A type of barley wine lees fungus and its application in biological herbicides and fungicides.

Abstract

This invention relates to the field of biocontrol bacteria technology, specifically to a type of barley brewing lees bacteria and its application in biological herbicides and fungicides, wherein the barley brewing lees bacteria is Bacillus subtilis (Bacillus subtilis). Bacillus subtle B9, whose 16S rDNA sequence is shown in SEQ ID NO:1, was deposited at the Guangdong Provincial Microbial Culture Collection Center on July 1, 2022, with accession number GDMCC No: 62595. The fermentation broth of *Bacillus oryzae* from this invention exhibits a 74.71% inhibitory effect on *Fusarium oxysporum* LM-C, and its inhibition rates on shoot length, root length, and fresh weight of *Rhizopus spp.* and *Oxalis spp.* seedlings all exceed 78%, making it suitable for preparing microbial herbicides.

Description

Technical Field

[0001] This invention relates to the field of biocontrol bacteria technology, specifically to a type of barley wine lees bacteria and its application in biological herbicides and fungicides. Background Technology

[0002] In modern agricultural production, chemical pesticides are widely used for the control of crop diseases, pests, and weeds due to their advantages of high efficiency, rapid application, and low cost, playing a vital role in increasing crop yields and income. However, the long-term use and abuse of chemical pesticides not only have adverse effects on the ecological environment and the health of humans and animals, but the resulting pesticide residues also threaten the safety of agricultural products. Furthermore, they can enhance the resistance of diseases, pests, and weeds to pesticides, making control more difficult.

[0003] To promote the green, high-quality, and sustainable development of agricultural production in my country, biological control is currently receiving significant attention due to its safety, environmental friendliness, and long-lasting effectiveness. Among these methods, utilizing microorganisms is one of the most effective approaches for the biological control of crop diseases, pests, and weeds. These microbial resources are frequently developed into various microbial pesticides in agricultural production. These pesticides are not only safe and harmless with high specificity, but also natural products that can decompose naturally, effectively preventing the development of pesticide resistance in plant diseases, pests, and weeds. There have been numerous reports on the development of weed-suppressing microbial resources. Louarn et al. found that extracts from arbuscular mycorrhizal fungi significantly inhibited the germination of black bean seeds. Xie Yangjun et al. screened a strain of Aspergillus A from the rhizosphere soil of allelopathic rice, which significantly inhibited the growth of barnyard grass at 100 and 200 times dilution of the fermentation broth. Abubakar et al. screened four strains of Pseudomonas, which showed good performance in inhibiting the growth of weeds such as wild oats and small-seeded canary grass. This phenomenon is thought to be due to the production of cyanide by the strains, which inhibits the metabolism of weed roots, thus exhibiting weed-suppressing activity. However, in agricultural production, many microbial strains often exhibit various problems such as mutation, degeneration, difficulty in colonization, and reduced antibacterial ability, which increases the difficulty of biological control. Therefore, while ensuring the preservation of strains, it is particularly important to actively screen out superior strains from nature that have a broad antibacterial spectrum, strong stress resistance, and high stability.

[0004] Highland barley (Hordeum vulgare L. var. nudum Hook. f.) is an annual crop belonging to the genus Hordeum in the family Poaceae. It is a distinctive crop in Qinghai Province, my country, and is rich in nutrients. Highland barley wine, brewed from highland barley, is a local specialty agricultural product. The brewing process often produces distiller's grains, which are rich in protein, carbohydrates, crude starch, and various trace elements, providing an excellent nutritional environment for microbial growth.

[0005] Currently, research on the biocontrol effects of Bacillus spp. derived from barley distiller's grains has been reported. Li Wei et al. isolated and screened three Bacillus strains from barley distiller's grains that exhibited highly efficient herbicidal activity and inhibitory effects against Sclerotinia sclerotiorum, the pathogen of rapeseed. Jia Pengli et al. screened a Bacillus berleis JZ3-1-15 strain from barley distiller's grains that showed inhibitory activity against the pathogen of potato dry rot. Shen Shuo screened three Bacillus strains derived from barley distiller's grains that showed inhibitory activity against potato virus Y. Therefore, isolating and screening strains with biocontrol effects from distiller's grains and their on-site production environment is of practical significance. Summary of the Invention

[0006] To address the aforementioned problems, this invention provides a barley wine lees fungus and its application in biological herbicides and fungicides.

[0007] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0008] The barley wine lees bacteria of this invention are Bacillus subtilis (B. subtilis) Bacillus subtilis B9, whose 16S rDNA sequence is shown in the appendix below (SEQ ID NO: 1), was deposited on July 1, 2022, at the Guangdong Provincial Microbial Culture Collection Center (GDMCC No: 62595). The depositary address is: 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, Guangdong Academy of Sciences, Postcode: 510070.

[0009] The fermentation broth of *Bacillus oryzae* of this invention exhibits a 74.71% inhibitory effect on *Fusarium solani* LM-C, and also strongly inhibits seed germination and seedling growth of wild rapeseed and wild oats, making it suitable for preparing microbial herbicides. Specifically, the fermentation broth of *Bacillus oryzae* inhibits the sprout length, root length, and fresh weight of wild rapeseed and wild oat seedlings by more than 78%.

[0010] The barley wine lees strain of this invention can be used to prepare a fungicide, which exhibits an inhibition rate of 74.71% against Fusarium solani LM-C.

[0011] This invention enriches the strain resources for the use of microorganisms to control plant diseases and lays the foundation for further research on their antibacterial mechanism and field disease control effects. Attached Figure Description

[0012] Other features, objects, and advantages of the present invention will become more apparent from the following detailed description of non-limiting embodiments with reference to the accompanying drawings:

[0013] Figure 1 For the screening of antagonistic bacteria.

[0014] Figure 2The inhibitory effects of seven antagonistic bacteria on the growth of Fusarium solani LM-C were investigated.

[0015] Figure 3 The inhibition rate of seven antagonistic bacteria against Fusarium solani LM-C is shown.

[0016] In the figure: different lowercase letters indicate that the differences between different treatments reached the 0.05 significance level.

[0017] Figure 4 The effects of seven antagonistic bacteria on the mycelium of Fusarium solani LM-C;

[0018] In the diagram: A: Normally growing Fusarium solani mycelium; B: Fusarium solani mycelium inhibited by B1; C: Fusarium solani mycelium inhibited by B3; D: Fusarium solani mycelium inhibited by B4; E: Fusarium solani mycelium inhibited by B6; F: Fusarium solani mycelium inhibited by B7; G: Fusarium solani mycelium inhibited by B8; H: Fusarium solani mycelium inhibited by B9.

[0019] Figure 5 The colony morphology and Gram staining of seven strains of *Bacillus oryzae* were obtained.

[0020] Figure 6 shows the phylogenetic tree of 16S rDNA from seven strains of *Distillers' Grains*.

[0021] Figure 7 The effects of three strains of barley distillers' grains on the seed germination of free-growing rapeseed and wild oats.

[0022] In the figure: different lowercase letters indicate that the differences between different treatments reached the 0.05 significance level. Detailed Implementation

[0023] The present invention will now be described in detail with reference to specific embodiments. These embodiments will help those skilled in the art to further understand the present invention, but do not limit the invention in any way. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention. These all fall within the scope of protection of the present invention.

[0024] Test data

[0025] 1.1 Test Materials

[0026] Test bacteria: Nine bacterial strains (all isolated from barley liquor mash) preserved in our laboratory were used, numbered B1, B2, B3, B4, B5, B6, B7, B8, and B9; Test pathogenic fungi: Three pathogenic fungi isolated and purified from quinoa diseases were used, numbered LM-A: Neosporidium calfii ( Neocamarosporium calvescens ), LM-C: Fusarium solani ( Fusarium solani), LM-D: Oat Endometriosis ( Drechslra avenacea The tested weed seeds were: wild oat seeds (Avena fatua L.) and wild rapeseed seeds (Brassica napus L.). Both were provided by the Institute of Plant Protection, Qinghai Academy of Agricultural and Forestry Sciences.

[0027] Test media: LB solid medium: 10.0 g peptone, 3.0 g meat extract, 5.0 g lactose, 0.024 g bromothymol blue, 20.0 g agar, 1000.0 mL distilled water, sterilized at 121℃ for 30 min; Modified Martin solid medium: 5.0 g peptone, 2.0 g yeast extract, 20.0 g glucose, 0.5 g magnesium sulfate, 1.0 g dipotassium hydrogen phosphate, 20.0 g agar, 1000.0 mL distilled water, sterilized at 121℃ for 30 min; LB liquid medium: same composition as LB solid medium, but without agar, sterilized at 115℃ for 30 min.

[0028] 1.2 Test Methods

[0029] 1.2.1 Strain Isolation

[0030] Weigh 10.00 g of naturally fermented barley lees into a 250 mL Erlenmeyer flask containing 90 mL of sterile water. Incubate at 28℃ and 180 r / min with constant temperature shaking for 30 min. Dilute the flask 10-fold using a serial dilution method to obtain 10... -3 10 -4 10 -5 Three different dilutions of bacterial suspension were prepared, with 0.1 mL of each dilution placed in the center of an isolation medium plate. The isolation media included Gao's No. 1, Modified Martin, and LB solid medium. The plates were spread evenly and incubated at 26°C for 7 days. Single colonies were picked from the plates and streaked repeatedly until single colonies were isolated.

[0031] 1.2.2 Flat Plate Standoff Test

[0032] Under aseptic conditions, nine strains of the tested bacteria were inoculated onto new LB solid medium by streaking with an inoculation loop; the resulting fungal mycelial cakes were then inoculated onto new Martin solid medium for activation culture.

[0033] Preliminary screening: The four-point method using mycelial cakes was used to screen for *Bacillus subtilis* strains exhibiting antagonistic effects against the pathogen. This method allows for the simultaneous study of the inhibitory effects of three *Bacillus subtilis* strains on one pathogen. On a clean bench, a metal punch was used to create holes in the activated cultured test pathogen. The resulting mycelial cakes, with a diameter of 5 mm, were picked up with tweezers and placed upside down in the center of a new LB solid medium. Similarly, a 5 mm diameter metal punch was used to create mycelial cakes from the activated cultured test bacteria. Mycelial cakes from three different strains were inoculated at three different locations 2 cm from the central mycelial cake. The control consisted of LB plates inoculated only with the test pathogen. The inoculation was repeated twice. The plates were incubated at 26°C. Once the control had completely covered the plate, the antagonistic effects of the different test bacteria against the test pathogen were observed and photographed.

[0034] Inhibitory effect of biocontrol bacteria on pathogens: To more accurately calculate the inhibition rate of different *Bacillus subtilis* strains obtained from the initial screening against pathogens, the screened pathogens were used as target bacteria, and a five-point method was used for repeated plate confrontation experiments. Using the same method of inoculating with bacterial cakes, 5 mm diameter pathogen bacterial cakes were inoculated into the center of a new LB agar plate, and bacterial cakes of the same biocontrol strain were inoculated into four different locations within 2 cm of the pathogen. A control plate containing only pathogens served as the inoculation, and the experiment was repeated three times. The plates were incubated at 26℃ until the control strain completely covered the plate. The diameter of the pathogenic fungal colonies was measured using the cross-cross method, the results were recorded and photographed, and the inhibition rate was calculated using the following formula:

[0035] Inhibition rate % = (Coronavirus diameter of control group - Coronavirus diameter of treatment group) / (Coronavirus radius of control group - Diameter of mycelial cake) × 100.

[0036] 1.2.3 Observation of antagonistic fungal hyphae

[0037] Biocontrol bacteria with inhibitory effects on pathogenic fungi were screened through plate confrontation experiments. To understand the inhibition mechanism of biocontrol bacteria on pathogenic fungi, further observation of the surface morphology and growth of pathogenic fungal hyphae is needed. In the confrontation experiment plates, hyphae at the edge of pathogenic fungal colonies that were inhibited by *Dendrobium oryzae* were used as the treatment group. In plates inoculated only with pathogenic fungi, hyphae at the colony edge were selected as the control group. Cube samples with a volume of less than 1 cubic centimeter were cut with a knife and placed in 2 mL sterile centrifuge tubes. 2.5% glutaraldehyde solution was added, and the tubes were sealed with sealing film to prevent leakage. The samples were stored and fixed at 4℃ for at least 12 hours. After fixation, the samples were sent to Hangzhou Yanqu Information Technology Co., Ltd. for processing and coating, followed by observation and photography using a scanning electron microscope.

[0038] 1.2.4 Strain morphology and molecular biological identification

[0039] Morphological identification: The selected antibacterial bacteria were inoculated into LB plates and cultured. The morphological characteristics of the colonies were observed and photographed. After culturing at 37°C for 16 h, a portion of the bacterial sample was picked up with a toothpick for Gram staining. The bacterial morphology, hyphae, and presence or absence of spores of the strain were observed under an optical microscope and photographed.

[0040] Molecular biological identification: DNA from antagonistic *Distillers haematomarginatus* was extracted using the traditional phenol-chloroform extraction method. PCR amplification was performed using universal primers for bacterial 16S rDNA. The primer sequences were: 27F: 5'-AGAGTTTGATCCTGGCTCAG-3', 1492R: 5'-CTACGGCTACCTTGTTACGA-3'. PCR amplification conditions were: 98℃ pre-denaturation for 30 s; 98℃ denaturation for 10 s, 55℃ annealing for 30 s, 72℃ extension for 1.5 min, for a total of 30 cycles; final extension at 72℃ for 5 min. The amplified products were subjected to 1.5% agarose gel electrophoresis, and the results were observed using blue light imaging. After purification, the products were sequenced using an ABI3730-XL sequencer. The above steps were completed by Shaanxi Aiyouji Biotechnology Co., Ltd.

[0041] Blast alignment was performed on the 16S rDNA splice sequences of seven strains of *Bacillus subtilis* from the NCBI nucleic acid database. A subset of strains with high homology to the identified strains were selected from the GenBank database, and phylogenetic trees were constructed using MEGA 7.0 software to understand the kinship relationships among the strains.

[0042] 1.2.5 Preparation of Fermentation Broth for Strains

[0043] On a clean bench, holes were punched in the B1, B8 and B9 ferns using a 5 mm diameter punch. The punched flasks were then placed in sterilized LB liquid culture medium, with 4 flasks in each conical flask. After sealing, the flasks were placed in a shaking incubator for 7 days (28℃, 180 r / min) to obtain the fermentation broth of the ferns.

[0044] 1.2.6 Determination of the bioactivity of *Distillers' grains*

[0045] Under aseptic conditions, the prepared fermentation broths of the three types of *Distillers' grains* were filtered through rapid filter paper to obtain the original fermentation broth. The original broth was then diluted 10-fold to obtain 10-fold diluted fermentation broths. Uniformly sized and plump wild rapeseed and wild oat seeds were selected. For the germination test, the wild oat seeds were dehulled, disinfected with a 2.5% sodium hypochlorite solution for 15 minutes, repeatedly rinsed with sterile water, and then germinated in sterile water at 25°C for 24 hours. A layer of filter paper was placed at the bottom of a sterilized glass petri dish. Using a 5 mL pipette, the original fermentation broth and the 10-fold diluted broth were added to the petri dish, ensuring the filter paper was thoroughly wetted. Ten seeds were evenly placed in each glass petri dish. Then, 5 mL of the original fermentation broth and a 10-fold dilution of the bacterial strain were pipetted into the corresponding petri dishes for germination testing. Water culture served as a control. Each treatment was replicated three times. The seeds were incubated at 25℃ in a light germination chamber for 7 days, with 5 mL of the corresponding bacterial solution added to the petri dishes every 3 days. Germination potential of the rapeseed and wild oat seeds was measured on day 3. Germination rate, seedling shoot length, root length, and seedling fresh weight were measured on day 7. Data were accurately recorded and photographed.

[0046] 1.2.7 Data Processing

[0047] Data on germination potential, germination rate, shoot length, root length, and seedling fresh weight of wild oats and wild rapeseed were collected using Microsoft Excel 2021. Significance analysis of these biological indicators was performed using DPS 9.01 statistical analysis software, and multiple comparisons were conducted using the LSD method. P A value <0.05 was considered statistically significant between treatments. Data were expressed as mean ± standard error and plotted using Origin Pro 9.0 software.

[0048] 2 Results and Analysis

[0049] 2.1 Antagonistic effect of highland barley distillers' grains on pathogens

[0050] Initial screening using plate contrast agents revealed that seven strains of *Distillers' grains* (B1, B3, B4, B6, B7, B8, and B9) were all resistant to *Fusarium solani* (the causal agent of solanaceous diseases). F. solani LM-C exhibits antagonistic activity (as shown in Table 1). The inhibitory effects of seven antagonistic bacteria on LM-C of Fusarium solani are as follows: Figure 1 As shown. By Figure 2It was found that the inhibition rates of the seven biocontrol bacteria against *Fusarium solani* were all significantly higher than those of the control. Biocontrol bacteria B9 showed the strongest inhibitory effect on *Fusarium solani* LM-C, with an inhibition rate of 74.71%, followed by B1 and B8, with inhibition rates of 73.92% and 73.73%, respectively, significantly higher than the other four *Fusarium* strains. In contrast, strain B7 showed the weakest inhibitory effect on *Fusarium solani* LM-C, with an inhibition rate of 63.33%, significantly lower than the inhibitory effects of strains B1, B3, B8, and B9.

[0051] Table 1. Antagonistic effects of nine strains of *Bacillus subtilis* on pathogenic fungi.

[0052]

[0053] Note: "+" indicates that the biocontrol bacteria has an inhibitory effect on pathogens, and "-" indicates that the biocontrol bacteria has no inhibitory effect on pathogens.

[0054] 2.2 Scanning electron microscopy observation of the antagonistic effect of *Dendrobium oryzae* on LM-C of *Fusarium solani*.

[0055] Under a scanning electron microscope, the mycelium of Fusarium solani growing normally was observed to be relatively thick and uniform, growing neatly, and the surface of the mycelium did not show obvious damage, depressions, or shriveling. Figure 4 A). Under a 10 μm scale, compared to the control, *Fusarium solani* LM-C exhibited mycelial curvature and entanglement under the antagonistic effects of seven different strains. Under the inhibitory effects of strains B2, B8, and B9, the mycelial surface of LM-C showed depressions and wrinkles (Figs. B, G, and H); under the influence of strain B3, the mycelial surface was severely damaged, with mycelial shrinkage, uneven thickness, and degeneration (Fig. C); under the influence of strains B4, B6, and B7, the LM-C mycelia were deformed, shriveled, and shrunken into sheets, with almost all contents disappearing, and some mycelia had attached substances on their surface (Figs. D, E, and F). Under a 1 μm scale, the surface morphology of the hyphae can be observed more clearly. The hyphae of the control group are smooth and rounded, while the hyphae of the experimental group show severe granularity, and some hyphae have a lot of attached substances. The hyphae have large cracks (Figures B, C, F), indicating that strains B1, B3, and B7 may have secreted some kind of lysing substance, which has a dissolving effect on the surface of the hyphae.

[0056] 2.3 Identification of strains

[0057] 2.3.1 Morphological observation of the strain

[0058] On LB agar plates, the colonies of strain B1 are round with irregular edges, no raised areas, light green in color, smooth and opaque on the surface, with short rod-shaped cells, a small number of spores, and are Gram-positive, occurring singly, in pairs, or in chains. The colonies of strains B3, B4, B6, B7, B8, and B9 have rough, opaque surfaces and irregular edges. Under a microscope, the cells are all rod-shaped, Gram-positive, and produce spores. The colonies of strains B3, B4, and B6 are milky white, with cells occurring singly or in pairs. Strain B7 is light yellow, with cells mostly singly or in pairs, and a few arranged in chains. The colonies of strains B8 and B9 are both light green, with B8 mostly singly and B9 mostly arranged in chains.

[0059] 2.3.2 Molecular biological identification of the strain

[0060] Sequence alignment was performed in MEGA 7 software, and the phylogenetic tree of the seven distillers' grains strains was constructed by expanding 1000 times using the neighbor-joining (NJ) method. Figure 6A In the middle, Bacillus oryzae B1 and Bacillus sp Gathered into one, B3 and Bacillus subtilis Gathered into one, B4 and Bacillus velezensis They clustered together. However, strains B6, B7, B8, and B9 clustered together, making it impossible to understand the genetic evolution of these four strains. Using the method described above, a phylogenetic tree of the four strains B6, B7, B8, and B9 was constructed. Figure 6B In the middle, the B6, B7, and B8 bacteria of highland barley wine lees respectively interacted with... Bacillus tequilensis , Bacillus velezensis , Bacillus subtilis They cluster into the same branch; Figure 6C In the middle, Bacillus subtilis B9 and Bacillus sp Gathered together, specifically with Bacillus subtilis They belong to the same branch. Therefore, based on the morphological identification of the strain, *Bacillus oryzae* B1 is a Bacillus (…). Bacillus The accession number is KU159257.1; strains B3, B8, and B9 are all Bacillus subtilis ( Bacillus subtilis The accession numbers are LC602907.1, MT111002.1, and DQ990038.1, respectively; B4 and B7 are Bacillus belyssus ( Bacillus velezensis The accession numbers are MN062956.1 and MF662486.1, respectively; B6 is Bacillus thymicus ( Bacillus tequilensis ), with login number MT501810.1.

[0061] 2.4 In-vessel seed germination test

[0062] 2.4.1 Effects of bacterial solution on seed germination

[0063] Three strains of *Fusarium solani* (B1, B8, and B9) with the strongest inhibitory effect against *Fusarium solani* were selected. Seed germination experiments were conducted to investigate the bioactivity of these three strains. Figure 7 It can be seen that after treatment with fermentation broths of strains B1, B8, and B9, the germination potential and germination rate of wild rapeseed and wild oat seeds did not reach the control level, and the differences were statistically significant. P <0.05%. After the fermentation broth was diluted 10 times, the three strains of *Distillers' grains* had no significant effect on the germination potential and germination rate of free-growing rapeseed seeds, while strains B1 and B8 showed significant inhibitory effects on the germination potential and germination rate of wild oat seeds, and strain B9 had a significant effect on the germination rate of wild oat seeds. P <0.05). Analysis revealed that the fermentation broth of three strains of *Bacillus subtilis* (barley lees fermentation broth) inhibited the germination of wild rapeseed and wild oat seeds. Under dilution conditions, the three strains showed more significant inhibitory effects on wild oat seed germination, with strain B8 exhibiting the strongest effect, achieving a 100% inhibition rate. Strain B9 showed the strongest inhibitory effect on the germination of wild rapeseed seeds.

[0064] 2.4.2 Effects of bacterial culture on seedling growth

[0065] Table 2 shows that the fermentation broths of strains B1, B8, and B9 all had a strong inhibitory effect on the growth of the tested weed seedlings, with inhibition rates of over 78% on seedling shoot length, root length, and fresh seedling weight. Compared with the control, the differences were statistically significant. P <0.05). Among them, strain B8 showed the best inhibitory effect on the growth of wild oat seedlings, with an inhibition rate of 100%. Strain B9 showed a high inhibitory effect on the growth of free-growing rapeseed seedlings, with inhibition rates of over 93% for all seedling indicators. Under the condition of 10-fold dilution of fermentation broth, the three strains of *Distillers' grains* had different degrees of influence on the growth of free-growing rapeseed and wild oat seedlings. Among them, the effect on the root growth of free-growing rapeseed seedlings was the greatest, all showing significant inhibition of root growth. The inhibition rates of root length by strains B1, B8, and B9 were 89.38%, 85.80%, and 78.83%, respectively. Strain B9 had the strongest inhibitory effect on shoot length and seedling fresh weight of free-growing rapeseed seedlings, while strain B8 showed a promoting effect. For wild oat seedlings, strain B8 had the strongest inhibitory effect on shoot length, while strain B1 had the strongest inhibitory effect on root length and seedling fresh weight, and strain B9 showed a promoting effect on seedling fresh weight. In summary, strains B9 and B1 showed the best growth-inhibiting effects on free-growing rapeseed seedlings and wild oat seedlings, respectively.

[0066] Table 2. Effects of three strains of *Distillers' grains* on the growth of free-growing rapeseed and wild oat seedlings.

[0067]

[0068] Note: The numbers in parentheses indicate the inhibition rates of different treatments on seedling shoot length, root length, and fresh weight. Different lowercase letters indicate that the differences between different treatments reached a significance level of 0.05.

[0069] In this experiment, three strains of *Bacillus oryzae* with the highest inhibition rates were selected. Treatment of wild rapeseed and wild oat seeds with their fermentation broth resulted in significantly lower germination potential, germination rate, shoot length, root length, and seedling fresh weight compared to the control. The inhibitory effect on wild oat seed germination was particularly strong. In the growth of wild rapeseed seedlings, the inhibition rates of all three strains were above 85%, and in wild oat seedlings, the inhibition rates were above 78%. When the fermentation broth was diluted 10-fold, the inhibitory effect of the three strains on wild rapeseed and wild oat decreased, but a significant inhibitory effect on the root length of wild rapeseed remained. Strain B9 showed the best inhibitory effect on the growth of wild rapeseed seedlings, while strain B1 showed a better inhibitory effect on the growth of wild oat.

[0070] The specific embodiments of the present invention have been described above. It should be understood that the present invention is not limited to the specific embodiments described above, and those skilled in the art can make various modifications or variations within the scope of the claims, which do not affect the essence of the present invention.

Claims

1. A type of barley distiller's grains mold, characterized in that, The barley wine lees bacteria are Bacillus subtilis (B. subtilis). Bacillus subtilis B9 was deposited at the Guangdong Provincial Center for Microbial Culture Collection on July 1, 2022, with accession number GDMCC No: 62595.

2. The application of the highland barley distiller's grains bacteria as described in claim 1, characterized in that, It can be used to prepare microbial herbicides; the fermentation broth of this barley wine lees bacteria has a strong inhibitory effect on the germination of wild rapeseed and wild oat seeds and the growth of seedlings.

3. The application as described in claim 2, characterized in that: The fermentation broth of this barley distillers' yeast inhibited the growth of seedling shoots, roots, and fresh weight of wild rapeseed and wild oats by more than 78%.

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