Isolation, purification, and identification of antifungal protein produced by Bacillus subtilis SL-44 and anti-fungal resistance in apple

Apple anthracnose is a fruit fungal disease that is currently recognized as one of the most severe threats to apples worldwide. In this study, antifungal protein from Bacillus subtilis SL-44 was isolated, purified, identified, and applied for Colletotrichum gloeosporioides control. The antagonistic experiment showed that SL-44 had an excellent broad spectrum against plant pathogenic fungi. The optimal fermentation conditions were as follows: initial pH was 7, inoculum volume was 2%, and rotational speed was 180 r/min. The optimized yield of antifungal protein increased by 45.83% compared with that before. The crude protein was isolated and purified by (NH4)2SO4 precipitation, DEAE-Sepharose Fast Flow, and Sephadex G-100 column chromatography. LC–MS analyzed that antifungal protein was likely to be a novel protein with a molecular weight of 42 kDa. The mechanism revealed that the antifungal protein may disrupt the cell wall structure of C. gloeosporioides and function as its antifungal action. Additionally, antifungal protein significantly alleviated the size of the lesion to more than 70% in the apple infection protection test. In conclusion, antifungal protein has remarkable potential in developing fungicides for the biological control of apple anthracnose. 1. B. subtilis SL-44 had broad-spectrum antagonism against plant pathogenic fungi. 2. The optimal fermentation conditions for extracting antifungal protein were optimized. 3. The antifungal protein is a novel protein with a molecular weight of 42 kDa. 4. The mechanism of antifungal protein may disrupt the cell wall structure of C. gloeosporioides.


Introduction
Apple (Malus domestica) is a significant economic fruit grown in China. Shaanxi is the primary producer and exporter, accounting for one-quarter of China and oneseventh of the world (Shah et al. 2022). Unfortunately, fruits are mechanically damaged during harvesting, transportation, and storage. Because the nutrients and volatiles in the damaged areas stimulate conidia germination, they are vulnerable to infection by pathogenic fungi parasitic on the fruit surface (Naets et al. 2020). Traditionally, synthetic fungicides consisting of carbendazim, thiabendazole, or imidazole have been widely used to arrest fruit decay (Bordoh et al. 2020). However, excessive application of synthetic fungicides has led to drug resistance of pathogens (Gur et al. 2021). In addition, the concern about the impact of fungicide residues on the environment and food safety has gradually increased (Wahab et al. 2022); as a result, there is an urgent need to develop biological methods to solve the problem.
Studies have shown that antagonistic microorganisms have been widely used to control soil-borne fungal diseases (Volynchikova and Kim 2022, Yin et al. 2022Zhang et al. 2020). Among the antagonistic microorganisms, Bacillus subtilis are the most promising candidate for use as a biocontrol agent because of its high growth rate, and ability to form endospores Responsible Editor: Diane Purchase and produce various antimicrobial compounds (Miljakovic, Marinkovic, and Balesevic-Tubic, 2020). B. subtilis MBI 600 reduced Botrytis cinerea mycelial growth and was able to suppress the disease incidence in cucumber plants (Samaras et al. 2021). B. subtilis NCD-2 can reduce the severity of clubroot disease by 50% . Therefore, antagonistic microorganisms have been used for biological control because they can resist pathogenic microorganisms and improve crop yield (Liang et al. 2021). The biocontrol mechanisms of B. subtilis include producing many antimicrobial substances, synthesizing extracellular enzymes, competing for nutrition and niche, and inducing systemic resistance of plants to pathogens (Caruso et al. 2022, Zhang andCheng 2022). Exceptionally, antifungal protein may be the main influential antifungal factor that inhibits the production of mycotoxins, conidia germination, and hyphal growth .
Antifungal protein is an emerging area of research due to its unique mechanism of action, excellent stability, and rare induced resistance to target pathogenic species (Toth et al. 2020). During its growth, B. subtilis secretes a very diverse library of antifungal proteins to inhibit the growth of pathogenic microbes. Besides, the antifungal protein gene was introduced into plant fungi or endophytic fungi to construct highly effective biocontrol engineering bacteria (Mathipa-Mdakane and Thantsha 2022). Whether it is directly used as a natural biochemical or expressed in animals and crops, it seems to be a promising and available candidate for bio-fungicides (Cruz-Martin et al. 2020). The purified fraction V of B. subtilis XB-1 may be a member of the chitosanase family, and the diameter of the maximum pathogen inhibition zone was 4.15 cm (Ren et al. 2019). β-glucanase in the B. subtilis CW14 genome was heterogeneously expressed in Pichia pastoris and had an antifungal effect on Aspergillus ochraceus to 97.1% . The antifungal protein MG-3A may be a serine protease with a molecular weight of about 48 kDa, which can effectively extend the shelf life of loquat fruit to 25 days (Yan et al. 2021).
However, the development and application of B. subtilis as a biological agent remain challenging despite many efforts to date. Firstly, the growth of bacteria and the production of secondary metabolites are susceptible to the influence of culture conditions, for instance, initial pH, inoculum volume, and rotational speed (Boukaew et al. 2022;Dey et al. 2020). Secondly, the secondary metabolites are unstable, with low yield and high purification cost (Rodríguez Estévez et al. 2020). In addition, the study of genome mining showed that the genome of B. subtilis has a large number of unexplored silent secondary metabolite biosynthesis gene clusters ; this indicates that the B. subtilis genome is still a valuable source of new drugs (Steinke et al. 2021). The gene (AtR100 and AtR472) have strong antimicrobial activity and broad-spectrum resistance to Xanthomonas oryzae pv. oryzicola, Clavibacter fungi, and B. cinerea (Fu et al. 2020).
In this study, B. subtilis SL-44 and C. gloeosporioides were used as the main subjects of the research. Firstly, the effect of SL-44 on the growth of pathogenic fungi was studied. Secondly, the optimal fermentation conditions for the extraction of antifungal protein were obtained by single-factor experiment and Box-Behnken design. Thirdly, the antifungal protein was extracted, purified, and identified by precipitation with (NH 4 ) 2 SO 4 , DEAE-Sepharose Fast Flow column, Sephadex G-100 column, and LC-MS. Finally, the antifungal mechanism was revealed, and the antifungal protein was applied to control the infection of C. gloeosporioides in apple fruit.

Antagonism assays
The antagonistic effect of SL-44 on pathogenic fungi was tested with minor modifications based on Li et al. (2021). Briefly, the microbial antagonism experiment was carried out in Petri plates with a diameter of 83 mm containing 20 mL of PDA medium. A hyphal plug with a diameter of 9 mm was cut from the cultured pathogenic strain and placed in the center of the plate. Four filter papers or holes were placed 22 mm from the center of the Petri plates. 10 µL of SL-44 (10 8 CFU/mL) was transferred to filter papers or holes and an equal amount of sterile water was used as a control. The average value of the inhibitory zone (the distance from the center of the filter paper or hole to the edge of the mycelium of the pathogen) was used to evaluate the antifungal effect of the sample. The inhibitory rate was calculated as (1-inhibitory zone in the treatment/colony diameter in the control) × 100.

Single-factor experiment
Many factors affect the level of microbial fermentation, among which conditions such as initial pH, inoculum volume, and rotational speed have a great influence on the growth of microorganisms and the yield of products (Bühlmann et al. 2022). The single-factor experiment was conducted to preliminarily determine the impact of initial pH, inoculum volume, and rotational speed on the effect of antifungal. In the experiment, set different values for each factor: initial pH (5,7,9), inoculum volume (1%, 2%, 3%), and rotational speed (160 rpm, 180 rpm, 200 rpm). The diameter of the inhibitory zone was set as a test indicator. The yield of antifungal protein in the fermentation broth was determined by precipitation with (NH 4 ) 2 SO 4 .

Box-Behnken design (BBD)
Design expert software (8.0.6) was used to design and analyze the experiment. The 17-run Box-Behnken design was used to optimize the parameters of the fermentation process of SL-44. The diameter of the inhibitory zone was set as the response value, and the values of three independent variables ( X 1 , initial pH; X 2 , inoculum volume; X 3 , rotational speed) and three levels were 1 (high), 0 (medium), and − 1 (low), respectively (Table S1). The equation of second-order polynomial mode is as below: where Y represents the response; 0 was a constant term coefficient; i , ii , and ij were the coefficients of the first, quadratic, and interaction terms, respectively; X i and X j were the independent variables. X i X j and X 2 i denoted the quadratic and interaction terms, respectively.

Extraction of the antifungal protein
The 2% of SL-44 seed culture was added to LB medium and incubated with shaking at 180 rpm for an additional 72 h at 30℃. The fermentation broth was centrifuged for 20 min (4℃, 12000 × g), and the supernatant was collected and filtered with a 0.22 µm filter membrane. The antifungal protein was precipitated from the supernatant by slow addition of (NH 4 ) 2 SO 4 to 40% saturation, and the mixture was stored at 4℃ overnight. The precipitated sample was centrifuged for 20 min (4℃, 12000 × g) and the antifungal protein was dissolved in 20 mmol/L Tris-HCl buffer (pH 8.4). To remove (NH 4 ) 2 SO 4 , the solution was dialyzed for 48 h and the buffer was changed every 4 h. The solution was concentrated in a freeze dryer (He Fan Instrument Co., Shanghai) and filtered through a 0.22 µm filter membrane which was further purified.

Purification of the antifungal protein
The crude protein was purified by anion exchange chromatography, gel filtration chromatography, and polyacrylamide gel electrophoresis. DEAE-Sepharose Fast Flow column (Bolshi Technology Co., Beijing) was vertically fixed in the AKTA pure 25 system (GE Corporation, USA), and 20 mmol/L Tris-HCl buffer (pH 8.4) with about 5 times the bed column volume was used to balance the chromatographic column. When balanced with the baseline, the flow rate was adjusted to 0.3 mL/min and the injection was started. The buffer solution elutes the non-adsorbed protein at a flow rate of 0.6 mL/min; then, gradient elutes the protein adsorbed on the column with 20 mmol/L Tris-HCl buffer (pH 8.4) containing 0 mol/L and 1.0 mol/L NaCl, collects the protein elution solution of each peak with 4 mL tubes, detects their antifungal activity respectively, and collects the active solution for freeze-drying and concentration. The active solution was loaded onto the Sephadex G-100 column (Maclean Biochemical Technology Co., Shanghai) pre-equilibrated with 20 mmol/L Tris-HCl buffer (pH 8.4) at a flow rate of 0.2 mL/min for gel chromatography, the protein was eluted with 20 mmol/L Tris-HCl buffer (pH 8.4), and the subsequent steps were as described above. SDS-PAGE was performed on the protein solution with antifungal activity.

LC-MS
Lysis solution (8 mol/L Urea/100 mmol/L Tris-Cl) was added to the sample, and dithiothreitol (DTT) was added after sonication in a water bath and incubated for 1 h at 37℃. Then, iodoacetamide (IAA) was added and the alkylation reaction was performed at room temperature and in the dark to close the sulfhydryl groups. The protein concentration was determined by the Bradford method (Karimi et al. 2022). After reduction and alkylation, 100 mmol/L Tris-HCl solutions were added to the sample, and the urea concentration was diluted to below 2 mol/L. Trypsin was added at a mass ratio of 1:50 of the enzyme to protein and incubated overnight at 37℃ for enzyme digestion. The next day, TFA was added to terminate the digestion and the supernatant was desalted with Sep-Pak C18. The supernatant was dried and stored at − 20℃ for freezing (Mikulášek et al. 2021). Mass spectrometry data were acquired using a Q Exactive HF mass spectrometer in tandem with an UltiMate 3000 RSLCnano liquid phase LC-MS. Peptide samples were dissolved by the loading buffer, aspirated by an autosampler, and separated by an analytical column (75 µm × 25 cm, C18, 1.9 µm, 120 Å). A 30-min analytical gradient was established using two mobile phases (mobile phase A: 0.1% formic acid, 3% DMSO, and mobile phase B: 0.1% formic acid, 3% DMSO, 80% ACN). The flow rate of the liquid phase was set to 300 nL/min. The mass spectra were acquired in DDA mode, with each scan cycle containing one full MS scan (R = 60 K, AGC = 3e6, max IT = 25 ms, scan range = 350-1500 m/z) and 20 subsequent MS/MS scans (R = 15 K, AGC = 1e5, max IT = 50 ms). The HCD collision energy was set to 27, the screening window of the quadrupole was set to 1.4 Da, and the dynamic exclusion time for ion repeat acquisition was set to 24 s. Mass spectrometry data were searched by MaxQuant (V1.6.6) software, using the database search algorithm Andromeda (Tyanova et al. 2016). The database used for the search was the custom proteome reference database Proteome (containing 4260 protein sequences). The main search parameters were as follows: Oxidation (M), Acetyl (Protein N-term) for variable modifications; Carbamidomethyl (C) for immobilization modifications; Trypsin/P for enzymatic cleavage; the primary mass spectrometry match tolerance was set to 20 ppm for the initial search and 4.5 ppm for the primary search; the secondary mass spectrometry match tolerance was set to 20 ppm. The search results were filtered by 1% FDR at the protein and peptide levels, and protein entries with anti-library proteins, contaminating proteins, and proteins with only one modified peptide were removed, and the remaining identification information was used for subsequent analysis.

Electron microscopy
FlexSEM 1000 Scanning Electron Microscope (Hitachi, Tokyo, Japan) and HT7800 Transmission Electron Microscope (Hitachi, Tokyo, Japan) were used to observe the morphological changes and ultrastructure of the sample. Samples were taken from mycelium at the edge of the inhibitory zone (5 mm × 5 mm). Hyphae were fixed in 2.5% glutaraldehyde solution at 4℃ overnight and then rinsed with 0.1 mol/L PBS buffer (pH 7.2) for 10 min (three times). The rinsed hyphae were fixed in 1% osmic acid solution for 1 h and then rinsed with 0.1 mol/L PBS buffer (pH 7.2) (three times). Next, the sample was dehydrated with ethanol solutions of 30%, 50%, 60%, 70%, 80%, and 90% for 10 min and then treated with 100% ethanol twice for 20 min. TEM sample was prepared and observed as described (Pang et al. 2021).

Minimum inhibition concentration (MIC)
MIC of antifungal protein was determined using the micro double dilution method (Schön et al. 2021). 50 µL of potato dextrose broth (PDB) containing the antifungal protein (2.81 mg/mL) was mixed with 50 µL of C. gloeosporioides spore suspension (10 6 spores/mL). The final concentration of antifungal protein in each well was 0, 1, 2,4,8,16,32,64,128, and 256 µg/mL. MIC was determined by incubating at 30℃ for 48 h. The lowest antifungal protein with no visible spore growth at the bottom of the well was used as MIC.

Ergosterol content
The content of ergosterol was determined according to . In brief, 100 µL of C. gloeosporioides was added to a PDB medium containing 0, 32, 64, and 128 µg/mL of antifungal protein and incubated at 28℃ for 4 days, and the mycelium was collected by centrifugation. The mycelium was suspended in ethanol-KOH (25% w/v, 3 mL) and a water bath at 85℃ for 1 h. 1 mL of distilled water and 3 mL of N-heptane were added and vortexed for 3 min; then, the heptane layer was separated and diluted with ethanol, and finally scanned by spectrophotometer (Shimadzu, Japan) at 240-300 nm.

Protein and nucleic acid leakage
500 mg of C. gloeosporioides was resuspended in 50 mL of PBS (pH 7.8, containing 0, 32, 64, and 128 µg/mL of antifungal protein) and incubated at 30℃, 180 rpm. The changes in absorbance values at 260 nm and 280 nm at different times were measured by a spectrophotometer (Xue et al. 2019).

Malondialdehyde (MDA) content
The mycelium of C. gloeosporioides was resuspended in PBS (pH 7.8, containing 0, 32, 64, and 128 µg/mL of antifungal protein) and incubated at 30℃, 180 rpm. 500 mg of mycelium treated with antifungal protein was incubated with 10 mL of PBS (pH 7.8, containing 2% polyvinylpyrrolidone and 1 mM EDTA) in an ice bath to form a homogenate and centrifuged at 12,000 × g and 4℃ for 15 min. 2 mL of supernatant was mixed with 2 mL of 0.67% thiobarbituric acid and boiled for 30 min. After the solution was cooled to room temperature, the mixture was centrifuged at 12,000 × g for 15 min. The supernatant was collected and the absorbance values were measured at 532 nm, 600 nm, and 450 nm (Handayani et al. 2019).
V t represents the total volume of the extract, V s represents the volume of the extract used for the determination, and W represents the sample mass.

Apple trials
Apple fruits with no wound and uniform size, color, and maturity were selected for follow-up experiments. The assay was based on the method described by Zheng et al. (2023) with some modifications. Apple fruit was soaked in 90% ethanol for 1 min and 3% sodium hypochlorite (NaOCl) for 60 s for surface disinfection and finally rinsed with sterile distilled water (three times). Four punctures were performed on the surface of apple fruits with a sterile needle to produce lesions (2 mm × 3 mm). 5 µL of conidial suspension of C. gloeosporioides and 10 μL of antifungal protein prepared in 20 mmol/L Tris-HCl buffer (pH 8.4) were inoculated at each wound site, and an equal volume of 20 mmol/L Tris-HCl buffer (pH 8.4) was used as a control. All apple fruits were transferred to a sterile plastic container and incubated in an artificial climate chamber at 25℃ and high humidity (about 60%) for 15 days. The diameter of the rot around each wound was recorded.

Statistical analysis
Origin 8.5 was used for data processing and displayed as mean ± standard error (SE). SPSS 19.0 (Chicago, IL, USA) analyzed statistical significance by one-way analysis of variance (ANOVA). When p ˂ 0.05, the difference was statistically significant.

SL-44 can efficiently inhibit the growth of pathogens
The suppressive activity of SL-44 against plant pathogenic fungi was tested on PDA plates (Fig. 1). Results showed that the hyphae of C. gloeosporioides reached the edge of plates in the control group, and its growth rate decreased after 5 to 6 days with SL-44 inoculation. In the next few days, the inhibitory zone of C. gloeosporioides did not change, remaining at an average of around 15.03 mm (Fig. 1a). This indicated that SL-44 may produce diffusive metabolites that efficiently inhibited the growth of C. gloeosporioides. Statistical analysis showed that SL-44 could reduce the average inhibitory rate of C. gloeosporioides in the plates by 81.89% (Fig. 1c). To determine whether the inhibitory effect of SL-44 can be broad-spectrum, other plant pathogenic fungi, including R. solani, A. alternata, F. oxysporum, and B. cinerea, were tested in the manner described above. Results showed that all plant pathogenic fungi were nearly spread over the entire surface in the control group, while their colonies were remarkably reduced in the treatment group (Fig. 1a). The growth of the F. oxysporum was decreased by SL-44, with an average inhibitory zone of 10.88 mm after 10 days, corresponding to an inhibitory rate of 86.89%, followed by B. cinerea, with an average inhibitory zone of 6.18 mm. SL-44 had a weak inhibitory effect on R. solani and A. alternata because they were also suppressed with an inhibitory zone of 4.12 mm and 5.07 mm. In general, SL-44 showed broad-spectrum antagonism and excellent control effects against a variety of plant pathogenic fungi. B. amyloliquefaciens 3-5 inhibited all 10 pathogenic fungi tested, with a 60% inhibition level when the mass ratio of 3-5 to nutrient soil was 10% ). Studies have shown that most Bacillus can produce a variety of antimicrobial substances, which can inhibit the growth of pathogens. Such for antifungal enzymes, the main antifungal mechanism is to change the permeability of the membrane by degrading the cell wall, causing membrane damage, so the cell content flows out and dies (Mota et al. 2022).
To further observe the inhibitory effect of SL-44 on the growth of the pathogen, the morphology of the pathogen treated by SL-44 was observed by SEM (Fig. 1b). Results showed that the mycelium of the control group was relatively smooth; its cytoplasm was transparent, uniform, and spaced; and the tips of the mycelium were tapered. However, the mycelium treated by SL-44 changed significantly compared with the normal mycelium, with the middle and tip of the mycelium expanded, twisted, or deformed, and finally broken to undergo lysis (Fig. 1b). Those changes indicate that SL-44 may produce antifungal substances to act on the mycelium of the pathogen, causing mycelial cells to become swelling, deformed, and eventually lysis, thus inhibiting the growth and reproduction of pathogen. This may be related to the fact that these antimicrobial substances contain enzymes that can lyse the cell walls or cell membranes of the pathogen, while the specific mechanism of their inhibition needs to be further investigated. Bacilysin can disrupt P. sojae hyphae so that B. velezensis FZB42 can efficiently inhibit the growth of P. sojae and other Phytophthora species (Han et al. 2021

Optimal fermentation conditions obtained by single-factor experiment and BBD
The yield and activity of antimicrobial substances in Bacillus fermentation products are mainly influenced by the fermentation conditions, so optimizing the fermentation conditions is an important way to improve the yield of antimicrobial substances (Hassan et al. 2020). B. subtilis grows logarithmically in the early stage, gradually accumulates metabolites in the middle stage, and dies when it reaches stable in the later stage (Ahsan et al. 2022). SL-44 already produced antimicrobial substances at 24 h, and as time went on, the number of antimicrobial substances continues to increase, reaching the maximum antifungal activity at 72 h. However, the antifungal activity decreased significantly after 72 h (Fig. 2a). This is probably because the growth of SL-44 entered a recession period, releasing a large number of enzymes (such as protease, lipase, etc.), and degrading part of antimicrobial substances, resulting in their reduced activity (Hjellnes et al. 2021). The growth and reproduction of bacteria need a suitable living environment, and when it is too high or too low, the biochemical reaction of bacteria will be affected; thus, the growth of bacteria will be inhibited (Balakrishnan et al. 2021). The antifungal activity was the best at pH 7 (Fig. 2b). It is the natural pH of the culture medium, which does not need to be adjusted by HCl or NaOH. This condition is suitable for the biosynthesis of antimicrobial substances ). The antifungal activity first improved with the increase of the amount of inoculation, reaching the peak when the amount of inoculation is 2%, and then showing a downward trend (Fig. 2c). The reason may be that the production of antimicrobial substances continues to increase over time, but because SL-44 is an aerobic bacterium, the number of bacteria in the culture medium is too much, resulting in the rapid consumption of nutrients, which makes the number of antifungal substances continue to decrease (Tao et al. 2022). The speed of the shaker is responsible for regulating the amount of dissolved oxygen. Under the same conditions, too high or too low speeds are not conducive to bacterial growth and reproduction (Ciobanu et al. 2020). With the continuous increase of rotational speed, the antifungal effect continues to increase; when the rotational speed exceeded 180 r/min, the antifungal activity decreases (Fig. 2d). Considering the actual operation, the optimum rotational speed of SL-44 fermentation was 180 r/min.
Based on the single-factor test results, the Box-Behnken design was carried out with initial pH, inoculum volume, and rotational speed as independent variables and the inhibitory zone as response value (Fig. 2e). Design-Expert 8.0.6 software is used to fit Table S2 into multiple regression analysis to obtain regression equation.
The regression model significance test and ANOVA were performed and the results are shown in Table S3. As seen in the above table, the regression model was highly significant (p model < 0.0001), the inhibitory zone misfit term was significant (p = 0.0009 < 0.05), the model had a good fit, and the experiment error was small. The coefficient of determination, R 2 = 0.9821, indicated a good correlation between the measured and predicted values of Y. Over 98.21% of the experimental values could be explained by this equation. The factors affected the degree of the inhibitory zone differently, A (0.1996) > B (0.5264) > C (0.9359); AB, AC, and BC were not significant (p > 0.05); the secondary terms A 2 , B 2 , and C 2 were significant (p < 0.0001).
By analyzing the regression model equation, it was concluded that the optimal fermentation conditions of SL-44 were initial pH 7, inoculum volume 2.03%, and rotational speed 180.09 rpm. To verify the degree of optimization of fermentation conditions, the antifungal activity and antifungal protein yield of the optimized fermentation conditions were compared and analyzed. Under the original fermentation conditions, the inhibitory zone was 11.95 mm, and about 0.24 g of antifungal protein could be obtained per liter of fermentation broth; the optimized inhibitory zone can reach 14.02 mm, which is 17.32% higher than that before optimization. 0.35 g extract of antifungal protein can be obtained per liter of fermentation broth, which is 45.83% higher than that before optimization.

Isolation, purification, and identification of the antifungal protein
To explore the structure and function of the antifungal protein, which can be better applied in agricultural production, it is necessary to isolate and purify the antifungal protein.
The fermentation supernatant was precipitated with 40% saturated (NH 4 ) 2 SO 4 to obtain crude protein. (NH 4  precipitation can effectively separate the antifungal protein from other miscellaneous proteins. Figure 3a-b show that 40% saturated (NH 4 ) 2 SO 4 precipitated protein has obvious antifungal activity. The dialyzed precipitated protein was filtered and loaded on a DEAE-Sepharose FastFlow column for separation. A peak (P1) appeared at the beginning of the elution with 20 mmol/L Tris-HCl buffer (pH 8.4), and then a high peak (P2) appeared with 20 mmol/L Tris-HCl buffer (pH 8.4) containing 1.0 mol/L NaCl (Fig. 3e). The analysis found that P1 is the unabsorbed fraction, indicating that fraction contains proteins that are uncharged or positively charged at the buffer. P2 contains a protein fraction that is strongly adsorbed, indicating that this fraction contains proteins with a large negative charge at the buffer and an isoelectric point bias towards acidity. The activity assay showed that P2 had an antifungal activity with an inhibition zone diameter of 11.67 mm, while P1 had no antifungal activity (Fig. 3c). Results indicated that P1 contained no active substance and P2 contained active protein. P2 was passed through a Sephadex G-100 column, one absorption peak was eluted (Fig. 3f), P2-1 was found to have antifungal activity with an inhibition zone diameter of 12.35 mm (Fig. 3d), and SDS-PAGE analysis of P2-1 yielded a single band with a molecular weight of 42 kDa (Fig. 3g). Protein yields of various purification steps are shown in Table 1. The protein secreted by B. subtilis XB-1 was extracted by 70% saturation (NH 4 ) 2 SO 4 and purified by Sephadex G-25 gel filtration column, and SDS-PAGE showed a single band with a molecular weight of about 43 kDa (Ren et al. 2019).
MaxQuant (V1.6.6) software was used for the retrieval of mass spectrum data, and the database was a custom proteome reference database proteome (containing 4260 protein sequences). The mass spectrum search results based on P2-1 are shown in Table S4; a total of 14 proteins were    identified, of which 7 proteins had quantitative information. Based on the mass spectrometry identification results, three proteins were initially identified with high confidence ( Fig. 3h-j), namely Uncharacterized YefB (accession number: O34574), ATP synthase subunit alpha (accession number: P37808), and Vegetative catalase (accession number: P26901). The Uncharacterized protein YefB (accession number: O34574) was analyzed using Prot Param software. A total of 300 amino acids were detected, the molecular weight is 35.046 kDa, and the theoretical isoelectric point is 9.10. Amino acid sequences of peptides of protein: IIEINEKLNDK which was assigned to the precursor 443.91 m/z. So, we concluded that this protein encodes an unknown protein and is considered a novel antifungal protein. In addition, it was found that the total number of positively charged residues (arginine and lysine) in the sequence was found to be 52 and the total number of negatively charged residues (aspartic acid and glutamic acid) was found to be 43. The predicted half-life is 30 h in mammalian reticulocytes, > 20 h in yeast, and > 10 h in Escherichia coli when cultured in vitro. Predict all protein domains and functional sites, and extract the annotation information of GO (Fig. S1A). The cellular process and metabolic processes are the most active in the biological process; the plasma membrane has obvious advantages in cellular components; binding and catalytic activity play a major role in molecular function. Genes involved in various metabolic pathways in the P2-1 proteome sequence were analyzed by KEGG (Fig. S1B). A large number of genes were involved in the metabolism of various substances, 17.14% in the metabolic pathways and 8.57% in the biosynthesis of secondary metabolites. The abundant metabolic pathways provide the essential carbon and nitrogen sources for the growth of SL-44 and the synthesis of secondary metabolites .
Depending on the synthesis pathway of antimicrobial substances, they can be divided into two categories: ribosomal synthesis pathway and non-ribosomal synthesis pathway. Some Bacillus subtilis can also produce some active antimicrobial proteins; the mechanism of inhibition of these proteins is to destroy the cell wall to cause deformation of the mycelium so that the spores germinate abnormally and inhibit its growth and reproduction (Rismondo and Schulz 2021). The transfer of genes encoding such antimicrobial proteins into plants to make them exhibit the corresponding antimicrobial properties is now one of the research priorities.

Mechanism of action of antifungal protein for controlling apple anthracnose
The MIC of antifungal protein was measured in 96-well plates using a twofold dilution method to assess its effect on the growth of C. gloeosporioides. Results showed that the MIC of antifungal protein on C. gloeosporioides was 64 µg/mL. To investigate the mechanism of inhibition of C. gloeosporioides by antifungal protein, TEM found that the untreated C. gloeosporioides cell wall was discernible full cytoplasm and intact organelle morphology (Fig. 4a). However, after treatment with the antifungal protein, cell wall rupture was observed with disorganized cytoplasmic structure and efflux of contents (Fig. 4b) (Liu et al. 2020). The rupture of the cell wall leading to the extravasation of cellular contents should be responsible for the damage to the mycelial structure shown in Fig. 1b. Studies have shown that antifungal protein targets different constituents in the cell wall of plant pathogenic fungi and disrupts the cell wall to resist the pathogenic fungal (Aliyeva-Schnorr et al. 2022;Kim et al. 2021). Ergosterol is an important component of microbial cell membranes and plays an important role in ensuring cell membrane integrity, membrane fluidity, and cellular material transport (Jiang and Lin 2022). The ergosterol content on the plasma membrane of C. gloeosporioides cells decreased significantly in a dosedependent manner with increasing concentrations of antifungal protein treated (Fig. 4c), suggesting that antifungal protein can affect the structural integrity of C. gloeosporioides cell membranes and thus their biological functions. The OD260 of the control supernatant did not change significantly with the increase of time (Fig. 4d). However, the OD260 of the supernatant of the 1MIC-treated group increased slowly in the first 120 min, and then remained stable at about 0.8 after 150 min until 180 min, indicating that the mycelial nucleic acid leakage of 1MIC treated became more and more serious with the extension of time. The trend of OD260 of supernatant of the 2MIC-treated group was similar to that of the 1MICtreated group. Throughout the treatment time, the OD260 of supernatant of the 2MIC-treated group was higher than that of the 1MIC-treated group, indicating that the high concentration of antifungal protein treatment could cause more serious damage to the cell membrane of C. gloeosporioides mycelium, which led to more nucleic acid leakage. Figure 4e shows the protein leakage from C. gloeosporioides mycelium treated with different concentrations of antifungal protein. The supernatant OD values of the control group did not change significantly throughout the treatment period, while the supernatant OD values of the 1MIC-treated group gradually increased at 120 min and were significantly higher than those of the control group (p < 0.05), after which the changes were not significant. In contrast, the supernatant OD values in the 2MIC-treated group were significantly higher than those in the control group throughout the treatment period (p < 0.05). These changes may be attributed to increased cell permeability and disruption of membrane integrity, usually implying leakage of electrolytes and loss of inclusions (Guo et al. 2020). MDA is the most important lipid peroxidation metabolite and is a key parameter reflecting the level of lipid peroxidation in an organism (Mousavi et al. 2020). The MDA content of C. gloeosporioides increases with the increase of antifungal protein concentration. When the antifungal protein concentration was 64 and 128 µg/mL, the MDA content of C. gloeosporioides was 7.33 and 9.46 nmol/g mycelia, which were 1.48 and 1.91 times higher than that of the control, respectively (Fig. 4f). Therefore, the mechanism of inhibition of C. gloeosporioides by antifungal protein may be by disrupting the membrane integrity of C. gloeosporioides mycelium, allowing the release of nucleic acids and proteins, which leads to impaired cell function and loss of viability. The effect of the antifungal protein on the progression of the lesion caused by C. gloeosporioides in apple fruit is shown in Fig. 4g-j. Fifteen days after inoculation of C. gloeosporioides on the surface of apples, dark brown spots with an average diameter of 18.36 ± 0.06 mm appeared on the surface (Fig. 4h). In the section, the rot gradually spread because C. gloeosporioides invaded the internal tissues. Eventually, the damaged area spread over almost the entire apple. The pathogen caused a distinct dark brown circular rot around the puncture point of the apple, and the surface of the lesion had a musty odor. In addition, conidia were observed on the wound surface. After the application of crude protein, the size and color of the lesions were significantly slowed down (Fig. 4i). As shown in Fig. 4j, the application of antifungal proteins significantly reduced the size of lesions induced by C. gloeosporioides to 13.67 ± 0.12 mm (more than 70%) in lesion diameter compared to untreated apples. Results showed that the antifungal proteins could effectively inhibit the growth of C. gloeosporioides and protect the apple from disease infection. Antifungal proteins may affect not only conidial germination but also any stage between conidial germination and infection, ultimately allowing disease mitigation (Arya et al. 2022). Therefore, antifungal proteins are significantly effective against apple diseases and can be widely used in agricultural production environments.

Conclusions
SL-44 could inhibit the growth of pathogenic strains and exhibit a broad-spectrum antifungal effect. The optimal fermentation conditions were as follows: initial pH was 7, inoculum volume was 2%, and rotational speed was 180 r/ min. Under these conditions, the yield of antifungal protein increased by 45.83% compared to that before optimization. The antifungal protein was likely a novel protein with a molecular weight of approximately 42 kDa. It exerts its antifungal effect by disrupting the structure and function of the cell walls. Meanwhile, it can reduce the content of ergosterol and affect the structure and stability of the membrane. Furthermore, it significantly alleviated the size of the lesion to more than 70% in the apple infection protection test. Therefore, antifungal protein may be a new and safe way to inhibit the development of apple anthracnose.