa Kühn, causal agent of wheat dwarf bunt

Abstract

Tilletia controversa Kühn (TCK) is the causal agent of dwarf bunt of wheat, a destructive disease in wheat-growing regions of the world. The role of Meja, SA and Meja + SA were characterized for their control of TCK into roots, coleoptiles and anthers. The response of the defence genes PR-10a, Catalase, COI1-1, COII-2 and HRin1 was upregulated by Meja, SA and Meja + SA treatments, but Meja induced high level of expression compared to SA and Meja + SA at 1, 2, and 3 weeks in roots and coleoptiles, respectively. The severity of TCK effects in roots was greater at 1 week, but it decreased at 2 weeks in all treatments. We also investigated TCK hyphae proliferation into coleoptiles at 3 weeks and into anthers to determine whether hyphae move from the roots to the upper parts of the plants. The results showed that no hyphae were present in the coleoptiles and anthers of Meja-, SA- and Meja + SA-treated plants, while the hyphae were located on epidermal and sub-epidermal cells of anthers. In addition, the severity of hyphae increased with the passage of time as anthers matured. Bunted seeds were observed in the non-treated inoculated plants, while no disease symptoms were observed in the resistance of inducer treatments and control plants. Plant height was reduced after TCK infection compared to that of the treated inoculated and non-inoculated treatments. Together, these results suggested that Meja and SA display a distinct role in activation of defence genes in the roots and coleoptiles and that they eliminate the fungal pathogen movement to upper parts of the plants with the passage of time as the anthers mature.

Figure 1

Time course of PR-10a, catalase and C0I1-1 transcript expression in roots in treated inoculated (inducer + TCK), treated non-inoculated (inducer), non-treated inoculated (TCK) plants. Plants receiving no inducer of resistance or TCK inoculation were used as controls. Treated mean application of inducers of resistance and inoculated mean application of TCK are shown. The treatments within the time interval followed by the same letters are not statistically significant using Tukey’s LSD test (P < 0.05).

Figure 2

Time course of CoI1-2, HRin1 and PR-10a transcript expression in roots and coleoptiles in treated inoculated (inducer + TCK), treated non-inoculated (inducer), non-treated inoculated (TCK) plants. Plants receiving no inducer of resistance or TCK inoculation were used as controls. Treated mean application of inducers of resistance and inoculated mean application of TCK are shown. The treatments within the time interval followed by the same letters are not statistically significant using Tukey’s LSD test (P < 0.05).

Figure 3

Time course of COI1-1, CoI1-2 and HRin1 transcript expression in coleoptiles in treated inoculated (inducer + TCK), treated non-inoculated (inducer), non-treated inoculated (TCK) plants. Treated mean application of inducers of resistance and inoculated mean application of TCK are shown. Plants receiving no inducer of resistance or TCK inoculation were used as controls. The treatments within the time interval followed by the same letters are not statistically significant using Tukey’s LSD test (P < 0.05).

Figure 4

Infestation of TCK in wheat roots and coleoptiles as indicated by staining with WGA-AF 488 (for hyphae) and propidium iodide (for root cell). Plants receiving no inducer of resistance or TCK inoculation were used as controls. (a) At 1 week, hyphae excessively occupied rhizodermal and cortical cells of the roots. Presence of hyphae was visualized in treated inoculated (TCK + inducers), non-treated inoculated (TCK), and control plants. (Meja) Presence of hyphae in Meja-treated plants. Hyphae moved within the cortical cells. After penetrating the cortical cells, the hyphae moved into the endodermis and vascular bundles. (SA) Hyphae appeared in the epidermal cells of SA-treated roots. (Meja + SA) Hyphae were observed in the epidermal cells of Meja + SA-treated roots. (TCK) The epidermal structures were highly infested in the non-treated inoculated (TCK) plants compared to all other treated plants and controls. (Control) No hyphae were observed in the root structures of control plants. Scale bars = 50 µm, 50 µm, 50 µm, 25 µm and 75 µm for Meja, SA, Meja + SA, TCK and control, respectively. (b) (Meja) Presence of hyphae in Meja-treated plants. Hyphae moved within the cortical cells and penetrated the cortical cells. (SA) Long hyphae appeared on the cortical and sub-epidermal cells of SA-treated roots. (Meja + SA) Hypha were observed on cortical cells in Meja + SA-treated roots. (TCK) The sub-epidermal structures were highly infested in the non-treated inoculated (TCK) plants compared to all other treated and control plants. (Control) No hyphae were observed in the root structures of the control plants. Scale bars = 25 µm, 50 µm, 50 µm, 25 µm and 50 µm for Meja, SA, Meja + SA, TCK and control, respectively. (c) (Meja) Hyphae were not present in Meja-treated plants. (SA) In the SA treatment, small amounts of hyphae were observed. Hyphae did not wrap around the sub-epidermal cells properly. (Meja + SA) There was no presence of hyphae in Meja + SA-treated plants. (TCK) The cortical cells were ruptured in the non-treated inoculated plants, indicating that the hyphae moved from the roots to other upper parts of the inoculated control plants. (Control) No hyphae were observed into root structures in the mock plants. Scale bars = 50 µm, 25 µm, 50 µm, 25 µm and 50 µm for Meja, SA, Meja + SA, TCK and control, respectively.

Figure 5

TCK is present on the epidermal and sub-epidermal cells of the anther. WGA-AF488 appeared green in live fungal tips, while PI appeared red colour in dead anther cells. (a–c) Hyphae were located on the EPI cells (scale bars = 25 µm). (d–f) Hyphae were located on the EN cells (scale bars = 7.5 µm). (g–i) Hyphae were located on the ML cells, scale bars = 10 µm. (j–l) Hyphae were located on the PMCs (scale bars = 75 µm).

Figure 6

TCK is present on the epidermal and sub-epidermal cells of the anthers in non-treated inoculated (TCK) and treated inoculated (inducers + TCK) plants compared to control plants. WGA-AF488 appeared green in hyphae, while PI appeared red in dead anther cells. (a–c) There were no hyphae on the EPI, EN and PMCs of anthers in Meja-treated plants (scale bars = 50 µm, 25 µm and 100 µm, respectively). (d–f) There were no hyphae on the EPI, EN and PMCs of anthers in SA-treated plants (scale bars = 50 µm, 25 µm and 50 µm). (g–i) Similarly, we did not observe hyphae on the EPI, EN and PMCs in Meja + SA-treated plants (scale bars = 25 µm, 50 µm and 50 µm, respectively). (j–l) Hyphae were located on the EPI, EN and PMCs in non-treated inoculated (TCK) anthers (scale bar = 25 µm, 25 µm and 50 µm, respectively.

Figure 7

Plant height of the treated inoculated, TCK-inoculated and control plants was measured and analysed using a LSD test (Statistix 8.1 software). Twelve replicates were used for comparing the plant height of treated inoculated, control and TCK-inoculated plants. Different letters represent the statistical significance among the different treatments.