Bacillus velezensis NEAU-L05 and application thereof as well as antibacterial cyclic lipopeptide Iturin A and application thereof

Abstract

The invention relates to the technical field of microorganisms, in particular to bacillus velezensis NEAU-L05, application of the bacillus velezensis NEAU-L05, antibacterial cyclic lipopeptide Iturin A and application of the antibacterial cyclic lipopeptide Iturin A. The bacillus velezensis NEAU-L05 disclosed by the invention is preserved in the China General Microbiological Culture Collection Center, the address is Institute of Microbiology, Chinese Academy of Sciences, 3 #, 1 # yard, West Beichen Road, Chaoyang District, Beijing, the preservation date is December 19, 2024, and the preservation number is CGMCC No.33130. The bacillus velezensis NEAU-L05 disclosed by the invention can be used for preparing the bacillus velezensis NEAU-L05. The bacteriostasis rate of the bacterial strain to botrytis cinerea of phytopathogen is 67.2%. The bacteriostatic cyclic lipopeptide ItuinA separated from the strain can improve the storage performance of berries, and a basis is provided for development of multiple biocontrol microbial agents with excellent performance.

Description

Technical Field

[0001] The present invention relates to the field of microbial technology, in particular to a strain of Bacillus velezensis NEAU-L05 and applications thereof, and an antibacterial cyclic lipopeptide Iturin A and applications thereof. Background Art

[0002] As the third generation of emerging commercial fruit of the genus Lonicera Linn. in the family Caprifoliaceae, blue indigo fruit (Lonicera caerulea) is one of the characteristic small berry tree species in the cold zone.

[0003] The pathogen of blue indigo fruit gray mold is Botrytis cinerea, also known as Botrytis cinerea. It can infect grape inflorescences, leaves, and young fruits, and can also endanger mature fruits, thereby compromising the entire fruit and vegetable industry chain from planting to fruit storage, causing significant economic losses. During the planting stage, chemical pesticides of various structural types have been the main force in the prevention and control of blue indigo fruit gray mold. Representative chemical pesticides include the early and widely used carbendazim and iprodione, and the more recently promoted use of boscalid and azoxystrobin. Although these chemical pesticides can effectively control grape gray mold, their relatively simple mechanisms of action and the high genetic variability of grape gray mold fungi have led to the continuous emergence of resistant strains in the field, creating a vicious cycle of excessive chemical pesticide use and excessive pesticide residues. During the fruit storage stage, due to the more stringent requirements for pesticide residues, the contradiction between blue indigo fruit gray mold prevention and control and chemical pesticide use is even more prominent.

[0004] Compared to chemical fungicides, identifying promising microbial strains for controlling plant diseases has become a key area of ​​future green pesticide development. To date, some strains of bacteria such as Streptomyces and fungi such as Trichoderma and Penicillium have been successfully developed into commercial fungicides. Microbial agents offer advantages such as good biodegradability, high safety, and resistance to drug resistance, making them crucial for the development of environmentally friendly, ecological agriculture.

[0005] The prior art has not found a microbial product that has both antibacterial effect and is beneficial to fruit preservation. Based on this, the present invention is proposed. Summary of the Invention

[0006] The object of the present invention is to provide a microbial product which has both antibacterial effect and is beneficial to fruit preservation.

[0007] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0008] The present invention provides a strain of Bacillus velezensis NEAU-L05, which is deposited in the General Microbiology Center of the China Culture Collection Administration, located at the Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, with a deposit date of December 19, 2024, and a deposit number of CGMCC No. 33130.

[0009] The present invention also provides the use of the Bacillus velezensis NEAU-L05 in preparing products for inhibiting plant pathogens;

[0010] The plant pathogen is Botrytis cinerea.

[0011] The present invention also provides the use of the Bacillus velezensis NEAU-L05 in fruit preservation;

[0012] The fruit is a berry.

[0013] The present invention also provides an antibacterial cyclic lipopeptide IturinA, which is obtained by fermenting the Bacillus velezensis NEAU-L05 and then separating it.

[0014] The present invention also provides a method for preparing the antibacterial cyclic lipopeptide IturinA, comprising the following steps:

[0015] (1) inoculating the Bacillus velezensis NEAU-L05 into a fermentation medium and culturing the medium on a shaking platform for 44 to 52 hours to obtain a fermentation product;

[0016] (2) centrifuging the fermentation product for 15 to 25 minutes, collecting the supernatant, adjusting the pH of the supernatant to 1.8 to 2.2, and then standing at 2 to 6° C. for 8 to 12 hours to obtain an intermediate product;

[0017] (3) centrifuging the intermediate product for 8 to 12 minutes, collecting the precipitate, mixing the precipitate with methanol, and extracting to obtain a methanol extract;

[0018] (4) rotary evaporating the methanol extract to obtain a crude extract of the fermentation supernatant;

[0019] (5) The crude extract of the fermentation supernatant was separated and purified by HPLC, and the fractions from 14 to 25 min were collected to obtain the antibacterial cyclic lipopeptide Iturin A.

[0020] Preferably, the fermentation medium uses water as a solvent and comprises the following components at the following concentrations:

[0021] Maltose 14-18g / L, beef extract 12-18g / L, magnesium sulfate 6-10g / L;

[0022] The pH of the fermentation medium is 6.5 to 6.8;

[0023] The rotation speed of the shaking culture is 150-170 rpm;

[0024] The shaking incubation temperature is 23-27°C;

[0025] The centrifugal speed in step (2) is 3800-4200 rpm;

[0026] The centrifugal speed in step (3) is 7000-9000 rpm;

[0027] The temperature of the rotary evaporation in step (4) is 35-39°C.

[0028] The present invention also provides the use of the antibacterial cyclic lipopeptide IturinA in the preparation of products for inhibiting plant pathogens;

[0029] The plant pathogen is Botrytis cinerea.

[0030] The present invention also provides the use of the antibacterial cyclic lipopeptide IturinA in the preparation of fruit preservation;

[0031] The fruit is a berry.

[0032] The present invention also provides a biocontrol agent comprising any one or both of the following:

[0033] (1) the Bacillus velezensis NEAU-L05 and its fermentation supernatant;

[0034] (2) The antibacterial cyclic lipopeptide IturinA.

[0035] The present invention also provides a fruit preservative comprising any one or two of the following:

[0036] (1) the Bacillus velezensis NEAU-L05 and its fermentation supernatant;

[0037] (2) The antibacterial cyclic lipopeptide IturinA.

[0038] The present invention provides a strain of Bacillus velezensis NEAU-L05 and its application, and an antibacterial cyclic lipopeptide IturinA and its application. The bacterial agent of the present invention is Bacillus velezensis NEAU-L05, which is deposited in the General Microbiology Center of the China Culture Collection Administration, located at the Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, with a deposit date of December 19, 2024, and a deposit number of CGMCC No. 33130.

[0039] The Bacillus velez NEAU-L05 of the present invention has an antibacterial rate of 67.2% against the plant pathogen Botrytis cinerea. After fermenting Bacillus velez NEAU-L05, the fermentation supernatant was found to effectively improve the storage performance of blue honeysuckle fruit in an experiment to improve the storage performance of blue honeysuckle fruit. Test data showed that the rot rate of blue honeysuckle fruit treated with this substance was 17.67%, a 15.66% reduction compared to the negative control group (33.33%) and an 80.66% reduction compared to the positive control group (98.33%). The antibacterial cyclic lipopeptide Iturin A can significantly reduce the weight loss rate of fruit and preserve the nutritional components in the fruit. This provides a basis for developing a biocontrol agent with multiple excellent properties, including good degradation performance, high safety, low resistance to drug resistance, and environmental friendliness.

[0040] Preservation Instructions

[0041] Bacillus velezensis NEAU-L05 is deposited in the General Microbiology Center of China Culture Collection Administration, located at Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing. The deposit date is December 19, 2024, and the deposit number is CGMCC No. 33130. BRIEF DESCRIPTION OF THE DRAWINGS

[0042] Figure 1 The morphological characteristics of strain NEAU-L05 (A is the colony morphology; B is the Gram staining result of the strain; C is the scanning electron micrograph of the strain);

[0043] Figure 2 The inhibitory effect of strain NEAU-L05 on Botrytis cinerea (A represents the control group of Botrytis cinerea LDGS-3; B represents the results of the plate confrontation experiment of strain NEAU-L05 against Botrytis cinerea);

[0044] Figure 3 is the phylogenetic tree of strain NEAU-L05;

[0045] Figure 4is the genomic characteristics of strain NEAU-L05;

[0046] Figure 5 The antibacterial effect of the crude extract of strain NEAU-L05 on Botrytis cinerea LDGS-3 (A represents the blank control of Botrytis cinerea LDGS-3, B represents the methanol control, and C represents the antibacterial effect of the crude extract of strain NEAU-L05);

[0047] Figure 6 HPLC analysis diagram (the upper diagram is for wavelength λ1 = 210 nm; the lower diagram is for wavelength λ2 = 254 nm);

[0048] Figure 7 is the antibacterial activity of each component (where A represents the blank control of Botrytis cinerea LDGS-3; B represents the methanol control; C represents the component of 1 to 13 min; D represents the component of 14 to 25 min);

[0049] Figure 8 This is the mass spectrum of LC-MC positive ion mode;

[0050] Figure 9 The dynamic changes of the decay rate of blue indigo fruit (A is the decay rate; B is the control effect);

[0051] Figure 10 The dynamic changes of ascorbic acid content in blue loquat fruit in different groups;

[0052] Figure 11 The dynamic changes of flavonoids content in different groups were shown;

[0053] Figure 12 The dynamic changes of total phenolic content in Lonicera edulis in different groups. DETAILED DESCRIPTION

[0054] The solutions provided by the present invention are described in detail below with reference to the embodiments, but they should not be understood as limiting the scope of protection of the present invention.

[0055] Example 1

[0056] Isolation, identification and preservation of strain NEAU-L05

[0057] 1. Culture medium configuration

[0058] LB medium is used for the isolation and purification of biocontrol strains;

[0059] PDA medium is used for the activation and cultivation of pathogens;

[0060] The specification for making solid culture medium is to add 20.0g agar per 1L of liquid culture medium.

[0061] LB medium: 5.0 g yeast extract powder, 10.0 g tryptone, 5.0 g sodium chloride, 1.0 L distilled water, sterilized at 121°C for 20 min;

[0062] PDA medium: 200.0 g potato, 20.0 g glucose, 20.0 g agar powder, 1.0 L distilled water, sterilized at 121°C for 20 min.

[0063] 2. Isolation and purification of strain NEAU-L05

[0064] Rhizosphere soil from the 1-10 cm surface of healthy blue indigo plants was collected from the Xiangyang Base of Northeast Agricultural University and stored at 4°C until use. 0.3 g of each soil sample was weighed, added to 30 mL of sterile water, and incubated in a 30°C constant-temperature shaker for 30 minutes. 30 μL was then spread on LB solid medium and incubated in a 30°C incubator for 48 hours. After single colonies grew on the plate, sterile toothpicks were used to select individual colonies of varying morphology and streaked onto LB solid medium plates for purification. The colonies were then incubated in a 30°C constant-temperature incubator until single colonies of consistent morphology emerged. The strain, NEAU-L05, was finally isolated from the rhizosphere soil of healthy blue indigo plants.

[0065] The morphological characteristics of the colonies, Gram staining results and scanning electron microscopy results are as follows: Figure 1 shown.

[0066] Figure 1 Single colonies of strain NEAU-L05 are pale yellow or milky white, opaque, with irregular, jagged edges and a typical rough surface texture of considerable thickness. Microscopic examination of strain NEAU-L05 after Gram staining revealed it to be Gram-positive (purple). Observation of strain NEAU-L05 using a SU8010 scanning electron microscope at an accelerating voltage of 5.0 kV and a magnification of 100,000x revealed a rod-shaped cell surface with a rough, uneven texture and no apparent flagella or pili.

[0067] 3. Determination of antibacterial activity of strain NEAU-L05

[0068] The antibacterial activity of strain NEAU-L05 was determined by the plate confrontation method using Botrytis cinerea LDGS-3 (Haohao Yan et al. First Report of Postharvest Fruit Rot Caused by Botrytis cinerea on Blue Honeysuckle (Loniceracaerulea L.) Fruit in China. Plant Disease, 2024, 108(2): 527.) as the target. The pathogen colony was punched with a sterile puncher with a diameter of 5 mm, and the bacterial cake was transferred to the center of the PDA culture plate. The NEAU-L05 single colony was spot-grafted on the plate at two opposite points 3 cm away from the center of the plate using the cross-cross method. The control group was not spot-grafted. Three parallel experiments were set up and cultured at 28°C. The pathogen on the plate not inoculated with the antagonistic strain (control group) grew to the edge of the plate for investigation. The control colony diameter and the treated colony diameter of the target bacteria were measured and expressed as the inhibition rate.

[0069] Inhibition rate (%) = [(control colony diameter - treated colony diameter) / (control colony diameter - 5 mm)] × 100%. Experimental results: Figure 2 shown.

[0070] The results showed that strain NEAU-L05 had a strong inhibitory effect on Botrytis cinerea LDGS-3, with an inhibition rate of 67.2%. Bacteria NEAU-L05 was able to inhibit the mycelial growth of Botrytis cinerea.

[0071] 4. Identification of strain NEAU-L05

[0072] (1) Physiological and biochemical characteristics of the strain

[0073] Physiological and biochemical tests were performed on strain NEAU-L05, referring to the methods in the Common Bacterial Systematic Identification Manual and the Bergey's Manual of Bacterial Identification. The results are shown in Table 1.

[0074] Table 1 Physiological and biochemical results of strain NEAU-L05

[0075] Project indicators result Project indicators result Gram stain + NaCl 1% + Citrate utilization test - NaCl 4% + H2S generation test - NaCl 8% + Methyl red test - Growth temperature 4℃ - VP test + Growth temperature 10℃ + Indole test - Growth temperature 30℃ + Catalase test - Growth temperature 50℃ + Nitrate reduction test + Hydrolysis of starch + Gelatin utilization test + Lecithinase - Malonate utilization experiment + Protease + Tryptophan deaminase test - Cellulase +

[0076] Note: “+”: positive reaction; “-”: negative reaction.

[0077] (2) 16S rDNA sequencing

[0078] The genomic DNA of strain NEAU-L05 was extracted and used as a template for PCR amplification using universal primers for bacterial 16S rDNA. The amplified product was recovered and sequenced. The universal primers are shown in SEQ ID NOs. 1-2.

[0079] SEQ ID NO.1 Upstream primer: 5'-AGAGTTTGATCCTGGCTCAG-3';

[0080] SEQ ID NO.2 downstream primer: 5'-GGTTACCTTGTTACGACTT-3'.

[0081] The sequence of 16S rDNA obtained by sequencing is shown in SEQ ID NO.3.

[0082] SEQ ID NO.3:

[0083]

[0084] (3) Submit the sequenced to GeneBank for homology comparison.

[0085] Experimental results: Figure 3 Strain NEAU-L05 shared 100% homology with Bacillus velezensis strain OR 594189. Based on its morphological, physiological and biochemical characteristics and 16S rRNA sequence analysis, strain NEAU-L05 was identified and named Bacillus velezensis NEAU-L05.

[0086] Example 2

[0087] Analysis of antibacterial substances in Bacillus velezensis NEAU-L05

[0088] 1. Whole genome sequencing and analysis of strain NEAU-L05

[0089] Whole-genome sequencing: Bacillus velezensisNEAU-L05 was cultured until its exponential growth phase, and the cells were centrifuged to obtain a pellet. The cells were cryopreserved and sent to Shanghai Paisonno Biotechnology Co., Ltd. for DNA extraction, whole-genome sequencing, and assembly. The results showed that the whole genome of Bacillus velezensisNEAU-L05 consists of a 4,202,506-bp circular chromosome with a GC content of 46.03% and 4,152 coding genes. The genome was uploaded to the NCBI database and received the upload number: SUB15249868. Genome circle map ( Figure 4 ) is a visualization of the genome of strain NEAU-L05.

[0090] Secondary metabolites produced by Bacillus play an important role in inhibiting plant pathogens and improving plant resistance. To verify the secondary metabolite production ability of strain NEAU-L05, the genome of strain NEAU-L05 was predicted using antiSMASH. A total of 13 secondary metabolite biosynthesis gene clusters were identified, accounting for 21.17% of the NEAU-L05 genome. Nine of the 13 gene clusters were identified as the synthesis gene clusters of substances such as bacinapeptin, surfactin, butirosinA, macrolactinH, bacillaene, fengycin, plipastatin, bacillomycin D, mycosubtilin, iturin, difficidin, bacillibactin, paenibactin, amylocyclicin, and bacilysin. Among them, 6 of the above 9 identified gene clusters have 100% similarity with the gene clusters synthesized by macrolactinH, bacillaene, fengycin, plipastatin, bacillomycinD, mycosubtilin, difficidin, bacillibactin, paenibactin, amylocyclicin, and bacilysin, respectively. Four other gene clusters were identified, including one with 50% similarity to the bacinapeptin synthesis gene cluster, one with 91% similarity to the surfactin synthesis gene cluster, one with 88% similarity to the iturin synthesis gene cluster, and one with 7% similarity to the butirosin A synthesis gene cluster. In addition, four gene clusters were not found to have similar clustering by antiSMASH analysis (Table 2).

[0091] Table 2 Secondary metabolite gene clusters of strain NEAU-L05

[0092]

[0093] 2. Preparation, antibacterial effect and component analysis of the antibacterial cyclic lipopeptide IturinA

[0094] Strain NEAU-L05 was fermented on a large scale (conditions: fermentation medium, water as solvent including maltose 15.94 g / L, beef extract 15.00 g / L, magnesium sulfate 8.03 g / L, pH 6.61, temperature 25°C, inoculation amount 3%, rotation speed 160 rpm, fermentation time 48 h), with a total fermentation volume of 20 L.

[0095] Secondary metabolite isolation method: Ferment a large volume of strain NEAU-L05 (20 L) and centrifuge at 4000 rpm for 20 minutes at 4°C. The supernatant was collected to obtain the NEAU-L05 fermentation supernatant. The supernatant was placed in a beaker, the pH adjusted to 2.0 with concentrated hydrochloric acid, and refrigerated at 4°C for 10 hours. After centrifugation at 8000 rpm for 10 minutes, the supernatant was removed and the solution was extracted by adding methanol in multiple portions and shaking until the methanol solution was colorless. All methanol extracts were combined and evaporated on a rotary evaporator at 37°C. The extract was reconstituted with 20 mL of methanol and filtered through a 0.22 μm filter to obtain a crude extract of the NEAU-L05 fermentation supernatant.

[0096] The antibacterial activity of crude extracts from the fermentation supernatant of strain NEAU-L05 was determined using the Oxford cup method. The blank control was treated with Botrytis cinerea LDGS-3. The methanol control group used methanol instead of the crude extract from the fermentation supernatant of strain NEAU-L05. The experimental group used crude extracts from the fermentation supernatant of strain NEAU-L05.

[0097] The test results are as follows Figure 5 As shown in the results, after 5 days of cultivation, the inhibition rate of the solvent methanol was 31.5% ± 2.3, and the inhibition rate of the crude extract of the fermentation supernatant reached 94.6% ± 2.2. The results showed that the crude extract of the fermentation supernatant exhibited a significant inhibitory effect on the gray mold LDGS-3 (p < 0.05), indicating that the crude extract of the fermentation supernatant was rich in secondary metabolites with high antibacterial activity.

[0098] The crude extract of the fermentation supernatant of strain NEAU-L05 was separated and purified by semi-preparative liquid chromatography (HPLC). The HPLC analysis spectrum is shown in FIG. Figure 6 After gradient elution, the fractions were combined into two components (0-13, 14-25 min) according to the peak time. The antibacterial activity of each component was evaluated by the Oxford cup method. After 5 days of culture, the results were as follows: Figure 7 As shown in the figure, the 14-25 min fraction had the most significant inhibitory effect on Botrytis cinerea, indicating that this fraction was enriched with core antimicrobial active ingredients.

[0099] The structure of the purified components (14-25 min group samples) was analyzed by liquid chromatography-mass spectrometry (LC-MS) to determine the core antibacterial active substances: LC-MS spectrum Figure 8 The ion peak is 1043.6[M+H] + It can be seen that the molecular weight of the substance is 1042.6 and the retention time is 17.45min. It is inferred that the substance is the lipopeptide Iturin A (iturin A).

[0100] Example 3

[0101] Experiment on improving low temperature storage performance of blue loquat fruit by fermentation supernatant of strain NEAU-L05

[0102] 1. Effect of the fermentation supernatant of strain NEAU-L05 on the decay rate of blue indigo fruit during storage Figure 9 Fresh, intact, mechanically undamaged Lonicera edulis fruits were selected as experimental materials for this study. The fruit surface pretreatment process was as follows: first, the fruit was soaked in 2% NaClO for 2 minutes, then rinsed twice with sterile distilled water and air-dried in a clean bench for 1 hour. Finally, a standardized wound with a diameter of 2 mm and a depth of 2 mm was created at the fruit base under a sterile operating environment. Three treatment groups were set up: the negative control (CK) consisted of pretreated Lonicera edulis fruits inoculated with 10 μL of 10% fermentation medium and then, 2 hours later, inoculated with 10 μL of sterile water; the positive control (GS) consisted of pretreated Lonicera edulis fruits inoculated with 10 μL of 10% fermentation medium and then, 2 hours later, inoculated with 10 μL of a pathogen spore suspension; and the treated group (CFS) consisted of pretreated Lonicera edulis fruits inoculated with 10 μL of 10% fermentation supernatant of strain NEAU-L05 and then, 2 hours later, inoculated with 10 μL of a pathogen spore suspension. The treated fruits were then transferred to a culture dish covered with moistened sterile filter paper and stored in an incubator at a relative humidity of 85% and a temperature of 25±1°C for 5 days. Three replicates were set up for each group (each group contained 20 fruit samples). The number of rotten blue indigo fruits was observed and recorded every other day.

[0103] The decay rate of blue honeysuckle in each treatment group increased over time. The CFS group, inoculated with the pathogen, significantly inhibited the decay rate (p < 0.05). After the third day, the decay rate in the GS group increased rapidly, while the decay rates in the CK and CFS groups increased more slowly. After five days of storage, the decay rate in the CFS group (17.67%) was significantly lower than that in the CK group (33.33%) and the GS group (98.33%). This indicates that the fermentation broth of strain NEAU-L05 can significantly inhibit the decay of blue honeysuckle (p < 0.05).

[0104] The fermentation medium uses water as a solvent and includes 15.94 g / L maltose, 15.00 g / L beef extract, 8.03 g / L magnesium sulfate, and pH 6.61.

[0105] The preparation method of pathogen spore suspension is as follows: Botrytis cinerea LDGS-3 was inoculated on PDA medium, cultured for 5 days, added with appropriate amount of sterile water, scraped off the mycelium and filtered with sterile gauze, and the spore suspension was adjusted to 1×10 6 The fermentation supernatant was prepared by inoculating a single colony of NEAU-L05 into 100 mL of fermentation medium, culturing for 48 hours, and then centrifuging at 4000 rpm and 4°C for 20 minutes to obtain the fermentation supernatant, which was then stored in a refrigerator at 4°C until use.

[0106] 2. Effects of the fermentation supernatant of strain NEAU-L05 on nutritional indicators of blueberry

[0107] like Figure 10 As shown, the ascorbic acid content in the CK and GS groups decreased during storage, with the CK group showing higher ascorbic acid content than the GS group. The ascorbic acid content in the CFS group initially increased and then decreased, with the decline being more gradual. On day 5, the ascorbic acid contents in the CK, GS, and CFS groups were 57.98%, 39.69%, and 76.71% of their initial values ​​on day 0, respectively. These results indicate that fermentation broth from strain NEAU-L05 can maintain a high ascorbic acid content in Lonicera edulis.

[0108] Depend on Figure 11 As can be seen, the flavonoid content in the CK and GS groups decreased during storage, while the flavonoid content in the CFS group initially increased and then decreased, with a slower rate of decline. During storage, the flavonoid content in the CFS group remained significantly higher than that in the CK and GS groups (p < 0.05). On day 5, the flavonoid content in the CK, GS, and CFS groups was 49.53%, 30.63%, and 74.11% of the initial value on day 0, respectively. These results indicate that treatment with the fermentation broth of the biocontrol strain NEAU-L05 effectively maintains the flavonoid content in Lonicera edulis and promotes flavonoid production.

[0109] Depend on Figure 12 During storage, the total phenolic content in the CK and GS groups decreased, while that in the CFS group initially increased and then decreased. On day 5, the total phenolic content in the CK, GS, and CFS groups was 39.09%, 27.35%, and 60.18% of the initial value on day 0, respectively. These results indicate that treatment with the fermentation broth of the biocontrol strain NEAU-L05 effectively maintains the total phenolic content in Lonicera edulis.

[0110] As can be seen from the above examples, the present invention provides a strain of Bacillus velezensis NEAU-L05 and its application and an antibacterial cyclic lipopeptide IturinA and its application. The bacterial agent of the present invention is Bacillus velezensis NEAU-L05, which is deposited in the General Microbiology Center of the China Culture Collection of Microorganisms, at the Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, with a deposit date of December 19, 2024, and a deposit number of CGMCC No. 33130. The antibacterial rate of Bacillus velezensis NEAU-L05 of the present invention against the plant pathogen Botrytis cinerea is 67.2%. After fermenting Bacillus velezensis NEAU-L05, the fermentation supernatant was found to effectively improve the storage performance of blue honeysuckle fruit in an experiment to improve the storage performance of blue honeysuckle fruit. Experimental data showed that the fruit rot rate of blue loquat fruit treated with this substance was 17.67%, a 15.66% decrease compared to the negative control (33.33%) and an 80.66% decrease compared to the positive control (98.33%). The antibacterial cyclic lipopeptide Iturin A significantly reduced fruit weight loss while preserving nutrients. This provides a basis for developing a biocontrol agent with multiple excellent properties, including excellent degradation performance, high safety, resistance to drug resistance, and environmental friendliness.

[0111] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as within the scope of protection of the present invention.

Claims

1. A strain of Bacillus velezensis NEAU-L05, characterized in that: It is deposited in the General Microbiology Center of China Culture Collection Administration, located at Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing. The deposit date is December 19, 2024, and the deposit number is CGMCC No. 33130.

2. Use of the Bacillus velezensis NEAU-L05 according to claim 1 in the preparation of a product for inhibiting plant pathogens; The plant pathogen is Botrytis cinerea.

3. Use of the Bacillus velezensis NEAU-L05 according to claim 1 in fruit preservation; The fruit is a berry.

4. An antibacterial cyclic lipopeptide IturinA, characterized in that The product is obtained by fermenting the Bacillus velezensis NEAU-L05 as claimed in claim 1 and then separating the fermented product.

5. A method for preparing an antibacterial cyclic lipopeptide IturinA, characterized in that: The steps include: (1) inoculating the Bacillus velezensis NEAU-L05 described in claim 1 into a fermentation medium and culturing on a shaking platform for 44 to 52 hours to obtain a fermentation product; (2) centrifuging the fermentation product for 15 to 25 minutes, collecting the supernatant, adjusting the pH of the supernatant to 1.8 to 2.2, and then standing at 2 to 6° C. for 8 to 12 hours to obtain an intermediate product; (3) centrifuging the intermediate product for 8 to 12 minutes, collecting the precipitate, mixing the precipitate with methanol, and extracting to obtain a methanol extract; (4) rotary evaporating the methanol extract to obtain a crude extract of the fermentation supernatant; (5) The crude extract of the fermentation supernatant was separated and purified by HPLC, and the fractions from 14 to 25 min were collected to obtain the antibacterial cyclic lipopeptide Iturin A.

6. The preparation method according to claim 5, characterized in that The fermentation medium uses water as a solvent and includes the following components in the following concentrations: Maltose 14-18g / L, beef extract 12-18g / L, magnesium sulfate 6-10g / L; The pH of the fermentation medium is 6.5 to 6.8; The rotation speed of the shaking culture is 150-170 rpm; The shaking incubation temperature is 23-27°C; The centrifugal speed in step (2) is 3800-4200 rpm; The centrifugal speed in step (3) is 7000-9000 rpm; The temperature of the rotary evaporation in step (4) is 35-39°C.

7. Use of the antibacterial cyclic lipopeptide IturinA according to claim 4 in the preparation of products for inhibiting plant pathogens; The plant pathogen is Botrytis cinerea.

8. Use of the antibacterial cyclic lipopeptide IturinA according to claim 4 in the preparation of fruit preservation; The fruit is a berry.

9. A biological control agent, characterized in that: Include any one or both of the following: (1) Bacillus velezensis NEAU-L05 and its fermentation supernatant according to claim 1; (2) The antibacterial cyclic lipopeptide Iturin A according to claim 4.

10. A fruit preservative, characterized in that: Include any one or both of the following: (1) Bacillus velezensis NEAU-L05 and its fermentation supernatant according to claim 1; (2) The antibacterial cyclic lipopeptide Iturin A according to claim 4.

Images