Table 1.
Effects of AA and BHAM at inhibitory concentrations on the MICs of PCZ and ICZ against R. oryzae spores in RPMI 1640 medium.
Figure 1.
Intracellular ROS accumulation in R. oryzae germlings treated with PCZ or ICZ plus the mitochondrial inhibitors AA and BHAM, germlings treated with only AA and BHAM, and untreated control germlings stained with DHR-123 detected using fluorescence microscopy and a spectrophotometer.
(A) Staining with DHR-123 showing increased red fluorescence. DIC, differential interference contrast. (B) Relative fluorescence of cells stained with DHR-123. The fluorescence was detected with excitation at 500 nm and emission at 550 nm. (C) SOD activity of treated and untreated control R. oryzae cells. P values for this and subsequent figures are denoted as follows: *, p<0.05, **, p<0.001, and ***, p<0.0001, while NS, p>0.05, compared to AA+BHAM treated controls. The experiments were performed in triplicate and repeated three times. Error bars indicate standard deviations for this and subsequent figures.
Figure 2.
Changes in ΔΨm and plasma membrane depolarization in R. oryzae germlings triggered by treatment with PCZ or ICZ combined with AA and BHAM.
(A) Stains of germlings with Rh-123 showing increased green fluorescence, indicating loss of ΔΨm. (B) Staining of germlings with DiBAC showing increased green fluorescence, indicating loss of viability owing to increased membrane permeability. DIC, differential interference contrast. (C) Relative fluorescence of germlings stained with Rh-123. (D) Percentages of R. oryzae germlings stained with DiBAC. *, p<0.05, ***, p<0.0001, while NS, p>0.05, compared to AA+BHAM treated controls.
Figure 3.
Effect of treatment with PCZ or ICZ combined with AA and BHAM on cyt c release from mitochondria in R. oryzae cells.
Treatment with PCZ or ICZ in combination with AA and BHAM produced lower cyt c levels in mitochondria than did treatment with AA and BHAM alone. Release of cyt c to the cytosol was assessed by measuring the absorbance at 550 nm with a Beckman DU-640 spectrophotometer.
Figure 4.
Effect of treatment with PCZ or ICZ in combination with AA and BHAM on caspase-like activity in R. oryzae germlings as determined using FITC-VAD-FMK probe.
(A) Caspase-like activity as visualized under a fluorescence microscope. DIC, differential interference contrast. (B) Relative fluorescence of germlings stained with FITC-VAD-FMK. Untreated control cells did not exhibit any fluorescence. ***, p<0.0001, NS, p>0.05, compared to AA+BHAM treated controls.
Figure 5.
Representative fluorescent images of R. oryzae cells treated with PCZ or ICZ in combination with AA and BHAM, cells treated with only AA and BHAM, and untreated control cells.
(A) Annexin V/PI double staining showing apoptotic cells (green fluorescence) and necrotic cells (red fluorescence). DIC, differential interference contrast. (B) Percentages of cells displaying annexin V/PI double staining. **, p<0.001, and ***, p<0.0001, compared to AA+BHAM treated controls.
Figure 6.
DNA and nuclear fragmentation in R. oryzae germlings treated with PCZ or ICZ in combination with AA and BHAM, germlings treated with only AA and BHAM, and untreated control germlings visualized using fluorescence microscopy.
(A) TUNEL/PI staining DNA fragmentation of R. oryzae germlings. (B) DAPI staining of nuclear condensation of R. oryzae germlings. DIC, differential interference contrast. (C and D) Percentages of R. oryzae germlings displaying loss of membrane integrity (PI+), DNA strand breaks (TUNEL+), and chromatin condensation (DAPI+). **, p<0.001, and ***, p<0.0001, while NS, p>0.05, compared to AA+BHAM treated controls.