Cryptococcus laurentii was isolated from the surfaces of apple fruits in a previous experiment (Qin et al., 2004) and grown in YPD broth (10 g yeast extract, 20 g peptone, and 20 g dextrose in 1 L water). Yeast cultures with an initial concentration of 1 × 10⁵ cells/mL were incubated at 26°C on a rotary shaker at 200 rpm for 17 h to reach the mid-log phase. Penicillium expansum was isolated from naturally infected apple fruits. It was routinely cultured on potato dextrose agar plates for 14 days at 25°C. Fungal spores were harvested by flooding the surface of the culture with sterile distilled water, followed by filtration through four layers of sterile cheesecloth. The number of spores in the resulting suspension was calculated using a hemocytometer. Before inoculation, the spore concentration in sterile distilled water was adjusted to 1 × 10⁴/mL.

Peach fruits (Prunus persica L. Batsch) at commercial maturity were harvested from an orchard in Beijing and immediately transported to the laboratory. Fruits without blemishes or rot were selected based on uniformity of size. Selected fruits were surface-disinfected with 2% (v/v) sodium hypochlorite for 2 min, rinsed with tap water, and air-dried prior to further use.

The median lethal concentration of H₂O₂ for C. laurentii was determined according to the methods of Chen et al. (2015). Cells in the mid-log phase were obtained by centrifugation. After being washed twice with sterile distilled water, yeast cells were resuspended in fresh YPD medium to a final concentration of 5 × 10⁷ cells/mL. H₂O₂ was added to each yeast culture to final concentrations of 0, 100, 200, 300, and 400 mM. Following incubation for 90 min (150 rpm, 26°C), yeast cells of each sample were collected and adjusted to 1 × 10⁶ cells/mL. To analyze survival rates, a 50 μL yeast sample was spread on a YPD solid plate. The plates were subsequently observed under a light microscope (Carl Zeiss, Oberkochen, Germany). The effects of treatment time with H₂O₂ on yeast viability were determined using a plate assay according to the methods of Liu et al. (2011b). Yeast cell viability was expressed as a percentage of the colony number following H₂O₂ treatment, relative to that without treatment. For each treatment, there were three replicates and the experiment was performed twice.

Intracellular ROS was detected using a 10 μM 2′,7′-dichlorodihydrofluorescein diacetate (DCFH-DA) oxidant-sensitive probe (Molecular Probes, Eugene, OR, USA). DCFH-DA was added to the yeast suspension and incubated in the dark at 37°C for 30 min. After being washed twice with PBS, yeast cells were examined under a microscope (Zeiss Axioskop, Oberkochen, Germany) using a 485-nm excitation and 530-nm emission filter combination. Three independent experiments were performed.

The fluorescence intensity of C. laurentii cells was determined using a fluorescence microplate reader (Synergy H4, BioTek, Winooski, VT, USA). Yeast samples were washed twice with N-2-hydroxyethylpiperazine-N-2’-ethanesulfonic acid (HEPES) buffer (pH 7.0) and incubated with 10 μM DCFH-DA at 37°C for 30 min. The samples were then washed twice with HEPES buffer and diluted to an optical density (OD) at 600 nm of 1.4. Yeast samples (200 μL/well) were then added to a 96-well dark microplate, and fluorescence was analyzed using a fluorescence microplate reader with an excitation wavelength of 492 nm and an emission wavelength of 527 nm. Three replicate wells were analyzed for each treatment, and the experiment was performed twice.

Biocontrol performance of C. laurentii against P. expansum was determined on peach fruits. C. laurentii cells at mid-log phase were either treated with 300 mM H₂O₂ for 90 min as described above or left untreated. Peach fruits were punctured at the equatorial line (three wounds per fruit) using a sterile nail, and first inoculated with 10 μL C. laurentii cell suspension (5 × 10⁷ cells/mL) and then with 10 μL P. expansum spore suspension (1 × 10⁴ spores/mL). Treated fruits were placed in plastic boxes. Each tray was enclosed with a polyethylene bag to maintain high humidity (about 95% relative humidity), and stored at 25°C. Disease incidence and lesion diameters of the fruits were recorded after 3, 4, and 5 days. Each treatment comprised three replicates with ten fruits per replicate, and the experiment was performed twice.

To discriminate between viable, necrotic, and apoptotic cells, flow cytometric measurements of Hoechst 33342/PI double stained yeast cells were carried out using a MoFlo XDP Cell Sorter (Beckman Coulter, Brea, CA, USA). Stained cells were analyzed using laser-based flow cytometry systems. Yeast cells at the mid-log phase were collected and treated with 300 mM H₂O₂ for 90 min. After being washed twice with PBS, the treated and untreated yeast cells were incubated with 10 μg/mL PI and 5 μg/mL Hoechst 33342 for 20 min in the dark. PI-positive yeast cells indicated damaged plasma membranes and the presence of necrotic cells. PI-negative and Hoechst 33342-positive yeast cells were considered apoptotic (Hong et al., 2007). The cell density of each sample was maintained at approximately 1 × 10⁶ cells/mL. The sample flow rate was 700 cells/s. A total of 20,000 cells were measured for each sample.

Rhodotorula glutinis, an oxidative stress-sensitive yeast, was used as a positive control in the analysis of antioxidant systems. For the enzyme activity assay, yeast cells were collected by centrifugation at specific intervals (0, 0.5, 1, 2, and 3 h) after being treated with a moderately lethal concentration of H₂O₂ and washed twice with PBS. The yeast cells were disintegrated with glass beads through vibration on a vortex mixer. SOD and CAT were extracted with 50 mM PBS (pH 7.0, 1 mM EDTA, and 2 mM phenylmethanesulfonyl fluoride). The reaction mixture (3 mL) with SOD contained 50 mM PBS, 13 mM methionine, 75 μM nitroblue tetrazolium, 10 μM EDTA, 2 μM riboflavin, and 50 μL enzyme extract. The mixtures were illuminated by light (4000 lx) for 20 min, and the absorbance was determined at 560 nm. Identical solutions held in the dark served as blanks. One unit of SOD activity was defined as the amount of enzyme causing 50% inhibition in the nitroblue tetrazolium reduction. The reaction mixture (1.5 mL) with CAT consisted of 1.4 mL H₂O₂ (40 mM) and 100 μL enzyme extract. The decomposition of H₂O₂ was determined based on the decline in absorbance at 240 nm. One unit of CAT activity was defined as the decomposition of 1 μM H₂O₂ per min. The activity of both enzymes was expressed as U/mg protein.

Extracts for the total glutathione assay were prepared according to the methods of Ellman (1959), with minor modifications. Cells of R. glutinis and C. laurentii at the mid-log phase were collected and treated with 30 and 300 mM H₂O₂ for 90 min, respectively. The cells were then harvested by centrifugation at 8000 × g for 3 min, washed three times with sterile distilled water, resuspended in PBS (pH 7.0), and extracted by vortexing with glass beads. The extracts were used for the total glutathione assay. Total glutathione content was determined using the GSH and GSSG Assay Kit S0053 (Beyotime, Shanghai, China) and 5, 5′-dithio-bis-nitrobenzoic acid. GSSG was reduced to GSH by glutathione reductase and NADPH. The absorbance was monitored at 412 nm and the results were expressed as mM/g.

The effects of exogenous GSH on yeast cell viability following H₂O₂ treatment were determined according to the methods of Liu et al. (2011b), with slight modifications. Yeast cells with an initial concentration of 1 × 10⁵ cells/mL were supplemented with GSH to yield final concentrations of 1 and 10 mM. After overnight cultivation, yeast cells at the mid-log phase were harvested by centrifugation at 8000 × g for 3 min and washed three times with fresh YPD to remove any residual GSH. Yeast cells were then resuspended in fresh YPD medium to a final concentration of 5 × 10⁷ cells/mL, and C. laurentii cells were treated with 300 mM H₂O₂ for 90 min. Cell viability was evaluated by the aforementioned methods.

All statistical analyses were performed using the SPSS version 13 software (SPSS Inc., Chicago, IL, USA). Data were analyzed using one-way ANOVA, and comparisons between means were performed using the Duncan’s multiple range test. Differences at P < 0.05 were considered significant.

Cell viability was measured following exposure of C. laurentii to increasing doses of H₂O₂ (ranging from 0 to 400 mM) and treatment intervals in YPD liquid media. Oxidative stress induced by H₂O₂ significantly inhibited cell viability in a dose- and time-dependent manner, and exposure to 300 mM H₂O₂ over 90 min was moderately lethal to cells (about 50% inhibitory) (Figures 1 and 2). Based on these results, 300 mM H₂O₂ over 90 min was selected as the appropriate concentration and interval to promote oxidative stress for subsequent studies.

Intracellular ROS production was measured by detection of fluorescence by DCFH-DA, which could be converted to highly fluorescent dichlorofluorescein in the presence of intracellular ROS. The results showed that H₂O₂ treatment could significantly induce the accumulation of intracellular ROS in C. laurentii (Figure 3A). Under conditions of oxidative stress induced by 300 mM H₂O₂, the percentage of ROS-positive cells was 46.6%. In contrast, only 5.6% of C. laurentii cells that were not subjected to exogenous oxidative stress showed visible ROS accumulation (Figure 3B). The results obtained in the analysis of fluorescence intensity were consistent with these findings. Treatment with 300 mM H₂O₂ significantly increased the intensity of dichlorofluorescein fluorescence in C. laurentii cells (Figure 3C).

In comparison with the control, non-H₂O₂-treated C. laurentii cells effectively inhibited postharvest decay caused by P. expansum on peach fruits (Figure 4A). Notably, the addition of 300 mM H₂O₂ significantly reduced biocontrol activity of C. laurentii. After 4 days, disease incidence in peach fruits treated with C. laurentii cells that had not been subjected to oxidative stress was 30%; whereas disease incidence in H₂O₂-treated samples was as high as 76% (Figure 4B). Moreover, the efficiency of C. laurentii cells in inhibiting lesion expansion was also significantly suppressed under exogenous oxidative stress (Figure 4C). These results suggest that exogenous oxidative stress could exert significant influence on biocontrol efficiency of C. laurentii. Thus, improving oxidative stress tolerance in C. laurentii is essential to enhancing its biocontrol efficiency.

Necrosis and apoptosis of C. laurentii cells were demonstrated using Hoechst 33342 and PI staining. Necrotic yeast cells with a damaged plasma membrane were detectable, based on their high red fluorescence due to PI uptake (Figure 5A). Following exposure to 300 mM H₂O₂ for 90 min, the integrity of the plasma membrane of C. laurentii cells was reduced to 75%, and the number of stained apoptotic yeast cells was increased to 29% (Figure 5B). However, damage to the plasma membrane and apoptosis were not significant in C. laurentii cells that had not been subjected to H₂O₂ treatment (Figures 5A,B).

For further analysis, necrosis and apoptosis in C. laurentii cells exposed to H₂O₂ were examined by flow cytometric analysis. On the dot plots, each cell is represented by a single dot (Figure 5C). When cells were stained with a combination of Hoechst 33342 and PI, three cell populations were observed: viable (R1), apoptotic (R2), and necrotic (R3) (Figure 5C). In the absence of H₂O₂-induced oxidative stress, the vast majority of C. laurentii cells was viable; however, following treatment with 300 mM H₂O₂, the percentage of viable cells was reduced to 46%, and the percentage of apoptotic and necrotic cells was 35 and 16%, respectively. These results are consistent with those of the survival analysis of C. laurentii under oxidative stress. These findings suggest that exogenous oxidative stress induced by 300 mM H₂O₂ could lead to apoptosis of C. laurentii cells that is primarily responsible for the decline in viability under oxidative stress.

To resist the effects of oxidative stress and maintain cellular homeostasis, cells have evolved two sophisticated antioxidant systems, the enzymatic (e.g., CAT and SOD) and non-enzymatic antioxidant (e.g., glutathione and vitamins) defense systems. CAT and SOD are two major antioxidant enzymes involved in the enzymatic antioxidant defense system. Previous studies have shown that treatment with 30 mM H₂O₂ for 90 min has a moderately lethal effect on R. glutinis cells (about 50% inhibitory). In contrast, the same concentration of H₂O₂ has no significant effects on the viability of C. laurentii (data not shown), indicating that in comparison to R. glutinis, C. laurentii shows greater resistance to exogenous oxidative stress. To determine the reasons for this higher oxidative tolerance of C. laurentii, the enzyme activities of CAT, SODe and total glutathione of R. glutinis and C. laurentii under moderately lethal oxidative stress (R. glutinis: 30 mM H₂O₂; C. laurentii: 300 mM H₂O₂), were assessed. CAT activity in C. laurentii was strongly induced by H₂O₂ and maintained at a higher level than it was in R. glutinis. The activity level in C. laurentii was approximately eightfold that in R. glutinis (Figure 6A). Similarly, H₂O₂ treatment was able to induce SOD activity in C. laurentii. In contrast, exogenous oxidative stress showed slight inhibitory effects on SOD activity in R. glutinis (Figure 6B). These data indicate that enzymatic antioxidants are major contributors to the overall antioxidant capacity of C. laurentii under exogenous oxidative stress.

Glutathione also plays an important role in cellular antioxidant defenses. More than 90% of the total glutathione pool was in the reduced form (GSH) and the remainder was in the oxidized form (glutathione disulfide, GSSG). As shown in Figure 6C, total glutathione levels in C. laurentii cells remained relatively stable under H₂O₂-induced oxidative stress. However, this form of oxidative stress showed an obvious inductive effect on total glutathione levels in R. glutinis. In terms of resistance to oxidative stress, the difference between C. laurentii and R. glutinis might be dependent on the various antioxidant systems.

Glutathione is considered the main ROS scavenger in cells (Schafer and Buettner, 2001). We evaluated the effects of exogenous GSH on the cell viability and biocontrol performance of C. laurentii under exogenous oxidative stress. Adding GSH to the culture medium could suppress the accumulation of intracellular ROS and enhance the tolerance of C. laurentii to H₂O₂-induced oxidative stress. When treated with 10 mM GSH, the percentage of C. laurentii cells exhibiting visible ROS staining was reduced by 23% (Figures 7A,B). When 10 mM GSH was added to YPD medium, the cell viability of C. laurentii was improved by 19% under moderately lethal oxidative stress (Figure 7C). Furthermore, we validated the beneficial effects of exogenous GSH on the biocontrol efficiency of C. laurentii against blue mold on peach fruits. Treatment with 1 mM GSH improved biocontrol efficiency of C. laurentii at the earlier intervals following inoculation (Figures 8A,B), whereas 10 mM GSH improved biocontrol efficacy of C. laurentii throughout the entire experiment. On days 4 and 5 post inoculation, the lesion diameters were reduced by 24 and 13%, respectively, following treatment with 10 mM GSH (Figures 8A,B).