β-Glucan Enhances the Biocontrol Efficacy of Marine Yeast Scheffersomyeces spartinae W9 against Botrytis cinerea in Strawberries

The marine yeast Scheffersomyeces spartinae W9 is a promising biocontrol agent for gray mold caused by Botrytis cinerea in strawberries. Improving the biocontrol efficacy of S. spartinae W9 is necessary for its commercial application. In this study, different concentrations of β-glucan were added to the culture medium to evaluate its effect on the biocontrol efficacy of S. spartinae W9. The results showed that 0.1% β-glucan could increase the biocontrol effect of S. spartinae W9 against B. cinerea in strawberries and in vitro. We found that adding 0.1% β-glucan to the culture medium promoted the growth of S. spartinae W9 in wounds of strawberries, enhanced biofilm formation ability, and secreted more β-1,3-glucanase. In addition, 0.1% β-glucan increased the survival rate of S. spartinae W9 under oxidative, thermal, osmotic, and plasma membrane stressors. Transcriptome analysis revealed 188 differential expressed genes in S. spartinae W9 cultured with or without 0.1% β-glucan, including 120 upregulated and 68 downregulated genes. The upregulated genes were associated with stress response, cell wall formation, energy production, growth, and reproduction. Thus, culturing with 0.1% β-glucan is an effective way to improve the biocontrol ability of S. spartinae W9 against gray mold in strawberries.


Introduction
Gray mold caused by Botrytis cinerea is the primary fungal disease of strawberry fruit during planting and postharvest storage [1]. Chemical fungicides have been used as the most effective method to control gray mold for decades. However, frequent use of chemical fungicides has increased the resistance of fungal pathogens and has had a negative influence on human health and environmental safety [2,3]. In recent years, researchers have been actively seeking alternatives to chemical fungicides. Biological control using microbial agents is considered to be a promising candidate in the management of postharvest diseases. Antagonistic yeasts are widely used to control postharvest diseases because of their biosafety, well-defined mechanism of action, and good commercial potential [4][5][6]. A variety of antagonistic yeasts have been reported to control gray mold decay of fruits, including Pichia guilliermondii [7], Wickerhamomyces anomalus [8], Metschnikowia pulcherrima [9], Candida pseudolambica [10], Metarhizium anisopliae [11], Scheffersomyeces spartinae [12], and so on. However, antagonistic yeasts have not been able to achieve the effect of traditional chemical fungicides in controlling postharvest diseases of fruit [13]. Therefore, it is important to improve the biocontrol efficacy of antagonistic yeasts.
Adding polysaccharides to the culture media may increase the biocontrol activity of antagonistic yeasts. β-glucan is a natural polysaccharide and the main compo-nent of yeast cell walls and has various biological activities [14]. β-glucan has been reported to improve various stress tolerances and biocontrol efficacy of Cryptococcus laurentii (Papiliotrema laurentii) [15,16] and enhance the biocontrol efficacy of Cryptococcus podzolicus (Saitozyma podzolica) [16] against postharvest decay of apples [17] and pears [18] by upregulating the genes related to cell wall synthesis and increasing its tolerance to oxidative stress. S. spartinae W9 is an antagonistic yeast isolated from the intertidal zone marine sediment by our laboratory and a promising biocontrol agent for gray mold in strawberries [12]. To promote the commercial application process, improving the biocontrol efficacy of S. spartinae W9 is urgent. Whether β-glucan can enhance the biocontrol efficacy of marine yeast S. spartinae W9 has yet to be studied.
Therefore, this study aimed to investigate the effect of β-glucan on the biocontrol efficacy of marine yeast S. spartinae W9 and reveal its possible mechanism. In this study, we investigated (1) the effect of different concentrations of β-glucan on antagonistic activities of S. spartinae W9 against B. cinerea in vitro and in strawberry fruit, (2) the effect of β-glucan on the colonization ability and biofilm forming ability of S. spartinae W9, (3) the effect of β-glucan on extracellular hydrolase activity of S. spartinae W9, (4) the effect of β-glucan on various forms of stress tolerance of S. spartinae W9, and (5) the effect of β-glucan on the transcriptome of S. spartinae W9.

Antagonistic Yeast and Fungal Pathogen
The antagonistic yeast S. spartinae W9 was preserved in our laboratory and had previously been isolated from the marine sediment in the South China Sea [12]. Consistent with the method used by  and Zhao et al. (2020) [17,18], the activated yeast was suspended in NYDB (nutrient yeast dextrose broth medium) or NYDB supplemented with 0.1%, 0.5%, 1%, and 2% β-glucan (w/v), and incubated in an oscillating incubator at 28 • C for 24 h.
B. cinerea was isolated from infected strawberry fruit and preserved in our laboratory. The strain was maintained and propagated on PDA (potato dextrose agar medium). Spore suspensions were washed from the mycelium, cultured for 7 d, and adjusted to 1 × 10 5 spores mL/L using a hemocytometer.

Fruit
Commercially mature strawberries (Fragaria ananassa Duch. cv. Zhangji) were harvested from a greenhouse in the Meishan District, Ningbo, China. Strawberries with uniform color and size and absence of visual injury or lesion were selected for experiments. The strawberries were wiped with 75% ethanol (v/v) for disinfection and air-dried at 20 • C [19].

Biocontrol Efficacy of S. spartinae W9 Cultured with Different Concentrations of β-Glucan against B. cinerea in Strawberries
This experiment was conducted according to the method described by Fu et al. (2015) [15]. A uniform wound was made at the strawberry equator with a sterilized nail (3 mm deep × 3 mm wide), and each wound was inoculated with 10 µL of any one of the following solutions: (1) sterile water (control), (2) S. spartinae W9 cell suspension cultured in NYDB medium (1 × 10 8 cells/mL), or (3) S. spartinae W9 cell suspension cultured in NYDB medium with 0.1%, 0.5%, 1%, and 2% β-glucan (w/v) (1 × 10 8 cells/mL). After two hours, each wound was injected with 10 µL of B. cinerea spore suspension (1 × 10 5 spores/mL). Then all strawberries were stored in a thermostatic chamber at 20 • C with 90% relative humidity. The incidence of gray mold and the diameter of each lesion were observed every day. Every treatment was replicated three times, with 20 fruits selected at random in each replicate.

Antagonistic Activity of S. spartinae W9 Cultured with β-Glucan against B. cinerea In Vitro
For the mycelia growth test, 100 µL of S. spartinae W9 suspensions (cultured in NYDB and NYDB with 0.1%, 0.5%, 1%, and 2% β-glucan) at a concentration of 1 × 10 5 cells/mL was spread into the PDA medium. A total of 100 µL of sterile water was spread to the PDA and used as the control. Afterwards, a 9-mm diameter plug taken from the edge of the actively growing B. cinerea colony was inoculated into the center of each PDA plate. The plates were incubated at 28 • C for 5 d, and the mycelia diameters were measured daily. The experiment consisted of three replicates, with 5 plates per replicate.
For the spore germination test, cell suspensions of 1 × 10 9 cells/mL S. spartinae W9 (cultured in NYDB and NYDB with 0.1%, 0.5%, 1%, and 2% β-glucan) were prepared. Then 200 µL of yeast suspension was mixed with 200 µL of B. cinerea (1 × 10 6 spores/mL) spore suspension and added into a 1.6-µL PDB medium. An amount of 200 µL sterile water mixed with 200 µL B. cinerea spore suspension was used as the control. The spores were incubated for 6 h at 28 • C and 180 rpm on a shaker. The germination rate of B. cinerea spores were counted by light microscopy and hemocytometer. At least 100 spores were observed in each treatment, with 3 replicates per treatment.

Population of Yeasts in Strawberry Wounds
In accordance with the method of Zou et al. (2022) [10], strawberries were wounded as described in 2.3 and then inoculated with S. spartinae W9 or 0.1% β-glucan-treated S. spartinae W9. All strawberries were stored at 20 • C with 90% relative humidity. The amount of yeast at the strawberry fruit wounds was measured at 0 h, 12 h, 24 h, 48 h, and 72 h. The fruit wound tissue was removed with a sterile blade and homogenized in a mortar containing 10 mL of sterile water. A total of 100 µL of the homogenate diluted to the appropriate concentration was spread on NYDA and incubated at 28 • C for 48 h to count the number of colonies. The number of yeast colonies at each wound was expressed as log 10 CFU/wound. The experiment consisted of three replicates, with three strawberries per replicate.
2.6. The Effect of 0.1% β-Glucan on the Growth of S. spartinae W9 S. spartinae W9 cells (1 × 10 8 cells/mL) were cultured in NYDB or NYDB with 0.1% βglucan at 28 • C. The growth of S. spartinae W9 was recorded by measuring the absorbance at 600 nm at 12 h, 24 h, 36 h, and 48 h. Three biological replicates were available for each treatment.

Determination of Biofilm Formation
The biofilm formation of S. spartinae W9 was assessed using the method of Qiu et al. (2022) [20] with some modifications. An amount of 100 µL of S. spartinae W9 suspension (1 × 10 7 cells/mL) was pipetted into 50 mL of YNB medium and cultured for about 12 h. Yeast precipitate was obtained by centrifugation at 8000 rpm, 4 • C, for 5 min and was washed twice to adjust the concentration of yeast to 1 × 10 8 cells/mL. Equal amounts of yeast suspension were injected into YNB or YNB containing 0.1% β-glucan and then were incubated at 28 • C, 75 rpm for 12, 18, 24, 36, and 48 h. For biofilm assay, the culture solutions were washed twice with phosphate buffer and treated with methanol for 15 min. Then, 200 µL of 0.4% crystalline violet was added for staining, and after 40 min, the excess dye was removed and washed with sterile water. Lastly, 200 µL of 33% glacial acetic acid was added and incubated for 30 min, and the supernatant was used to measure the absorbance at 590 nm.

Measurement of Extracellular Hydrolases
The ability of S. spartinae W9 to produce extracellular hydrolases, mainly including chitinase and β-1,3-glucanase (GLU), was determined by an extracellular hydrolase assay plate [21]. Chitinase and CLU can decompose the colloidal chitin and laminarin, respec-tively. If S. spartinae W9 secreted chitinase and CLU, the hyaline rings would appear in the plates.
According to the result of an extracellular hydrolase assay plate, we determined the GLU activity of S. spartinae W9. S. spartinae W9 cultured in NYDB or NYDB with 0.1% βglucan were sampled at 12,16,20,24,36, and 48 h. The fermentation broth was centrifuged at 6000 rpm for 10 min, and the supernatant was filtered through a sterile membrane for enzyme assay. The GLU activity was determined using the 3, 5-dinitrosalicylic acid method [22], and the absorbance at 540 nm was measured to calculate GLU activity. One unit (U) of GLU was defined as the amount of the enzyme required to produce 1 nmol of glucose per minute, and the results were expressed as U/mL. Each group contained three independent replicates.
To determine the tolerance of S. spartinae W9 to high temperatures, the yeast suspensions were placed into a water bath at 40 • C, 45 • C, and 50 • C for 30 min. The heat-treated yeast suspensions were cooled at room temperature for 10 min and diluted to the appropriate concentration. Then, 0.1 mL of each yeast suspension was spread on a NYDA plate to calculate the survival rate.
S. spartinae W9 or β-glucan-treated S. spartinae W9 yeast suspensions were injected into NYDB (control) and a selective medium (NYDB-4% NaCl, NYDB-7% NaCl, NYDB-10% NaCl, NYDB-1.25mM Yeasts were incubated at 28 • C and 180 rpm for 24 h and then diluted to the appropriate concentration to spread on NYDA plates. All plates were incubated at 28 • C for 48 h, and the numbers of yeast colony were recorded. The survival rates of S. spartinae W9 or β-glucan-treated S. spartinae W9 without stress treatment were used as respective controls. Each treatment contained three independent replicates.

Transcriptomic Analysis
RNA from S. spartinae W9 cultured in NYDB with or without 0.1% β-glucan was isolated using TRIzol (Thermofisher, MA, USA) according to the manufacturer's instructions. The mRNA with PolyA (polyadenylation) was specifically captured and fragmented with a magnesium ion interruption kit (NEBNext R Magnesium RNA Fragmentation Module, cat. E6150S, New England Biolabs, MA, USA). The fragmented RNA was synthesized into cDNA. Then, double-stranded DNA were synthesized and repaired. The fragment sizes were screened and purified using magnetic beads, and the cDNA library was enriched with PCR. RNA-seq was performed by LC-Bio Technology CO., Ltd. Hangzhou, China. Genes with a fold change in expression level ≥ 1.2 and adjusted p < 0.01 were identified as differentially expressed genes (DEGs). The DEGs' data was analyzed for GO and KEGG enrichment analysis.

Real-Time Quantitative PCR
RNA extracted from S. spartinae W9 cultured in NYDB with or without 0.1% β-glucan were first reverse transcribed to cDNA. RT-qPCR was performed using ChamQ Universal SYBR qPCR Master Mix (Vazyme, Nanjing, China). Twelve DEGs were randomly selected for validation by qPCR, and ACT1 was used as an internal reference gene. Relative gene expression was calculated according to the 2 −∆∆Ct method. Table 1 contains a list of the selected genes and the specific primers. Table 1. Primers used for RT-qPCR analysis of S. spartinae genes.

Statistical Analysis
SPSS statistics software version 20 was used to analyze the data. When the number of comparisons within the group was three or more, the data were compared with the mean by implementing Duncan's multiple analysis using ANOVA; when the number of comparisons within the group was two, the mean was compared using the independent samples t-test. Significance was assessed when p < 0.05. Data were expressed as mean ± standard deviation.

Biocontrol Efficacy of S. spartinae W9 Cultured with β-Glucan against B. cinerea in Strawberries
As shown in Figure 1A, gray mold decay of strawberries in all groups was observed at the fourth day after inoculation, and S. spartinae W9 cultured with or without β-glucan reduced the disease incidence of strawberries. S. spartinae W9 cultured with 0.1% β-glucan significantly reduced the disease incidence when compared to yeast without β-glucan after the fourth day (p < 0.05). At the end of storage, the disease incidence was 96.7% in the control group, 63.3% in the S. spartinae W9 group, and only 40% in the 0.1% β-glucantreated S. spartinae W9 group ( Figure 1A,B). Adding 0.1% β-glucan into NYDB effectively improved the biocontrol efficacy of S. spartinae W9.

In Vitro Inhibition of S. spartinae W9 Cultured with β-Glucan against B. cinerea
As shown in Figure 2A, β-glucan enhanced the antagonistic effect of S. spartinae W9 against B. cinerea in vitro. On the fourth day, the mycelia of B. cinerea in the control group covered almost the whole plate, while B. cinerea mycelia in the other groups were significantly inhibited (p < 0.05). Compared to the S. spartinae W9 cultured without β-glucan, 0.1% and 1% β-glucan were effective in increasing the inhibition of B. cinerea mycelial growth, for which the diameter of B. cinerea plaques in the S. spartinae W9 treatment group was 23.4 mm, while the diameters of the 0.1% and 1% β-glucan-induced S. spartinae W9 treated groups were 18.8 mm and 18 mm, respectively.

In Vitro Inhibition of S. spartinae W9 Cultured with β-Glucan against B. cinerea
As shown in Fig. 2A, β-glucan enhanced the antagonistic effect of S. spartinae W9 against B. cinerea in vitro. On the fourth day, the mycelia of B. cinerea in the control group covered almost the whole plate, while B.cinerea mycelia in the other groups were significantly inhibited (p < 0.05). Compared to the S. spartinae W9 cultured without β-glucan, 0.1% and 1% β-glucan were effective in increasing the inhibition of B. cinerea mycelial growth, for which the diameter of B. cinerea plaques in the S. spartinae W9 treatment group was 23.4 mm, while the diameters of the 0.1% and 1% β-glucan-induced S. spartinae W9 treated groups were 18.8 mm and 18 mm, respectively. Figure 2B shows that S. spartinae W9 cultured with or without β-glucan significantly inhibited spore germination of B. cinerea (p < 0.05). S. spartinae W9 cultured with different concentrations of glucan showed different inhibitory effects on spore germination of B. cinerea. The germination rate of B. cinerea spores in 0.1% β-glucan-treated S. spartinae W9  Figure 2B shows that S. spartinae W9 cultured with or without β-glucan significantly inhibited spore germination of B. cinerea (p < 0.05). S. spartinae W9 cultured with different concentrations of glucan showed different inhibitory effects on spore germination of B. cinerea. The germination rate of B. cinerea spores in 0.1% β-glucan-treated S. spartinae W9 was 9.5%, which was significantly lower than the 17.1% in the S. spartinae W9 group (p < 0.05). Only 0.1% β-glucan increased the ability of S. spartinae W9 to inhibit spore germination of B. cinerea.

Effects of 0.1% β-Glucan on the Growth of S. spartinae W9 In Vitro
S. spartinae W9 yeasts were cultured in NYDB and NYDB containing 0.1% β-gluc at a concentration of 0.1% for 48 h, respectively. The OD600 of S. spartinae W9 in both med showed no significant difference (p > 0.05; Table 2). The results indicated that 0.1% β-gl can had no significant effect on the growth of S. spartinae W9 in vitro.

Effects of 0.1% β-Glucan on Colonization Ability in Strawberries and Biofilm Forming Ability of S. spartinae W9
S. spartinae W9 cultured with or without β-glucan was able to grow rapidly in t wounds of strawberries, and its population levels reached the highest value at 24 h (Figu 3A). The population of the β-glucan-induced S. spartinae W9 increased from 6.1 log10 CF wound −1 to 7.7 log10 CFU wound −1 at 24 h, which was significantly higher than that S.spartinae W9 cultured in NYDB (p < 0.05). The growth rate of the β-glucan-treated spartinae W9 within 24 h was higher than S. spartinae W9 cultured in NYDB.
Crystal violet staining was used to further evaluate the biofilm formation capacity S. spartinae W9. During the 24 h of yeast culture, the biofilm formation capacity remain

Effects of 0.1% β-Glucan on the Growth of S. spartinae W9 In Vitro
S. spartinae W9 yeasts were cultured in NYDB and NYDB containing 0.1% β-glucan at a concentration of 0.1% for 48 h, respectively. The OD 600 of S. spartinae W9 in both media showed no significant difference (p > 0.05; Table 2). The results indicated that 0.1% β-glucan had no significant effect on the growth of S. spartinae W9 in vitro.

Effects of 0.1% β-Glucan on Colonization Ability in Strawberries and Biofilm Forming Ability of S. spartinae W9
S. spartinae W9 cultured with or without β-glucan was able to grow rapidly in the wounds of strawberries, and its population levels reached the highest value at 24 h ( Figure 3A). The population of the β-glucan-induced S. spartinae W9 increased from 6.1 log 10 CFU wound −1 to 7.7 log 10 CFU wound −1 at 24 h, which was significantly higher than that of S. spartinae W9 cultured in NYDB (p < 0.05). The growth rate of the β-glucantreated S. spartinae W9 within 24 h was higher than S. spartinae W9 cultured in NYDB.

Effects of 0.1% β-Glucan on Extracellular Hydrolase of S. spartinae W9
The extracellular hydrolase assay plates showed that S. spartinae W9 could secrete GLU but not chitinase ( Figure 4A). The 0.1% β-glucan could not induce S. spartinae W9 to secrete chitinase. The extracellular GLU activities of S. spartinae W9 cultured in NYDB with 0.1% β-glucan were significantly higher than those of S. spartinae W9 cultured in NYDB during the culture period ( Figure 4B), indicating that β-glucan could induce S. spartinae W9 to secrete more GLU. (B) GLU activity of S. spartinae W9 cultured in NYDB or NYDB containing 0.1% β-glucan. Asterisks indicate a significant difference between S. spartinae W9 cultured with or without 0.1% β-glucan identified by the t-test (p < 0.05).

0.1% β-Glucan Increased the Survival rate of S. spartinae W9 under Different Stresses
S. spartinae W9 cells harvested from NYDB with 0.1% β-glucan showed higher survival rates under oxidative, thermal, osmotic, and plasma membrane stresses, compared with the yeast cells harvested from NYDB ( Figure 5). The survival rate of S. spartinae W9 under 10 mM H2O2, 10% NaCl, 1% SDS, and 50 °C were 66.2%, 68.2% 67.7%, and 76.3%, respectively, while those of β-glucan-treated S. spartinae W9 were 87.4%, 71.0%, 70.6%, Crystal violet staining was used to further evaluate the biofilm formation capacity of S. spartinae W9. During the 24 h of yeast culture, the biofilm formation capacity remained stable in each group ( Figure 3B). The OD 590 of S. spartinae W9 cultured with β-glucan was consistently higher than that of S. spartinae W9 cultured without β-glucan. This indicated that β-glucan promoted the formation of biofilm for S. spartinae W9 and enhanced its biofilm formation capacity.

Effects of 0.1% β-Glucan on Extracellular Hydrolase of S. spartinae W9
The extracellular hydrolase assay plates showed that S. spartinae W9 could secrete GLU but not chitinase ( Figure 4A). The 0.1% β-glucan could not induce S. spartinae W9 to secrete chitinase. The extracellular GLU activities of S. spartinae W9 cultured in NYDB with 0.1% β-glucan were significantly higher than those of S. spartinae W9 cultured in NYDB during the culture period ( Figure 4B), indicating that β-glucan could induce S. spartinae W9 to secrete more GLU.

Effects of 0.1% β-Glucan on Extracellular Hydrolase of S. spartinae W9
The extracellular hydrolase assay plates showed that S. spartinae W9 could secrete GLU but not chitinase ( Figure 4A). The 0.1% β-glucan could not induce S. spartinae W9 to secrete chitinase. The extracellular GLU activities of S. spartinae W9 cultured in NYDB with 0.1% β-glucan were significantly higher than those of S. spartinae W9 cultured in NYDB during the culture period ( Figure 4B), indicating that β-glucan could induce S. spartinae W9 to secrete more GLU.  (B) GLU activity of S. spartinae W9 cultured in NYDB or NYDB containing 0.1% β-glucan. * indicate a significant difference between S. spartinae W9 cultured with or without 0.1% β-glucan identified by the t-test (p < 0.05).

RNA-seq Analysis and RT-qPCR Validation
A total of 40.87 G clean reads were obtained from S. spartinae W9 cultured in NYDB with or without β-glucan. The Q30 levels were both higher than 70%, indicating that the base quality was up to standard. GC contents were both above 40%, and the percentage of valid reads exceeded 95%, which indicated that the data were reliable. By comparing the gene expression levels between S. spartinae W9 cultured in NYDB with and without β-glucan, 188 genes were identified as DEGs, including 120 upregulated genes and 68 downregulated genes.
A total of 172 DEGs were able to be annotated in the GO database and were enriched into three major categories ( Figure 6A). In the biological process category, the DEGs were involved in translation, the biological process, the oxidation-reduction process, and so on. The highly enriched DEGs of the cellular components were involved in cell organelles and cell membranes. The subclass with the most enriched DEGs by molecular function was the structural constituent of ribosome (34 DEGs) subclass, followed by RNA binding (12 DEGs).
The DEGs were classified into five categories by KEGG enrichment analysis ( Figure 6B). Among them, the pathway with the most DEGs was translation, involving 40 genes. The second pathway was the carbohydrate metabolism pathway, with 13 DEGs. RT-qPCR was performed on 12 genes to validate the results of RNA-seq. The tran script levels of all selected genes determined by RT-qPCR were consistent with those ob tained using RNA-seq (Figure 7).

Discussion
A bottleneck in the development of antagonistic yeasts has been the limited biocontrol effect in practical application. Therefore, it is very important to enhance the biocontrol efficacy of antagonistic yeasts. It has been reported that β-glucan could improve the biocontrol efficacy of C. laurentii and C. podzolicus against postharvest blue mold of pears [24]. In the present study, our results also confirmed that β-glucan was an effective elicitor to improve the biocontrol ability of S. spartinae W9 on gray mold of strawberry fruit. The 0.1% β-glucan enhanced the biocontrol efficacy and antagonistic activity of S. spartinae W9 against B. cinerea (Figures 1 and 2). Despite the differences in PDA medium and in fruit [25], the increased antagonistic effects of S. spartinae W9 by β-glucan were consistent. Therefore, we selected 0.1% β-glucan to culture S. spartinae W9 and reveal the involved mechanism.
The key factor affecting the biocontrol activity of antagonistic yeasts has been proposed to be the rate of colonization [26]. Yeasts rapidly consume limited nutrients, thereby inhibiting spore germination of fungal pathogens [27]. This study showed that the addition of β-glucan to the medium had no effect on the growth and reproduction of S. spartinae W9 in vitro, while β-glucan could promote the colonization of S. spartinae W9 in strawberry wounds within 24 h of storage ( Figure 3A).  also found that β-glucan increased the growth ability of C. podzolicus in apple wounds [28]. Di Francesco et al. (2017) found that the colonization ability of yeast on the wounds has a certain correlation with the biofilm-forming ability [28]. High-density yeast forms exopolysaccharide capsules, which can more firmly adhere to fruit wounds and prevent pathogen infection [29,30]. Our results showed that the addition of 0.1% β-glucan to the culture medium effectively improved the biofilm formation capacity of S. spartinae W9 ( Figure 3B). β-glucan improved the colonization ability in strawberries and biofilm forming ability of S. spartinae W9, which inhibited the growth of B. cinerea, thus reducing the incidence of gray mold in strawberries. Our findings are similar to the previous reports, which showed that some exogenous nutrients could induce antagonistic yeasts to enhance the biocontrol efficacy by improving the growth ability at fruit wounds and biofilm formation ability [31,32].
The ability of antagonistic yeast to resist pathogens also depends on the secretion of extracellular hydrolase. GLU can hydrolyze glucan, which is the major component of fungal cell walls, leading to cell malformation and cytoplasmic leakage, and is one of the possible mechanisms of biocontrol of antagonistic yeasts [33]. GLU is an inducible hydrolase enzyme, and the presence of β-glucan could induce the secretion of GLU from antagonistic yeasts. Our results showed that S. spartinae W9 could secret GLU, and 0.1% β-glucan induced the higher extracellular GLU activities ( Figure 4). This suggests that 0.1% β-glucan could induce S. spartinae W9 to secrete more GLU, which might be an important factor for the enhanced biocontrol efficacy of S. spartinae W9. This finding is consistent with the report of Zhao et al. (2020), which showed that higher GLU activity of C. podzolicus is an important factor for the increased biocontrol effect of C. podzolicus cultured with β-glucan [17,18].
In practical application environments such as orchards and postharvest storage, various abiotic stressors could decrease the biocontrol efficacy of antagonistic yeasts [34]. Thus, improving the ecological adaptability and resistance of antagonistic yeasts is an effective way to ensure their biocontrol efficacy. SDS is an organic compound that penetrates cell membranes, causes damage to the plasma membrane, and limits cell growth [35,36]. Oxidative adversity was simulated using hydrogen peroxide because a fruit wound is an environment with high levels of reactive oxygen species [37], which cause oxidative damage to the cell membrane component membrane lipids, thereby destroying the integrity of the cell [38]. NaCl is a common osmotic stressor, and high NaCl has the effect of inhibiting cell growth [39]. The present study showed that the growth of S. spartinae W9 was significantly inhibited under these stressors, while S. spartinae W9 cultured with 0.1% β-glucan could enhance tolerance under these stressors ( Figure 5). Fu et al. (2015) also found that 0.5%-β-glucan-induced C. laurentii can survive better under heat and oxidative stress [15].
These findings suggest that β-glucan could improve the stress tolerance of antagonistic yeasts and help to improve their biocontrol efficacy in practical applications.
The transcriptome analysis was conducted to explore the molecular mechanism of β-glucan, enhancing the biocontrol efficacy of S. spartinae W9. The results showed that β-glucan caused 188 DEGs, and the DEGs were related to cell wall formation and cell integrity, energy production, growth, and reproduction ( Figure 6). Some upregulated DEGs are associated with stress response, including GRE2, CYS3, AOX2, and ERG3. GRE2 has been reported to be induced in yeast cells in response to oxidative stress [40] and osmotic stress [41]. Similarly, elevated AOX2 implies an acceleration of oxidative processes in mitochondria and limitation of ROS production [42]. The upregulation of the ERG3 gene due to β-glucan was beneficial for maintaining the stability of yeast cell plasma membranes and improving the resistance of yeast to oxidative stress [43]. CYS3 can play a protective role for yeast in a state of oxidative stress and promote the efficiency of yeast cell proliferation [44][45][46]. The upregulation of GRE2, CYS3, AOX2, and ERG3 could explain the increased oxidative stress tolerance of S. spartinae W9 cultured with 0.1% β-glucan.
The yeast cell wall serves a variety of biological functions, including protecting against environmental stresses. It also affects the permeability of the cell wall when it is attacked by substances that disrupt cell integrity [47]. The genes TDH1 and ECM4 have been shown to be involved in cell wall structure [48,49]. Compared to the control, the higher expression of TDH1 and ECM4 in S. spartinae W9 cultured with 0.1% β-glucan promoted cell wall biosynthesis and biocontrol efficacy.
Intracellular energy is heavily depleted when yeast cells are under abiotic stress [50]. ACH1 provides acetyl coenzyme A (acetyl-CoA) to mitochondria by transferring the CoA group to acetate, which may accelerate metabolism in the TCA cycle and glycolysis [51]. The gene ADH1 encodes alcohol dehydrogenase, which is the primary enzyme for the reoxidation of NADH in yeast. Zhong et al. (2021) showed that inactivation of ADH1 caused the accumulation of NADH, resulting in the imbalance of NADH/NAD and growth retardation [52]. The upregulation of ACH1 and ADH1 in S. spartinae W9 cells cultured with 0.1% β-glucan led to higher colonization ability in strawberry wounds and improved biocontrol efficacy. In addition, upregulation of the RNR2 gene required for DNA repair may be helpful for maintaining cellular integrity [53]. In brief, β-glucan increased the energy metabolism and cellular integrity of S. spartinae W9 cells.
The transcriptome analysis demonstrated that the 0.1 % β-glucan-induced S. spartinae W9 achieved better proliferation and enhanced biological activity by altering cellular processes, energy metabolism, and stress responses. Similarly, Zhao et al. (2020) used transcriptomic analysis to reveal that β-glucan accelerated the synthesis of cell walls and energy of C. podzolicus, enhanced its antioxidant ability, and thus improved the biological control efficacy [18].

Conclusions
Adding 0.1% β-glucan into the culture medium significantly enhanced the biocontrol efficacy of S. spartinae W9 against B. cinerea in strawberries. β-glucan improved the colonization ability of yeast in strawberries, the biofilm forming ability, and the stress tolerance of S. spartinae W9. β-glucan also upregulated the genes involved in stress responses, cell wall formation, energy metabolism, growth, and reproduction of S. spartinae W9.