An endophytic rice bacillus AN6 and its application

Abstract

This invention belongs to the field of microbial technology, specifically relating to a type of endophytic Bacillus belye AN6 in rice and its applications. Specifically, this invention provides a Bacillus belye AN6, classified and named as follows: Bacillus velezensis This invention provides a novel rice endophytic Bacillus bereaves strain AN6. Through isolation, screening, identification, and experimental verification, it was found that this strain exhibits broad-spectrum resistance to various plant pathogens, including rice bacterial blight and rice blast. This strain has the ability to inhibit multiple plant pathogens and adsorb heavy metals, which is of great significance for the biological control of rice diseases and the development of green agriculture. (CCTCC accession number: M 20231886)

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:

[0005] Bacillus belyssus AN6, classification and nomenclature Bacillus velezensis Accession number CCTCC NO: M20231886.

[0006] 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.

[0007] 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).

[0008] 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.).

[0009] 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.

[0010] 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:

[0011] 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 .

[0012] Furthermore, the application of the aforementioned Bacillus belyssus AN6 or the aforementioned bacterial agent in siderogenic carriers.

[0013] 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.

[0014] Furthermore, the present invention provides a method for preventing or treating rice bacterial blight by applying the aforementioned fungicide.

[0015] Furthermore, the present invention provides a method for promoting high production of phytoestrogens by *Bacillus belyssus* AN6, comprising the following steps:

[0016] (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;

[0017] (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.

[0018] (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.

[0019] 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

[0020] Figure 1Biological 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.

[0021] 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.

[0022] 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 %.

[0023] Figure 4 The growth rate curve of AN6 strain in NB medium at 30℃ and 160rpm.

[0024] Figure 5 . AN6 protease detection results.

[0025] Figure 6 AN6 Ferrocarrier generation capacity test diagram.

[0026] 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.

[0027] 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.

[0028] Figure 9 . Xoo Scanning electron microscopy (SEM) and transmission electron microscopy (TEM) images of cell morphology. (A) Normal Xoo SEM images of cell morphology. Scale bar: 1.00 µm. (B) Under stress from AN6 fermentation supernatant, 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 stress from AN6 fermentation supernatant, Xoo TEM image of cell morphology. Scale bar: 200.00 nm.

[0029] Figure 10 AN6 Xoo Biological control effect. The indoor trial used the indica rice variety YX650. Prevention of AN6+. Xoo (Pre): Rice leaves after inoculation Xoo Spray AN6 bacterial suspension 12 hours before treatment; Xoo +AN6 (Tre): During vaccination Xoo AN6 bacterial suspension was sprayed 12 hours after inoculation. (A) Severity of rice bacterial blight 15 days after inoculation. (B) Statistical results of lesion length. ***P <0.001, ns, no statistical significance compared with the blank control. Statistical analysis was performed using GraphPad software, including one-way ANOVA and Dunnett's post-hoc test for multiple comparisons.

[0030] Figure 11 Inhibitory effects of AN6 on different pathogenic fungi (xg, F6, A4, F1, F15, YM1, 37-1, YSD4, YSD7, A5). (A) In vitro inhibitory activity of AN6, its volatile organic compounds, and fermentation supernatant on the mycelial growth of different pathogenic fungi. CK: Uninoculated fungal plates; Dual Culture Assay: Plate confrontation method to detect the inhibitory effect of AN6 on pathogenic fungi; VOCs Assay: Plate inversion method to detect the inhibitory effect of volatile organic compounds produced by AN6 on pathogenic fungi; CFS Assay: Inhibitory effect of aseptic fermentation filtrate of AN6 on pathogenic fungi. (B) Statistical results of the inhibitory effects of AN6 on different pathogenic fungi. **P <0.01; ***P<0.001; ns, no statistical significance compared with the blank control group. Statistical analysis was performed using GraphPad software, with one-way ANOVA and Dunnett's post-hoc test for multiple comparisons.

[0031] Figure 12 Inhibitory effects of AN6 on different pathogenic fungi (46-1, YZJ1, YKG1, YF2, YX, A2). (A) In vitro inhibitory activity of AN6, its volatile organic compounds, and fermentation supernatant on the mycelial growth of different pathogenic fungi. CK: Uninoculated fungal plates; Dual Culture Assay: Plate confrontation method to detect the inhibitory effect of AN6 on pathogenic fungi; VOCs Assay: Plate inversion method to detect the inhibitory effect of volatile organic compounds produced by AN6 on pathogenic fungi; CFSA Assay: Inhibitory effect of aseptic fermentation filtrate of AN6 on pathogenic fungi. (B) Statistical results of the inhibitory effects of AN6 on different pathogenic fungi. **P <0.01; ***P <0.001; ns, no statistical significance compared with the blank control group. Statistical analysis was performed using GraphPad software, with one-way ANOVA and Dunnett's post-hoc test for multiple comparisons. Detailed Implementation

[0032] The following will clearly and completely describe the concept and technical effects of this application in conjunction with embodiments, so as to fully understand the purpose, features and effects of this application. For the experimental methods, purchased goods, unless specific conditions are specified, shall be carried out under conventional conditions or conditions recommended by the manufacturer. Reagents or instruments used, unless the manufacturer is specified, can be commercially available conventional products. Unless otherwise defined herein, the scientific and technical terms used in connection with this invention should have the meaning commonly understood by one of ordinary skill in the art. Exemplary methods and materials are described below; however, similar or equivalent methods and materials described herein can also be used in the practice and testing of this disclosure.

[0033] Bacillus belesiensis ( Bacillus velezensis AN6, deposited at Wuhan University, Wuhan, China, was deposited at the China Center for Type Culture Collection (CCTCC) on October 13, 2023, with accession number CCTCC NO: M20231886. The viability of this strain was confirmed as viable on October 20, 2023.

[0034] The following are the standard culture media used in the implementation:

[0035] NA solid medium (g / L): 3 g beef extract, 5 g peptone, 10 g sucrose, 1 g yeast powder, 15 g agar powder, add water to a final volume of 1 L, pH 7.0–7.2, autoclave at 121℃ for 30 min;

[0036] NB liquid culture medium (g / L): 3 g beef extract, 5 g peptone, 10 g sucrose, 1 g yeast powder, add water to a final volume of 1L, pH 7.0–7.2, autoclave at 121℃ for 30 min;

[0037] PDA solid medium (g / L): 200g potato, 20g glucose, 15g agar powder, add water to a final volume of 1L, pH 7.0–7.2, autoclave at 121℃ for 30 min;

[0038] Example 1

[0039] 1. Isolation and purification of endophytic bacteria in rice:

[0040] Rice sample source: Rice leaves collected in June 2022 from a rice planting base in Wanqiao Town, Dali City, Yunnan Province.

[0041] Isolation of strains: Rice samples were surface-sterilized and cut into small pieces. The cut rice samples were then immersed in 75% alcohol for 20 seconds for disinfection, followed by immersion in 5% sodium hypochlorite for 1 minute. Finally, they were rinsed 3-5 times with sterile water and dried with sterile absorbent paper. The samples were then ground in a sterilized mortar with 1-2 mL of sterile water. The grinding solution was serially diluted to 10⁻⁶. -1 10 -2 10 -3 10 -4 10 -5 Five concentration gradients were used, with 100 μL of each concentration spread onto NA plates and incubated in a 28 °C incubator for 12–24 h.

[0042] Purification of the strain: Select single colonies with obvious inhibition zones, and purify them by streaking on NA plates using the triple-line method. Repeat the purification process to obtain pure culture strains. If the cultured colonies are consistent with the typical characteristics and their morphological characteristics are similar, it indicates that pure culture strains have been obtained. Number them sequentially.

[0043] Preservation of the strain: Inoculate a single colony of the pure culture strain into NB liquid medium and incubate at 28℃ and 180rpm for 12h. Then, take 1mL of the bacterial solution and mix it with 1mL of 50% sterile glycerol, and store it at -80℃ for a long time.

[0044] 2. The colony morphology, Gram staining, and scanning electron microscopy of AN6 were performed according to standard procedures.

[0045] 3. AN6's 16S rRNA, gyrA Genetic identification

[0046] Molecular identification of strains was performed using 16S rRNA, gyrA Genomic DNA was extracted from strain AN6 using a bacterial genomic DNA extraction kit (OMEGA). PCR amplification was performed using specific primers for the bacterial 16S rRNA gene: 27f (5'-GAGAGTTTGATCCTGGCTCAG-3') and 1492r (5'-ACGGATACCTTGTTACGACT-3'), and primers for the housekeeping gene gyrA: gyrA-1066r (5'-CAAGGTAATGCTCCAGGCATTGCT-3') and gyrA-42f (5'-CAGTCAGGAAATGCGTACGTCCTT-3'). The PCR reaction mixture (25 μL) consisted of: 1 μL template DNA, 0.5 μL primers (F / R), 12 μL PCR Buffer Mix, and 11 μL ddH2O. 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 product was sent to Beijing Qingke Biotechnology Co., Ltd. for sequencing analysis. The BLAST function of the NCBI website (National Center for Biotechnology Information (nih.gov)) was used to analyze the measured 16S rRNA... gyrA Homology analysis was performed on the sequences, sequence similarity analysis was performed using Geneious 8.0 software, and a phylogenetic tree was constructed using MEGA 11 software.

[0047] 4. Results

[0048] Images of the antibacterial effect of Bacillus belyssus AN6, colony morphology, Gram staining, and scanning electron microscopy (5.0kV 9.0mm × 6.00k) observations are shown below. Figure 1 As shown: AN6 has a significant inhibitory effect on rice bacterial blight pathogens; on LA medium, AN6 colonies are milky white, opaque, with wrinkled surfaces and irregular edges; Gram-positive, rod-shaped; under scanning electron microscopy, strain AN6 cells are rod-shaped with irregular wrinkles on the outer surface.

[0049] AN6's 16S rRNA, gyrAGenetic identification revealed that the 16S rRNA gene of AN6 is shown in SEQ ID NO. 1. Preliminary homology analysis of the AN6 16S rRNA sequence using BLAST from NCBI showed that strain AN6 is similar to... Bacillus velezensis CBMB205 (NR_116240) Bacillus velezensis FZB42 (NR_075005) Bacillus subtilis subsp. 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).

[0050] 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).

[0051] 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.

[0052] Example 2

[0053] 1. Determination of the growth rate of AN6

[0054] 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. 600The 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.

[0055] 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. AN6 was transferred 1:100 to 30 mL of NB medium and cultured at 28 °C with shaking. The fastest growth rate of AN6 was achieved between 7-10 h in NB liquid medium shake-culture flasks, and the growth rate tended to saturate after 13 h, with the highest growth concentration being OD. 600 ≈1.75, such as Figure 4 As shown.

[0056] 2. Detection of AN6 protease activity

[0057] The AN6 strain was cultured in NB medium with shaking for 10-12 h, then transferred to fresh NB medium at a ratio of 1:100 and cultured until OD reached. 600 ≈1.0. Collect 1 mL of bacterial culture, suspend the cells in sterile water, and adjust the OD value to 1.0. 600 To obtain a concentration of 1.0, drop 2 µL of bacterial suspension onto a NA plate containing 10% skim milk. After the bacterial suspension has dried, incubate the plate upside down in a 28 °C incubator for 24–36 h. Measure and compare the size of the hydrolytic clear zone around the colonies. The experiment was performed in triplicate.

[0058] The protease activity of AN6 strain was detected using the agar dilution method. AN6 strain was incubated in NB medium with shaking for 10–12 h, then transferred to fresh NB medium at a 1:100 ratio and cultured until OD reached [value missing]. 600 ≈1.0. Collect 1 mL of bacterial culture, suspend the cells in sterile water, and adjust the OD value to 1.0. 600 To reach 1.0, take 2 µL of bacterial suspension and drop it onto an NA plate containing 10% skim milk. After the bacterial suspension is dried, place the plate in a 28 ℃ constant temperature incubator and invert it for 24-36 h. Observe the size of the hydrolysis clear zone around the colony.

[0059] The effect of AN6 on protease activity is shown in the figure below. Figure 5 As shown, AN6 exhibits good protease activity.

[0060] 3. AN6 Ferric Carrier Production Capacity Testing

[0061] 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.

[0062] The effect diagram of AN6 iron production capacity is shown below. Figure 6 As shown, AN6 can generate ferrocarriers.

[0063] 4. AN6 Heavy Metal Adsorption Capacity Test

[0064] 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.

[0065] 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%.

[0066] Example 3

[0067] 1. Determination of the antagonistic activity of AN6 against rice bacterial blight pathogen.

[0068] 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.

[0069] 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.

[0070] 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, 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.

[0071] 2. Results of antagonistic activity assay

[0072] Agar dilution method for determining the effect of Bacillus belye AN6 on rice bacterial blight pathogen ( Xanthomonas oryzae pv. oryzae abbreviation Xoo Antagonistic activity of the test strain AN6 and the tested rice bacterial blight pathogens (X1, X2, X3, X11, X12, LC2, LC10) were inoculated in NB liquid medium and cultured at 28 ℃ with shaking at 180 rpm until OD. 600 ≈1.0, the tested rice bacterial blight pathogens (X1, X2, X3, X11, X12, LC2, LC10) were dissolved in NA medium at a ratio of 1:100 and poured to prepare inoculum 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 suspension was inoculated into each Oxford cup. Each pathogen was tested in triplicate. 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.

[0073] The antagonistic effect of AN6 against rice bacterial blight pathogen is shown in the figure below. Figure 7 As shown in the figure. The results showed that Bacillus belye AN6 inhibited the bacterial blight pathogens X1, X2, X3, X11, X12, LC2, and LC10 of rice by 73.60%, 70.71%, 70.28%, 48.07%, 71.71%, 73.02%, and 72.86%, respectively, indicating that AN6 has a significant antagonistic effect on rice bacterial blight pathogens.

[0074] 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.

[0075] 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.

[0076] 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.

[0077] 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.

[0078] 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.

[0079] 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.

[0080] 3. Efficacy test of AN6 against bacterial leaf blight in potted rice

[0081] 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.

[0082] Table 1 Treatment Design for Anti-effect Test

[0083]

[0084] 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.

[0085] Example 4

[0086] 1. Determination of the antagonistic activity of AN6 against pathogenic fungi

[0087] 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%.

[0088] Testing for pathogens:

[0089] Rice blast fungus: Magnaporthe oryzae (xg) Pyricularia oryzae (YX);

[0090] Alternaria: Alternaria tenuissima (F6) Alternaria alternata (F15);

[0091] Fusarium: Fusarium fujikuroi (37-1) Fusarium proliferatum (A2) Fusarium grasses (A5) Fusarium oxysporum (YM1);

[0092] Black spores: Nigrospora oryzae (YZJ1) Nigrospora spherica (YF2);

[0093] Black spores: Epicoccum latus (A4) Epicoccum sorghum (YKG1); and / or Helicobacter pylori: Bipolaris cynodontis (YSD7) Bipolar rice (YSD4);

[0094] Diplosporum cocovenenans: Lasiodiplodia theobromae (F1);

[0095] Dicreus: Nakataea oryzae (46-1).

[0096] The antagonistic activity of AN6 against different pathogenic fungi is as follows: 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.

[0097] Example 5: Fermentation culture of AN6 to produce high yield of nutrient (CAS No. 102577-03-7)

[0098] The fermentation culture of Bacillus belyss AN6 was optimized using fengycin content as the main evaluation index. The fermentation medium method was as follows:

[0099] (1) Preparation of rice bacterial blight pathogen ( Xanthomonas oryzae pv. rice 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;

[0100] (2) AN6 high-yield nutrient-rich medium (g / L): 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 autoclaved at 121℃ for 30 min.

[0101] (3) Bacillus belye AN6 was cultured in NB liquid medium at 28 ℃ with shaking at 180 rpm until OD. 600 ≈1.0, and then 10% AN6 inoculum was added to the Fengyuansu medium at a volume ratio, and cultured at 28 ℃ and 180 rpm for 48 h to obtain AN6 fermentation broth.

[0102] (4) Centrifuge the AN6 fermentation broth at 4℃ and 8000 r / min for 20 min, collect the supernatant, add 6 mol / L hydrochloric acid to the supernatant to adjust the pH to 2.00, let it stand overnight at 4℃, centrifuge at 4℃ and 8000 r / min for 20 min, collect the precipitate, add 10 ml / g methanol to the precipitate, and adjust the pH to 7.00 with 1 mol / L sodium hydroxide, extract for 4 h, centrifuge at 4℃ and 8000 r / min for 20 min, collect the supernatant, concentrate the supernatant by nitrogen blowing, and freeze dry at −90℃ to obtain crude lipopeptide extract. The crude lipopeptide extract was dissolved in methanol and centrifuged at 8000 r / min for 20 min at 4℃. The supernatant was collected, filtered through a 0.22 μm microporous membrane, and analyzed by high performance liquid chromatography to detect the content of Fengycin. Under these fermentation conditions, the Fengycin content increased from 0.26 g / L in NB liquid medium to 3.68 g / L. The improved medium significantly increased the Fengycin yield of Bacillus belyssus AN6.

[0103] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. The application of Bacillus belyssus AN6 in the remediation of heavy metal pollution, characterized in that, Classification and nomenclature of Bacillus belyss AN6 Bacillus velezensis The accession number is CCTCC NO: M 20231886, and the heavy metal is Ni or Cr.

2. The application of a microbial agent in the treatment of heavy metal pollution, characterized in that, The bacterial agent contains Bacillus belyssus AN6 as described in claim 1, and the heavy metal is Ni or Cr.

3. The application of a microbial fertilizer in the treatment of heavy metal pollution, characterized in that, The microbial fertilizer contains Bacillus yanhusuo AN6 as described in claim 1, and the heavy metal is Ni or Cr.

4. The application of a soil remediation agent in the treatment of heavy metal pollution, characterized in that, The soil remediation agent contains Bacillus berberis AN6 as described in claim 1, and the heavy metal is Ni or Cr.

5. A method for promoting high production of essential elements by Bacillus belyssus AN6 according to claim 1, characterized in that, Includes the following steps: (1) Preparation of rice bacterial blight pathogen Xoo Liquid fermentation filtrate: Inoculated with NB liquid medium to activate... Xoo The mycelial blocks 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.

Images