Rice endophytic bacillus velezensis AN6 and application thereof

Abstract

The invention belongs to the technical field of microorganisms, particularly relates to rice endophytic bacillus velezensis AN6 and application thereof, and particularly provides bacillus velezensis AN6 which is classified and named as bacillus velezensis and has the preservation number of CCTCC (China Center for Type Culture Collection) NO: M 20231886. The invention provides a novel rice endophytic bacillus velezensis strain AN6, through separation, screening, identification and experimental verification, it is found that the strain shows broad-spectrum resistance to various phytopathogens including rice bacterial leaf blight, rice blast and the like, the strain has the capacity of inhibiting various phytopathogens and has the capacity of adsorbing heavy metal, and the strain has the advantages of being capable of inhibiting various phytopathogens and capable of being used as a novel rice endophytic bacillus velezensis strain AN6. The method is of great significance to biological control of rice diseases and development of green agriculture.

Description

Technical Field

[0001] This invention belongs to the field of microbial technology, specifically relating to an endophytic Bacillus belye AN6 of rice and its applications. Background Technology

[0002] Rice ( Oryza sativa Rice (L.) is one of my country's most important grain crops, with a wide planting area, making it crucial to ensure and increase its yield. However, rice faces threats from various diseases during cultivation, such as bacterial blight, rice blast, and sheath blight. These diseases severely impact rice yield and quality, causing significant economic losses to agricultural production. Traditionally, using chemical pesticides to control rice diseases has been a relatively effective method. However, the long-term and large-scale use of chemical pesticides not only increases production costs, but some pesticide products may also introduce heavy metal impurities during production. For example, heavy metals such as arsenic, chromium, nickel, lead, and cobalt have been detected in glyphosate formulations, leading to a series of problems such as environmental pollution and increased pathogen resistance, threatening the stability of the ecosystem and human health. Therefore, finding greener and more eco-friendly methods for controlling rice diseases has become one of the important directions of current agricultural scientific research.

[0003] Against this backdrop, biological control has received widespread attention as an environmentally friendly method of disease control. Biological control utilizes microorganisms and their metabolites to inhibit pathogens and reduce disease occurrence. It offers advantages such as high efficiency, low toxicity, and safety, while also improving the soil microecological environment and enhancing crop disease resistance, making it an ideal alternative to chemical pesticides. In recent years, research has found that various endophytic bacteria have significant inhibitory effects on rice diseases. Among them, Bacillus species, due to their abundant metabolites, diverse mechanisms of action, and strong environmental adaptability, have become a research hotspot in the biological control of rice diseases. Bacillus can secrete active substances such as antibiotics, growth regulators, and enzymes, inhibiting the growth of various pathogenic microorganisms. Furthermore, Bacillus can induce systemic resistance in rice, enhancing the plant's self-defense capabilities. *Bacillus belye* is a specific example. (Bacillus velezensis) It has been a research hotspot in recent years, but most of the reports on its efficacy against single diseases are currently limited to Bacillus belyssus with broad-spectrum resistance, as well as biological control resources that can be used for environmental pollution control. Summary of the Invention

[0004] The main objective of this invention is to provide a novel rice endophytic Bacillus belye strain AN6 to address the problems existing in the prior art. Specifically, this invention provides the following technical solution: Bacillus belyssus AN6, classification and nomenclature Bacillus velezensis Accession number CCTCC NO: M20231886.

[0005] This invention provides a microbial agent containing *Bacillus belyssus* AN6. In some embodiments, the microbial agent may optionally include a bacterial strain, culture medium components (such as carbon source, nitrogen source, inorganic salts), carrier (such as clay, talc, kaolin, etc.), pH adjuster, surfactant (such as Tween 20, Tween 80), etc.

[0006] This invention provides a microbial fertilizer, wherein the inoculant contains *Bacillus belyssioides* AN6 or the inoculant itself. In some embodiments, the optional microbial fertilizer may also include other microbial strains with synergistic effects, organic matter (such as crop straw, humic acid, amino acids), inorganic nutrients (such as macroelements nitrogen, phosphorus, and potassium, and microelements calcium, magnesium, sulfur, silicon, boron, zinc, manganese, copper, iron, and molybdenum), and plant growth regulators (such as indoleacetic acid, cytokinin, and gibberellin).

[0007] This invention provides a soil remediation agent containing *Bacillus belyssus* AN6 or the aforementioned inoculant. In some embodiments, the optional soil remediation agent may also include soil conditioners (such as sawdust, hay, cut grass, vermiculite, leaves, wheat straw, sawdust, etc.), nutrients (such as ammonium sulfate, sodium chloride, potassium dihydrogen phosphate, ammonium nitrate, humic acid, etc.), organic carriers (wheat bran, peat moss, zeolite powder, etc.), and minerals (such as silica sand, silicon, iron, aluminum, silica gel, calcium sulfate, etc.).

[0008] This invention provides a bactericide containing *Bacillus belyssus* AN6 or the aforementioned bacterial agent. In some embodiments, the optional bactericide includes adjuvants (such as emulsifiers, dispersants, etc.), chelating agents (such as EDTA), chemical bactericides, or natural compounds.

[0009] This invention also provides the application of the aforementioned Bacillus berberis AN6 or the aforementioned fungicide in the prevention and control of plant diseases, wherein the pathogens causing the diseases include: Rice bacterial blight pathogen: Xanthomonas oryzae pv. oryzae ; and / or rice blast fungus: Magnaporthe oryzae , Pyricularia oryzae ; and / or Alternaria: Alternaria tenuissima , Alternaria alternata ; and / or Fusarium: Fusarium fujikuroi , Fusarium proliferatum , Fusarium graminearum , Fusarium oxysporum ; and / or Ichthyophthirius multifiliis: Nigrospora oryzae , Nigrospora sphaerica ; and / or Acinetobacter nigricans: Epicoccum latusicollum , Epicoccum sorghinum ; and / or Helicobacter pylori: Bipolaris cynodontis , Bipolaris oryzae ; and / or Diplosporum cocovenenans:Lasiodiplodia theobromae Diplosporum: Nakataea oryzae .

[0010] Furthermore, the application of the aforementioned Bacillus belyssus AN6 or the aforementioned bacterial agent in siderogenic carriers.

[0011] Furthermore, the application of the Bacillus vesiculosus AN6 or the soil remediation agent in the treatment of heavy metal pollution, wherein the heavy metal is Ni or Cr.

[0012] Furthermore, the present invention provides a method for preventing or treating rice bacterial blight by applying the aforementioned fungicide.

[0013] Furthermore, the present invention provides a method for promoting high production of phytoestrogens by *Bacillus belyssus* AN6, comprising the following steps: (1) Preparation of rice bacterial blight pathogen Xoo Liquid fermentation filtrate: Inoculated with NB liquid medium to activate... Xoo The bacteria were cultured at 28 ℃ with shaking at 180 rpm for 48 hours, and then... Xoo Bacterial culture, centrifuged, and filtered to obtain Xoo filtrate; (2) AN6 high-yield nutrient-rich medium: 25 g wheat flour, 15 g beef extract, 10 g peptone, 0.5 g FeSO4, 0.3 g MgSO4, 1 g NaCl; add 100 ml of... Xoo The filtrate was diluted with water to a final volume of 1L, pH 6.0, and then autoclaved for later use. (3) Bacillus berberis AN6 was inoculated into AN6 high-yield nutrient medium and fermented to obtain AN6 fermentation broth. The AN6 fermentation broth was separated and purified to obtain nutrient.

[0014] This invention provides a novel endophytic Bacillus belyssioides strain AN6 for rice. Through isolation, screening, identification, and experimental verification, it was found to exhibit broad-spectrum resistance to various plant pathogens, including rice bacterial blight and rice blast. This strain's ability to inhibit multiple plant pathogens is of great significance for the biological control of rice diseases and the development of green agriculture. Simultaneously, this strain has the ability to adsorb heavy metals nickel and chromium, making it suitable for soil remediation. Bacillus belyssioides strain AN6 provides a new option for the biological control of rice diseases and contributes to achieving environmentally friendly agricultural production methods. Attached Figure Description

[0015] Figure 1 Biological characteristics of strain AN6. (A) AN6's resistance to rice bacterial blight pathogen (… Xanthomonas oryzae pv. oryzae(A) Inhibitory effect of AN6; (B) AN6 cell morphology observed under a scanning electron microscope; (C) Colony morphology of AN6 strain on NA medium; (D) Gram staining morphology of AN6 strain.

[0016] Figure 2 Phylogenetic tree of AN6 strain and other Bacillus members constructed using the maximum likelihood method based on the 16S rRNA gene. The gene sequence of *Escherichia coli* U5 / 41 (NR_024570) was used as the root of the phylogenetic tree, and guiding consensus was inferred from 1000 replicates. The bootstrap value is expressed as a percentage.

[0017] Figure 3 . based on gyrA Genes, phylogenetic tree diagrams of AN6 strain and other Bacillus members constructed using the maximum likelihood method, inferred from 1000 replicates leading consensus, with expansion values ​​expressed as %.

[0018] Figure 4 The growth rate curve of AN6 strain in NB medium at 30℃ and 160rpm.

[0019] Figure 5 . AN6 protease detection results.

[0020] Figure 6 AN6 Ferrocarrier generation capacity test diagram.

[0021] Figure 7 AN6 against Xanthomonas oryzae Xanthomonas oryzae pv. Oryzae (A) Graph showing the in vitro antagonistic activity assay of AN6 against different sources. Xoo In vitro antagonistic activity of the strains. X1, X2, and X3 were isolated from rice bacterial blight samples from Baoshan, X11 and X12 from rice bacterial blight samples from Dali, and LC2 and LC10 from rice bacterial blight samples from Lüchun County, Honghe Prefecture. (B) AN6 against different sources Xoo Inhibition rate of bacterial growth. ***P <0.001, ns, no statistical significance compared with the control group. Statistical analysis was performed using GraphPad software, evaluated by one-way ANOVA, followed by Dunnett's post-hoc test for multiple comparisons.

[0022] Figure 8 The volatile organic compounds produced by AN6 were detected using the inverted plate method against Xanthomonas oryzae. Xoo The inhibition effect diagram. (A) Blank control. (B) The effect of volatile organic compounds produced by AN6 on... Xoo Growth inhibition effect.

[0023] Figure 9 . Xoo Scanning electron microscope (SEM) and transmission electron microscope (TEM) images of cell morphology. (A) Normal Xoo SEM images of cell morphology. Scale bar: 1.00 µm. (B) Under AN6 fermentation supernatant stress, Xoo SEM images of cell morphology. Scale bar: 1.00 µm. (C) Normal Xoo TEM images of cell morphology. Scale bar: 200.00 nm. (D) Under AN6 fermentation supernatant stress, Xoo TEM images of cell morphology. Scale bar: 200.00 nm.

[0024] Figure 10 . Biocontrol effect of AN6 on Xoo . Indoor test used indica rice variety YX650. AN6+ was prevented from infecting rice leaves by spraying AN6 bacterial suspension 12 hours before inoculation of Xoo (Pre): AN6 bacterial suspension was sprayed on rice leaves 12 hours before inoculation of Xoo bacterial suspension; AN6+ was treated by spraying AN6 bacterial suspension 12 hours after inoculation of Xoo (Tre): AN6 bacterial suspension was sprayed on rice leaves 12 hours after inoculation of Xoo bacterial suspension. (A) Disease incidence of rice white leaf blight 15 days after inoculation. (B) Statistical results of lesion length. ***P <0.001, not statistically significant compared with the blank control. Statistical analysis was performed using GraphPad software, by one-way ANOVA and Dunnett's multiple comparison post-test.

[0025] Figure 11 . Inhibition effect of AN6 on different pathogenic fungi (xg, F6, A4, F1, F15, YM1, 37-1, YSD4, YSD7, A5). (A) In vitro inhibition activity of AN6 and its volatile organic compounds and fermentation supernatant on mycelial growth of different pathogenic fungi. CK: fungal plates without inoculation of bacteria; Dual Culture Assay: plate confrontation method to detect the inhibition effect of AN6 on pathogenic fungi; VOCs Assay: plate upside-down method to detect the inhibition effect of volatile organic compounds produced by AN6 on pathogenic fungi; CFS Assay: inhibition effect of sterile fermentation filtrate of AN6 on pathogenic fungi. (B) Statistical results of inhibition effect of AN6 on different pathogenic fungi. **P <0.01; ***P <0.001; not statistically significant compared with the blank control. Statistical analysis was performed using GraphPad software, by one-way ANOVA and Dunnett's multiple comparison post-test.

[0026] Figure 12 . AN6 against different pathogenic fungi (46-1, YZJ1, YKG1, YF2, YX, A2). (A) In vitro inhibitory activity of AN6 and its volatile organic compounds and fermentation supernatant on mycelial growth of different pathogenic fungi. CK: fungal plates without inoculation of bacteria; Dual Culture Assay: plate confrontation method to detect the inhibitory effect of AN6 on pathogenic fungi; VOCs Assay: plate upside-down method to detect the inhibitory effect of volatile organic compounds produced by AN6 on pathogenic fungi; CFS Assay: inhibitory effect of sterile fermentation filtrate of AN6 on pathogenic fungi. (B) Statistical results of AN6 inhibitory effect on different pathogenic fungi. **P <0.01; ***P <0.001; ns, not statistically significant compared with the blank control group. Statistical analysis was performed using GraphPad software, one-way ANOVA was used, and Dunnett's multiple comparison post-hoc test was performed. DETAILED DESCRIPTION

[0027] The concept and resulting technical effects of the present application will be described below in conjunction with examples to clearly and completely understand the purpose, features and effects of the present application. In the test method, the purchased commodities are used according to the conventional conditions or the conditions recommended by the manufacturer, and the reagents or instruments not specified by the manufacturer can be obtained by purchasing the conventional products on the market. Unless otherwise defined herein, the scientific and technical terms used in conjunction with the present disclosure should have the meanings commonly understood by those of ordinary skill in the art, and the following describes exemplary methods and materials, but methods and materials similar or equivalent to those described herein can also be used in the practice and testing of the present disclosure.

[0028] Bacillus velezensis (Bacillus velezensis) Bacillus velezensis ) AN6, preservation address: Wuhan University, Wuhan, China, deposited with the China Center for Type Culture Collection (CCTCC) on October 13, 2023, preservation number CCTCC NO: M20231886, the survival of the strain was detected on October 20, 2023, and the result was survival.

[0029] The conventional medium in the implementation is as follows: NA solid medium (g / L): beef extract 3 g, peptone 5 g, sucrose 10 g, yeast powder 1 g, agar powder 15 g, add water to 1 L, pH 7.0-7.2, 121°C high pressure sterilization for 30 min; NB liquid medium (g / L): beef extract 3 g, peptone 5 g, sucrose 10 g, yeast powder 1 g, add water to 1 L, pH 7.0-7.2, 121 ℃ high pressure sterilization for 30 min; PDA solid medium (g / L): potato 200 g, glucose 20 g, agar powder 15 g, add water to 1 L, pH 7.0-7.2, 121 ℃ high pressure sterilization for 30 min; Example 1 1. Isolation and purification of rice endophytic bacteria: Rice sample source: rice leaf samples collected in Dali City Wanqiao Town Rice Planting Base, Yunnan Province in June 2022 Strain isolation: The rice sample was subjected to surface disinfection treatment and cut into small pieces. The cut rice sample was immersed in 75% alcohol for 20 s for disinfection, then transferred to 5% sodium hypochlorite for 1 min, and finally rinsed with sterile water for 3-5 times. The sample was placed in a sterilized mortar and ground with 1-2 mL sterile water. The grinding liquid was diluted to 10 -1 , 10 -2 , 10 -3 , 10 -4 , 10 -5 five concentration gradients, 100 uL of each concentration was inoculated on NA plate, and incubated in a 28 ℃ constant temperature incubator for 12-24 h.

[0030] Strain purification: single colonies with obvious inhibition zone were selected, and three-line method was used to purify on NA plate, and purified repeatedly to obtain pure culture strains. The culture colonies were consistent with the typical characteristics and the morphological characteristics tended to be consistent, indicating that the pure culture strain had been obtained, and was numbered in turn.

[0031] Strain preservation: the pure culture strain single colony was inoculated in NB liquid medium, and cultured in a 28 ℃, 180 rpm shaker for 12 h. 1 mL of bacterial liquid was taken and mixed with 1 mL of 50% sterile glycerol, and then stored at -80 ℃ for long-term preservation.

[0032] 2. Colony morphology, gram staining and scanning electron microscopy of AN6 were operated according to the conventional method 3. 16S rRNA, gyrA gene identification of AN6 Molecular identification of the strain was performed by 16S rRNA, gyrAGenomic DNA of strain AN6 was extracted using bacterial genomic DNA extraction kit (OMEGA) and PCR amplified with specific primers 27f (5'-GAGAGTTTGATCCTGGCTCAG-3') and 1492r (5'-ACGGATACCTTGTTACGACT-3') for bacterial 16S rRNA gene, primers gyrA-1066r (5'-CAAGGTAATGCTCCAGGCATTGCT-3') and gyrA-42f (5'-CAGTCAGGAAATGCGTACGTCCTT-3') for housekeeping gene gyrA. The PCR reaction system was (25 μL): 1 μL template DNA, 0.5 μL primers (F / R), 12 μL PCR Buffer Mix, 11 μL ddH2O. The PCR amplification conditions were: 95 °C for 5 min, 94 °C for 30 s, 60 °C for 30 s, 72 °C for 90 s, 30 cycles; 72 °C for 4 min. The purified PCR products were sent to Beijing Genesee Biotechnology Co., Ltd. for sequencing analysis. The homology analysis of the measured 16S rRNA, gyrA sequences was performed using the BLAST function of the NCBI website (National Center for Biotechnology Information (nih.gov)), the sequence similarity analysis was performed by software Geneious 8.0, and the phylogenetic tree was constructed using MEGA 11 software.

[0033] 4. Results The inhibition effect diagram, colony morphology, gram staining, and scanning electron microscope (5.0 kV 9.0 mm x 6.00 k) observation pictures of Bacillus velezensis AN6 are shown in the following table: Figure 1 AN6 has obvious inhibition effect on rice bacterial leaf blight; on LA medium, the colony of AN6 is milky white, opaque, the surface of the colony is wrinkled, and the edge is irregular; gram-positive, rod-shaped; under scanning electron microscope, the cells of strain AN6 are rod-shaped, and the outer surface has irregular wrinkles.

[0034] The 16S rRNA, gyrA gene of AN6 was identified. The 16S rRNA gene of AN6 is shown as SEQ ID NO. 1. The 16S rRNA sequence of AN6 was analyzed by preliminary BLAST homology analysis in NCBI, and it was found that strain AN6 was similar to Bacillus velezensis CBMB205 (NR_116240), Bacillus velezensis FZB42 (NR_075005), Bacillus subtilissubsp. subtilis 168 (NR_102783) Bacillus amyloliquefaciens NBRC 15535 (NR_041455) Bacillus amyloliquefaciens The 16S rRNA of strains such as MPA 1034 (NR_117946) showed high homology, reaching 99.79%. Phylogenetic tree construction revealed that strain AN6 and... Bacillus velezensis Clustered on the same branch (e.g.) Figure 2 (As shown).

[0035] AN6 gyrA The gene is shown in SEQ ID NO. 2. AN6. gyrA Preliminary homology alignment analysis of the gene sequence using BLAST from NCBI showed that AN6 is similar to... Bacillus velezensis UCD10600 (OP561957.1) Bacillus velezensis SBG9 (MN882071.1), Bacillus velezensis strains such as UCD10598 (OP561955.1) gyrA The gene sequences are most similar, with a homology of 98.89%. Phylogenetic tree results also show that AN6 is... Bacillus velezensis In the same branch (e.g.) Figure 3 (As shown).

[0036] AN6 was preserved and named Bacillus belye ( Bacillus velezensis AN6 was deposited at the China Center for Type Culture Collection (Wuhan University, Wuhan, China) on October 13, 2023, with accession number CCTCC NO: M20231886.

[0037] Example 2 1. Determination of the growth rate of AN6 After picking a single colony of AN6 and incubating it overnight in NB liquid medium, a certain amount of bacterial culture was collected to collect the bacterial cells. The cells were washed twice with sterile water, and the OD was adjusted. 600 The value was 1.0. The culture was transferred 1:100 to 30 mL of NB medium and incubated at 28 °C with shaking. OD was measured hourly until the bacteria entered the stationary phase. 600 Value. The experiment was set up with 3 replicates.

[0038] After picking a single colony of AN6 and incubating it overnight in NB liquid medium, a certain amount of bacterial culture was collected to collect the bacterial cells. The cells were washed twice with sterile water, and the OD was adjusted. 600was 1.0. The AN6 was inoculated in 30 mL NB medium at a ratio of 1:100 and cultured at 28 °C with shaking. The AN6 reached the maximum growth rate between 7-10 h and the growth rate tended to be saturated after 13 h. The highest growth concentration OD 600 ≈1.75, as shown in Figure 4 .

[0039] 2. Detection of protease activity of AN6 The AN6 strain was cultured in NB medium for 10-12 h, and then inoculated in fresh NB medium at a ratio of 1:100 and cultured. The culture was continued until the OD 600 ≈1.0. 1 mL of the bacterial solution was collected and suspended in sterile water to adjust the OD 600 to 1.0. 2 μL of the bacterial solution was dropped onto a NA plate containing 10% skim milk. After the bacterial solution was dried, the plate was placed in a 28 °C incubator and cultured for 24-36 h. The size of the hydrolysis transparent circle around the colony was measured and compared. Three replicates were set in the experiment.

[0040] The agar dilution method was used to detect the protease activity of AN6. The AN6 strain was cultured in NB medium for 10-12 h, and then inoculated in fresh NB medium at a ratio of 1:100 and cultured. The culture was continued until the OD 600 ≈1.0. 1 mL of the bacterial solution was collected and suspended in sterile water to adjust the OD 600 to 1.0. 2 μL of the bacterial solution was dropped onto a NA plate containing 10% skim milk. After the bacterial solution was dried, the plate was placed in a 28 °C incubator and cultured for 24-36 h. The size of the hydrolysis transparent circle around the colony was observed.

[0041] The effect of the protease activity of AN6 is shown in Figure 5 . The AN6 has good protease activity.

[0042] 3. Detection of siderophore production ability of AN6 The siderophore-producing ability of the strain was detected using CAS medium. First, CAS A solution was prepared: 1 mmol / L CAS (chrome azurite), 4 mmol / L CTMAB (cetyltrimethylammonium bromide), and 0.1 mmol / L FeCl3·6H2O. Then, CAS B solution was prepared: 0.1 mol / L phosphate buffer, pH 7.0. Finally, CAS C solution was prepared: 2 g sucrose, 3 g acid-hydrolyzed casein, 20 mL 1 mmol / L MgSO4, 1 mL 1 mmol / L CaCl2, and 18–20 g agar powder, pH 7.0. CAS A, B, and C solutions were sterilized at 115°C for 15 min. 5 mL of CAS A solution and 0.5 mL of CAS B solution were added to 100 mL of CAS C solution to prepare CAS culture plates. An Oxford cup was placed in the center of the CAS culture plate, and 50 μL of LAN6 bacterial culture (OD) was added. 600 (≈2.0), place in a 28 ℃ constant temperature incubator and incubate for 2-4 days, then observe whether a transparent zone forms around the colony.

[0043] The effect diagram of AN6 iron production capacity is shown below. Figure 6 As shown, AN6 can generate ferrocarriers.

[0044] 4. AN6 Heavy Metal Adsorption Capacity Test NiCl2·6H2O and K2Cr2O7 were added to NB liquid culture medium respectively to make Ni 2+ Cr 6+ The concentration was 15 mg / L, and the solution was introduced into the OD. 600 1 ml of 1.0 bacterial culture was placed in a 28 ℃ constant temperature incubator and shaken at 180 rpm for 3 days. The culture medium was then filtered through a 0.45 μm microporous membrane to separate the bacterial cells from the culture medium. The Ni and Cr contents in the filtrate were determined by flame atomic absorption spectrophotometry (operation reference HJ 491-2019). The experiment was repeated three times.

[0045] After three days of cultivation, the average Ni content was measured to be 3.5 mg / kg, and the adsorption treatment efficiency of strain AN6 for heavy metal Ni was 76.67%. The average Cr content was measured to be 4.9 mg / kg, and the adsorption treatment efficiency of strain AN6 for heavy metal Cr was 67.33%.

[0046] Example 3 1. Determination of the antagonistic activity of AN6 against rice bacterial blight pathogen. The antagonistic activity of Bacillus belyceta AN6 against rice bacterial blight pathogen was determined by the agar dilution method: The test strain AN6 and the tested rice bacterial blight pathogens (X1, X2, X3, X11, X12, LC2, LC10) were inoculated into NB liquid medium and cultured at 28 ℃ with shaking at 180 rpm until OD... 600 ≈1.0, the test pathogens (X1, X2, X3, X11, X12, LC2, LC10) were dissolved in NA medium at a ratio of 1:100 and poured to prepare bacterial plates. Then, an Oxford cup with a diameter of 6 mm was placed in the center of the NA plate, and 50 μL of AN6 bacterial solution was inoculated into each Oxford cup. Each pathogen was replicated in 3 times. The plates were placed in an incubator at 28℃ and incubated for 48 h. The size of the transparent inhibition zone around the colony was observed and recorded.

[0047] The method of inverted plate testing for the volatile substances produced by Bacillus belye AN6 against rice bacterial blight pathogens Xoo Antagonistic activity: Test strain AN6 and tested pathogenic bacteria Xoo Inoculated into NB liquid medium and cultured at 28 ℃ with shaking at 180 rpm until OD. 600 ≈1.0, Remove the caps from two 9 cm diameter petri dishes, invert them bottom to top, and prepare NA agar plates for both dishes. Spread 100 μL of Bacillus belye AN6 suspension onto the bottom dish, and inoculate 2 μL of cultured test pathogen bacteria in the center of the top dish. Xoo Two dishes were inverted together, sealed with sealing film, and incubated at 28°C for 3 days. The diameter of the tested pathogenic bacteria was measured using the cross-sectional method. The control group consisted of tested pathogenic bacteria that were not inverted. Xoo Grown on NA medium, with 3 replicates for each treatment.

[0048] AN6 aseptic fermentation supernatant for rice bacterial blight Xoo Effects on cell growth: AN6 and rice bacterial blight pathogen Xoo Inoculated into NB liquid medium and cultured at 28 ℃ with shaking at 180 rpm until OD. 600 ≈1.0, take 1 mL of AN6 bacterial culture, centrifuge at 8000 rpm for 5 min to collect the supernatant, and filter twice using a 0.22 μm bacterial filter to obtain AN6 sterile fermentation supernatant; in the experimental group, add AN6 sterile fermentation supernatant to fresh NB liquid culture medium at a ratio of 1:100, and add 100 μL of rice bacterial blight pathogen. Xoo The control group used 100 μL of rice bacterial blight pathogen. Xoo The bacterial culture was added to fresh NB liquid culture medium and incubated at 28 ℃ with shaking at 180 rpm until the control group's OD was reached. 600≈1.0, each take 1 mL of bacteria liquid to collect bacteria, according to the standard steps of scanning electron microscope to prepare samples for SEM observation, TEM observation, image acquisition and photography.

[0049] 2. Antagonistic activity determination results Agar dilution method for determining the antagonistic activity of Bacillus velezensis AN6 on Xanthomonas oryzae pv. oryzae (Xoo) Xanthomonas oryzae pv. oryzae , referred to as Xoo : the test strain AN6 and the test Xanthomonas oryzae pv. oryzae (X1, X2, X3, X11, X12, LC2, LC10) were inoculated in NB liquid medium and cultured at 28℃ with 180 rpm shaking until OD 600 ≈1.0, the test Xanthomonas oryzae pv. oryzae (X1, X2, X3, X11, X12, LC2, LC10) was dissolved in NA medium at a ratio of 1:100 to make a bacteria plate, then a 6 mm diameter Oxford cup was placed in the center of the NA plate, 50 μL of AN6 bacterial liquid was added into each Oxford cup, each pathogenic bacteria was repeated for 3 times, and it was placed in a 28℃ incubator, after 48h of culture, the size of the transparent inhibition zone around the colony was observed and recorded.

[0050] The antagonistic effect of AN6 on Xanthomonas oryzae pv. oryzae is shown in Figure 7 . The results show that the inhibition rates of Bacillus velezensis AN6 on Xanthomonas oryzae pv. oryzae X1, X2, X3, X11, X12, LC2, LC10 are 73.60%, 70.71%, 70.28%, 48.07%, 71.71%, 73.02%, 72.86% respectively, indicating that AN6 has significant antagonistic effect on Xanthomonas oryzae pv. oryzae.

[0051] The method of plate upside down was used to detect the antagonistic activity of volatile substances produced by Bacillus velezensis AN6 on Xanthomonas oryzae pv. oryzae Xoo : the test strain AN6 and the test pathogenic bacteria Xoo were inoculated in NB liquid medium and cultured at 28℃ with 180 rpm shaking until OD 600 ≈1.0, two 9 cm diameter culture dishes were removed from the dish cover and the dish bottom was placed upside down together, both the upper and lower dishes were made into NA medium plates, 100 μL of Bacillus velezensis AN6 bacterial suspension was coated on the lower dish, 2 μL of the cultured test pathogenic bacteria Xoo was inoculated in the middle of the upside down dish, the two dishes were placed together and sealed with a sealing film, then it was placed in a 28℃ incubator for 3 days, the growth diameter of the test pathogenic bacteria was measured by cross method. The control group was the test pathogenic bacteria Xoo growing on NA medium, each treatment was repeated for 3 times.

[0052] Bacillus belye AN6 produces volatile substances that affect rice bacterial blight pathogens. Xoo The inhibition effect diagram is as follows Figure 8 As shown. The results indicate that the pathogenic bacteria treated by inverting the container... Xoo The growth rate was much lower than that of the control group. After 3 days of growth, the diameter of the pathogenic bacteria in the experimental group was 0.5 cm, while that in the control group was 1.0 cm, indicating that the volatile substances produced by AN6 have a significant antagonistic effect on rice bacterial blight pathogens.

[0053] Effects of aseptic fermentation supernatant of Bacillus belyss AN6 on cell growth of rice bacterial blight pathogen: AN6 and rice bacterial blight pathogen Xoo Inoculated into NB liquid medium and cultured at 28 ℃ with shaking at 180 rpm until OD. 600 ≈1.0, take 1 mL of LAN6 bacterial culture, centrifuge at 8000 rpm for 5 min to collect the supernatant, and filter twice using a 0.22 μm bacterial filter to obtain AN6 sterile fermentation supernatant; in the experimental group, add the AN6 sterile fermentation supernatant to fresh NB liquid culture medium at a ratio of 1:100, and add 100 μL of rice bacterial blight pathogen. Xoo The control group used 100 μL of rice bacterial blight pathogen. Xoo The bacterial culture was added to fresh NB liquid culture medium and incubated at 28 ℃ with shaking at 180 rpm until the control group's OD was reached. 600 ≈1.0, take 1 mL of bacterial culture from each sample to collect bacterial cells, and prepare samples according to the standard procedure for scanning electron microscopy for SEM observation, TEM observation, image acquisition and photography.

[0054] AN6 and Xoo Inoculated into NB liquid medium and cultured at 28°C with shaking at 180 rpm until OD... 600 The concentration was increased to approximately 1.0. Then, 1 mL of the AN6 suspension was centrifuged at 8000 rpm for 5 minutes, the supernatant was collected, and filtered twice using a 0.22 μm bacterial filter to obtain sterile AN6 fermentation supernatant. In the experimental group, the sterile AN6 fermentation supernatant was added to fresh NB liquid culture medium at a ratio of 1:100, and 100 μL of [unspecified ingredient] was added. Xoo Suspension. In the control group, 100 μL of... Xoo The suspension was added to fresh NB liquid medium. Both groups were incubated at 28°C with shaking at 180 rpm until the OD of the control group reached its maximum. 600 The culture was then collected from each group, with a volume of 1.0. The culture was then observed using a scanning electron microscope (Hitachi Regulus 8100; Hitachi, Ltd., Japan) and a transmission electron microscope. Xoo Cell morphology.

[0055] AN6 aseptic fermentation supernatant for rice bacterial blight Xoo The effect of cell growth is shown in the figure. Figure 9 As shown. The shake culture results indicate that the rice bacterial blight pathogen grew in NB liquid medium supplemented with AN6 sterile fermentation supernatant. Xoo The growth rate is extremely low. Scanning electron microscopy observations show that, under normal conditions, Xoo The cell surface is smooth and maintains a typical rod-shaped morphology. Figure 9 A). In contrast, under culture conditions containing aseptic fermentation supernatant of AN6, Xoo The cells exhibited marked morphological abnormalities, including elongation and deformation, indicating membrane stress and structural damage. Figure 9 B). Transmission electron microscopy observations further support these findings, showing that under normal conditions, [the following text appears to be unrelated and possibly a separate sentence fragment: "...grown..."] Xoo The cells exhibited a complete ultrastructure with a well-defined cell wall and no obvious abnormalities. Figure 9 C). However, cells treated with AN6 supernatant showed significant morphological changes, including indistinct or ruptured cell wall structures and plasmolysis-like phenomena, indicating severe physiological stress and, in some cases, cell lysis. Figure 9 D). Combining SEM and TEM results, the aseptic fermentation supernatant of AN6 can significantly damage... Xoo The abnormal cell structure leads to significant morphological abnormalities and physiological dysfunction. These findings provide direct morphological evidence for the antagonistic activity of AN6.

[0056] Based on the environmental factors that can cause changes in bacterial morphology and the antagonistic activity of Bacillus belyssus AN6, it is speculated that antibiotics or antibacterial substances are present in the aseptic fermentation supernatant of AN6.

[0057] 3. Efficacy test of AN6 against bacterial leaf blight in potted rice The experiment was conducted in a light box at a constant temperature of 28 ℃ and a relative humidity of 70%. Rice plants planted for 60 days were selected for the inoculation experiment; the rice variety was "YX65". The rice bacterial blight pathogen (… Xoo Bacillus yanhusuo and Bacillus bereaves AN6 were inoculated into NB liquid medium and cultured at 28 ℃ with shaking at 180 rpm until the logarithmic growth phase. The cells were collected by centrifugation at 8000 rpm for 5 min and rinsed with sterile water. Xoo AN6 bacterial suspension diluted to OD 600 ≈1.0. Xoo Inoculation was performed using the leaf-cutting method, with sterile scissors dipped into the leaf. XooRice leaves were pruned 2 cm from the leaf tip. The antagonistic bacteria AN6 were inoculated using a spray method. The diluted AN6 bacterial suspension was dispensed into sprayers and evenly sprayed onto the rice leaf surface, ensuring the bacterial solution did not pool and run downhill. Four different treatment groups were set up for the efficacy test, as detailed in Table 1.

[0058] Table 1 Treatment Design for Anti-effect Test

[0059] The effect of AN6 on the control of bacterial leaf blight in potted rice is as follows: Figure 10 As shown, spraying AN6, whether for prevention or control, has a control effect on rice bacterial blight. The endophytic bacteria AN6 on the surface have a biocontrol effect on the rice bacterial blight pathogen.

[0060] Example 4 1. Determination of the antagonistic activity of AN6 against pathogenic fungi Three different methods were used to determine the antagonistic activity of AN6 against different pathogenic fungi. The Dual Culture Assay involved inoculating a 5 mm diameter pathogenic fungal block onto the center of a PDA plate, and spotting 5 μL of AN6 suspension (OD) approximately 3 cm to the left and right of the pathogenic fungus. 600 =1.0); Volatile Organic Compounds Assay (VOCs Assay): The inhibitory effect of volatile organic compounds produced by AN6 on pathogenic fungi was measured using the double-plate inverted method. Specifically, two 9cm diameter petri dishes were placed together with their bottoms facing each other, the lower dish containing NB medium. 200 μL of fresh AN6 bacterial suspension (OD200) was taken. 600 =1.0) Spread evenly on agar plates, with PDA medium on top, and inoculate the center of each plate with a fresh pathogenic fungal block (5 mm in diameter). Invert the plates together. Aseptic fermentation supernatant method (CFS Assay): AN6 bacterial suspension (OD) 600 After centrifugation (=1.0), the supernatant was collected and filtered through a 0.22 μm bacterial filter to obtain sterile fermentation supernatant. This supernatant was dissolved in PDA medium at a 1:100 ratio to prepare plates. Fresh pathogenic fungal blocks (5 mm in diameter) were inoculated into the center of each plate. Plates inoculated only with pathogenic fungal blocks served as the control group. Each treatment was repeated three times. The plates were incubated at 28°C for 5 days. The diameter of the pathogenic fungal colonies in the control group and the diameter of the pathogenic fungal colonies extending towards the antagonistic bacteria in the treatment group were measured. The inhibition rate was calculated using the following formula: Inhibition rate = (Control group indicator fungal colony diameter - Treatment group indicator fungal colony diameter) / Control group indicator fungal colony diameter × 100%.

[0061] Testing for pathogens: Rice blast fungus: Magnaporthe oryzae (xg)Pyricularia oryzae (YX); Alternaria alternata: Alternaria tenuissima (F6), Alternaria alternata (F15); Fusarium oxysporum: Fusarium fujikuroi (37-1), Fusarium proliferatum (A2), Fusarium graminearum (A5), Fusarium oxysporum (YM1); Exserohilum rostratum: Nigrospora oryzae (YZJ1), Nigrospora sphaerica (YF2); Cladosporium herbarum: Epicoccum latusicollum (A4), Epicoccum sorghinum (YKG1); and / or Pythium aphanidermatum: Bipolaris cynodontis (YSD7), Bipolaris oryzae (YSD4); Lasiodiplodia theobromae: Lasiodiplodia theobromae (F1); Diaporthe phaseolorum: Nakataea oryzae (46-1).

[0062] The antagonistic activity of AN6 against different pathogenic fungi is shown in Table 1. Figure 11 , Figure 12As shown, from left to right, the results and inhibition rates of the control (CK), dual culture assay, volatile organic compound (VOC) assay, and aseptic fermentation supernatant (CFSA assay) are presented. The dual culture assay showed that AN6 inhibited the mycelial growth of pathogenic fungi by 38.09%–84.24%. Compared with the control group, AN6 showed inhibition rates exceeding 80% against xg, 46-1, and YZJ1. The highest inhibition rate was observed against xg (84.24±1.05%), followed by YZJ1 (81.24±2.10%). The VOC assay showed that the VOCs produced by AN6 inhibited the mycelial growth of pathogenic fungi by 0–54.44%, with the highest inhibition rate against F6 (54.44±2.71%), followed by A4 (53.21±6.00%). The results of the aseptic fermentation supernatant (CFS Assay) assay showed that AN6 and its aseptic fermentation supernatant inhibited the mycelial growth of pathogenic fungi by 25.46%–89.17%. Compared with the control group, AN6 and its aseptic fermentation supernatant showed inhibition rates of over 80% against pathogens F15 and YF2, with the highest inhibition rate against YF2 (89.17±0.72%), followed by F15 (86.27±0.98%). AN6 showed varying degrees of inhibition against all 16 pathogenic fungi using three different detection methods. It exhibited particularly strong inhibitory effects in the plate confrontation method and the aseptic fermentation supernatant method, with maximum inhibition rates of 84.24% and 89.17%, respectively. These results indicate that strain AN6 has high inhibitory potential against the 16 tested pathogenic fungi, highlighting its potential as a candidate strain for biocontrol.

[0063] Example 5: Fermentation culture of AN6 to produce high yield of nutrient (CAS No. 102577-03-7) The fermentation culture of Bacillus belyss AN6 was optimized using fengycin content as the main evaluation index. The fermentation medium method was as follows: (1) Preparation of rice bacterial blight pathogen ( Xanthomonas oryzae pv. oryzae abbreviation Xoo Liquid fermentation filtrate: Inoculated with NB liquid culture medium to activate... Xoo The bacteria were cultured at 28 ℃ with shaking at 180 rpm for 48 hours, and then... Xoo The bacterial culture was centrifuged at 8000 rpm for 5 min, and the supernatant was collected and filtered twice using a 0.22 μm filter to obtain the final product. Xoo filtrate; (2) AN6 fengycin high-yield medium (g / L): wheat flour 25 g, beef extract 15 g, peptone 10 g, FeSO4 0.5 g, MgSO4 0.3 g, NaCl 1 g; add 100 ml of Xoo filtrate, add water to 1 L, pH 6.0, 121℃ high-pressure sterilization for 30 min; (3) B. velezensis AN6 is cultured in NB liquid medium at 28℃ with 180 rpm shaking until OD 600 ≈1.0, then 10% AN6 strain is inoculated into the fengycin medium at a volume ratio, and the culture is shaken at 28℃ and 180 rpm for 48 h to obtain AN6 fermentation broth.

[0064] (4) The AN6 fermentation broth is centrifuged at 8000 r / min for 20 min at 4℃, and the supernatant is collected. 6 mol / L hydrochloric acid is added to the supernatant to adjust the pH to 2.00, and it is placed at 4℃ overnight. It is centrifuged at 8000 r / min for 20 min at 4℃, and the precipitate is collected. 10 ml / g of methanol is added to the precipitate, and 1 mol / L sodium hydroxide is used to adjust the pH to 7.00, and it is extracted for 4 h. It is centrifuged at 8000 r / min for 20 min at 4℃, and the supernatant is collected. The supernatant is concentrated by nitrogen blowing, and freeze-dried at −90℃ to obtain a crude lipopeptide extract. The crude lipopeptide extract is dissolved in methanol, and centrifuged at 8000 r / min for 20 min at 4℃. The supernatant is collected, filtered through a 0.22 μm microporous filter, and analyzed by high-performance liquid chromatography to detect the fengycin content. Under this fermentation condition, the fengycin content is increased from 0.26 g / L in NB liquid medium to 3.68 g / L. The improved medium significantly improves the fengycin yield of B. velezensis AN6.

[0065] The above only describes the preferred embodiments of the present application, and it should be noted that those skilled in the art can make several improvements and refinements without departing from the principles of the present application, and these improvements and refinements should also be considered within the protection scope of the present application.

Claims

1. Bacillus velezensis AN6, characterized in that, Classification Bacillus velezensis , Accession No. CCTCC NO: M 20231886.

2. An inoculant characterized in that, The bacterial agent contains the Bacillus velezensis AN6 according to claim 1.

3. A microbial fertilizer, characterized by, The bacterial agent contains the Bacillus velezensis AN6 according to claim 1, or the bacterial agent according to claim 2.

4. A soil remediation agent, characterized by, The soil remediation agent contains the Bacillus velezensis AN6 according to claim 1, or the bacterial agent according to claim 2.

5. A bactericide characterized by comprising: The fungicide contains the Bacillus velezensis AN6 according to claim 1, or the bacterial agent according to claim 2.

6. Use of Bacillus velezensis AN6 according to claim 1 or the bactericide according to claim 5 for the control of plant diseases, characterized in that, The pathogenic bacteria causing the disease include: Xanthomonas oryzae pv. oryzae: Xanthomonas oryzae pv. oryzae ; and / or Magnaporthe grisea: Magnaporthe oryzae , Pyricularia oryzae ; and / or Alternaria alternata: Alternaria tenuissima , Alternaria alternata ; and / or Fusarium sp.: Fusarium fujikuroi , Fusarium proliferatum , Fusarium graminearum , Fusarium oxysporum ; and / or Mycosphaerella fijiensis: Nigrospora oryzae , Nigrospora sphaerica ; and / or Sphaceloma nigrum: Epicoccum latusicollum , Epicoccum sorghinum ; and / or Pythium aphanidermatum: Bipolaris cynodontis , Bipolaris oryzae ; and / or Aspergillus niger: Lasiodiplodia theobromae ; Aspergillus clavatus: Nakataea oryzae .

7. The Bacillus velezensis AN6 according to claim 1 or the bacterial agent according to claim 2 is used for producing siderophores.

8. The use of B. velezensis AN6 of claim 1 or the soil remediator of claim 4 in the remediation of heavy metal pollution, characterized in that, The heavy metal is Ni or Cr.

9. A method for preventing or treating rice bacterial leaf blight, characterized by, The fungicide according to claim 5 is applied.

10. A method of promoting high production of weissella paramesenteroides AN6 of claim 1, characterized by, The method comprises the following steps: (1) Preparation of Xanthomonas oryzae pv. oryzae Xoo Liquid fermentation filtrate: NB liquid medium was used to inoculate activated bacteria, and the bacteria were cultured at 28°C with 180 rpm shaking for 48 h. Bacterial solution was obtained, centrifuged, and filtered to obtain the filtrate. Xoo Xoo Xoo filtrate.​​ (2) AN6 high-yield Fengyuan culture medium: wheat flour 25 g, beef extract 15 g, peptone 10 g, FeSO4 0.5 g, MgSO4 0.3 g, NaCl 1 g; add 100 ml of Xoo The filtrate was diluted to 1 L with water, pH 6.0, and autoclaved for standby use. (3) The Bacillus velezensis AN6 is inoculated into the AN6 high-yield prodigiosin culture medium, and AN6 fermentation liquor is obtained through fermentation culture; and the prodigiosin is obtained through separation and purification of the AN6 fermentation liquor.

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