To determine

adhesion ability, the total number of germlings incubated for 24 h in the circles was first counted under the microscope and then washed by dipping in distilled water 100 times vertically to remove the detached infection structures. Subsequently, the remaining germlings in the corresponding circle were counted again. Adhesion ability was assessed by the percentage of the number of the germlings that remained in comparison with the number before washing. All experiments were repeated three times. Droplets of M. oryzae Br48 spore suspension and enzymes (20 μL each) were inoculated on wheat leaves and placed in the dark in a moistened box at 25 °C. Six hours after incubation, the inoculated seedlings were gently washed with running water. The seedlings were incubated for a further 3 days and symptoms were observed. Disease symptoms

Selleckchem AZD8055 click here were evaluated by the severity of the inoculated spot as follows: 5 – typical spore suspension lesion (control), 4 – 70% of control, 3 – 50% of control, 2 – 20% of control, 1 – 10% of control, 0 – no symptoms. Experiments were repeated three times. For SEM, droplets of a 20-μL spore suspension were inoculated on wheat leaves and from 6 to 24 hpi the droplets were replaced by each enzyme solution (20 μL) and the seedlings incubated in an environment-controlled room with fluorescent lighting at 25 °C up to 25 hpi. The inoculated seedlings were then gently washed under running water. The washed leaves were cut to approximately 1 × 1 cm and fixed with a freeze-drying method (Nemoto et al., 1992). The specimens were placed in a freeze-drying copper container (Nissin EM) that was designed for fungi and the container submerged in liquid nitrogen until its surface was completely frozen. The container with specimen was placed in a freeze-drying machine (Nissin EM) to evaporate the ice crystals of container completely. The specimens were retrieved from

the container and fixed with Tryptophan synthase osmium tetroxide vapor for 2 h. Subsequently, the specimens were coated with platinum by an ion-sputtering device (E-1010; Hitachi), and three pieces of leaf were observed in every treatment (200 germlings or vestiges of the presence of the germlings were evaluated for each leaf) with SEM (S-3500N; Hitachi). The spores were incubated on plastic substrates for 0, 1, or 6 h, and each sample then subjected to treatment with each enzyme. In the enzyme treatments at 0 hpi, most of the spores germinated on the substrate (data not shown). However, appressorium formation was significantly inhibited (<50%) by the treatment with β-1,3-glucanase, α-mannosidase, β-mannosidase, lipase, α-chymotrypsin, pepsin, pronase E, trypsin, and collagenases (crude, type I type 4, type V, and type N-2), and was moderately inhibited (65–75%) by the treatment with protease or gelatinase B (Fig. 1).