Bacillus paralicheniformis and application of volatile substances thereof

Abstract

This invention discloses a strain of *Bacillus paralicheniformis* IFST-745 and the application of its volatile substances. *Bacillus paralicheniformis* IFST-745 was deposited on May 4, 2023, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC No. 27245. The volatile substances produced by this strain IFST-745 can inhibit the growth of various plant pathogenic fungi in vitro. It can effectively inhibit *Penicillium fingernail* infection of citrus and *Fusarium verticillatum* infection of corn kernels. It also has good control effects on green mold of citrus fruits and ear rot of corn. This invention provides a reference and more options for biocontrol agents of postharvest fungal diseases, and has significant application value.

Description

Technical Field

[0001] This invention relates to the field of microbial control technology, specifically to the application of a strain of Bacillus paralicheniformis IFST-745 and its volatile substances in disease control. Background Technology

[0002] Statistics show that approximately 20-25% of fruits and vegetables worldwide are infected by plant pathogenic fungi during post-harvest handling each year, leading to mold and rot, resulting in significant resource waste and economic losses. Most pathogenic fungi causing fruit and vegetable mold not only cause disease but also produce various mycotoxins, posing a serious threat to the health of animals and humans who ingest them. Fungi belonging to the genera *Aspergillus*, *Fusarium*, and *Penicillium* are particularly harmful. Among these toxic compounds, aflatoxin, ochratoxin A, deoxynivalenol, fumonisin, patulin, and patulin are the most important and dangerous mycotoxins in agriculture and the food industry. The presence of toxin-producing fungi not only leads to a decline in crop quality and yield but also threatens public health through contaminated food.

[0003] Bacillus sp., a diverse genus of bacteria, possesses characteristics such as rapid reproduction, strong resistance, mature fermentation processes, and long shelf life, making it an important source of biocontrol strains. Bacillus primarily exerts its biocontrol potential by competing with pathogenic fungi for nutrient space, secreting antimicrobial substances, and inducing plant resistance. Among these, volatile substances produced during Bacillus metabolism are significant biocontrol factors. It has been reported that volatile substances produced by various Bacillus species have a certain inhibitory effect on the vegetative growth of different postharvest pathogenic fungi. Compared with traditional pesticides, these volatile small-molecule compounds do not leave obvious residues on the fruit surface and can exhibit effects similar to traditional fungicides. Fumigating postharvest fruit with volatile organic compounds (VOCs) produced by Bacillus or artificially applied mixtures of volatile small molecules can eliminate or inhibit pathogens during transportation and storage, representing an effective method for low-residue control of postharvest diseases. In recent years, with the continuous improvement of agricultural sustainability and public concern about the use of hazardous chemicals, biological control is increasingly becoming an alternative to fungicides, and Bacillus VOCs are expected to become an effective biological control agent. Summary of the Invention

[0004] The purpose of this invention is to provide a strain of Bacillus paralicheniformis IFST-745, which can inhibit the growth of mycelia of various pathogenic fungi. The volatile substances produced by this strain can also inhibit the growth of various postharvest pathogenic fungi and control them, thus enriching the biocontrol bacterial library and providing more options for the biological control of postharvest fungal diseases. This invention addresses the problems of limited types of existing biocontrol bacteria for postharvest diseases, restricted selection, narrow antibacterial spectrum, poor efficacy, and poor environmental adaptability.

[0005] To achieve the above objectives, this invention provides a strain of Bacillus paralicheniformis IFST-745, which was deposited on May 4, 2023, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC No. 27245.

[0006] The Bacillus paralichrysogenum IFST-745 provided by this invention can be used for post-harvest disease control in crops.

[0007] Furthermore, the aforementioned diseases are plant pathogenic fungi, including one or more of the following: Fusarium verticillatum, Fusarium graminearum, Aspergillus flavus, Phytophthora citrinum, Fusarium solani, Penicillium digitatum, and Botrytis cinerea.

[0008] The present invention also provides a biocontrol agent comprising the above-mentioned Bacillus paralichrysiformis IFST-745 and / or volatile substances produced therefrom that have antibacterial activity.

[0009] Furthermore, the volatile substances contained in the above include one or more of the following: n-butanol, isoamyl alcohol, 1-decyl alcohol, 2-phenylethanol, hexyldecyl alcohol, butyl 2-methylbutyrate, isopropyl palmitate, diisobutyl phthalate, acetic acid, 3-methylvaleric acid, 2-amino-6-methylbenzoic acid, 1,2,4,5-tetramethylbenzene, phenol, 3,5-di-tert-butylphenol, 2-methylpyrazine, 2,5-dimethylpyrazine, 2-ethyl-5-methylpyrazine, 2,3,5-trimethylpyrazine, hexadecane, heptadecane, 6-methyl-2-heptanone, 2-decanone, 2-nonadecanone, o-aminoacetophenone, 3,4-dimethylacetophenone, paraformaldehyde, and chamomile blue.

[0010] The biocontrol agent provided by this invention can be used for the prevention and control of citrus green mold and / or corn ear rot.

[0011] The present invention relates to Bacillus paralichrysogenus IFST-745 and its applications. This strain and its volatile substances can inhibit a variety of pathogens and have the following advantages:

[0012] 1. The *Bacillus paralichrysogenus* IFST-745 provided by this invention was isolated from kimchi samples. The volatile substances it produces can inhibit the mycelial growth of various pathogens, including *Fusarium verticillatum*, *Fusarium graminearum*, *Aspergillus flavus*, *Phytophthora nicotinae*, and *Penicillium digitatum*, with inhibition rates of 43.53%, 26.53%, 27.56%, 25.88%, and 42.15%, respectively. This demonstrates that *Bacillus paralichrysogenus* IFST-745 has good and safe application prospects in the preparation of biocontrol agents that inhibit the growth of pathogens.

[0013] 2. The present invention also provides a group of volatile substances produced by Bacillus paralichrysiformis IFST-745 that can inhibit the growth of pathogens, and it has been determined that 13 of these substances have an inhibitory effect on the mycelial growth of Fusarium verticillatum and Penicillium digitatum. At the same time, these 13 volatile substances also have antibacterial effects on Botrytis cinerea, Phytophthora nicotineum, Aspergillus flavus and Fusarium graminearum.

[0014] 3. The 13 volatile substances with antibacterial activity provided by this invention can be used to prevent and control corn ear rot caused by Fusarium oxysporum and citrus green mold caused by Penicillium digitatum. Among them, chamomile, 3,5-di-tert-butylphenol, 2-decanone, acetic acid and phenol have significant inhibitory effects on citrus green mold caused by Penicillium digitatum. At the same time, all 13 substances can significantly reduce the incidence of corn ear rot, and have significant disease control effects. Attached Figure Description

[0015] Figure 1 This is the plate inhibition result of the target bacteria screened in this invention against Fusarium verticillatum.

[0016] Figure 2 This is the phylogenetic tree result of IFST-745 based on gyrA in this invention.

[0017] Figure 3 This is the result of the dual-plate-on-plate inhibition of various pathogens by IFST-745 in this invention.

[0018] Figure 4 The results show the inhibitory effects of different volatile compounds generated by IFST-745 on Fusarium verticillatum in this invention.

[0019] Figure 5 The results show the inhibition of Penicillium fingering by different volatile compounds generated by IFST-745 in this invention.

[0020] Figure 6 The results show the inhibitory effect of VOCs generated by IFST-745 in this invention on the mycelial growth of other pathogenic fungi.

[0021] Figure 7This invention demonstrates the control effect of 13 volatile compounds with potent antibacterial activity generated by IFST-745 on green mold disease in citrus caused by Penicillium finger.

[0022] Figure 8 This invention describes the control effect of 13 volatile compounds with strong antibacterial activity generated by IFST-745 on corn ear rot caused by Fusarium oxysporum. Detailed Implementation

[0023] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0024] Note: All reagents and consumables not specifically described in this experiment are conventional reagents and consumables, and all methods not specifically described are conventional operating methods in this field.

[0025] Partial culture medium:

[0026] LB liquid medium: 10 parts tryptone, 5 parts yeast extract, 10 parts NaCl, and sterile H2O to make up to 1000 parts. Adjust the pH to 7, autoclave at 121℃ for 20 minutes, and store for later use.

[0027] LB solid medium: Add 15 g / L agar powder to LB liquid medium, autoclave at 121℃ for 20 min and store for later use.

[0028] PDA medium: Wash and peel potatoes, cut them into small pieces, weigh 200 portions, add 800 portions of sterile H2O, boil for 25 minutes, filter through four layers of gauze, add 20 portions of glucose and 15 portions of agar powder to the filtrate, continue heating and stirring evenly, cool, add sterile H2O to make up to 1000 portions, autoclave at 121℃ for 20 minutes, and store for later use.

[0029] Example 1: Isolation and Identification of Bacillus paralichrysum IFST-745

[0030] This embodiment provides the isolation process of Bacillus paralichrysogenum IFST-745, as detailed below:

[0031] (1) Isolation and purification of the strain: Under aseptic conditions, a small amount of kimchi sample was placed in a 90 mL Erlenmeyer flask containing sterile physiological saline and shaken thoroughly. A sterile inoculating loop was used to collect the suspension of the sample, and it was serially diluted with physiological saline (to 10⁻⁶ ppm each time). -4 10 -5 10 -610 -7 Each concentration of dilution was streaked evenly onto LB agar plates, with each concentration gradient repeated three times. The plates were then incubated at 30°C for 2 days. Single colonies with different culture characteristics, colors, shapes, and textures were selected from the plates and purified on LB agar plates. The purified single colonies obtained by streaking multiple times on LB agar plates were numbered and stored.

[0032] (2) Preparation of bacterial culture: The bacterial strains isolated and purified in (1) above were inoculated into LB liquid medium and cultured at 37℃ and 180 rpm for 16-18 h to obtain the culture solutions of each strain; the bacterial culture concentration was adjusted to 10. 9 CFU / mL, store at 4°C for later use.

[0033] (3) Preparation of pathogen spore suspension: Fresh pathogens cultured on PDA medium were picked, and mycelia were transferred to sterile water and thoroughly mixed and shaken. The concentration of the pathogen spore suspension was adjusted to 1×10⁻⁶ using a hemocytometer. 6 The spores were prepared at a concentration of 1 spore per mL for later use, thus obtaining a spore suspension of each pathogen. This suspension was stored at 4°C for later use.

[0034] (4) Screening of biocontrol strains with antibacterial effects: Using Fusarium verticillatum as an indicator bacterium, the strains isolated and purified from kimchi samples were screened for volatile antibacterial active substances through a double-plate mating test. 20 mL of LB solid medium was poured into each plate. After the medium cooled and solidified, 10 μL of the strain culture solution from (2) was added to the center of the LB medium. Another number of plates were prepared, and 20 mL of PDA medium was poured into each plate. After cooling and solidification, 0.7 mm of Fusarium verticillatum mycelium was taken with a punch and inoculated into the center of the PDA medium. The plates were mated with the bacteria on the LB medium and sealed with sealing film. The control group was treated with no inoculated strains on the LB plates. Each treatment was repeated 3 times and cultured at 25℃ for 5 days. The strains with significantly limited vegetative growth of Fusarium verticillatum compared with the control group were selected, and the inhibition rate was calculated. The plate inhibition results of the target bacteria against Fusarium verticillatum are as follows. Figure 1 As shown in the figure, A represents the control group, and B represents the experimental group inoculated with the selected strain. The inhibition rate was calculated based on the inhibition zones, and a strain with strong antibacterial effect was obtained and named IFST-745, with an inhibition rate of 39.7%.

[0035] (5) Identification of IFST-745: Based on the methods described in Bergey's Manual of Bacterial Identification, 8th Edition and the Manual of Systematic Identification of Common Bacteria, the colony morphology and physiological and biochemical characteristics of this bacterium were identified as follows:

[0036] Morphological characteristics: After culturing single colonies of IFST-745 on LB solid medium for 2 days, the colonies are 3-4 mm in diameter, milky white, and raised. Gram staining of IFST-745 produces a blue-purple color, indicating Gram-positive bacteria. Scanning electron microscopy reveals that the bacteria are rod-shaped, approximately 1.90-3.94 μm in length.

[0037] The results of the physiological and biochemical characteristic tests are shown in Table 1 below. It can be seen that strain IFST-745 can produce catalase, but not oxidase; the urease, gelatin hydrolysis, casein hydrolysis, starch hydrolysis and β-galactosidase activities of IFST-745 are all positive; while Tween 20, Tween 40, Tween 80, stachyose and inositol are all negative.

[0038] Table 1 Physiological and biochemical characteristics of IFST-745

[0039]

[0040] Molecular biological identification: The IFST-745 genome was extracted using the Tiangen Bacterial Genome Extraction Kit. PCR amplification of the gyrA gene was performed using universal primers for Bacillus spp. gene identification. The amplified products were recovered using a MEGAX biogel recovery kit, ligated into the pMD18-TVector plasmid, and sent to Sangon Biotech Co., Ltd. for sequencing. The sequencing results were compared and analyzed using NCBI's BLAST tool. After obtaining relevant gene sequence information, a phylogenetic tree of the two genes was constructed using MEGAX. The sequencing results were then assembled using DNAstar and the vector sequence was removed. The results were uploaded to the Basic Local Alignment Search Tool (BLAST, https: / / blast.ncbi.nlm.nih.gov / Blast.cgi) in The National Center for Biotechnology Information (NCBI) for homology comparison analysis. The gyrA gene sequence was compared using BLAST, and similar sequences were selected for sequence alignment analysis. A phylogenetic tree was constructed using MEGAX. The IFST-745 phylogenetic tree based on gyrA is shown below. Figure 2 As shown in the figure. Further analysis of the phenotypic characteristics of IFST-745 indicates that IFST-745 is a strain of Bacillus paralicheniformis.

[0041] Based on the above characteristics, IFST-745 was identified as *Bacillus paralicheniformis*. This strain was deposited on May 4, 2023, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, 100101, China, with accession number CGMCC No. 27245 and taxonomical name *Bacillus paralicheniformis*.

[0042] Example 2: Determination of the antibacterial activity of IFST-745

[0043] This embodiment provides the inhibitory effect of volatile substances produced by Bacillus paralichrysogenus IFST-745 on various pathogens. The method for determining the antibacterial ability is as follows:

[0044] Bacillus paralichrysogenum IFST-745 was inoculated into LB liquid medium and cultured at 37℃ and 180 rpm for 16-18 h to obtain a suspension of IFST-745; the bacterial concentration was adjusted to 10. 9 CFU / mL, store at 4°C for later use.

[0045] *Fusarium verticillatum*, *Fusarium graminearum*, *Fusarium solani*, *Aspergillus flavus*, *Phytophthora nicotinae*, *Penicillium fingernail*, and *Botrytis cinerea* were activated from a -80℃ freezer, followed by a double-plate inoculation experiment. The procedure was performed on a clean bench. 20 mL of LB solid medium was poured into each plate, and after cooling and solidification, 10 μL of IFST-745 suspension was dropped into the center of the plate. Separately, a 0.7 mm mycelial disc of each of the seven pathogens was inoculated into the center of a PDA medium plate and inverted onto the LB medium inoculated with IFST-745 for the double-plate inoculation experiment, and sealed with sealing film. A control group was used, with LB plates without IFST-745 inoculation. Each treatment was repeated three times, and the plates were incubated at 25℃ for 5 days. The colony diameter of each pathogen was measured, and the inhibition rate was calculated. The double-plate inhibition results of IFST-745 against multiple pathogens are shown below. Figure 3 As shown, where, Figure 3 In the diagram, A represents a schematic diagram of a flat plate fastening experiment. Figure 3 B in the figure represents the results of the cross-hatching experiment for seven pathogens. Figure 3The value of C in the figure represents the inhibition rate of the pathogen. It can be seen that *Bacillus paralichrysiformis* IFST-745 exhibits inhibition rates of 43.53%, 26.53%, 27.56%, 25.88%, and 42.15% against five pathogens: *Fusarium solani*, *Fusarium graminearum*, *Aspergillus flavus*, *Phytophthora xanthoides*, and *Penicillium digitatum*, respectively. However, its inhibition rates against *Fusarium solani* and *Botrytis cinerea* are relatively low, at only 9.42% and 6.14%, respectively. This indicates that the volatile organic compounds (VOCs) produced by IFST-745 have a good inhibitory effect on *Fusarium solani*, *Penicillium digitatum*, *Aspergillus flavus*, *Fusarium graminearum*, and *Phytophthora xanthoides*. Therefore, this strain IFST-745 and its produced VOCs can be used to prepare biocontrol agents for the control of various pathogenic fungi.

[0046] Example 3: Generation, Collection and Identification of Volatile Antibacterial Substances of IFST-745

[0047] I. The method for generating and collecting volatile antibacterial substances from Bacillus paralichrysiformis IFST-745 is as follows:

[0048] VOCs produced by *Bacillus paralichrysogenum* IFST-745 were collected using headspace solid-phase microextraction gas chromatography-mass spectrometry (HS-SPME-GC-MS). Single colonies of IFST-745 were picked and placed in LB broth to prepare a 10⁻⁶ concentration. 9 After CFU / mL, the sample was transferred to a 20 mL headspace vial and incubated at 37°C for 16–18 h, with LB broth without inoculation serving as a control under the same conditions. Extraction was performed using a polydimethylsiloxane (Carboxen / PDMS, 75 μm coating thickness, Supelco) extraction fiber head. The extraction head was baked at 260°C for 60 min, then stirred at 30°C and subjected to headspace adsorption of the IFST-745 sample for 50 min. Once extraction reached equilibrium under these conditions, the fiber was immediately injected into the gas chromatograph inlet (heated to 250°C) for VOCs desorption and chromatographic analysis.

[0049] II. The method for determining the volatile antibacterial substances of Bacillus paralichrysogenus IFST-745 is as follows:

[0050] VOCs were analyzed using gas chromatography (GC) with a mass spectrometer (MS) detector and an autosampler for SPME vials. VOCs were separated in an HP5-MS column (30 m × 0.25 mm, 0.25 μm). The oven temperature was initially set at 40 °C for 3 min, then increased to 260 °C at a rate of 8 °C / min and held for 5 min. Helium was used as the carrier gas, and the injection port temperature was maintained at 250 °C. The mass spectrometer was operated in 70 eV electron ionization mode, with the ion source temperature fixed at 230 °C and the sensor line temperature at 260 °C, using continuous scans from 50 m / z to 500 m / z. VOCs were identified by comparing mass spectra and retention times with real control compounds and with mass spectra from a NIST library. The relative abundance of each component was calculated using peak area normalization. At least 27 volatile substances were found to be produced by Bacillus paralichrysum IFST-745, namely n-butanol, isoamyl alcohol, 1-decyl alcohol, 2-phenylethanol, hexyldecyl alcohol, butyl 2-methylbutyrate, isopropyl palmitate, diisobutyl phthalate, acetic acid, 3-methylvaleric acid, 2-amino-6-methylbenzoic acid, 1,2,4,5-tetramethylbenzene, phenol, 3,5-di-tert-butylphenol, 2-methylpyrazine, 2,5-dimethylpyrazine, 2-ethyl-5-methylpyrazine, 2,3,5-trimethylpyrazine, hexadecane, heptadecane, 6-methyl-2-heptanone, 2-decanone, 2-nonadecanone, o-aminoacetophenone, 3,4-dimethylacetophenone, paraformaldehyde, and chamomile blue, as detailed in Table 2 below.

[0051] Table 2 Total Volatile Matter

[0052]

[0053] Example 4: Determination of the antibacterial activity of VOCs generated by IFST-745 against Fusarium oxysporum and Penicillium digitatum.

[0054] The antibacterial activity of volatile substances produced by Bacillus paralichrysogenum IFST-745 against Fusarium oxysporum and Penicillium digitatum was determined by the following method:

[0055] A total of 25 volatile substances were purchased from *Bacillus paralichrysogenus* IFST-745 in Example 3. 2,3,5-Trimethylpyrazine and 1,2,4,5-Tetramethylbenzene were not available. The specific VOCs exhibiting inhibitory activity against *Fusarium verticillatum* and *Penicillium digitatum* were determined using a double-plate mating experiment. *Fusarium verticillatum* and *Penicillium digitatum* were removed from a -80°C freezer, activated, and allowed to fully colonize the plates before the double-plate mating method was used for the experiment. A filter paper disc was placed in the center of the petri dish, and 10 μL of a single pure substance was added to it. A new petri dish was prepared, and 20 mL of PDA medium was poured in. A bacterial cell (approximately 7 mm in diameter) of the pathogen was placed in the center of the petri dish and then inverted onto the petri dish containing the filter paper. A control group had an equal volume of sterile saline added to the filter paper. The dishes were sealed with sealing film and incubated at 25°C for 5 days. Each treatment was repeated three times. The colony diameter of the pathogen in the treated and control plates was recorded, and the inhibition rate of the volatile substances against the pathogen was calculated. The inhibition results of different volatile compounds produced by IFST-745 against *Fusarium verticillatum* are shown below. Figure 4 As shown; the inhibitory effects of different volatile compounds produced by IFST-745 on Penicillium fingering are as follows. Figure 5 As shown, a total of 13 substances exhibited inhibitory effects on the mycelial growth of *Fusarium verticillatum* and *Penicillium fingernail*. Their inhibition rates against *Fusarium verticillatum* were as follows: 2,5-dimethylpyrazine 18.82%, 2-methylpyrazine 23.92%, 6-methyl-2-heptanone 28.24%, chamomile blue 33.33%, 3,5-di-tert-butylphenol 43.53%, 2-decanone 28.00%, 1-decanol 66.47%, phenethyl alcohol 72.94%, o-aminoacetophenone 87.06%, 3-methylvaleric acid 91.76%, and 3,4-dimethylacetophenone 91.76%. Acetic acid and phenol both had an inhibition rate of 91.76%; the inhibition rates against Penicillium fingerlings were as follows: 2,5-dimethylpyrazine 50.59%, 2-methylpyrazine 91.76%, 6-methyl-2-heptanone 30.59%, chamomile blue 25.88%, 3,5-di-tert-butylphenol 64.71%, 2-decanone 27.06%, 1-decanol 91.76%, phenethyl alcohol 38.24%, o-aminoacetophenone 91.76%, 3-methylvaleric acid 91.76%, 3,4-dimethylacetophenone 91.76%, acetic acid 91.76%, and phenol 91.76%.

[0056] Example 5: Determination of the inhibitory effect of VOCs generated by IFST-745 on the mycelial growth of other pathogenic fungi.

[0057] The inhibitory effect of volatile substances produced by Bacillus paralichrysogenus IFST-745 on the hyphal growth of other pathogenic fungi was determined by the following method:

[0058] The effect of single-component VOCs produced by *Bacillus paralichrysogenus* IFST-745 on the mycelial growth of *Botrytis cinerea*, *Aspergillus flavus*, *Phytophthora nicotineae*, and *Fusarium graminearum* was determined using the double-plate inverted method. The four pathogens were activated after being taken from a -80°C freezer. After the plates were fully colonized, a 7 mm diameter mycelial cake was placed in a petri dish containing PDA medium. A separate petri dish with a filter paper disc in the center was prepared, and 13 single-component VOCs identified in Example 4 as having inhibitory effects on *Fusarium verticillatum* and *Penicillium digitatum* were added to the filter paper. The inoculated petri dish was then inverted on top, sealed with sealing film, and incubated at 25°C for 5 days. Each treatment was repeated three times. The colony diameter of the pathogens in the treatment and control groups was recorded, and the inhibition rate of the volatile substances on the pathogens was calculated. The results of the inhibitory effect of VOCs produced by IFST-745 on the mycelial growth of other pathogenic fungi are shown below. Figure 6 As shown, where, Figure 6 In this context, A represents the result of the snapping experiment. Figure 6 B in the figure represents the statistical results of inhibition rate. It can be seen that the seven single-component VOCs that have strong inhibitory effects on *Fusarium oxysporum* and *Penicillium digitatum* also have good inhibitory effects on other pathogens. In addition, unlike *Fusarium oxysporum* and *Penicillium digitatum*, 2-decanoate also showed strong inhibitory effects in the inhibition experiments against these four pathogens, with inhibition rates as high as 63.53%, 53.33%, and 63.49% against *Botrytis cinerea*, *Phytophthora nicotine*, and *Fusarium graminearum*, respectively. This indicates that phenol, 3,4-dimethylacetophenone, o-aminoacetophenone, and phenylethanol all have good broad-spectrum inhibitory effects on pathogens.

[0059] Example 6: Determination of the half-maximum effective concentration (EC50) of the volatile active ingredient produced by IFST-745 against Fusarium oxysporum and Penicillium digitatum.

[0060] The half-maximum effective concentration (EC50) of the volatile active ingredient produced by Bacillus paralichrysogenus IFST-745 against Fusarium oxysporum and Penicillium digitatum was determined by the following method:

[0061] Using *Fusarium verticillatum* and *Penicillium digitatum* as indicator fungi, the procedure was performed on a clean bench. 20 mL of PDA solid medium was poured into each plate, and after cooling and solidification, 5 μL of the pathogen from Example 1 (3) was added to the center of the PDA medium at a concentration of 1 × 10⁻⁶. 6A spore suspension of 10 spores / mL was prepared. After standing for a few minutes, the suspension was inverted onto a plate lid containing filter paper. Different amounts (0.75, 1.5, 3, 6, 9 μL) of the volatile substance with an inhibition rate greater than 60% from Example 5 were added to the center of the plate lid, ensuring the concentration of the volatile substance in the petri dish was controlled at 10, 20, 40, 80, and 120 μL / L. The plate was then quickly sealed with sealing film. An equal volume of sterile water was added to the filter paper as a control group. Each treatment was repeated three times, and the plates were incubated at 25°C for 5 days. The colony diameter of the pathogen in the treated and control plates was recorded, and the inhibition rate of the volatile substance against the pathogen was calculated. The results are shown in Table 3 below.

[0062] Table 37 shows the inhibition rates of 37 single VOCs against two pathogens.

[0063]

[0064] Application Example 1: Application of 13 volatile substances with antibacterial activity in the control of corn ear rot and citrus green mold.

[0065] This embodiment provides the application of the above-mentioned 13 volatile substances with antibacterial activity in the prevention and control of corn ear rot and citrus green mold. The application method is as follows:

[0066] After thoroughly washing healthy, uniformly sized oranges with clean water, rinse them a second time with sterile water. Then, disinfect the surface with a 75% ethanol solution. Once the ethanol has evaporated from the fruit surface, use a sterile needle to make a 5mm x 5mm wound in the center of the orange. Finally, apply 10μL of a 10% ethanol solution to the wound. 6 A spore suspension of *Penicillium fingering* was prepared. For the control group, sterile water was added dropwise. After complete saturation, the suspension was placed on a shelf in a 3L desiccator. A vial containing 1 mL of a single pure substance was placed at the bottom, and the solution was sealed with white petroleum jelly. Three fruits were used in each treatment group, and the experiment was repeated three times. The fruit was placed at 25℃ for 7 days. The diameter of the pathogen was observed and measured, and the incidence rate was calculated. The control effects of 13 volatile compounds with strong antibacterial activity produced by IFST-745 on citrus green mold caused by *Penicillium fingering* were as follows: Figure 7 As shown, where, Figure 7 In the figure, A represents the control effect on oranges infected with citrus green mold. Figure 7 In the figure, B represents the statistical result of the inhibition rate. Figure 7 It is known that chamomile, 3,5-di-tert-butylphenol, 2-decanone, acetic acid and phenol have significant inhibitory effects on citrus green mold caused by Penicillium finger, but acetic acid can cause large-scale browning on the surface of oranges.

[0067] Take fresh corn kernels and soak their surface twice in 75% ethanol, 3 minutes each time; then wash them 5 times with sterile water to remove the ethanol. Place the sterilized corn kernels into 60 mm agar plates, 15 kernels per plate, and add 10 μL of 10% ethanol to each kernel. 6 A suspension of *Fusarium verticillatum* spores per mL was prepared. Small plates containing corn kernels were then placed into 90 mm agar plates. Simultaneously, a filter paper disc with 10 μL of a single pure substance added was placed at the edge of the larger agar plate, ensuring the filter paper did not contact the small plates. Sterile water was added to the filter paper disc as a control. After 5 days of incubation, disease development was observed. The control effects of 13 volatile compounds with potent antibacterial activity produced by IFST-745 on corn ear rot caused by *Fusarium verticillatum* were as follows: Figure 8 As shown, where Figure 8 In the figure, A represents the control effect on corn kernels infected with corn ear rot. Figure 8 In the figure, B represents the relative biomass of *Fusarium verticillatum* in maize kernels of each group. Figure 8 It can be seen that all 13 volatile substances can effectively inhibit the disease development of Fusarium verticillatum in maize, among which 3-methylvaleric acid has the best inhibitory effect.

[0068] Although the present invention has been described in detail through the preferred embodiments above, it should be understood that the above description should not be considered as a limitation of the present invention. Various modifications and substitutions to the present invention will be apparent to those skilled in the art after reading the above description. Therefore, the scope of protection of the present invention should be defined by the appended claims.

Claims

1. A strain of Bacillus paralicheniformis IFST-745, characterized in that, The classification number of this bacterium is Bacillus paralicheniform The bacteria was deposited on May 4, 2023, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC No. 27245.

2. The *Bacillus paralichrysogenum* IFST-745 as described in claim 1 for the prevention and control of *Penicillium fingernail* (… Penicillium fingered Citrus green mold caused by *Fusarium moniliforme* or by *Fusarium verticillatum* (… Fusarium verticillium Application in corn ear rot caused by )

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