Bacillus velezensis and application thereof

Abstract

The application belongs to the technical field of Chinese medicine plant disease biological control, and particularly relates to a bacillus velezensis and application thereof. Bacillus velezensis The nsu-59 strain is preserved in the China Center for Type Culture Collection, and the preservation number is CCTCC NO: M 2025573. The bacillus velezensis provided by the application can not only prevent and treat the anthracnose of trichosanthes fruit, but also promote the seed germination of trichosanthes, and has a broad-spectrum antifungal effect on various Chinese medicine plant pathogenic fungi such as anthracnose, fusarium, trichoderma and diaporthe, has low prevention and treatment cost, is green and environment-friendly, will not cause drug resistance, and has great potential to be developed into a Chinese medicine plant biocontrol agent.

Description

Technical Field

[0001] This invention belongs to the field of biological control technology for diseases caused by traditional Chinese medicine, specifically involving a strain of Bacillus belye and its application. Background Technology

[0002] Trichosanthes kirilowii, also known as Gualou, is a perennial vine belonging to the Cucurbitaceae family. Its roots, fruits, and seeds are all used medicinally. Modern research shows that Trichosanthes kirilowii has effects such as clearing heat and resolving phlegm, relieving chest congestion, and promoting bowel movements. It also exhibits good biological activity in lowering blood sugar, anti-tumor activity, and antibacterial activity. In recent years, the artificial cultivation of Trichosanthes kirilowii has developed on a large scale in my country. However, in artificial cultivation, Trichosanthes kirilowii is often affected by continuous cropping obstacles. The core impact is the disruption of the synergistic balance between soil, crop, and microorganisms, leading to deterioration of soil physical and chemical properties, imbalance of soil nutrients, accumulation of autotoxic substances, proliferation of harmful microorganisms, and reduction of beneficial microorganisms. Ultimately, this results in stunted growth, reduced yield, and poorer quality of Trichosanthes kirilowii, while also exacerbating the occurrence of pests and diseases. The main pests and diseases affecting Trichosanthes kirilowii are fungal diseases, commonly including anthracnose, root-knot nematode disease, and leaf spot. Among these, *Anthracnose* (a fungus) is particularly prevalent. Colletotrichum sp . Anthracnose caused by the disease is widespread and difficult to control, becoming an important factor limiting the yield and quality of Trichosanthes kirilowii.

[0003] Currently, the main agricultural practices in Trichosanthes kirilowii production include crop rotation, selection of resistant varieties, tillage, and field sanitation, combined with the application of chemical fungicides such as carbendazim, chlorothalonil, and propiconazole for control. For example, reference 1 (Hu Xianhai et al., Occurrence patterns and control methods of Trichosanthes kirilowii anthracnose, *Modern Agricultural Science and Technology*, November 2006) discloses chemical control: 50% thiophanate-methyl wettable powder at a dilution of 600 times can be used. Use fungicides such as 600x dilution of 1% carbendazim wettable powder, 300x dilution of 80% mancozeb wettable powder, 600x dilution of 70% mancozeb wettable powder, 600x dilution of 75% chlorothalonil wettable powder, 500x dilution of 25% carbendazim wettable powder, 4000x dilution of 25% cymoxanil emulsifiable concentrate, 200x dilution of 2% antifungal agent (Nongkang 120 aqueous solution), or 200x dilution of 2% Wuyimycin aqueous solution, etc. Spray promptly for prevention and control at the initial stage of disease or when the weather conditions are conducive to disease development. However, long-term use of chemical agents can easily lead to drug resistance in pathogens, resulting in excessive pesticide residues on materials, and can also disrupt the soil microecological balance, causing the control effect to decline year by year.

[0004] Biological control of Trichosanthes kirilowii, a disease caused by continuous cropping, is one of the important directions to ensure the sound and healthy development of the Trichosanthes kirilowii industry. For example, reference 2 (Xu Weifang et al., the disease control ability and mechanism of biocontrol bacteria HX0037 against Trichosanthes kirilowii anthracnose, Biotechnology Bulletin, 2024, Vol. 40, Issue (4): 228-241.) discloses the determination of the effect of HX0037 on Trichosanthes kirilowii anthracnose using the plate confrontation method. Colletotrichum gloeosporioides ), Jujube anthracnose ( Collettrichum coccodes ), pear anthracnose ( Colletotrichum fructicola ) and apple anthrax ( Cryptosporiopsis malicorticis The antagonistic activity of *Bacillus amyloliquefaciens* HX0037 was evaluated using an in vitro inoculation method to control anthracnose on leaves and fruits of *Trichosanthes kirilowii*. The results showed that *Bacillus amyloliquefaciens* HX0037 exhibited good inhibitory ability against the growth of anthracnose pathogens, and it holds promise for further development as a biopesticide for the green control of anthracnose in *Trichosanthes kirilowii*. Additionally, CN115029268A discloses a strain of *Streptomyces venezuelae* and its application in controlling crop diseases. This *Streptomyces venezuelae* is... Streptomyces venezuelae S2-2, this strain has a good inhibitory effect on *Trichosanthes kirilowii* anthracnose, significantly reducing the infection rate, and showing good results in in vitro experiments. Overall, there are few reports on biocontrol technologies, and there is currently a lack of biocontrol agents with industrial application prospects, which limits the development and application of this technology. Summary of the Invention

[0005] To address the aforementioned problems, this invention provides a strain of Bacillus belyceae and its applications. This strain possesses broad-spectrum antifungal activity, capable of controlling various plant pathogenic fungi, including those causing anthracnose in Trichosanthes kirilowii, and also promotes seed germination in Trichosanthes kirilowii. It has the potential to be developed into a biocontrol agent, solving the problems of continuous cropping obstacles and anthracnose disease encountered in current Trichosanthes kirilowii production, and improving the quality and yield of Trichosanthes kirilowii.

[0006] On the one hand, the present invention provides a strain of Bacillus belye. Bacillus velezensis The nsu-59 strain is deposited at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC NO: M 2025573.

[0007] On the other hand, the present invention provides the aforementioned fermentation broth of Bacillus belyssus or an extract thereof. Preferably, the extract is obtained by solid-liquid separation of the Bacillus belyssus fermentation broth, acid precipitation of the liquid obtained from the solid-liquid separation, and further alcohol extraction of the precipitate obtained from the acid precipitation. More preferably, the extract is an antifungal lipopeptide.

[0008] On the other hand, the present invention provides the volatile metabolites of the aforementioned Bacillus belyssus, specifically, volatile organic gases (VOCs) produced during the metabolism of strain NSU-59.

[0009] On the other hand, the present invention provides a microbial agent comprising the aforementioned *Bacillus belye*, fermentation broth, extract, or volatile metabolites. The dosage form of this microbial agent is not specifically limited and can be a liquid or solid agent, with a viable bacteria content of approximately 1 × 10⁻⁶. 9 CFU / mL.

[0010] On the other hand, the present invention provides the application of the aforementioned *Bacillus belye*, fermentation broth, extract, volatile metabolites, or inoculant in the treatment of plant pathogens, wherein the plant pathogen is any one or a combination of two or more of the following genera: *Anthracis*, *Fusarium*, *Trichoderma*, or *Metacarpa*, preferably *Anthracis*, *Fusarium*, *Trichoderma*, and *Metacarpa*. Preferably, the *Anthracis* pathogen is *Colletotrichum gloeosporioides* (…). Colletotrichum gloeosporioides ) and cryptic anthrax bacteria ( Colletotrichum aenigma The pathogen of the Fusarium genus is Fusarium graminearum ( ); Fusarium graminearum Trichoderma pathogens are green Trichoderma ( ); Trichoderma virens The pathogen of the genus *Phyllostachys* is *Phyllostachys prawnae* (…). Diaporthe compacta ).

[0011] On the other hand, the present invention provides the application of the aforementioned Bacillus belye or fermentation broth or extract or volatile metabolite or bacterial agent in the prevention and control of Trichosanthes kirilowii anthracnose.

[0012] On the other hand, the present invention provides the application of the aforementioned Bacillus vesiculosus, or fermentation broth, or extract, or volatile metabolite, or bacterial agent in promoting the germination of Trichosanthes kirilowii seeds.

[0013] The beneficial effects of this invention are as follows: This invention provides a strain of Bacillus belye, an endophytic bacterium found in the fruit of Trichosanthes kirilowii ( Bacillus velezensis It can not only prevent and control anthracnose of Trichosanthes kirilowii, but also promote the germination of Trichosanthes kirilowii seeds. It also has a broad-spectrum antifungal effect against a variety of pathogens of Chinese medicinal plants, including Anthracnose, Fusarium, Trichoderma and Echinochloa. It is low-cost, environmentally friendly and does not produce drug resistance, and has great potential to be developed into a biocontrol agent for Chinese medicinal plants. Attached Figure Description

[0014] 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 one by one below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0015] Figure 1 Schematic diagrams of morphological and physiological biochemical identification of strain NSU-59; A: Colony morphology on LB medium; B: Gram staining image; C: Spore staining image; D: Cellulase detection image; E: Hemolysis detection image.

[0016] Figure 2 A schematic diagram of the phylogenetic tree of strain nsu-59 based on 16S rDNA; Figure 3A schematic diagram of the phylogenetic tree of strain nsu-59 based on single-copy homologous genes; Figure 4 The antagonistic effect of the aseptic fermentation supernatant of strain NSU-59 on 14 plant pathogens is shown in the figure. Figure 5 The image shows the antibacterial effect of strain NSU-59 against two types of Trichosanthes anthracnose pathogens as determined by a plate test. Figure 6 The image shows the antibacterial effect of volatile gases from strain NSU-59 on two types of anthracnose pathogens of Trichosanthes kirilowii, as detected by two-divided plates. Figure 7 The graph shows the antibacterial activity of NSU-59 fermentation broth extract against two types of Trichosanthes anthracnose pathogens. Figure 8 The image shows the safety effect of strain NSU-59 on tissue culture seedlings of Trichosanthes kirilowii. Figure 9 The control effect of NSU-59 bacterial suspension on anthracnose of detached leaves (A) and anthracnose of fruit of Trichosanthes kirilowii (B) is shown in the figure. Figure 10 The effect of NSU-59 bacterial suspension and sterile supernatant on the germination of Trichosanthes kirilowii seeds is shown in the figure. Figure 11 The graph shows the control effect of strain NSU-59 on anthracnose in potted Trichosanthes kirilowii; A: diseased group; B: group treated with carbendazim; C: bacterial suspension 1×10 9 CFU / mL treatment group; D: bacterial suspension 1×10 8 CFU / mL treatment group; E: bacterial suspension 1×10 7 CFU / mL treatment group; F: bar chart of control effect.

[0017] Figure 12 The following graphs illustrate the disease control and growth promotion effects of strain NSU-59 on Trichosanthes kirilowii under field conditions: A: Field diagram of Trichosanthes kirilowii at different growth and development stages; B: Bar graph of leaf anthracnose disease index; C: Bar graph of leaf anthracnose control efficiency; D: Bar graph of fruit anthracnose disease index; E: Bar graph of fruit anthracnose control efficiency; F: Trichosanthes kirilowii fruit from different treatment groups; G: Bar graph of Trichosanthes kirilowii fruit weight. Detailed Implementation

[0018] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, 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, 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.

[0019] In the following embodiments: Potato glucose agar medium (PDA medium): 200.00 g potato, 20.00 g glucose, 15.00~20.00 g agar, 1000.00 mL distilled water.

[0020] Luria-Bertani solid medium (LB medium): 10.00 g tryptone, 5.00 g yeast extract, 10.00 g NaCl, 15.00~20.00 g agar, 1000.00 mL distilled water.

[0021] Water agar medium (WA medium): 15.00~20.00 g agar, 1000.00 mL distilled water.

[0022] Nutrient agar medium (NA medium): 10.00 g peptone, 3 g beef extract, 5.00 g NaCl, 15.00~20.00 g agar, 1000.00 mL distilled water.

[0023] Gao's I medium (GA medium): soluble starch 20.00 g, KNO3 1.00 g, NaCl 0.50 g, K2HPO4 0.50 g, MgSO4 0.50 g, FeSO4 0.01 g, agar 15.00~20.00 g, distilled water 1000.00 mL.

[0024] Czapek's medium (CZA medium): FeSO4 0.01 g, KCl 0.50 g, agar 15.00 g, distilled water 1000.00 mL.

[0025] Potato glucose broth (PDB) medium: 200.00 g potato, 20.00 g glucose, 1000.00 mL distilled water.

[0026] Example 1: Isolation and Preservation of NSU-59 Strains 1. Strains Isolation The strain of this invention is a Bacillus belysinus isolated from the fruit of Trichosanthes kirilowii cultivation base in Chuanwen, Anqing City, Anhui Province. Bacillus velezensisThe isolation method is as follows: Healthy *Trichosanthes kirilowii* fruits were routinely disinfected with 5% sodium hypochlorite and 75% alcohol sequentially. After confirming thorough surface disinfection, endophytic bacteria were isolated from the fruits using tissue isolation and dilution plating methods. Fruit tissues were placed on six different culture media: potato dextrose agar, Luria-Bertani solid medium, water agar, nutrient agar, Gao's I medium, and Czapek's agar, and incubated at 25°C. Observations were made daily. Once colonies appeared, the bacteria were purified using the streak plating method. Pure cultures were preserved using glycerol and stored at -80°C for later use.

[0027] 2. Strain preservation information: Bacillus belyssus nsu-59 ( Bacillus velezensis (NSU-59) was deposited on March 24, 2025 at the China Center for Type Culture Collection (Address: Wuhan University, Luojia Mountain, Bayi Road, Wuchang District, Wuhan, Hubei, China, 430072, China, Tel: 027-68754052), accession number: CCTCC NO: M 2025573.

[0028] Example 2: Morphological characteristics and physiological and biochemical detection of strain nsu-59 The NSU-59 strain appears as round, smooth, milky-white, opaque colonies on LB solid medium, with a viscous consistency. The colonies are uniform and tightly bound to the medium. Figure 1 A). Gram staining showed that strain NSU-59 is a rod-shaped Gram-positive bacterium ( Figure 1 B), spore staining revealed a single oval spore in the center of the bacterial cell ( Figure 1 C). The NSU-59 strain can utilize glucose, sucrose, and mannitol, but not lactose and maltose; it spreads and grows on semi-solid agar medium, indicating its motility; it is negative for indoleacetic acid, methyl red, VP, Simon's citrate, hydrogen sulfide, and urea tests; it can produce cellulase (…). Figure 1 D), but cannot produce siderophores; hemolytic test is negative ( Figure 1 E), see Table 1 below (Table 1).

[0029] Table 1. Physiological and biochemical detection results of NSU-59 strain

[0030] Example 3: Molecular biological identification of strain nsu-59 16S ribosomal deoxyribonucleic acid (16S rDNA) extraction from strain nsu-59: Total DNA from strain nsu-59 was extracted using DNA extraction buffer (PrepMan Ultra Sample Preparation Reagent kit, Applied Biosystems). The 16S rDNA sequence was amplified by polymerase chain reaction (PCR) using the universal primers 27F / 1492R, yielding a 1459 bp sequence. This sequence was submitted to the National Center for Biotechnology Information (NCBI) gene database and obtained the GenBank accession number OR392941. The obtained sequence was aligned using NCBI BLAST, and a phylogenetic tree was constructed using relevant sequences from homologous species. Phylogenetic analysis based on 16S rDNA showed that strain nsu-59 shared over 98% similarity with several strains of *Bacillus belyssiensis*, and was located on the smallest branch of the phylogenetic tree with *Bacillus belyssiensis* accessions ON680836, ON797328, and OR794123. Figure 2 ).

[0031] Furthermore, the steps for constructing a phylogenetic tree based on single-copy orthologous genes are as follows: Complete genomes of several biocontrol-functional Bacillus strains were downloaded from the NCBI GenBank database and annotated using Prokka 1.14.6 software; subsequently, single-copy orthologous genes were identified using OrthFinder 2.5.4 software based on the obtained protein sequences; finally, a phylogenetic tree was constructed using the maximum likelihood method with IQ-Tree software version 2.2.0.3, and a bootstrap test with 1000 replicates was set to evaluate the partial support. The results showed that strain nsu-59 and Bacillus bereaves NJN-6 (CP007165) belong to the same minimal clade (…). Figure 3 ).

[0032] Based on its morphological, physiological, and biochemical characteristics, as well as the phylogenetic analysis results based on the 16S rDNA gene and a single copy of orthologous genes, strain nsu-59 was finally identified as Bacillus belyesensis and named [name missing]. Bacillus velezensis nsu-59.

[0033]

[0034] Example 4: Detection of the antibacterial spectrum of NSU-59 strain The antimicrobial spectrum of strain NSU-59 was detected using the mycelial growth rate method: NSU-59 strain was inoculated into LB liquid medium and fermented on a shaker at 28℃ and 180 r / min for 12 h to obtain the seed culture. Fresh seed culture was inoculated into LB liquid medium at a 1% inoculation rate and fermented on a shaker at 28℃ and 180 r / min for 72 h to obtain the fermentation supernatant. After fermentation, the supernatant was centrifuged at 12000 r / min for 30 min and filtered through a 0.22 µm filter to obtain the sterile fermentation filtrate. The sterile fermentation filtrate of NSU-59 strain was added to PDA medium at a ratio of 5% to prepare drug-containing plates. Fourteen different pathogenic bacteria of traditional Chinese medicine were inoculated with mycelial cakes (5 mm in diameter) in the center of each plate. The control group did not include the sterile fermentation filtrate of antagonistic bacteria. Each experiment was repeated three times. The pathogens tested in this experiment were: *Colletotrichum gloeosporioides* NFD-5 (… Colletotrichum gloeosporioides NFD-5), Cryptoanthritis NFD-6 ( Colletotrichum aenigma NFD-6), Anthracnose TJ ( Colletotrichum coccodes TJ), Anthracnose MK ( Colletotrichum mangiferae MK), Fusarium oxysporum TRF-2 ( Fusarium fujikuroi TRF-2), Fusarium graminearum QSSD-1 ( Fusarium graminearum QSSD-1), Fusarium solani SH18 ( Fusarium solani SH18), Trichoderma harzianum SH2 ( Trichoderma harzianum SH2), Trichoderma viride SH4 ( Trichoderma virens SH4), Penicillium lilacinus NST-9 ( Penicillium janthinellum NST-9), Sclerotinia sclerotiorum QMRH-8 ( Sclerotinia sclerotiorum QMRH-8), Coccidioides buergerianum QSRD-2 ( Macrophomina phaseolina QSRD-2), Chilodonella stearothermiae NFD-23 ( Stagonosporopsis sp. NFD-23), Ligustrum lucidum NFD-36 ( Diaporthe compacta (NFD-36). After all treatment plates were incubated at 25°C for 10 days, the diameter of the pathogenic fungal colonies was determined using the cross-hatching method, and the inhibition rate was calculated using the following formula: Inhibition rate (%) = (Coronary diameter of control group - Colony diameter of treatment group) / Colony diameter of control group × 100%.

[0035] The results showed that strain NSU-59 exhibited varying degrees of antifungal activity against 14 pathogenic fungi. Figure 4The NSU-59 strain not only exhibits good inhibitory effects against anthracnose fungi, but also against plant pathogens such as Fusarium, Trichoderma, Penicillium, and Sclerotinia. In particular, it shows inhibition rates of 72.47%, 80.78%, 76.67%, 78.64%, and 89.61% against *Colletotrichum gloeosporioides* NFD-5, *Colletotrichum jujuba* TJ, *Fusarium graminearum* QSSD-1, *Trichoderma viride* SH4, and *Ligustrum lucidum* NFD-36, respectively. This demonstrates that the NSU-59 strain possesses broad-spectrum antifungal activity and has great potential for development into a biocontrol agent.

[0036] Example 5: Detection of antagonistic activity of strain nsu-59 against Trichosanthes anthracnose pathogen. The antagonistic activity of NSU-59 bacterial suspension against two Anthracnose pathogens of Trichosanthes kirilowii was detected using a plate confrontation experiment. Pathogen cakes (Colletotrichum gloeosporioides NFD-5 or Anthracnose cryptogenioides NFD-6) with a diameter of 5 mm were inoculated into the center of fresh potato dextrose agar plates. Parallel streaks were made on both sides of the cakes using a fresh NSU-59 bacterial suspension cultured for 24 h, with the streaks positioned 2.5 cm from the center of the PDA plate. The control group was not inoculated with antagonistic bacteria. Each experiment was repeated three times. After incubating all treatment plates at 25℃ for 10 days, the diameter of the pathogenic fungal colonies was determined using the cross-cross method. The inhibition rate was calculated using the following formula: Inhibition rate (%) = (Coronary diameter of control group - Colony diameter of treatment group) / Colony diameter of control group × 100%.

[0037] The results showed that strain NSU-59 exhibited excellent antagonistic activity against *Colletotrichum gloeosporioides* NFD-5 and *Colletotrichum cryptosporioides* NFD-6, with inhibition rates of 55.98% and 69.61%, respectively, demonstrating inhibition of hyphal growth and disruption of hyphal structure. Figure 5 ).

[0038] Example 6: Detection of the antagonistic activity of organic volatile gases from strain NSU-59 against anthrax bacteria. The antagonistic effect of the NSU-59 strain on volatile organic compounds (VOCs) was detected using the bipartite plate confrontation method. The specific method included: in the experimental group, a 5 mm diameter pathogenic fungal cake (Colletotrichum gloeosporioides NFD-5 or Anthracnose cryptica NFD-6) was inoculated on one side of the bipartite plate away from the center point, and the antagonistic bacterial suspension was streaked in a "Z" shape on the other side of the bipartite plate. The control group was not inoculated with antagonistic bacteria. The plates were incubated at 25℃ for 5 days, and the colony radius of the pathogenic fungi was measured to calculate the inhibition rate. The experiment was repeated 3 times.

[0039] Inhibition rate (%) = (Coronation radius of pathogenic fungi in control group - Colony radius of pathogenic fungi in experimental group) / Colony radius of pathogenic fungi in control group × 100% The results showed that, compared with the control group, the mycelial growth of the pathogenic fungus in the experimental group was significantly inhibited, with an inhibition rate of 44.81%. Microscopic observation of the mycelia in both the control and experimental groups revealed that the mycelia in the control group were uniform and smooth, while the mycelia in the experimental group showed swelling. Figure 6 This result indicates that VOCs produced by antagonistic bacteria inhibit the growth of pathogens by altering the hyphal structure of the pathogens.

[0040] Example 7: Inhibitory effect of antifungal lipopeptides from strain NSU-59 on anthrax bacteria. After inoculating strain NSU-59 into LB liquid medium and fermenting for 72 h, the supernatant was collected by centrifugation, and 6 mol / L hydrochloric acid was slowly added while stirring to adjust the pH to 2.0. After overnight precipitation at 4°C, the precipitate was collected by centrifugation, dried in an oven at 55°C, dissolved in methanol, and the filtrate was collected by vacuum filtration. The extract was concentrated under reduced pressure at 45°C and 100 r / min using a rotary evaporator to obtain antifungal lipopeptides (CLPs), and the net weight was calculated. The antifungal lipopeptides (CLPs) were dissolved in methanol to prepare 10 mg / mL and 20 mg / mL CLPs solutions. The antifungal activity of the antifungal lipopeptide extract was evaluated using a disc diffusion assay: 10 μL of CLPs solution was dropped onto filter paper discs, placed 2 cm from the center of a PDA plate, and a bacterial cake of NFD-5 or NFD-6 was inoculated at the center of the plate. Filter paper discs with 10 μL of methanol were used as controls, and the diameter of the inhibition zone was measured. The assay was repeated three times.

[0041] The results showed that the antifungal lipopeptides of strain nsu-59 significantly inhibited the hyphal growth of pathogens NFD-5 and NFD-6. The antifungal effect of 20 mg / mL CLPs was superior to that of 10 mg / mL CLPs, with inhibition rates of 56.32% and 56.50% for NFD-5 and NFD-6, respectively, at 20 mg / mL. Microscopic examination indicated that the antifungal lipopeptides of strain nsu-59 inhibited pathogen growth by altering the hyphal structure of the pathogens. Figure 7 ).

[0042] Example 8: Safety test of strain nsu-59 on Trichosanthes kirilowii tissue culture seedlings The effect of co-culturing NSU-59 strain with Trichosanthes kirilowii tissue culture seedlings on the growth of the seedlings was observed: NSU-59 strain seed culture was inoculated into PDB medium and cultured at 28℃ and 180 r / min for 24 h. The bacterial suspension was then diluted with sterile water to a concentration of approximately 5 × 10⁻⁶. 8CFU / mL was prepared for use; the NSU-59 strain was inoculated into PDB medium and cultured at 28°C and 180 r / min for 72 h on a shaker. The fermentation broth was centrifuged at 12000 r / min for 30 min. The supernatant was filtered through a 0.22 μm microporous membrane for sterilization and diluted 10 times for use.

[0043] Take tissue culture seedlings with uniform growth: ① Inoculate with sterile water; ② Inoculate with NSU-59 bacterial suspension; ③ Inoculate with NSU-59 sterile filtrate. During the experiment, use a sterile syringe to inject 50 μl of the solution into the root of each seedling. Each group has 4 replicates (each bottle contains 6 seedlings, and each bottle is one replicate). Place the seedlings in a 25℃ artificial climate chamber for 9 h of light and 15 h of darkness. Observe the growth of the seedlings daily, and record the growth on day 10.

[0044] The results showed that after 10 days of culture, the growth of Trichosanthes kirilowii tissue culture seedlings treated with NSU-59 bacterial suspension and sterile filtrate was basically the same as that treated with sterile water, and the survival rate of the tissue culture seedlings was 100%, indicating that NSU-59 strain had no toxic effect on Trichosanthes kirilowii tissue culture seedlings. Figure 8 ).

[0045] Example 9: Inhibitory effect of strain NSU-59 on isolated anthrax The efficacy of in vivo biocontrol was tested using detached leaves and fruits of *Trichosanthes kirilowii*. Leaves and fruits were divided into three treatment groups: CK group (normal control): leaves were inoculated with PDA blank agar blocks, and fruits were inoculated with sterile water; T1 group (pathogen-only inoculation): leaves were inoculated with *Trichosanthes kirilowii* anthracnose mycelium grown on PDA medium for 7 days, and fruits were inoculated with *Trichosanthes kirilowii* anthracnose spore suspension grown on PDA medium for 7 days; T2 group (co-inoculation of biocontrol bacteria and pathogen): leaves and fruits were first sprayed with a biocontrol strain suspension, and after the surface dried, leaves were inoculated with pathogen mycelium, and fruits were inoculated with pathogen spore suspension. Each group was repeated three times. The treated leaves were placed in WA medium and cultured continuously at 25℃ in a light incubator for 6 days; the fruits were sealed with plastic wrap and also cultured at 25℃ in a light incubator for 9 days. Pathogen infection status of *Trichosanthes kirilowii* leaves and fruits was observed and recorded regularly, and the control effect was calculated using the following formula. Lesion area = π × [(lesion length / 2 + lesion width / 2) / 2] 2 Control effect (%) = (area of ​​lesions in group T1 of Trichosanthes kirilowii – area of ​​lesions in group T2 of Trichosanthes kirilowii) / area of ​​lesions in group T1 of Trichosanthes kirilowii × 100%.

[0046] The results showed that the leaves in the sterile water control group remained intact, while the leaves in the diseased group showed large areas of white and brownish mycelium. The leaves in the NSU-59 strain treatment group also showed white and brownish mycelium, but the colony area was significantly suppressed compared to the diseased group, and the control effect was 79.97%. Figure 9A); In the sterile water control group, the fruits remained intact and bright green, while the fruits in the diseased group showed extensive rot and yellowing. Compared to the diseased group, the rotten area of ​​the fruits in the NSU-59 strain-treated group was significantly reduced, and almost no yellowing was observed, with a control effect of 77.48% ( Figure 9 B). The results showed that the antagonistic bacterium nsu-59 had a good inhibitory effect on anthracnose in both leaves and fruits of Trichosanthes kirilowii, and has great potential for the control of anthracnose in Trichosanthes kirilowii.

[0047] Example 10: Effect of strain 10nsu-59 on the germination of Trichosanthes kirilowii seeds Surface disinfection of Trichosanthes kirilowii seeds: Select plump and uniform Trichosanthes kirilowii seeds, wash them with clean water, soak them in 75% alcohol for 2 minutes for surface disinfection, rinse them 3 times with sterile water, and set aside.

[0048] Following the method described in "Example 4", fermentation supernatant of strain NSU-59 was prepared. The supernatant was diluted 10-fold, 100-fold, and 1000-fold with sterile water, and the resulting dilutions were stored at 4°C for later use. The prepared bacterial suspension was centrifuged at 8000 r / min for 10 min, and the bacterial precipitate was collected. After washing the bacterial precipitate three times with sterile water, a solution with a concentration of approximately 1×10⁻⁶ was prepared. 9 CFU / mL bacterial suspension. Dilute the bacterial suspension with sterile water 10, 100 and 1000 times, and store the resulting dilutions at 4°C for later use.

[0049] Treatment of Trichosanthes kirilowii seeds with fermentation broth and bacterial suspension: Surface-sterilized Trichosanthes kirilowii seeds were soaked in sterile fermentation supernatant diluted 10, 100, and 1000 times, and 1×10⁻⁶ bacterial suspensions. 8 CFU / mL, 1×10 7 CFU / mL and 1×10 6 The seeds were placed in a CFU / mL bacterial suspension in a clean bench for 24 h, with sterile water immersion as a control. The seeds of *Trichosanthes kirilowii* (a type of orchid) soaked for 24 h were spread evenly on petri dishes (9 cm in diameter) containing sterile moistened filter paper, with 20 seeds per dish and 5 replicates per treatment. The petri dishes were placed at 25℃ under a light intensity of 200 μmol·m⁻². -2 s -1 The seeds were incubated in an intelligent light-controlled incubator with a photoperiod of 12 h / 12 ​​h (light / dark) and a relative humidity of 80%. Sterile water was sprayed daily to maintain humidity for 20 consecutive days. Starting from the germination of the first seed, the number of germinating seeds was counted daily, and the number of germinations on the 8th day was taken as the germination potential. Once the germination was stable, the germination rate of the *Trichosanthes kirilowii* seeds was calculated, and 8 seedlings were randomly selected from each culture dish to measure the seedling radicle length, plumule length, and fresh weight. Germination potential (%) = (Number of germinated seeds on the 8th day after inoculation / Number of tested seeds) × 100%; Germination rate (%) = Number of germinated seeds on day 16 after inoculation / Number of seeds tested × 100%.

[0050] The results showed that both the NSU-59 bacterial suspension and the fermentation supernatant improved the germination potential and germination rate of *Trichosanthes kirilowii* seeds. The highest germination potential was observed at 100-fold dilution of the NSU-59 bacterial suspension and 1000-fold dilution of the supernatant, reaching 45.00% and 48.33%, respectively. The highest germination rate (78.33%) was observed at 10-fold dilution of the NSU-59 bacterial suspension, and the highest germination rates (66.67%) were observed at both 10-fold and 1000-fold dilutions of the NSU-59 supernatant. Treatment with a 1000-fold dilution of the bacterial suspension resulted in a maximum radicle length of 16.05 cm, significantly higher than the sterile water control group. Treatment with a 100-fold dilution of the supernatant also showed good growth-promoting effects, with the radicle reaching 14.57 cm. The longest plumule length was 2.49 cm with the 1000-fold dilution of the bacterial suspension, followed by 2.38 cm with the 10-fold dilution of the supernatant, both significantly better than the sterile water control group. The hypocotyl length reached a maximum of 4.89 cm with the 10-fold and 1000-fold dilutions of the bacterial suspension, with lengths of 4.79 cm and 4.85 cm respectively. The highest seed fresh weight was 0.75 g with the 10-fold dilution of the bacterial suspension, significantly higher than other treatment groups and the control group. The fresh weight also increased significantly by 0.58 g with the 10-fold dilution of the supernatant. Figure 10 (Table 2).

[0051] Overall, the bacterial suspension and fermentation supernatant of strain NSU-59 at different dilutions can effectively promote the growth of the radicle, plumule, and hypocotyl of Trichosanthes kirilowii seedlings and increase the fresh weight of the seedlings. Among them, the 1000-fold dilution of the bacterial suspension has the most prominent effect on promoting the growth of the radicle and plumule, while the 10-fold dilution of the bacterial suspension has the most significant effect on increasing the fresh weight.

[0052] Table 2. Statistical data on the effects of different concentrations of supernatant and bacterial suspension of strain NSU-59 on the germination indicators of Trichosanthes kirilowii seeds.

[0053] Note: Different lowercase letters indicate significant differences between treatments. P <0.05) Example 11: Test on the control effect of strain 11nsu-59 on anthracnose in potted Trichosanthes kirilowii This experiment investigated the effect of strain NSU-59 on the control of anthracnose on potted Trichosanthes kirilowii leaves using conventional spraying. Trichosanthes kirilowii tissue culture seedlings with uniform growth were selected, and the following three treatment groups were established: Group T1 (pathogen-only inoculation): Leaves were inoculated with a suspension of Trichosanthes kirilowii anthracnose spores; Group T2 (co-inoculation of biocontrol bacteria and pathogen): The leaves were evenly sprayed with bacterial suspensions diluted 10, 100, and 1000 times until the liquid dripped evenly, and then incubated in an artificial climate chamber at 25℃ and 90% humidity for 24 h, followed by inoculation with pathogen spore suspension as in Group T1; Group T3 (co-treatment of chemical pesticide and pathogen): Leaves were first sprayed with a chemical pesticide (carbendazim diluted 1000 times), and then incubated in an artificial climate chamber at 25℃ and 90% humidity for 24 h, followed by inoculation with pathogen spore suspension as in Group T1. All three treatment groups were treated by pricking the leaves with a needle before inoculation with the pathogen, with five biological replicates per treatment. After the disease developed, the disease incidence was observed and recorded, and the disease index and control effect were calculated. The disease severity was classified into five levels based on the degree of leaf infection; the grading criteria and calculation formula are as follows: Grade 0: No disease spots on the entire leaf (or fruit); Grade 1: Only a few brown spots on diseased leaves (or fruits); Grade 2: Disease spots cover less than 25% of the leaf (or fruit) area; Grade 3: Lesions cover 26% to 50% of the leaf (or fruit) area; Grade 4: Lesions cover 51% to 75% of the leaf (or fruit) area; Level 5: Disease spots cover more than 75% of the leaf (or fruit) area and the fruit is completely rotten; Disease index (%) = ∑ (number of diseased leaves × average representative level of the disease) / (total number of leaves in the surveyed plants × highest disease level) × 100%; Prevention and control efficacy (%) = (Control disease index - Treatment disease index) / Control disease index × 100% The results showed that different concentration gradients of NSU-59 bacterial suspensions could effectively prevent infection of *Trichosanthes kirilowii* seedlings by *Trichosanthes kirilowii* anthracnose. A few yellowish-brown spots appeared on the leaf surface of the chemically treated group. Figure 11 B); In the water treatment group, the leaves of *Trichosanthes kirilowii* showed extensive infection and leaf withering, and the number of infected leaves was relatively large. Figure 11 A); and the leaves of the Trichosanthes kirilowii treated with NSU-59 bacterial suspension showed less leaf surface infection and leaf withering compared with the water control group ( Figure 11 C, 11D, 11E). The concentration is 1×10⁻⁶. 8 The group treated with CFU / mL of NSU-59 bacterial suspension showed the highest control effect. Figure 11 D), reaching 72.83% ( Figure 11 F).

[0054] Example 12: Control of field anthracnose in Trichosanthes kirilowii by strain 2nsu-59 The experimental site was the Wanghe Trichosanthes kirilowii base in Qianshan City, Anhui Province (plots that had been continuously cropped for the past two years). The soil types and fertility were similar, and there were three experimental plots (180 m² each). 2 The level of field management is consistent.

[0055] The field experiment had three treatment groups: (1) nsu-59 strain diluted 100 times in fermentation broth (1×10 8 (2) Pesticide group used in normal agricultural production (prochloraz diluted 1000 times); (3) Water control group. Each treatment group was replicated 3 times, with 5 Trichosanthes kirilowii plants per replicate, and each replicate was a plot covering 20 m². 2 The experiment was conducted in a randomized block design. The experiment took place during the flowering and fruiting period of *Trichosanthes kirilowii*, with each treatment group receiving three spraying treatments in early May, mid-August, and early October. Field disease and yield surveys: By the end of November, when the *Trichosanthes kirilowii* fruit harvest was completed, data on healthy fruits, diseased fruits, and single fruit weight were collected for each treatment group. The disease index of fruits and leaves and the control effect were calculated according to the grading standards in Example 11.

[0056] The results showed that the NSU-59 strain treatment group had a good control effect on anthracnose of Trichosanthes kirilowii and a certain promoting effect on the growth of Trichosanthes kirilowii. Ten days after the first application, the leaves of the water-treated group showed a small amount of yellowing and wilting at the leaf margins, while there were no significant changes in the pesticide group and the NSU-59 strain treatment group. Ten days after the second application, some leaves of the NSU-59 strain treatment group showed yellowing and wilting, with a lower degree of disease than the water-treated group, and a higher fruit setting rate. The pesticide group had a small amount of disease on its leaves and a higher fruit setting rate. Figure 12 A); Ten days after the third application of the pesticide, the weight of individual fruits was measured. The average weight of individual fruits in the NSU-59 strain treatment group was 724.85 g, slightly better than the average weight of 700.75 g in the pesticide group, and much higher than the 519.79 g in the water treatment group. Figure 12 F, Figure 12 G); Field leaf disease statistics showed that the disease indexes of the water-treated group and the pesticide-treated group were 65.60% and 29.60%, respectively, while the leaf disease index of the NSU-59 strain-treated group was 38.33%. Figure 12 B), compared to the water treatment group, the disease index decreased by 41.57%, and the disease control effect on leaves was 41.56%. The leaf control effect of the pesticide treatment group was 54.88%. There was no statistically significant difference in the control effect between the NSU-59 strain treatment group and the pesticide group. P >0.05)( Figure 12C). In the field disease statistics, the disease index of Trichosanthes kirilowii fruit in the water treatment group was 33.91%, while the disease index of Trichosanthes kirilowii fruit in the pesticide treatment group was 14.12%. Figure 12 (D), while the disease index of the fruit treated with NSU-59 decreased by 47.45% compared to the water-treated group. The control effect of NSU-59 on the fruit was 47.43%, while the control effect of pesticide on the fruit was 58.35%. Figure 12 E, Table 3).

[0057] Table 3. Statistical data on the effects of NSU-59 bacterial suspension on the growth indicators of Trichosanthes kirilowii in the field.

[0058] Note: Different lowercase letters indicate significant differences between treatments. P <0.05) Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A strain of Bacillus belesii, characterized in that, The Bacillus belesis is Bacillus velezensis The nsu-59 strain is deposited at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC NO: M2025573.

2. The fermentation broth of Bacillus belye as described in claim 1, or an extract thereof.

3. The fermentation broth of Bacillus belye according to claim 2, or an extract thereof, is characterized in that, The extract is obtained by solid-liquid separation of Bacillus belye fermentation broth, acid precipitation of the liquid obtained from the solid-liquid separation, and further alcohol extraction of the precipitate obtained from the acid precipitation.

4. The volatile metabolites of Bacillus belyssus as described in claim 1.

5. A microbial agent, characterized in that, The microbial agent comprises Bacillus bellis as described in claim 1, or the fermentation broth or extract as described in any one of claims 2-3, or the volatile metabolite as described in claim 4.

6. The application of Bacillus belyssus of claim 1, or the fermentation broth or extract of any one of claims 2-3, or the volatile metabolite of claim 4, or the inoculant of claim 5, in the fight against plant pathogens, wherein the plant pathogen is any one or a combination of two or more of the following: Anthracnose pathogens, Fusarium pathogens, Trichoderma pathogens, or Echinochloa pathogens.

7. The application according to claim 6, characterized in that, The plant pathogens mentioned are fungi of the genera *Anthracis*, *Fusarium*, *Trichoderma*, and *Metacarpa*.

8. The application according to claim 6 or 7, characterized in that, The anthrax bacteria mentioned are *Colletotrichum gloeosporioides* (… Colletotrichum gloeosporioides ) and cryptic anthrax bacteria ( Colletotrichum aenigma The Fusarium pathogen mentioned is Fusarium graminearum ( ); Fusarium graminearum The Trichoderma pathogen mentioned is *Trichoderma viride* ( ). Trichoderma virens The pathogen of the genus *Metacarpa* is *Metacarpa privet* (…). Diaporthe compacta ).

9. The use of Bacillus berberis as described in claim 1, or the fermentation broth or extract as described in any one of claims 2-3, or the volatile metabolite as described in claim 4, or the inoculum as described in claim 5, in the prevention and control of Trichosanthes kirilowii anthracnose.

10. The application of Bacillus berberis as described in claim 1, or the fermentation broth or extract as described in any one of claims 2-3, or the volatile metabolite as described in claim 4, or the inoculum as described in claim 5, in promoting the germination of Trichosanthes kirilowii seeds.

Images