Evaluating Rice Blast Fungus Virulence

For spray inoculation, conidial suspension (10 ml) containing Tween 20 (250 ppm) and conidia harvested from 12-day-old cultures on OMA plate (1–5×10⁵ conidia/ml) was sprayed onto four-weeks old susceptible rice seedlings (Oryza sativa cv. Nakdongbyeo). Inoculated plants were placed in a dew chamber at 25°C for 24 hours in the dark, and then transferred back to the growth chamber with a photoperiod of 16 hours using fluorescent lights [74] . Disease severity was assessed at seven days after inoculation. The %DLA was recorded to permit more accurate evaluation of the virulence of the mutants. Photographs of diseased rice leaves including eight centimeter long leaf blades were taken. The number of pixels under lesion areas and healthy areas of diseased leaves was calculated by Axiovision image analyzer with the photographs. For microscopic observation of penetration and infectious growth on rice tissue, excised rice leaf sheath of Nakdongbyeo were prepared as previously described [30] (link),[42] and inoculated by conidia suspension (1×10⁴ conidia/ml) on the adaxial surface. After 24, 48 and 96 hours incubation in a moistened box, the sheaths were trimmed to remove chlorophyll enriched plant parts. Remaining epidermal layer of mid vein (three to four cell layers thick) were utilized for microscopic observations. Inoculation on onion epidermis was performed as previously described [75] (link). Fixation and aniline blue staining of rice sheath and onion epidermis were performed as previously described [75] (link). Samples were incubated in lactophenol at room temperature for 1hour and directly mounted with 70% glycerin or transferred into 0.01% aniline blue for 1hour and destained with lactophenol. For 3, 3′-diaminobenzidine (DAB, Sigma, D-8001) staining, samples were incubated in 1mg/ml DAB solution (pH 3.8) at room temperature for 8 hours and destained with clearing solution (ethanol∶acetic acid = 94∶4, v/v) for 1 hour. For observation and scoring penetration rate and IH development, conidia suspension were dropped on onion epidermis and incubated for 72 hours in moistened culture plate. Samples were fixed and stained as rice sheath described above. Extensive IH from single appressoria with no (or scatterd) callose were scored as normal IH, relative short and attenuated IH with accumulated callose were scored as retarded IH, appressorium developing very short IH or penetration peg with strong callose were scored as blocked IH, and appressorium without IH and callose deposition were scored no penetration.