The antagonistic yeast, D. hansenii M13, was originally isolated from the surface of mango fruit and identified by its general morphology and DNA sequence of the ITS region of ribosomal DNA (Leaw et al., 2006). It was cultured in a yeast-peptone-dextrose (YPD) broth (10 g of yeast extract, 20 g of peptone, and 20 g of dextrose in 1 L of water). Twenty milliliters of YPD was placed in a 50-mL conical flask and inoculated with D. hansenii at an initial concentration of 10⁵ cells/mL determined using a hemocytometer. Yeast cultures were incubated at 25°C on a rotary shaker at 200 r.p.m. for 16 h.

The fungal pathogens, B. cinerea and P. expansum, were isolated from infected fruit and maintained on potato dextrose agar (PDA) at 4°C. To reactivate the culture and verify its pathogenicity, the pathogens were inoculated into wounds of kiwifruit fruit and re-isolated onto PDA once the infection was established. Spore suspensions of the two pathogens were obtained from 2-week-old PDA cultures incubated at 25°C. The spore number was calculated with a hemocytometer, and the concentration was adjusted to 1 × 10⁴ spores/mL with sterile distilled water.

Kiwifruit (Actinidia deliciosa cv. Hayward) were harvested at commercial maturity. The average quality parameters at the time of harvest were: 9.8 Brix, 67 N firmness, and 96 g fresh weight per fruit. Fruits without wounds or rot were selected based on uniformity of size, disinfected with 2% (v/v) sodium hypochlorite for 2 min, rinsed with tap water, and air-dried. These fruits were used in the biocontrol assay.

Yeast cultures were grown overnight and the culture tubes were then centrifuged at 8,000 × g for 3 min. The yeast cells were subsequently washed three times with sterile distilled water to remove any residual medium, centrifuging the cells between each wash (Liu et al., 2012). Washed cells were resuspended in the same volume (20 mL) of fresh YPD, supplemented with mannitol or sorbitol at a final concentration of 0.1 M and incubated at 25°C for 2 h on a rotary shaker at 200 rpm. The selected concentration of mannitol and sorbitol was based on preliminary experiments. Control cells were subjected to the same process, but in a medium that was not supplemented with mannitol or sorbitol. Cells were harvested by centrifugation at 8,000 × g for 3 min and washed three times with sterile distilled water in order to remove any residual medium. The mannitol-treated (MT), sorbitol-treated (ST) and non-treated (NT) control yeast samples were suspended in water at 1 × 10⁷ cells/mL and used in the subsequent analyses.

The effect of polyol pretreatment on tolerance of D. hansenii to oxidative and high-temperature stress was measured as previously described (Deveau et al., 2010), with slight modification. To measure oxidative stress tolerance, a 10 mL sample of MT, ST, or NT (control) yeast at a concentration of 1 × 10⁷ cells/mL was placed in a 50-mL conical flask and exposed to oxidative stress conditions, 30 mM H₂O₂, at 25°C for 30 min on a rotary shaker at 200 rpm. To assay high temperature tolerance, 1 mL of MT, ST, or NT (control) yeast cells (1 × 10⁷ cells/mL) was placed into several 1.5 mL Eppendorf tubes. The tubes were then placed in a 40.5°C water bath for 30 min, and manually shaken once every 5 min. At designated time points, 50 μL of serial 10-fold dilutions of the samples were spread on YPD agar plates. The plates were incubated at 25°C for 3 days, and then the number of Colony-Forming Units (CFUs) per plate was determined. Survival rates were expressed as a percentage of the number of colonies produced after the oxidative or high-temperature stress treatments relative to the number of CFUs produced from samples prior to each treatment (Liu et al., 2012). Three replicates were used for each treatment, and each experiment was repeated three times.

The oxidant-sensitive probe, 2,7-dichlorodihydrofluorescein diacetate (H₂DCFDA; Invitrogen, Eugene, OR, United States), was used to assess the intracellular accumulation of ROS in yeast cells (Liu et al., 2011). Yeast cell samples were collected from cultures exposed to 30 mM H₂O₂ or 40.5°C for 30 min. The cell samples taken prior to exposure to the oxidative or heat stress served as time 0. Yeast cell samples were washed with phosphate buffered saline (PBS) buffer (pH 7.0) and resuspended in the same buffer containing 25 μM H₂DCFDA (dissolved in dimethyl sulfoxide). The suspension was incubated in the dark at 30°C for 1 h. After washing twice with PBS buffer, spores were examined under a FV3000 confocal microscope (Olympus, Tokyo, Japan) equipped with a UV-light source using a 488 nm excitation and 510 nm emission filter combination. Ten fields of view from each slide (at least 200 cells) were randomly chosen and the number of cells producing visible levels of ROS was counted. The ROS level was calculated as a percentage (number of fluorescing cells divided by number of cells present in the bright field image × 100). Three biological replicates were used for each sample in each assay, and the experiments were repeated three times.

Total RNA from yeast samples (MT, ST, and NT) was extracted, treated with DNase, and purified using an Ultrapure RNA Kit (CWBIO, Beijing, China) according to the manufacturer’s instructions. RNA quality was evaluated by gel electrophoresis and spectrophotometric analysis (Waltham, MA, United States). First-strand cDNA was synthesized using a TransScript One-Step gDNA Removal and cDNA Synthesis SuperMix kit (TransGen Biotech, Beijing, China). The resulting cDNA was used for RT-qPCR analysis following the manufacturer’s protocol. Briefly, each RT-qPCR reaction was carried out in a 20 μL reaction including 10 μL of GoTaq qPCR Master Mix (Promega, Madison, Wisconsin, United States) and 200 nmol of each gene-specific primer. The RT-qPCR was conducted on a qTOWER 2.2 (Analytik Jena AG, Germany) using the following cycling conditions: 95°C for 30 s; 40 cycles of 95°C for 5 s followed by 58°C for 15 s, 72°C for 10 s. The expression level of two target genes, CAT1 and SOD1, was analyzed using gene-specific primers (Table 1). Transcript levels of 18S rRNA gene served as an internal control. The 2^–ΔΔCT method was used to calculate relative expression (Livak and Schmittgen, 2001). To ensure that single products were amplified, each PCR reaction was subjected to a melting curve analysis of the amplification products. PCR products were cloned and sequenced to verify their identity. There were three biological replicates and three technical replicates for each treatment, and the experiment was repeated three times.

Yeast cell samples were collected from cultures exposed to 40.5°C or 30 mM H₂O₂ for 30 min. Samples of yeast cells taken prior to exposure to the oxidative or heat stress served as time 0. Yeast cells in the collected samples were disrupted in liquid nitrogen and suspended in chilled potassium phosphate buffer (0.1 M, pH 7.4). The cell homogenate was centrifuged at 10,000 × g for 20 min at 4°C and the supernatant was used for the enzyme assays. The activity of catalase (CAT) and superoxide dismutase (SOD) was measured using a commercial assay kit (Nanjing Jiancheng Bioengineering Institute, Nanjing, China), and expressed as U per mg protein. Protein content was determined using the Bradford assay with bovine serum albumin used to construct a standard curve (Bradford, 1976). One unit of CAT activity was defined as the decomposition of 1 μmol H₂O₂ per second in the reaction system, while one unit of SOD activity was defined as the amount of enzyme causing 50% inhibition in the nitro blue tetrazolium (NBT) reduction (Reverberi et al., 2005; Liu et al., 2011). Three biological replicates were used for each sample in each assay, and the experiments were repeated three times.

Intracellular mannitol and sorbitol were extracted as described in a previous study (Hua et al., 2015) with slight modification. Yeast cell samples were collected by centrifugation at 8,000 × g for 3 min and resuspended in HPLC-grade water. The cell samples were then disrupted with the use of a Q125 sonicator (QSonica, Newtown, CT, United States) for 2 min, boiled for 5 min, and then cooled to room temperature. After vortexing, the mixture in each tube was centrifuged at 1,840 × g for 10 min. The supernatant was filtered through a 0.2 μm filter membrane before determination of mannitol and sorbitol levels using an HPLC equipped with a carbohydrate analysis column and a refractive index detector (Agilent Series 1200, Agilent Technologies, Santa Clara, CA, United States). For mannitol, the mobile phase was acetonitrile-water (75:25 in volume) at 1 mL/min (Liu et al., 2009). For sorbitol, the mobile phase was acetonitrile-water (80:20 in volume) at 0.8 mL/min (Hua et al., 2015). Mannitol or sorbitol was quantified using a standard (Sigma-Aldrich, Shanghai, China) with a linear response range of 0.05–10 mg mL^–1. Intracellular mannitol and sorbitol concentrations were presented as μmol per gram fresh weight of yeast cells (Abadias et al., 2000). Three biological replicates were used for each sample in each assay and the experiments were repeated three times.

Three wounds (4 mm deep × 3 mm wide) were made on the equator of each kiwifruit with a sterile nail, and a 10 μL suspension of MT or ST and NT D. hansenii cells (1 × 10⁷ cells/mL) was administered to each wound to characterize the population dynamics of D. hansenii in wounds of kiwifruit fruit. Fruit samples were collected one hour after their initial application to the wounds and then daily for a period of 4 days. Yeast populations were measured as described by Liu et al. (2012). Briefly, yeasts were recovered by removing ten samples of wounded tissues with a cork borer (1 cm diameter × 1 cm deep). Samples were then ground with a mortar and pestle in 10 mL sterile distilled water. Subsequently, 50 μL of serial 10-fold dilutions were spread on YPD agar plates. Samples taken at 1 h after treatment served as time 0. Fruits stored at 25°C were assessed each day for 4 days. Colonies were counted after incubation of YPD agar plates at 25°C for 2 days and expressed as the Log10 CFU per wound. There were three biological replicates evaluated for each treatment, and the experiment was repeated three times.

The determination of biocontrol efficacy was determined as described by Wang et al. (2018). Three wounds (4 mm deep × 3 mm wide) were made on the equator of each fruit with a sterile nail. A 10 μL suspension of NT, MT, or ST cells of D. hansenii (1 × 10⁷ cells/mL) was administered to each wound. Sterile distilled water served as a control. After fruits were air-dried for 2 h, 10 μL of either P. expansum or B. cinerea suspension (1 × 10⁴ spores/mL) were inoculated into each wound. Treated fruits were placed in a covered plastic food tray. Each tray was enclosed with a polyethylene bag in order to maintain high humidity (approximately 95% RH) and stored at 25°C. Disease incidence and lesion diameter of kiwifruit fruits were recorded after 4 days. Each treatment contained three biological replicates with ten fruits per replicate and the experiment was repeated three times.

All statistical analyses were performed using SPSS version 20.0 (SPSS Inc., United States) software. Data with a single variable (treatment) were analyzed by a one-way ANOVA, and mean separations were performed using a Duncan’s multiple range test. Differences at P < 0.05 were considered significant. Data presented in this paper were pooled across three independent repeated experiments.

Mannitol-treated (MT) or sorbitol-treated (ST) yeast cells exhibited significantly higher viability than non-treated (NT) cells. As presented in Figure 1, the viability of NT yeast cells was about 50%, after exposure to 30 mM H₂O₂ or 40.5°C for 30 min. In contrast, the viability of MT or ST yeast cells was significantly higher, after exposure to 30 mM H₂O₂ or 40.5°C for 30 min, respectively. These results demonstrate that yeast cells pretreated with mannitol or sorbitol had a statistically significant higher level of viability compared to NT cells.

At time 0 (the time point after the 2-hour pretreatment but prior to exposure to the oxidative or high-temperature stress), the percentage of MT and ST cells, as well as NT cells, exhibiting ROS was less than 10% (Figure 2). This percentage increased when cells were exposed to either a 30-minute treatment of 30 mM H₂O₂ or 40.5°C. Both MT and ST cells exhibited a significantly lower percentage of cells exhibiting ROS compared to NT cells under each specific stress.

Results of the RT-qPCR analysis on the expression of CAT1, SOD1 transcripts in MT, ST, and NT cells of D. hansenii is presented in Figure 3. The RT-qPCR analysis of gene expression of yeast samples at time 0 (the time point after the 2-hour pretreatment but prior to exposure to the oxidative or high-temperature stress) indicated that pretreatment of yeast cells with sorbitol or mannitol upregulated the expression of CAT1 and SOD1 in D. hansenii cells. The expression of CAT1 was significantly higher in MT and ST cells than in NT cells exposed to 30 mM H₂O₂ (30 min). Similarly, the expression of CAT1 in cells exposed to 40.5°C (30 min) yeast cells was also elevated (Figure 3A). The expression of SOD1 in pretreated cells was significantly higher than in NT cells exposed to 30 mM H₂O₂ (30 min). The expression of SOD1 in cells exposed to 40.5°C (30 min) was also significantly higher in MT and ST cells than in NT cells (Figure 3B).

The activity of catalase and SOD activity were measured in the pretreated and control samples in response to the oxidative and high-temperature stress treatments. Results indicated that CAT and SOD activity was significantly higher in MT and ST yeast cells than in NT yeast cells (Figure 4). The activity of these antioxidant enzymes, as with the level of expression of their corresponding genes, was significantly higher in yeast cells of D. hansenii pretreated with mannitol or sorbitol than in untreated cells prior to and after exposure (time 0 and after 30 min) to the subsequent high-temperature and oxidative stress. CAT activity in MT and ST cells in response to exposure to the high-temperature and oxidative stress was significantly higher than in NT cells (Figure 4A). SOD activity was also significant higher in MT and ST cells exposed to the high-temperature and oxidative stress than in NT cells at all of the evaluated time points (Figure 4B).

As indicated in Figure 5, the intracellular level of mannitol in MT cells was significantly higher than it was in ST and NT cells at time 0 (the time point after the 2-hour pretreatment but prior to exposure to the oxidative or high-temperature stress). Exposure to either a 30-minute treatment of 30 mM H₂O₂ or 40.5°C elevated intracellular mannitol content, with MT cells still exhibiting the highest mannitol content (Figure 5A). Similarly, ST cells had the highest intracellular sorbitol content at time 0 and under the stress conditions (Figure 5B).

All of the D. hansenii cultures (NT, MT, and ST) grew rapidly after they were administered to fruit wounds. The population of MT and ST cells, however, was significantly higher than the population of NT cells on each of 4 days following their inoculation into wounds (Figure 6). Importantly, the ability of yeast biocontrol agents to rapidly establish themselves and grow in fruit wounds once they are administered is considered a biocontrol trait that is essential for effective control.

As shown in Figure 7, the antagonistic yeast, D. hansenii, significantly reduced both disease incidence and lesion diameter of both blue mold and gray mold infections of kiwifruit caused by P. expansum and B. cinerea, respectively. Notably, the incidence of both blue and gray mold decay in fruit treated with NT cultures was about 20% lower than the control group where no yeast cells were administered, which had an incidence level of 100% for both pathogens. In contrast, disease incidence in kiwifruit treated with MT or ST cell groups was reduced significantly, relative to the NT control (Figure 7A). Correspondingly, lesion diameters caused by P. expansum and B. cinerea were significantly smaller on kiwifruit fruits inoculated with MT or ST cells, compared to lesion size in kiwifruit treated with NT cells (Figure 7B). These data indicate that D. hansenii significantly reduced both disease incidence and lesion size for both gray mold and blue mold pathogens, however, the use of yeast cells pretreated with mannitol or sorbitol further increased the level of control beyond the level observed for non-treated (NT) cells.