Endophytic bacillus velezensis and application thereof

Abstract

This invention discloses an endophytic Bacillus belye and its applications, relating to the field of biocontrol technology. This invention demonstrates good control efficacy against Alternaria spp. leaf spot disease of currants nigricans. The endophytic Bacillus belye is deposited at the China General Microbiological Culture Collection Center (CGMCC), with accession number CGMCC No. 30625. In field control, when the OD600 value of the fermentation broth of biocontrol bacterium SH1 was 0.5, the control efficacy reached 75.7%. When the concentration of the lipopeptide extract of biocontrol bacterium SH1 was 0.5 μg / mL, the control efficacy reached 80.8%. Bacillus belye SH1 exhibits good biocontrol potential in the control of Alternaria spp. leaf spot disease of currants nigricans, and further applied research should be strengthened.

Description

Technical Field

[0001] This invention relates to the field of biological control technology, and more specifically to an endophytic Bacillus belye and its application. Background Technology

[0002] Blackcurrant is one of the dominant tree species in cold regions of my country, with Heilongjiang Province having the largest planting area nationwide, reaching nearly 20,000 mu (approximately 1,333 hectares). Blackcurrant fruit is sweet and sour with a fragrant aroma, and can be processed into beverages, fruit wines, and flavorings, possessing significant nutritional, health, medicinal, and cosmetic value. However, with the continuous increase in the planting area and years of blackcurrant cultivation, leaf spot disease has become increasingly severe, leading to the loss of large amounts of leaves and fruit, affecting the quality and reducing yield. Furthermore, the causative agent is unclear, and control measures are far from ideal, making this a critical issue that urgently needs to be addressed for the industrial development of blackcurrant.

[0003] Currently, the main pathogens reported to cause leaf spot disease in blackcurrants are *Pseadopeziza ribis*, *Gloesporium ribis*, and *Septoria ribis*. However, a serious leaf spot disease that has not been previously reported has emerged in blackcurrant growing areas. At the horticultural experimental station of Northeast Agricultural University, it was found that affected leaves initially appear water-soaked, later turning light gray or grayish-white with irregular dark brown spots. In severe cases, the leaves curl, wither, and fall off, affecting fruit yield and quality. Sun et al. (2021) used primers ITS, GAPDH, and ACT to identify *Alternaria*, which causes ginger lily leaf spot disease. Li et al. (2023) used primers ITS, TEF-1α, and RPB2 to identify *Alternaria*, which causes peony red spot disease. Qi et al. (2024) used primers ITS, GAPDH, ACT and TEF-1α to identify Alternaria, which causes leaf spot disease in dandelion.

[0004] Therefore, providing a biocontrol strain for controlling diseases of blackcurrant plants is a problem that urgently needs to be solved by those skilled in the art. Summary of the Invention

[0005] In view of this, the present invention provides an endophytic Bacillus belye and its application.

[0006] To achieve the above objectives, the present invention adopts the following technical solution:

[0007] An endophytic Bailus velezensis, deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 30625, was deposited on May 14, 2024, at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, and is classified as Bailus velezensis.

[0008] A biological control agent comprising the endophytic Bacillus belye as described in claim 1.

[0009] Application of endophytic Bacillus belye in the control of Alternaria leaf spot disease of spruce.

[0010] Application of endophytic Bacillus belye in the control of plant leaf diseases, wherein the leaf disease pathogens include any one of the following: Duchesnea indicum leaf spot, Rhododendron leaf blight, Maple leaf spot, Elderberry leaf spot, Actinidia chinensis leaf blight, and Asters leaf spot.

[0011] As can be seen from the above technical solution, compared with the prior art, this invention discloses an endophytic Bacillus belye SH1, which has a good control effect on Alternaria spp. leaf spot disease of currants spp. In field control, when the OD600 value of the biocontrol bacterium SH1 fermentation broth is 0.5, the control effect reaches 75.7%. When the concentration of the lipopeptide extract of biocontrol bacterium SH1 is 0.5 μg / mL, the control effect reaches 80.8%. Bacillus belye SH1 shows good biocontrol potential in the control of Alternaria spp. leaf spot disease of currants spp., and further applied research should be strengthened. Attached Figure Description

[0012] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on the provided drawings without creative effort.

[0013] Figure 1 The field control effect of biocontrol bacterium SH1 suspension on blackcurrant leaf spot disease;

[0014] Figure 2 Antagonistic effect of biocontrol bacterium SH1 against 9 pathogenic fungi causing leaf diseases in plants;

[0015] Figure 3 Gram staining of biocontrol bacteria SH1 and colony and Gram staining;

[0016] Figure 4 Physiological and biochemical characteristics of biocontrol bacterium SH1;

[0017] Figure 5 A phylogenetic tree of the biocontrol bacterium SH1 was constructed based on the 16S rRNA gene.

[0018] Figure 6 Field control efficacy of biocontrol bacteria fermentation liquid against blackcurrant leaf spot disease;

[0019] Figure 7 Effects of SH1 lipopeptide extracts on the growth of Alternaria alternata hyphae;

[0020] Figure 8 Effects of biocontrol bacterium SH1 lipopeptide extract on the hyphal morphology of Alternaria alternata;

[0021] Figure 9 The effect of temperature on the antibacterial activity of SH1 lipopeptides;

[0022] Figure 10 The effect of pH on the antibacterial activity of SH1 lipopeptides;

[0023] Figure 11 Effects of ultraviolet irradiation on the antibacterial activity and stability of SH1 lipopeptide extract;

[0024] Figure 12 Chromatographic profile of lipopeptide extract of biocontrol bacterium SH1;

[0025] Figure 13 Antagonistic effect of the elution peak of the biocontrol bacterium SH1 lipopeptide extract on the growth of Alternaria alternata hyphae;

[0026] Figure 14 Matrix-assisted laser resolution mass spectra of SH1 lipopeptide extract. Detailed Implementation

[0027] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. 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.

[0028] Experimental materials

[0029] PDA medium: 200g potato; 20g glucose; 20g agar powder; 1000mL distilled water.

[0030] Beef extract peptone medium (NA): Ingredients: 3g beef extract; 10g peptone; 15g NaCl; 1000mL distilled water; pH 7.0-7.2;

[0031] Gram staining reagents: 95% ethanol solution, iodine solution, safranin counterstain, crystal violet stain.

[0032] Test media: NA medium, PDA medium, same as 2.5.1, LB medium.

[0033] LB liquid medium (LB): yeast extract 5g; peptone 10g; NaCl 10g; distilled water 1000mL; pH 7.0-7.2;

[0034] Physiological and biochemical test reagents: Novel microbial micro-biochemical assay tubes (Guangdong Huankai Biotechnology Co., Ltd.);

[0035] Fermentation medium for lipopeptides: 200g sucrose; 10g Na2HPO4; 3g KH2PO4; 3g NH4NO3

[0036] 2g; yeast extract 0.2g; MgSO4·7H2O 0.2g; MnSO4·4H2O 1μg; CaCl2

[0037] 0.7 μg; 1000 mL distilled water;

[0038] Example 1

[0039] Initial screening of biocontrol bacteria

[0040] Test pathogen: The most pathogenic Alternaria strain HLJ-4 was isolated from blackcurrant leaf spot disease in the experiment.

[0041] Method for isolating biocontrol bacteria: After surface disinfection, weigh healthy blackcurrant leaves and place them in a sterile petri dish. Add 5 mL of sterile water and grind into a slurry using a glass rod. Take 1 mL of the slurry and dilute it to a final volume with a measured amount of sterile water. -4 10 -5 10 -6 Take 100 μL of each sample and spread it evenly on NA plates. After the surface is dried, incubate at 30℃ for 2-3 days. Then, pick a single colony and transfer it to an NA slant tube and incubate at 30℃ for 2 days. Store for later use. Simultaneously, take blackcurrant leaves, add sterile water directly, and incubate at 26℃ and 150 rpm for 20 minutes on a shaker. Take 1 mL of the solution and dilute it to a measured volume of sterile water to a final concentration of 1:10. -4 10 -5 10 -6 Take 100 μL of each sample and spread it evenly on an NA plate. After the surface is dried, incubate at 30°C for 2-3 days. Then, pick a single colony and incubate it on an NA slant at 30°C for 2 days. Finally, store it in a refrigerator at 4°C for later use.

[0042] Screening of biocontrol bacteria: The plate confrontation culture method was used, with the pathogenic fungal isolate Alternaria alternata strain HLJ-4 as the target for control. Antagonistic bacteria were screened by spreading different biocontrol bacteria onto NA plates and incubating them at 28℃ for 48 hours. Two parallel lines were drawn with the target bacteria at a distance of 2.5 cm from the center of the PDA plate. A HLJ-4 bacterial dish (d = 0.5 cm) was inoculated in the center of the PDA plate, with each treatment replicated three times. After incubation at 25℃ in the dark for 6 days, the longest and shortest radii were measured, and the ratio of the longest to shortest radius was calculated. The best strains with a ratio greater than 2 were used for further screening.

[0043] Second screening of biocontrol bacteria

[0044] Using a transfer hook, a suitable amount of bacterial flora from the initial screening of biocontrol bacteria was picked up and activated in LB liquid medium for 12 hours. Then, 1 mL of the suspension was transferred to 50 mL of LB liquid medium and incubated at 28°C and 180 rpm for 24 hours. Appropriate amounts of physiological saline were added to adjust the bacterial concentration to 10⁸ CFU / mL, 10¹⁰ CFU / mL, and 10¹² CFU / mL, respectively, for later use. Each treatment consisted of 10 strains, with 3 replicates. A blank control group was sprayed with an equal volume of sterile water. 24 hours after spraying the biocontrol bacteria, the pathogen, Alternaria alternata strain HLJ-4, was spray-inoculated. After culturing on PDA plates for 5 days, the spores were rinsed with sterile water, filtered through double-layer gauze, and diluted to 1×10⁻⁶ using a hemocytometer (Shenzhen Jieshun Technology Industry Co., Ltd.). 5 After spraying with 1 spore / mL, the area was kept moist with plastic sheeting for 48 hours. Ten days after inoculation, the disease incidence in each treatment was investigated, and the disease index and control efficacy were calculated. The target biocontrol strain was determined based on the combined results of the initial and secondary screening for further testing.

[0045] Eight biocontrol bacteria were screened from 260 bacterial strains obtained from leaves infected with blackcurrant leaf spot disease. These bacteria showed good antagonistic effects against Alternaria alternata, the pathogen causing blackcurrant leaf spot disease. The results are shown in Table 1.

[0046] Table 1: Antagonistic Effects of Eight Biocontrol Bacteria on Alternaria alternifolia, the Pathogen of Blackcurrant Leaf Spot.

[0047]

[0048] Note: 'a' indicates the mean of the maximum / minimum radii over three replicates, ± standard error (SE). Values ​​labeled with different letters are significant at the p < 0.05 level.

[0049] As can be seen from Table 1, among the eight biocontrol bacteria, biocontrol bacteria No. 1, No. 3, No. 4, and No. 7 had better antibacterial effects. Among them, biocontrol strain No. 1 had the best antibacterial effect (named SH1) and had good biocontrol potential. It was selected as the target biocontrol strain for rescreening.

[0050] The field efficacy of biocontrol bacterium SH1 was determined by spraying the bacterial suspension until the leaf surface was saturated.

[0051] Table 2. Field control efficacy of biocontrol bacterium SH1 suspension against blackcurrant leaf spot.

[0052]

[0053] Note: The value of 'a' represents the mean ± standard error (SE) of three replicates. Values ​​labeled with different letters are significant at the p < 0.05 level.

[0054] The field control efficacy of biocontrol bacterium SH1 suspension against blackcurrant leaf spot disease is shown in [reference needed]. Figure 1 A) Blank control; B) 10 8 CFU / mL; C)10 10 CFU / mL; D)10 12 CFU / mL;

[0055] From Table 2 and Figure 1 It can be seen that as the concentration of the biocontrol bacterium SH1 suspension increases, the disease index decreases, and the control effect strengthens. The concentration of the biocontrol bacterium SH1 suspension was 10... 12 CFU / mL showed the best control efficacy against blackcurrant leaf spot, reaching 75.7%.

[0056] Determination of the antibacterial spectrum of biocontrol bacterium SH1

[0057] Nine common local pathogenic fungi of woody plants were selected: *Diaporthe eres* (dusted strawberry leaf spot fungus), *Sphaerulina azaleae* (rhododendron leaf blight fungus), *Colletotrichum acericola* (sugar maple leaf spot fungus), *Corynespora cassiicola* (elderberry leaf spot fungus), *Neopestalotiopsis rosae* (kiwi fruit leaf blight fungus), *Alternaria alternata* (Chinese aster leaf spot fungus), *A. alternata* (birch leaf spot fungus), *A. alternata* (weeping willow leaf spot fungus), and *A. alternata* (blue honeysuckle leaf spot fungus).

[0058] Using the confrontation culture method, pathogenic bacteria of nine leaf diseases were inoculated into the center of PDA plates (d=0.7cm), and the inhibition spectrum of the biocontrol bacterial strain with the best resistance was determined.

[0059] Table 3. Antagonistic effect of biocontrol bacterium SH1 against 9 pathogenic fungi causing foliar diseases in plants.

[0060]

[0061] Note: Data are expressed as the mean of the maximum / minimum radius over three replicates, ± standard error (SE). According to Duncan's multiple range test, different letter representations after the numerical values ​​are statistically significant at P < 0.05.

[0062] The antagonistic effect of biocontrol bacterium SH1 against 9 pathogenic fungi causing foliar diseases of plants is shown in [reference needed]. Figure 2 A) Duchesnea indicum leaf spot (Diaporthe eres); B) Rhododendron leaf blight (Sphaerulina azaleae); C) Sugar maple leaf spot (Colletotrichum acericola); F) Elderberry leaf spot (Corynespora cassiicola); E) Kiwifruit leaf blight (Neopestalotiopsis rosae); F) China aster leaf spot (Alternaria alternata); G) Birch leaf spot (A. alternata); H) Weeping willow leaf spot (A. alternata); I) Honeysuckle berry leaf spot (A. alternata);

[0063] From Table 3 and Figure 2 It can be seen that the biocontrol bacterium SH1 has a certain inhibitory effect on the pathogenic fungi causing leaf diseases of nine common woody plants. In particular, it has a strong antagonistic effect on the pathogenic fungi of Duchesnea seres, Sphaerulinaazaleae, Colletotrichum acericola, Corynesporacassiicola, Neopsestalotiopsis rosae, and Alternaria alternata, with the longest radius / shortest radius ratio being greater than 2. The results indicate that SH1 has a broad spectrum of inhibition, especially against Alternaria alternata, a leaf spot pathogen of various plants, and has a wide range of applications.

[0064] Morphological identification of biocontrol bacterium SH1

[0065] Morphological characteristics: A small amount of biocontrol bacteria was inoculated into LB liquid medium and incubated in a shaker at 28°C and 180 rpm for 24 hours. The bacterial suspension was then diluted 10... 8 CFU / mL (OD 600 = 0.1), take 15 μL and spread it evenly on NA plate, incubate at 30℃ for 2 days, and observe the colony morphology.

[0066] Gram staining: Biocontrol bacterial growth smeared on a glass slide was stained with crystal violet and safranin counterstaining solutions, respectively. After washing and drying, the color changes were observed under an optical microscope. Gram-positive bacteria appeared purple, while Gram-negative bacteria appeared red.

[0067] Gram staining and colony analysis of biocontrol bacterium SH1 are shown in [link to Gram staining data]. Figure 3 A) Gram staining; B) Colonies; such as Figure 3 As shown, after being cultured on LB solid medium at 37°C for 24 hours, strain SH1 formed milky white, round colonies with a dry, wrinkled surface and a raised center; it was a Gram-positive, short rod-shaped bacterium.

[0068] Physiological and biochemical reaction determination of biocontrol bacteria SH1

[0069] Physiological and biochemical tests: The tests were performed using physiological and biochemical test reagents and in accordance with the instructions for use.

[0070] Based on Bergey's Manual of Bacterial Identification and the Manual of Systematic Identification of Common Bacteria edited by Dong Xiuzhu and Cai Miaoying, the morphological and physiological biochemical characteristics of biocontrol bacteria were identified.

[0071] Table 4. Physiological and biochemical characteristics of biocontrol bacterium SH1

[0072]

[0073] Note: "+" represents a positive reaction; "-" represents a negative reaction.

[0074] Physiological and biochemical characteristics of biocontrol bacteria SH1 are shown in [link to relevant documentation]. Figure 4 A) Malonate; B) Mannitol; C) Fructose; D) Hydrogen sulfide test; E) Maltose; F) Gelatin liquefaction; G) Lactose; H) Glucose; I) Sorbitol; J) Rhamnose; K) Cellobiose; L) Nitrate reduction test;

[0075] From Table 4, Figure 4 It can be seen that this is a Gram-positive, anaerobic bacterium capable of producing nitrate reductase and catalase, but not hydrogen sulfide. It can utilize glucose and xylose, but not mannitol, fructose, maltose, lactose, sorbitol, arabinose, rhamnose, cellobiose, or sucrose. It is negative for malonate and positive for citrate; it hydrolyzes gelatin and starch; and it is positive for VP and MR tests.

[0076] Molecular identification of biocontrol bacterium SH1

[0077] DNA was extracted from the tested biocontrol bacteria using the Kangwei Century DNA Extraction Kit, and 16S rDNA amplification technology was employed. The primer sequences are as follows:

[0078] 1492R: 5'-GGTTACCTTGTTACGACTT-3', as shown in SEQ ID No. 1;

[0079] 27F: 5'-AGAGTTGATCCTGGCTCAG-3', as shown in SEQ ID No. 2;

[0080] Table 5 PCR reaction system

[0081]

[0082] Table 6 PCR amplification program reactions

[0083]

[0084] Electrophoresis detection: The amplification products were accurately detected using 1.0% agarose gel electrophoresis and then sent to a bioengineering company (Shanghai) for gene sequencing. The obtained sequences were compared online for homology in the NCBI database. A phylogenetic tree was constructed using Mega 7.0 software with a neighbor-joining method (parameter 1000).

[0085] The PCR product was amplified and sequenced to obtain a 1446 bp 16S rDNA product. Blast alignment was performed on the NCBI system, and SH1 showed 99.79% identity with Bacillus velezensis strain ACH16. The sequence was uploaded to GeneBank and obtained accession number OR857515. A phylogenetic tree was constructed using MEGA software.

[0086] A phylogenetic tree of biocontrol bacterium SH1 constructed based on the 16S rRNA gene is shown below. Figure 5 The results showed that the biocontrol bacterium SH1 and B. velezensis YC7010 (KP201498.1) belonged to the same branch and had 100% similarity, both being Bacillus beleises.

[0087] In summary, based on morphological identification, physiological and biochemical reaction identification, and molecular identification results combined with phylogenetic tree analysis, the biocontrol bacterium SH1 was identified as Bacillus velezensis.

[0088] Preparation of biocontrol bacteria SH1 lipopeptide extract

[0089] Antagonistic bacteria were added to LB liquid medium and cultured in a shaker at 37°C for 24 h. 5 mL of bacterial suspension was added to every 200 mL of lipopeptide fermentation medium and cultured in a shaker at 28°C and 180 rpm for 72 h. The above liquid was added to centrifuge tubes and balanced. The centrifuge speed was adjusted to 10000 rpm, and the supernatant obtained after centrifugation for 15 min was poured into an Erlenmeyer flask. The supernatant in the Erlenmeyer flask was adjusted to pH 2.0 with 6M hydrochloric acid and refrigerated at 4°C for 24 h, resulting in a precipitate at the bottom of the Erlenmeyer flask. The above liquid was added to centrifuge tubes and balanced. The mixture was centrifuged at 5000 rpm for 10 min, the supernatant was discarded, and the precipitate was dissolved in pH 2.0 HCl. The mixture was centrifuged again, and the precipitate was retained and dissolved in methanol. The solution was evaporated to dryness using a rotary vacuum evaporator adjusted to 35°C. An appropriate amount of methanol was added to dissolve the lipopeptide substance, which was then stored for later use.

[0090] Biocontrol effect of SH1 lipopeptide extract on blackcurrant leaf spot disease

[0091] Table 7. Field control efficacy of biocontrol bacterium SH1 lipopeptide extract against blackcurrant leaf spot.

[0092]

[0093] Note: The value of 'a' represents the mean ± standard error (SE) of three replicates. Values ​​labeled with different letters are significant at the p < 0.05 level.

[0094] The field control efficacy of biocontrol bacteria fermentation liquid against blackcurrant leaf spot disease is shown in [the figure]. Figure 6 A) Blank control; B) 317 μg / mL propiconazole·pyraclostrobin; C) 5 μL / mL lipopeptide extract

[0095] From Table 7 and Figure 6 It can be seen that the lipopeptide extract of biocontrol bacterium SH1 has a significant control effect on blackcurrant leaf spot disease, which is comparable to that of propiconazole·pyraclostrobin, with a control effect of 80.8%.

[0096] Determination of the antibacterial mechanism of lipopeptide extracts against Alternaria alternata

[0097] Effects of lipopeptide extracts on conidia production, germination and hyphal growth

[0098] Table 8. Effects of lipopeptide extracts from biocontrol bacterium SH1 on Alternaria alternata spore production, spore germination, and mycelial growth.

[0099]

[0100] The effect of SH1 lipopeptide extracts on the growth of Alternaria alternata hyphae is shown in the figure. Figure 7Lipopeptide concentration: A) Control; B) 1 μL / mL; C) 3 μL / mL; D) 5 μL / mL;

[0101] From Table 8, Figure 7 It can be seen that lipopeptide extracts at concentrations of 1 μL / mL, 3 μL / mL, and 5 μL / mL all have a certain inhibitory effect on spore production, spore germination, and mycelial growth of Alternaria alternata. Moreover, with the increase of lipopeptide extract concentration, the inhibition rate of spore production, spore germination, and mycelial growth of Alternaria alternata gradually increases. When the concentration of lipopeptide extract reaches 5 μL / mL, the inhibition rates on spore production, spore germination, and mycelial growth are 86.60%, 73.57%, and 52.15%, respectively.

[0102] Effects of lipopeptide extracts on the morphology of Alternaria alternata hyphae

[0103] (1) Effects on the growth of pathogenic mycelia

[0104] Lipopeptide extracts were added to quantitative PDA medium that had been melted and cooled to 45°C, with concentration gradients of 1 μL / mL, 3 μL / mL, and 5 μL / mL. The mixture was poured into petri dishes and cooled to solidify. A culture dish (d = 0.7 cm) containing pathogenic fungus HLJ-4 was inoculated in the center of the petri dish. Each treatment was repeated three times. The control group consisted of medium with an equal volume of sterile water. The dishes were incubated at 26°C for 5 days. The colony diameter was measured, and the inhibition rate was calculated using the same formula as in 2.4.1.

[0105] (2) Effects on the production of conidia by pathogens

[0106] The pathogenic fungus HLJ-4 was propagated in PDA plates until the colony diameter reached 5 cm. After removing the culture medium without mycelium growth, 20 mL of lipopeptide extracts of the same gradient concentration were added. The control group was treated with 20 mL of sterile water. The pathogenic mycelium on the surface of the culture medium was scraped off using a transfer hook and soaked in each treatment liquid for 20 min. The liquid was then discarded, and the plates were incubated in the dark at 26°C for 3 days. Each treatment was repeated 3 times. After 3 days, 20 mL of sterile water was added to the plates, and the newly formed mycelium on the surface of the culture medium was scraped off using a transfer hook. The spore suspension was obtained by filtering with sterile gauze. The concentration of conidia in different treatments was counted using a hemocytometer, and the spore production inhibition rate was calculated to determine the effect of lipopeptide extracts on the production of pathogenic conidia.

[0107] (3) Effect on the germination of pathogenic conidia

[0108] The pathogenic fungal spores (d = 0.7 cm) were inoculated at the center of a PDA plate and incubated at 26°C for 3 days. An appropriate amount of sterile water was poured into a petri dish, and the pathogenic hyphae on the surface of the medium were scraped off using a transfer hook. After shaking in an Erlenmeyer flask and filtering with sterile gauze, the spore suspension was adjusted to a concentration of 10⁵ spores / mL with sterile water. Appropriate amounts of the spore suspension were added to each well of a cell culture plate (24 wells). Lipopeptide extracts were added to adjust the final concentrations to three gradients: 1 μL / mL, 3 μL / mL, and 5 μL / mL, with a total volume of 2 mL. The control was a spore suspension without lipopeptide extract. Each treatment was repeated three times. The number of spores germinating in the control treatment was observed daily under an optical microscope. When the spore germination rate was above 60%, the number of germinating and non-germinating spores in each treatment was investigated, and the inhibition rate of pathogenic spore germination under different treatments was calculated.

[0109] (4) Effects on the hyphal morphology of pathogenic fungi

[0110] The lipopeptide extracts were diluted with sterile water to the same gradient concentrations as above, and 2 mL of each was added to a cell culture plate (24 wells). The propagated pathogenic fungus HLJ-4 was then inoculated into the center of the plate and cultured for 12 h. Newly formed hyphae on the edge of the colony in the PDA culture dish were picked and added to each concentration of lipopeptide extract. Sterile water without lipopeptide extract was used as a blank control. Each treatment was repeated 3 times and cultured in the dark at 26℃ for 24 h. The changes in the hyphal morphology of the pathogen under different concentration treatments were observed using an optical microscope.

[0111] The effect of biocontrol bacterium SH1 lipopeptide extract on the hyphal morphology of Alternaria alternata is shown in the figure. Figure 8 Lipopeptide concentration: A) Control; B) 1 μL / mL; C) 3 μL / mL; D) 5 μL / mL;

[0112] Depend on Figure 8 It can be seen that the untreated Alternaria alternata hyphae are relatively uniform in thickness and do not produce spores. After treatment with different concentrations of lipopeptide extract, the hyphae swell in the middle and at the tip, the hyphae become uneven in thickness and bend and fold, and abnormal spores are produced. There are bulges at the tip or sides. As the concentration of lipopeptide extract increases, the deformity becomes more obvious. The hyphae and spores rupture at the swollen sites, and the protoplasm is exposed.

[0113] Determination of the thermal stability of lipopeptides in biocontrol bacteria SH1

[0114] Lipopeptide extracts were placed in a series of temperature gradients (4℃, 26℃, 40℃, 60℃, 80℃, 90℃, and 100℃), and treated with a water bath for 20 min at each temperature. The treated lipopeptide extracts were added to a quantitative amount of PDA medium to a concentration of 5 μL / mL, and plates were prepared. The propagated pathogenic fungus HLJ-4 was then agarocidal (d = 0.7 cm) placed in the center of each plate and incubated at 26℃ in the dark for 5 days. The control was agar without lipopeptide extracts. Each treatment was repeated in triplicate. Colony diameters were measured, and the inhibition rate was calculated using the same formula as in section 2.4.1.

[0115] The effect of temperature on the antibacterial activity of SH1 lipopeptides is shown in the figure. Figure 9 : a. The numerical value represents the mean ± standard error (SE) of three replicates. Values ​​labeled with different letters are significant at the p < 0.05 level, and the same applies below.

[0116] from Figure 9 It can be seen that the antibacterial effect of biocontrol bacteria lipopeptide extracts did not change significantly when treated at different temperatures, indicating that lipopeptide extracts still have good thermal stability at higher temperatures.

[0117] Determination of acid-base stability of lipopeptides in biocontrol bacteria SH1

[0118] Lipopeptide extracts were adjusted to different pH values ​​(2, 3, 4, 5, 6, 7, 8, 9, 10, 11, and 12) using 1 mol / L HCl and 1 mol / L NaOH, respectively. The control consisted of untreated lipopeptide extract. The adjusted lipopeptides were then added to a quantitative amount of PDA medium to a concentration of 5 μL / mL. Plates were prepared, and a culture dish (d = 0.7 cm) of the propagated pathogenic fungus HLJ-4 was inoculated at the center of each plate. The plates were incubated at 26℃ in the dark for 5 days, with each treatment replicated in triplicate. Colony diameters were measured, and inhibition rates were calculated.

[0119] The effect of pH on the antibacterial activity of SH1 lipopeptides is shown in [reference needed]. Figure 10 ;

[0120] from Figure 10 It can be seen that the antibacterial effect is best at pH 7-8. Under strong acid and strong alkaline conditions, the antibacterial activity is significantly reduced, while a certain inhibitory effect can still be maintained in the pH range of 3-11. This indicates that such active substances have a certain degree of acid and alkali tolerance, but are not tolerant to strong acids and alkalis.

[0121] UV sensitivity determination of lipopeptides in biocontrol bacteria SH1

[0122] Lipopeptide extracts were poured into sterile petri dishes and placed 30 cm away from a 30W UV lamp for irradiation for 30 min, 60 min, and 90 min, respectively. The control consisted of untreated lipopeptide extracts. The different treatments of lipopeptides were added to a quantitative amount of PDA medium to a concentration of 5 μL / mL, and plates were prepared. A culture disc (d = 0.7 cm) of the pathogenic fungus HLJ-4 was inoculated at the center of each plate. The plates were incubated at 26℃ in the dark for 5 days. The control consisted of medium without lipopeptide extracts. Each treatment was repeated in triplicate. Colony diameters were measured, and the inhibition rate was calculated.

[0123] The effect of ultraviolet radiation on the antibacterial activity and stability of SH1 lipopeptide extract is as follows: Figure 11 ;

[0124] from Figure 11 It can be seen that the antibacterial rate of biocontrol bacteria lipopeptide extracts was not significantly affected by UV irradiation for different durations, indicating that the antibacterial active substances are not sensitive to ultraviolet light.

[0125] Purification and Identification of Antibacterial Active Substances from Biocontrol Bacterium SH1 Lipopeptide Extract

[0126] Purification of antibacterial active substances from biocontrol bacterium SH1 lipopeptide extract

[0127] The lipopeptide extract was spectrally scanned using a UV spectrophotometer, and its maximum absorption peak was determined in the range of 200–1100 nm.

[0128] The extract was filtered using a Φ=0.22μm bacterial filter, and the sterile filtrate was separated and purified using a protein purification chromatography system based on the NGC Quest100plus system. The protein chromatography column was equilibrated with PBS buffer, and 215nm was set as the detection wavelength, the flow rate was set to 1mL / min, and the detection time was 50min.

[0129] A full-wavelength scan of the lipopeptide extract of biocontrol bacterium SH1 was performed in the range of 200 nm to 1100 nm. The results showed that the maximum absorption peak of the lipopeptide extract appeared at a wavelength of 215 nm. Therefore, 215 nm was selected as the wavelength for protein purification analysis. After elution by molecular sieve according to mass, the chromatographic pattern of the lipopeptide extract of biocontrol bacterium SH1 is shown below. Figure 12 The 41st, 42nd, 47th, 49th, 51st and 52nd tubes were selected for the experiment.

[0130] Verification of the antibacterial activity of the elution peak of the biocontrol bacterium SH1 lipopeptide extract

[0131] The antibacterial activity of purified samples with different components against *Hymenochloa spp.* leaf spot pathogen was analyzed using the paper disc inhibition zone method. The pathogenic fungus was inoculated onto a PDA plate (d = 0.7 cm) at the center, and sterile filter paper discs were placed at equal distances from the center (2 cm from the center in all four directions). 10 μL of each purified component was added to each plate, with an equal volume of sterile water added as a control. The plates were incubated in the dark at 28°C for 3–5 days. The inhibition radius of the pathogenic fungus was measured, and the antibacterial activity of different components was evaluated.

[0132] To clarify the antibacterial activity of the elution peaks, plate confrontation experiments were conducted on tubes with different elution peaks. The antagonistic effect of the elution peak of the biocontrol bacterium SH1 lipopeptide extract on the growth of Alternaria alternata hyphae is shown in [the table below]. Figure 13 A represents the antagonistic effect of tubes 41, 42, and 47 and the water control on the growth of Alternaria alternata mycelia; B represents the antagonistic effect of tubes 49, 51, and 52 and the water control on the growth of Alternaria alternata mycelia; and C represents the antagonistic effect of tube 42 and the water control on the growth of Alternaria alternata mycelia.

[0133] like Figure 13 As shown, tubes 42, 49, 51, and 52 all exhibited good antibacterial activity, indicating a high content of antibacterial active ingredients. Tube 42, in particular, showed the best antibacterial effect and will be used for further detailed identification and analysis of the antibacterial active ingredients.

[0134] Mass spectrometry identification of the main antibacterial active ingredients of biocontrol bacterium SH1

[0135] The matrix-assisted laser desorption / ionization mass spectrometry (MALDI-TOF-MS) system used was the Ultraflex Extreme MALDI-TOF-TOF Mass Spectrometer from Bruker Daltonics, employing positive ion detection and reflectance mode. The procedure was commissioned to Shanghai Houji Biotechnology Co., Ltd., and the main antibacterial active substances in the target biocontrol bacterial strain were identified by combining the molecular weights and related information of Bacillus lipopeptides from published literature.

[0136] The matrix-assisted laser desorption / ionization time-of-flight mass spectrometry (MALDI-TOF-MS) spectroscopy analysis of the SH1 lipopeptide extract is shown in the figure below. Figure 14 ;

[0137] from Figure 14The mass spectrum shows nucleus-to-mass ratios (m / z) around 1008, 1022, 1036, 1050, 1057, 1436, 1450, and 1463. This suggests that the mass ratios of 1008, 1022, 1036, and 1050 form a homologue of C13-16 Surfactin A, C14-16 Surfactin B, or C13-16 Surfactin C; 1057 is C15 Iturin A; and 1436, 1450, and 1463 form a homologue of C14-16 Fengycin A. Analysis suggests that the sample may bind an H+ ion, and the -CH2 and -NH2 atoms in its structure may also bind or lose an H+ ion. Therefore, the measured molecular weight ±2 Da is reasonable. Thus, it is speculated that the antibacterial active substances mainly contain Surfactin, Iturin, and Fengycin.

[0138] The above description of the disclosed embodiments enables those skilled in the art to make or use the invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the invention. Therefore, the invention is not to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. An endophytic Bacillus belye, characterized in that, The endophytic Bacillus belysinus ( Bailus velezensis It is deposited at the China General Microbiological Culture Collection Center, with accession number CGMCC No. 30625.

2. A biological control agent, characterized in that, Includes the endophytic Bacillus belye as described in claim 1.

3. The application of the endophytic Bacillus belye as described in claim 1 in the control of Alternaria leaf spot disease of tussock currants, characterized in that, The blackcurrant leaf spot disease is caused by... Alternaria alternata Triggered.

4. The application of the endophytic Bacillus belye as described in claim 1 in the prevention and control of plant foliage pathogens, wherein the plant foliage pathogen is... Diaporthe eres , Sphaerulina azaleae , Colletotrichum acericola , Corynespora cassiicola , Neopestalotiopsis rosae and Alternaria alternata Any one of them.

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