Lactobacillus plantarum for inhibiting the pathogen of liang-gua-ben leaf spot and application thereof

Abstract

The present application relates to the field of biotechnology, and more particularly to a lactarius lactis for inhibiting the pathogenic bacteria of leaf spot disease of Angelica biserrata and application thereof, and provides the lactarius lactis (Irpex lacteus) with the Tibetan number CGMCC No.42470.The experimental results show that the strain has the abilities of dissolving phosphorus, releasing potassium and producing cellulase, has high inhibiting capacity on the pathogenic bacteria of leaf spot disease, i.e. alternaria alternata and gloeophyllum trabeum, and can be used in the promotion of growth and the prevention and treatment of leaf spot disease, and has wide application prospect.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and in particular to *Lactobacillus thuringiensis*, a fungus that inhibits leaf spot disease in Ligusticum chuanxiong, and its application. Background Technology

[0002] Liao Gao version ( Conioselinum smithii (H. Wolff) Pimenov & Kljuykov) is a perennial herb belonging to the genus *Ligustrum* of the family Apiaceae, and is one of the important traditional Chinese medicines in Northeast China. Due to its rhizome being rich in volatile oils and ferulic acid, it possesses effects such as dispelling wind and cold, removing dampness and relieving pain, and occupies an important position in the traditional Chinese medicine market. In recent years, with the sharp decline in wild *Ligustrum lucidum* resources due to long-term over-harvesting, the huge market demand has led to a year-on-year increase in artificial cultivation area. Due to factors such as climate change and field management, pest and disease problems of *Ligustrum lucidum* have become increasingly prominent, seriously restricting the healthy development of the industry. Among them, leaf spot disease caused by *Alternaria* fungi is one of the main diseases in *Ligustrum lucidum* cultivation. This disease is characterized by rapid spread and wide range of damage, mainly infecting the leaves, petioles, and stems of the plant. In the early stages of the disease, small brown or dark brown spots appear on the leaves, which then gradually expand, causing the leaves to wither and curl, eventually leading to the death of the entire plant. Leaf spot disease causes a significant decrease in the photosynthetic capacity of *Ligustrum lucidum*, hinders rhizome development, and reduces dry matter accumulation. In severe cases, diseases can cause large areas of plants to die in the field, resulting in a sharp decrease in the yield of medicinal materials by 30% to 50%, or even a complete crop failure. In addition, diseases can reduce the appearance quality and the content of effective ingredients in medicinal materials, directly and severely damaging the economic income of medicinal farmers.

[0003] Currently, leaf spot disease control mainly relies on chemical control, primarily using systemic fungicides and traditional broad-spectrum fungicides. While these methods are effective, they easily lead to problems such as pathogen resistance, pesticide pollution, and residues, and also kill beneficial insects. In contrast, biological control is safer, more environmentally friendly, and has a longer-lasting effect, effectively protecting plant health, targeting specific pathogens precisely, reducing environmental pollution, and protecting ecological balance. Biocontrol bacteria are an important pathway and tool for achieving biological control. However, research on the control of leaf spot disease in Ligusticum chuanxiong is relatively insufficient. Existing research mainly focuses on the isolation and identification of endophytic bacteria in Ligusticum chuanxiong and the control of other diseases. Therefore, exploring control methods for leaf spot disease in Ligusticum chuanxiong and researching the efficacy of novel endophytic bacteria in controlling the disease is of great significance. Summary of the Invention

[0004] In view of this, the technical problem to be solved by the present invention is to provide a *Lactobacillus thuringiensis* strain that inhibits the pathogen of leaf spot disease of *Ligusticum striatum* and has a certain growth-promoting function, and its application.

[0005] This invention provides Irpexlacteus LG-EN-N3 with accession number CGMCC No.42470.

[0006] Furthermore, the present invention provides a bacterial community including the aforementioned Irpex lacteus LG-EN-N3.

[0007] Furthermore, the present invention provides a microbial agent comprising Irpex lacteus or the bacterial flora described in the present invention.

[0008] In this invention, the dosage form of the microbial agent includes granules, liquids, and / or dry powders, and this invention does not limit the dosage form.

[0009] This invention provides the use of at least one of the following (I) to (III) in inhibiting plant pathogens: I) The lactobacillus LG-EN-N3 (Irpex lacteus) described in this invention; II) The bacterial flora described in this invention; III) The bacterial agent described in this invention.

[0010] Furthermore, the plants include those belonging to the Apiaceae family and the Ligusticum genus.

[0011] Furthermore, the plants of the Apiaceae family and the Ligusticum genus include Ligusticum liaotiensis.

[0012] The pathogens include leaf spot pathogens, gray mold pathogens, wilt pathogens and / or anthracnose pathogens.

[0013] Furthermore, the pathogen causing the leaf spot disease includes Alternaria microphylla; The anthrax bacteria include *Anthrax globulus*. The pathogen causing gray mold includes *Botrytis cinerea*. The pathogen causing wilt includes Fusarium oxysporum.

[0014] This invention provides the use of at least one of the following (I) to (III) in promoting plant growth (promoting growth) by lysing phosphorus and / or releasing potassium: I) The lactobacillus LG-EN-N3 (Irpex lacteus) described in this invention; II) The bacterial flora described in this invention; III) The bacterial agent described in this invention.

[0015] Furthermore, the growth promotion is achieved through phosphorus solubilization, potassium solubilization, cellulase degradation, nitrogen fixation, amylase production, and sucrase production.

[0016] Specifically, compared with other milkweed fungi, this invention has a stronger inhibitory effect on leaf spot pathogens, and also on other pathogens, such as Alternaria stenozoata (…). Alternaria angustiovoidea Alternaria alternata ( ) Alternaria alternata ), Soybean anthracnose spores ( Colletotrichum sojae ), multi-host anthrax bacteria ( Colletotrichum plurivorum ) and Vitaspora ( Colletotrichum vittalense It exhibits strong inhibitory effects on various plants, including tomatoes, peppers, and potatoes. Furthermore, in terms of growth promotion, its growth-promoting effect on various plants is significantly superior to other milky white fungi.

[0017] This invention provides *Irpex lacteus*, with Tibetan cultural relic number CGMCC No. 42470. Experiments show that, compared with existing technologies, this invention is the first to detect *Irpex lacteus*'s growth-promoting properties, including phosphorus solubilization, potassium solubilization, cellulose degradation, nitrogen fixation, and the production of amylase and sucrase. It exhibits excellent phosphorus solubilization, potassium solubilization, cellulose degradation, nitrogen fixation, and the production of amylase and sucrase. Simultaneously, this bacterium has a high inhibitory effect on the leaf spot pathogens *Alternaria spp.* and *Anthracnose*; it can be used for growth promotion and leaf spot disease control, improving the overall disease resistance of plants. It provides a good biocontrol resource for diseases caused by pathogenic fungi of *Ligusticum striatum*, and offers new materials for biological control, with broad application prospects. Attached Figure Description

[0018] Figure 1 The morphology of a single colony of the pathogenic bacterium Ligusticum liaoense after living on PDA medium for about 10 days is shown; where A is the front view of growth on PDA medium; B is the back view of growth on PDA medium. Figure 2 Microscopic morphology of spores of the pathogenic fungus Ligusticum liaoense; where A is a low-power field of view; and B is a high-power field of view. Figure 3 The Neighbor-Joining phylogenetic tree of strain LG-BB-2-3 was constructed based on the 16S rRNA gene sequence alignment results. Figure 4 The growth of healthy leaves of Ligusticum liaoense after inoculation with pathogenic strains (scale bar: 1:1.16). Figure 5 The morphology of a single colony of Ligusticum liaoense endophytic bacteria (N3) living on PDA medium for about 5 days; where A is the front view of growth on PDA medium; B is the back view of growth on PDA medium. Figure 6 Microscopic morphology of endophytic fungi (N3) from Ligusticum liaoense; Figure 7 The Neighbor-Joining phylogenetic tree of strain LG-EN-N3 was constructed based on the 16S rRNA gene sequence alignment results. Figure 8The diagram shows the qualitative detection ability of endophytic bacteria to solubilize phosphorus; where A is the front view of the culture medium; and B is the back view of the culture medium. Figure 9 The diagram shows the qualitative test results for the potassium-solubilizing ability of endophytic bacteria; where A is the front view of the culture medium; and B is the back view of the culture medium. Figure 10 The growth of endophytic bacteria on cellulose Congo red solid medium over 10 days is shown; where A is the front view of the medium and B is the back view of the medium. Figure 11 The growth of endophytic bacteria on cellulose Congo red solid medium after 15 days is shown; where A is the front view of the medium and B is the back view of the medium. Figure 12 The growth of endophytic bacteria on Assumption solid medium over 7 days is shown; where A is the front view of the medium and B is the back view of the medium. Figure 13 The growth of endophytic bacteria on amylase solid medium over 7 days is shown; where A is the front view of the medium and B is the back view of the medium. Figure 14 The growth of endophytic bacteria on sucrase solid medium over 7 days is shown; where A is the front view of the medium and B is the back view of the medium. Figure 15 The image shows the inhibitory effect of LG-EN-2-3, the pathogen of Ligusticum liaoense, on the endophytic bacteria LG-EN-N3; where A is the front view of the culture medium; and B is the back view of the culture medium. Figure 16 Show Irpex lacteus LG-EN-N3 and literature reports Irpex lacteus Bar chart comparing the inhibition rates against four pathogens; Figure 17 The effect of endophytic fungus LG-EN-N3 on the incidence of leaf spot disease in Ligusticum liaoense was shown. In the figure, A represents the growth and development of potted Ligusticum liaoense 25 days after being irrigated with pathogenic fungus LG-EN-2-3; B represents the growth and development of potted Ligusticum liaoense 25 days after being irrigated with a 1:1 solution of pathogenic fungus LG-EN-2-3 and endophytic fungus.

[0019] Biological Preservation Instructions

[0020] The biological material LG-EN-N3, classified as *Irpex lacteus*, was deposited on December 12, 2025, at the China General Microbiological Culture Collection Center (CGMCC), Institute of Microbiology, Chinese Academy of Sciences, No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC No. 42470.

[0021] Biological material LG-BB-2-3, classified as *Alternaria tenuissima*, was deposited on December 12, 2025, at the China General Microbiological Culture Collection Center (CGMCC), Institute of Microbiology, Chinese Academy of Sciences, No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC No. 42359. Detailed Implementation

[0022] This invention provides a *Lactobacillus subtilis* strain that inhibits the pathogen of leaf spot disease in *Ligusticum striatum* and its application. Those skilled in the art can refer to the content of this document and appropriately modify the process parameters to achieve the desired result. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included in this invention. The methods and applications of this invention have been described through preferred embodiments. Those skilled in the art can clearly modify or appropriately change and combine the methods and applications described herein without departing from the content, spirit, and scope of this invention to implement and apply the technology of this invention.

[0023] In this invention, the endophytic bacteria is *Lactobacillus lactis* LG-EN-N3.

[0024] The test materials used in this invention are all common commercially available products. The invention is further illustrated below with reference to embodiments: Example 1: Isolation and Identification of Leaf Spot Pathogen I. Methods 1. Pathogen screening In July 2025, *Ligusticum chuanxiong* plants exhibiting leaf spot disease symptoms were collected from the *Ligusticum chuanxiong* planting base of the Institute of Special Agricultural Products, Chinese Academy of Agricultural Sciences. The collected *Ligusticum chuanxiong* plants were repeatedly rinsed under tap water, placed on filter paper to air dry, and then, using sterile scissors, 4mm sections were cut at the junction of diseased and healthy tissue in a clean bench. 2 Diseased tissue was first disinfected with 75% ethanol for 1 minute, then rinsed 5 times with sterile water for 1 minute each time. Next, the tissue was soaked in 5% sodium hypochlorite solution for 5 minutes, followed by rinsing with sterile water 5 times for 1 minute each time. Finally, the tissue was placed on sterile filter paper to absorb excess moisture, and 5 pieces were inoculated onto PDA medium and cultured at 28°C in the dark. After colonies grew, single spores were isolated from the colonies using a sterile inoculation needle and transferred to PDA medium for further purification. Finally, one purified strain was selected as the pathogenic strain for Ligusticum chuanxiong leaf spot disease for related experiments. The obtained strain was cultured at 28°C for 5 days, and the colony morphology was observed for future use.

[0025] Prepare PDA culture medium according to the standard formula, heat to melt and stir evenly, pour into 250mL Erlenmeyer flasks, seal the flask mouth with breathable sealing film, and sterilize together with 1.5mL centrifuge tubes in an autoclave for about 30 minutes. In a clean bench, while still hot, aliquot the PDA culture medium into centrifuge tubes, tilt the centrifuge tubes at an angle of 15° to 30° on the table, and allow them to cool and solidify naturally (about 30 minutes). Take a 5mm diameter mycelial cake, mycelial side down, and inoculate it onto PDA slant culture medium, and store it in a 4℃ freezer for later use. Alternatively, take 0.75mL of bacterial suspension, add 0.25mL of sterile 30% glycerol to a 2mL cryovial, mix thoroughly, and then flash-freeze in liquid nitrogen at -80℃.

[0026] 2. Identification of leaf spot pathogen strains

[0027] (1) Morphological identification

[0028] Endophytic fungi of *Ligusticum striatum* were inoculated into PDA solid medium and incubated at 28°C for 7–15 days. The morphological characteristics of the colonies were observed, and their shape, surface gloss, edge morphology, and color were recorded. Hyphae were scraped from the pre-purified and isolated strain using a sterile inoculation needle to prepare temporary slides, and the spore morphology of the strain was observed under a 400× microscope.

[0029] (2) Molecular identification

[0030] Genomic DNA was extracted from the pathogen using a nucleic acid extraction kit from Beijing Box Biotechnology Co., Ltd. Using the extracted genomic DNA as a template, PCR amplification of pathogen strains 2-3 was performed using primers H3-1a (5'-actaagcagaccgcccgcagg-3', SEQ ID NO:1) and H3-1b (5'-gcgggcgagctggatgtcctt-3', SEQ ID NO:2). The PCR reaction system consisted of 12.5 μL of 2×Taq PCR Master Mix, 0.5 μL each of 10 μmol / L forward and reverse primers, 1 μL of template DNA (approximately 100 ng / μL), and ddH2O to a final volume of 25 μL. Ultrapure water was used as the negative control. The PCR program was as follows: 96℃ pre-denaturation for 2 min; 96℃ denaturation for 15 s, 55℃ annealing for 30 s, 75℃ extension for 35 s, and 72℃ extension for 2 min. A total of 30 cycles were performed. After the reaction, 2 μL of PCR product was loaded onto a 1.5% agarose gel and electrophoresed at 190V for 45 min. After photographing with a gel imaging system, the target bands were excised, recovered, and purified. The clearly defined PCR products were sent to Shanghai Sangon Biotech Co., Ltd. for sequencing. After sequencing, the sequence was compared with reference sequences in GenBank using the BLAST program in the NCBI database. Available standard sequences were compared using Mega 12.0 software, and a phylogenetic tree was constructed using neighbor-joining.

[0031] (3) Pathogenicity test of leaf spot disease

[0032] The Koch's postulates were used for verification. A leaf was punctured 1 cm from the leaf margin with an inoculation needle. Two to three colonies of mycelium were collected and inoculated at the puncture site. The leaf was then placed in a sterile petri dish with an appropriate amount of sterile water, and the water was changed every three days. The disease condition of the leaves was observed after 10 days.

[0033] II. Results

[0034] 1. Morphological identification of the pathogen of Ligusticum chuanxiong

[0035] The colonies are generally round or nearly round, dark green in color, opaque, and relatively soft in texture, with a fluffy, downy surface and relatively neat edges. The spores are oval, septate, and arranged in chains. The colony morphology is as follows... Figure 1 As shown, the microscopic images are as follows: Figure 2 As shown.

[0036] 2. Molecular identification of the pathogen of Ligusticum chuanxiong

[0037] The 16S rDNA sequence of pathogens 2-3 was amplified and sequenced. The sequencing results are as follows: (SEQ ID NO:3); The sequenced sequences were submitted to NCBI for BLAST alignment, and sequences of strains with high similarity were downloaded. A phylogenetic tree was constructed using Mega 12.0 software; see details below. Figure 3 . Figure 3 The 16S rDNA sequence alignment results showed that strain LG-BB-2-3 was similar to... Alternaria tenuissima The similarity was the highest. Therefore, based on a comprehensive analysis of morphological observations, molecular identification results, and 16S rDNA sequence comparison results, strain LG-BB-2-3 was determined to be *Alternaria viridis*, and was named *Alternaria viridis*. Alternaria tenuissima LG-BB-2-3.

[0038] The above-screened Alternaria microphylla ( Alternaria tenuissimaLG-BB-2-3 was deposited on December 12, 2025, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 42359. The deposit address is Institute of Microbiology, Chinese Academy of Sciences, No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing. The requesting institution for deposit was the Institute of Special Products, Chinese Academy of Agricultural Sciences.

[0039] 3. Pathogenicity determination of Ligusticum striatum pathogen

[0040] The results of the reconnection method are as follows: Figure 4 As shown, from Figure 4 It can be confirmed that by re-inoculating with LG-BB-2-2 strain, typical leaf spot symptoms appeared on Ligusticum leaves 10 days later, and the inoculated pathogen could be re-isolated from the diseased site.

[0041] Example 2: Isolation and identification of endophytic bacteria from Ligusticum chuanxiong

[0042] I. Methods

[0043] 1. Isolation of endophytic bacteria from Ligusticum chuanxiong

[0044] Healthy, vigorous 3-year-old Ligusticum striatum plants were collected and rinsed under running water to remove surface dirt. They were then rinsed five times with sterile water, blotted dry with sterile paper, and placed in a laminar flow hood for preliminary UV disinfection for 30 minutes. Intact leaves and stems were placed on sterile filter paper and cut into 1cm pieces using a sterile knife. 2 The tissue was cut into chunks. The cut plant tissue was immersed in 75% anhydrous ethanol solution for 1 minute, then rinsed 5 times with sterile water for 1 minute each time. Next, it was immersed in 5% sodium hypochlorite solution for 5 minutes, then rinsed 5 times with sterile water for 1 minute each time. The sterilized tissue chunks, segments, and slices were then placed sequentially into PDA medium, with 5 tissue chunks per petri dish. The sterile water from the final rinse was spread onto potato dextrose agar and incubated at 30°C. If no bacteria grew on the culture medium spread with the sterile water from the final rinse, the surface sterilization was considered thorough. After 7 days, uncontaminated tissue was harvested and cut into 0.25cm pieces. 2Small pieces (segments, slices) of tissue were inoculated into PDA medium, with each sterilization treatment repeated three times. Six pieces of tissue were inoculated into each petri dish. The dishes were incubated at 28°C, and colony growth was observed periodically (days 2, 5, and 7). Different single colonies were picked from the edge for purification and culture, yielding several endophytic bacterial discs. Antagonistic bacteria were screened using plate confrontation. The isolated pathogen of *Ligusticum chuanxiong* leaf spot (5 mm in diameter) was inoculated in the center of a PDA plate, and endophytic bacterial discs (5 mm in diameter) were symmetrically inoculated 1 cm away from the center. By comparing the growth of the pathogen, an endophytic bacterial strain with strong inhibitory activity against the growth of *Ligusticum chuanxiong* leaf spot pathogen was selected.

[0045] 2. Identification of endophytic bacterial strains

[0046] (1) Morphological identification

[0047] Endophytic fungi from *Ligusticum striatum* were inoculated into PDA solid medium and incubated at 28°C for 7–15 days. The morphological characteristics of the colonies were observed, and their shape, surface gloss, edge morphology, and color were recorded. Hyphae were scraped from the pre-purified and isolated strain using a sterile inoculation needle to prepare temporary slides, and the spore morphology of the strain was observed under a 400× microscope.

[0048] (2) Molecular identification

[0049] Genomic DNA of the pathogen was extracted using a nucleic acid extraction kit from Beijing Box Biotechnology Co., Ltd., and then analyzed using the universal fungal primer ITS1 (…). -tccgtaggtgaacctgcgg- (SEQ ID NO:4) and downstream primer ITS4 ( -tcctccgcttattgatatgc- PCR amplification was performed on endophytic strain N3 (SEQ ID NO:5). The PCR reaction system (20 μL) consisted of: 10 μL 2×PCR Master Mix, 1 μL each of forward and reverse primers (10 μmol / L), 2 μL template DNA (100 ng / μL), and 6 μL ddH2O. The reaction conditions were: 95℃ pre-denaturation for 5 minutes, followed by 30 cycles (95℃ for 30 seconds, 52℃~58℃ for 30 seconds, 72℃ for 45~60 seconds), and a final extension at 72℃ for 5 minutes to ensure complete reaction. The PCR amplification product was detected by electrophoresis on a 1.5% agarose gel, yielding a clear band of approximately 750 bp. The amplified fragment was excised and the amplification product was recovered using a gel extraction kit. The recovered product was sent to Shanghai Sangon Biotech Co., Ltd. for sequencing. After sequencing, the 16S rRNA sequence was compared with sequences in GenBank using the BLAST program for homology. The available standard sequences were compared in the Mega 12.0 software, and a phylogenetic tree was constructed using the neighbor-joining method.

[0050] 3. Detection of the life-promoting properties of endophytic bacteria

[0051] (1) Detection of phosphate-solubilizing ability of endophytic bacteria

[0052] The phosphorus-solubilizing ability of endophytic bacteria was qualitatively and quantitatively determined using NBRIP medium (10 g / L glucose, 5 g / L calcium phosphate, 5 g / L magnesium chloride (MgCl2·6H2O), 0.25 g / L magnesium sulfate heptahydrate (MgSO4·7H2O), 0.2 g / L potassium chloride (KCl), 0.1 g / L ammonium sulfate ((NH4)2SO4), and 15–18 g / L agar). Qualitative determination of endophytic bacteria was performed using NBRIP solid medium. 100 mL of NBRIP liquid medium was poured into a stainless steel saucepan, and 1.5 g of agar was added. The mixture was heated over low heat with constant stirring to prevent scorching or overflow until completely dissolved. The solution was then dispensed into Erlenmeyer flasks and autoclaved at 121°C (0.103 MPa–0.105 MPa) for 15 minutes. After sterilization, wait for the culture medium to cool to 50℃~60℃ (warm to the touch but not hot), then pour it into sterile culture dishes in a laminar flow hood. Next, use a punch to remove the purified endophytic bacterial bacterial discs, placing one disc in each sterile NBRIP solid culture dish, with three replicates. Incubate upside down at 28℃ for 7 days, observing whether the bacterial strain can grow in its culture medium. If it can grow, it indicates that the strain has phosphate-solubilizing ability.

[0053] Quantitative determination of endophytic bacteria was performed using NBRIP liquid medium. Purified endophytic bacterial strains were inoculated into NBRIP liquid medium, with three biological replicates. A blank control (CK) of the same volume was also included, and cultures were established under the same conditions. Cultures were performed at 28°C and 180 rpm for 7 days. After culture, the inoculated fermentation broth and CK culture were centrifuged at 10,000 rpm for 10 minutes at 4°C, and both were filtered through a 0.45 μm filter. 5 mL of the supernatant was transferred to a 50 mL volumetric flask, diluted with 25 mL of distilled water, and then 2 mL of 0.5 mol / L dilute sulfuric acid and 5 mL of molybdenum antimony reagent were added to each flask. The volume was then adjusted to 50 mL with distilled water, shaken well, and allowed to stand for 30 min. The supernatant from the uninoculated NBRIP liquid medium served as the blank matrix solution. This matrix solution was used to dilute phosphorus standards.

[0054] Take seven 50 mL volumetric flasks and add 5 mL of blank matrix solution to each flask. Add 0, 1, 2, 4, 6, 8, and 10 mL of 5 μg / mL phosphorus standard working solution sequentially to each flask, followed by approximately 25 mL of distilled water, then 2 mL of 0.5 mol / L dilute sulfuric acid, and finally 5 mL of molybdenum antimony colorimetric reagent. Dilute to 50 mL with distilled water and mix well to obtain phosphorus standard solutions with concentrations of 0, 0.1, 0.2, 0.4, 0.6, 0.8, and 1 μg / mL. After standing for 30 min, detect the phosphorus concentration using a UV spectrophotometer. Plot a standard curve with OD700 value on the x-axis and phosphorus content (μg / mL) on the y-axis to obtain a regression equation (R² ≥ 0.999). Measure the OD700 value using a UV-Vis spectrophotometer and calculate the phosphorus content per unit volume of fermentation broth using the obtained standard curve equation. Simultaneously, a blank control of uninoculated NBRIP liquid medium was measured to eliminate interference from the culture base and abiotic effects on phosphorus determination.

[0055] Phosphorus dissolution amount = phosphorus content of inoculated group - phosphorus content of uninoculated blank control group.

[0056] (2) Detection of potassium solubilization capacity of endophytic bacteria

[0057] The potassium-solubility of endophytic bacteria was qualitatively determined using Alexandrite silicate solid medium (5.0 g / L sucrose, 0.5 g / L magnesium sulfate heptahydrate, 2.0 g / L disodium hydrogen phosphate, 0.005 g / L ferric chloride, 0.1 g / L calcium carbonate, 1.0 g / L potassium feldspar powder, 15.0–18.0 g / L agar, with the final pH adjusted to 7.2 ± 0.2). 24.51 g (precise weight 24.505 g) of Alexandrite silicate dry powder medium was dissolved in 1000 mL of distilled water, dispensed into 250 mL Erlenmeyer flasks, and autoclaved at 121 °C for 15 min. After sterilization, the medium was allowed to cool to 50 °C–60 °C (warm to the touch but not hot) and poured into sterile petri dishes in a laminar flow hood. Use a punch to remove the mycelial cake of the endophytic strain. Place one mycelial cake in each petri dish and set up 3 replicates. Incubate upside down at 28°C for 7 days and observe whether it can grow in Alexandrite silicate solid medium. If it can grow, it indicates that the strain has potassium solubilizing ability.

[0058] The potassium-soluble capacity of endophytic bacteria was quantitatively measured using a liquid medium containing sparingly soluble potassium. Endophytic mycelia were inoculated into 250 mL Erlenmeyer flasks containing 50 mL of this medium, with three replicates and one blank control. After culturing at 28°C and 180 rpm for 7 days, the fermentation broth was centrifuged at 12000 rpm for 5 min. The supernatant was filtered through a 0.45 μm aqueous filter membrane, and the soluble potassium content in the supernatant was measured by flame emission spectrometry.

[0059] To construct a potassium standard curve: Accurately weigh 0.1907 g of dried potassium chloride (KCl, analytical grade), dissolve it in 1 mol / L ammonium acetate (NH4OAc) solution, and dilute to 1000 mL to obtain a potassium standard stock solution with a concentration of 100 μg / mL. Accurately pipette 0, 2.5, 5.0, 10.0, 15.0, 20.0, and 40.0 mL of the above solution into seven 100 mL volumetric flasks, respectively, and dilute to the mark with 1 mol / L NH4OAc solution, then mix well. This yields potassium standard solutions with concentrations of 0, 2.5, 5, 10, 15, 20, and 40 mg / L. Plot a standard curve with potassium concentration (mg / L) on the x-axis and flame photometer reading (emission intensity) on the y-axis, and draw the regression equation. The net dissolved potassium (mg / L) of the strain is obtained by subtracting the potassium concentration of the blank control from the potassium concentration measured in the supernatant.

[0060] (3) Detection of the cellulose degradation ability of endophytic bacteria

[0061] First, prepare the cellulose Congo red solid medium (5.0 g / L cellulose powder, 0.2 g / L Congo red, 1.0 g / L sodium nitrate (NaNO3), 0.5 g / L potassium dihydrogen phosphate (KH2PO4), 0.5 g / L dipotassium hydrogen phosphate (K2HPO4), 0.5 g / L magnesium sulfate heptahydrate (MgSO4·7H2O), 0.5 g / L potassium chloride (KCl), 0.5 g / L yeast extract, 0.5 g / L acid-hydrolyzed casein, 0.01 g / L ferrous sulfate (FeSO4), 15.0~18.0 g / L agar, pH: 7.0±0.1). Weigh 2.53 g of the dry cellulose Congo red medium powder, dissolve it in 100 mL of distilled water, stir thoroughly for 5 min, breaking up any clumps during stirring, dispense into containers, and autoclave at 121°C for 15 min. After sterilization, cool to 50℃~55℃ and pour approximately 20mL into sterile culture dishes in a laminar flow hood. After solidification, take an endophytic bacterial cake (5mm in diameter), mycelial side down, and inoculate it in the center of the aforementioned cellulose Congo red solid medium. Set up three replicates and incubate at 28°C for 7 days. Observe whether the endophytic bacteria can grow in the cellulose Congo red solid medium. If they can grow, it indicates that the strain has the ability to degrade cellulose.

[0062] (4) Detection of nitrogen fixation capacity of endophytic bacteria

[0063] Weigh 33.7g of Assabeth dry powder medium (10.0g / L mannitol, 0.2g / L potassium dihydrogen phosphate, 0.2g / L magnesium sulfate, 0.2g / L sodium chloride, 0.1g / L calcium sulfate, 5.0g / L calcium carbonate, 15.0~18.0g / L agar, adjusted pH to 6.8~7.0), add to 1000mL of distilled water, heat to boiling in a stainless steel pot and stir until completely dissolved. Dispense into containers and autoclave at 121℃ for 15min. After removal, cool to 50℃~55℃, and pour approximately 20mL into sterile petri dishes in a laminar flow hood. Inoculate endophytic bacterial discs (5mm) onto Assabeth solid medium plates, setting up 3 replicates. After incubation at 28℃ upside down for 7 days, observe whether the bacterial strains on the medium grow normally. If they grow, it indicates that the endophytic bacteria have nitrogen-fixing ability; otherwise, they do not.

[0064] (5) Detection of the ability of endophytic bacteria to produce amylase

[0065] Qualitative detection of endophytic bacterial production was performed using a solid amylase culture medium. 10g of soluble starch, 5g of peptone powder, 1g of yeast powder, 1g of dipotassium hydrogen phosphate, 0.5g of magnesium sulfate, and 20g of agar were weighed and poured into a stainless steel heating pot. 1L of distilled water was added and stirred thoroughly until completely dissolved. The volume was then adjusted to 1L. The solution was dispensed into 250mL Erlenmeyer flasks, sealed with breathable sealing film, and then autoclaved at 121°C for 15 minutes. After cooling to 50°C–55°C, approximately 20mL of the solution was poured into sterile petri dishes in a laminar flow hood and allowed to solidify. Endophytic bacterial pellets (5mm in diameter) with the mycelial side down were inoculated into the center of the solid amylase culture medium. Three replicates were performed. The culture was carried out at 28°C for 7 days, and the growth of the bacteria in the solid medium was observed. If growth was observed, it indicated that the strain had the ability to produce amylase.

[0066] Quantitative detection of endophytic amylase: Endophytic bacteria were inoculated into 50 mL of sterile PDB medium. After inoculation, the culture was carried out at 28℃ and 180 rpm for 7 days. After the culture was completed, the fermentation broth was centrifuged at 12000 rpm for 5 min. 2 mL of the supernatant and 2 mL of 1% soluble starch solution were mixed and reacted precisely in a 50℃ water bath for 30 min. After the reaction was completed, 2 mL of DNS reagent was immediately added, mixed, and then rapidly heated in a boiling water bath for 5 min to terminate the reaction and develop color. After cooling to room temperature, the volume was adjusted to 50 mL with deionized water. Using the control tube as a blank, the absorbance of the sample tube was measured at a wavelength of 540 nm. The absorbance value was substituted into the standard curve equation to calculate the mass (mg) of reducing sugar in the corresponding system. According to the formula, enzyme activity (U / mL) = (A×1000) / (MW×t×V); where A is the mass of reducing sugar, MW is the molecular weight of reducing sugar, t is the time of enzyme reaction, and V is the volume of crude enzyme solution added to the reaction system.

[0067] (6) Detection of sucrase production capacity of endophytic bacteria

[0068] First, prepare a solid culture medium for fungal sucrase detection. Weigh 10g sucrose, 5g peptone powder, 1g yeast powder, 1g dipotassium hydrogen phosphate, 0.5g magnesium sulfate, and 20g agar, and pour them into a stainless steel heating pot. Add 1L of distilled water and stir thoroughly until completely dissolved. Make up to 1L. Dispense into 250mL Erlenmeyer flasks and autoclave at 121°C for 15 minutes. After sterilization, cool to 50°C-55°C and pour approximately 20mL into sterile petri dishes in a laminar flow hood. After solidification, take an endophytic mycelial cake (5mm in diameter), mycelial side down, and inoculate it in the center of the solid culture medium containing sucrase. Perform three replicates and incubate at 28°C for 7 days. Observe whether the fungus can grow in the solid culture medium. If it can grow, it indicates that the strain has the ability to produce sucrase.

[0069] Quantitative detection of sucrase in endophytic bacteria: Endophytic bacteria were inoculated into 50 mL of sterile PDB medium. After inoculation, the culture was carried out at 28℃ and 180 rpm for 7 days. After the culture, the fermentation broth was centrifuged at 12000 rpm for 5 min, and 2 mL of the supernatant was taken and mixed with a 1% sucrose solution prepared with pH 5.0 citrate buffer. The mixture was then incubated in a 50℃ water bath for 30 min. Immediately after the reaction, 2 mL of DNS reagent was added, mixed, and the mixture was quickly heated in a boiling water bath for 5 min to terminate the reaction and develop color. After cooling to room temperature, the volume was adjusted to 50 mL with distilled water and mixed. Using the control tube as a blank, the absorbance of the sample tube was measured at a wavelength of 540 nm. The absorbance value was substituted into the standard curve to calculate the mass A (mg) of reducing sugar in the reaction system. Sucrase activity is calculated using the formula: Enzyme activity (U / mL) = (A×1000) / (MW×t×V), where A: mass of reducing sugar, MW: molecular weight of reducing sugar, t: enzyme reaction time, and V: volume of crude enzyme solution added to the reaction system.

[0070] II. Results

[0071] 1. Morphological identification of endophytic fungi in Ligusticum chuanxiong

[0072] Photographs of plate cultures of Ligusticum liaothomsonii endophytic bacteria strains are shown below. Figure 5 As shown, the colonies are generally round or nearly round, milky white in color, with fluffy hyphae on the surface, and a loose texture. They spread outward from the center in an "umbrella-like" pattern, forming obvious radial textures, with fine white hyphae extending outward from the edges. Figure 6 The fungal spores are oval or rod-shaped, nearly colorless and transparent, with a smooth surface.

[0073] 2. Molecular identification

[0074] The 16S rDNA sequence of endophytic bacterium N3 was amplified and sequenced. The sequencing results are as follows: (SEQ ID NO:6); The sequenced sequences were submitted to NCBI for BLAST alignment, and sequences of strains with high similarity were downloaded. A phylogenetic tree was constructed using Mega 12.0 software; see details below. Figure 7 The 16S rDNA sequence alignment results showed that strain LG-EN-N3 was similar to... Irpex lacteus The similarity was the highest. Therefore, based on the comprehensive analysis of morphological observations, molecular identification results, and 16S rDNA sequences, the results can be confirmed that strain LG-EN-N3 is *Brachys leucopsis*, and it is named *Brachys leucopsis*. Irpex lacteus LG-EN-N3.

[0075] 3. Detection of endophytic bacteria's ability to promote growth and degrade cellulose

[0076] (1) Detection of phosphate-solubilizing ability of endophytic bacteria

[0077] The isolated endophytic fungal discs were inoculated onto NBRIP solid medium and cultured for 7 days. Figure 8 As can be seen, endophytic fungi grew colonies on NBRIP solid medium, preliminarily proving that these endophytic bacteria have phosphate-solubilizing capabilities. Furthermore, using uninoculated NBRIP liquid medium as a blank control, a standard curve was plotted with OD700 value on the x-axis and phosphorus content (mg / L) on the y-axis. The standard curve is y = 1.9185x - 0.0929, R0. 2 The value was 0.9994, indicating that the detection method is highly feasible. The phosphorus content in the supernatant and uninoculated NBRIP liquid medium, determined by the molybdenum-antimony colorimetric method, was 148.74 mg / L and 4.468 mg / L, respectively. This performance is superior to that of previously reported *Lactobacillus subtilis* in terms of phosphorus solubility.

[0078] Table 1. Determination of Quantitative Phosphate Solubility of Endophytic Bacteria

[0079] Soluble phosphorus (mg / L) = 148.74 - 4.468 = 144.272 (mg / L)

[0080] (2) Detection of potassium solubilization capacity of endophytic bacteria

[0081] The isolated *Irpex lacteus* was inoculated into potassium-solubilizing liquid medium and cultured for 7 days. Figure 9 It was observed that this strain could grow normally and form colonies on Alexandrite silicate solid medium, preliminarily indicating its potassium-solubilizing ability. Further quantitative detection of soluble potassium content in the fermentation supernatant was performed using flame emission spectrometry. A standard curve was plotted with potassium standard solution concentration (μg / mL) as the x-axis and flame photometer emission intensity as the y-axis, yielding the regression equation y = 0.3318x + 0.1443, R² = 0.9998, indicating good linearity and reliable detection results. The potassium concentration in the fermentation supernatant of this strain reached 105.8 mg / L, confirming its strong potassium-solubilizing function. According to a literature search, there are currently no reports on the quantitative detection of potassium-solubilizing ability in *Eriocaulon buergerianum*, making this property the first discovery and verification in *Eriocaulon buergerianum*.

[0082] (3) Detection of the cellulose degradation ability of endophytic bacteria

[0083] The isolated endophytic fungi were inoculated into cellulose Congo red solid medium and cultured at 28°C for 10 and 15 days. The results are as follows: Figure 10 and Figure 11As shown, the endophytic fungi can grow normally and form colonies on cellulose Congo red solid medium, and clear degradation areas are observed, proving that the endophytic fungi have the ability to degrade cellulase.

[0084] (4) Detection of nitrogen fixation capacity of endophytic bacteria

[0085] Endophytic bacterial discs were inoculated onto Assumption agar plates, with three replicates. After incubation at 28°C inverted for 7 days, the results are as follows: Figure 12 As shown, the endophytic fungi can grow normally on Assumption solid medium, indicating that the fungi have nitrogen-fixing ability.

[0086] (5) Detection of the ability of endophytic bacteria to produce amylase

[0087] Endophytic fungal cakes were inoculated onto amylase solid medium and cultured at 28°C for 7 days. The results are as follows: Figure 13 The strain shown was able to grow normally. Its amylase-producing ability was further quantitatively verified using the DNS colorimetric method. Preparation of the glucose standard curve: Approximately 2.5 g of analytically pure glucose was weighed and placed in a weighing bottle, dried in an oven at 105 °C for 2 h, and cooled to room temperature. 2.000 g of the dried anhydrous glucose was accurately weighed, dissolved in a small amount of distilled water, transferred to a 1 L volumetric flask, and diluted to the mark with distilled water. The solution was thoroughly mixed to obtain a glucose standard stock solution with a concentration of 2 mg / mL. 2.5 mL of this stock solution was pipetted into a 50 mL volumetric flask, diluted to the mark with distilled water, and shaken well to obtain a glucose standard intermediate solution with a concentration of 100 μg / mL. Six 50 mL volumetric flasks were prepared by pipetting 0, 0.1, 0.2, 0.3, 0.4, and 0.5 mL of glucose standard intermediate solution into each flask. The solutions were then diluted to the mark with distilled water and shaken well to obtain a series of glucose standard solutions with concentrations of 0, 0.2, 0.4, 0.6, 0.8, and 1.0 μg / mL, respectively. The series solutions were subjected to colorimetric treatment using the DNS method, and the absorbance was measured at 540 nm. A standard curve was plotted with glucose concentration on the x-axis and absorbance on the y-axis. The calculated amylase activity of this endophytic fungus was 16.58 U / mL. Currently, there is no quantitative data on the amylase activity of *Lactobacillus thuringiensis* in the published literature; this result fills a gap in the literature. Furthermore, as a natural endophyte, an enzyme activity of 16.58 U / mL indicates the background level of this fungus's enzyme activity, demonstrating potential for further development and enhancement.

[0088] (6) Detection of sucrase production capacity of endophytic bacteria

[0089] Endophytic fungal mycelium was inoculated onto sucrase solid medium and cultured at 28°C for 7 days. The results are as follows: Figure 14As shown, the strain can grow normally, indicating that it has the ability to produce sucrase. Further quantitative detection was performed using the DNS colorimetric method. A glucose standard solution was prepared using the same procedure as the aforementioned amylase activity assay. A standard curve was plotted with glucose concentration on the x-axis and absorbance on the y-axis, and the absorbance was measured at 540 nm using the same colorimetric treatment. The calculated amylase activity of this endophytic fungus was 7.97 U / mL.

[0090] Example 3 Determination of the broad-spectrum antibacterial spectrum of *Lactobacillus leucocephala* LG-EN-N3

[0091] Alternaria stenozoata (U. stenozoata) was cultured using the plate confrontation method. Alternaria angustiovoidea Alternaria alternata ( ) Alternaria alternata ), Soybean anthracnose spores ( Colletotrichum sojae ), multi-host anthrax bacteria ( Colletotrichum plurivorum Vitaspora ( Colletotrichum vittalense ), extended Penicillium ( Penicillium expansum The mycelia were cultured separately on PDA medium. After the mycelia had covered the entire plate, mycelial blocks were punched out from the plate using a 5mm perforator. One mycelial block was taken, mycelial side down, and inoculated into the center of a new PDA plate. Using the mycelial block as the center, a 5mm mycelial block of *Lactobacillus leucovora* LG-EN-N3 was inoculated 2.5cm away from the center. Three replicates were set up and incubated in a 28℃ incubator for 7 days. The antibacterial effect was observed and recorded. The relative antibacterial rates of *Lactobacillus leucovora* LG-EN-N3 against various pathogens are shown in Table 2.

[0092] Table 2. Antifungal effect of *Lactobacillus sulfadiazine* LG-EN-N3 against five plant pathogenic fungi.

[0093]

[0094] Example 4: Growth-promoting effects of Lactobacillus LG-EN-N3 on tomatoes, peppers, potatoes, and wheat.

[0095] The experiment included a treatment group, a control group (CK), and a blank group, as follows: the treatment group consisted of plants inoculated with *Lactobacillus thuringiensis* bacterial suspension; the control group (CK) consisted of plants not inoculated with any inoculum; and the blank group consisted of plants inoculated only with inactivated *Lactobacillus thuringiensis* bacterial suspension. This was to eliminate interference from other components in the bacterial suspension (such as culture medium components) and ensure the accuracy of the experimental data. Preparation of the bacterial suspension: The activated *Lactobacillus thuringiensis* LG-EN-N3 strain was inoculated into liquid culture medium such as PDB, and after cultivation, diluted to a spore concentration of approximately 1 × 10⁻⁶. 6 CFU / mL, for later use.

[0096] Sampling and Index Measurement: After 5 weeks of cultivation, the entire plant was carefully removed, and the soil adhering to the root surface was gently washed off with clean water to avoid damaging the roots. Subsequently, various growth indicators were measured: plant height (from the base of the plant to the apical meristem) and root length (from the tip of the taproot to the base of the root) were measured using a ruler; stem diameter was measured using calipers. The relative growth rates of each treatment group were calculated based on the measured growth indicators, and the data were compared in tabular form to clearly present the effects of *Lactobacillus urinaria* LG-EN-N3 on the growth of different crops. The relative growth rate results of *Lactobacillus urinaria* LG-EN-N3 on the phenotypes of tomato, pepper, potato, and wheat are shown in Table 3.

[0097] Relative growth rate (%) = [(Treatment group average - Control group average)] / Control group average × 100%

[0098] Table 3. Relative growth rates of four crop growth indicators

[0099]

[0100] Example 5: Investigation on the inhibitory effect of endophytic fungi of Ligusticum chuanxiong on the pathogen of leaf spot disease.

[0101] I. Methods

[0102] 1. Flat Plate Standoff Experiment

[0103] A plate confrontation experiment was used to test the inhibitory effect of endophytic fungal strain N3 on the mycelial growth of *Alternaria alternata* 2-3, the pathogen causing leaf spot of *Ligusticum liaoense*. Both endophytic and pathogenic strains were activated and cultured separately on PDA plates at 28℃ for 5 days. Using a sterile pipette tip, 5 mm diameter mycelial cakes were collected from the edge of the uniformly growing colonies. In the confrontation experiment, pathogenic mycelial cakes were inoculated in the center of the PDA plate, and endophytic mycelial cakes were symmetrically inoculated approximately 1 cm from the edge of the plate. Three replicates were set up, with plates inoculated only with pathogenic mycelial cakes serving as a blank control. All plates were incubated upside down at 28℃. When the pathogen had essentially colonized the control plates, the growth of the pathogen in the confrontation group was observed and recorded, and the inhibition rate of endophytic fungi on pathogen growth was calculated.

[0104] Inhibition rate (%) = (Net pathogen growth in control group - Net pathogen growth in treatment group) / Net pathogen growth in control group × 100%

[0105] 2. Potted plant verification

[0106] Single colonies of pathogenic bacteria and endophytic bacteria were picked and inoculated into 30 mL of PDB liquid medium, respectively, and cultured at 28°C with shaking at 180 rpm for 12 h to obtain bacterial suspensions of pathogenic bacteria and endophytic bacteria. The bacterial suspensions were centrifuged at 12000 rpm for 4 min, and the bacterial pellet was collected and diluted with deionized water to a final concentration of 10. 8CFU / mL was used to obtain bacterial suspensions of two strains.

[0107] (1) Pour the pathogen suspension into the potted plants that are growing normally.

[0108] (2) A 1:1 mixture of pathogenic bacterial solution and endophytic bacterial solution was poured into pots for normal growth. The two pots were observed and analyzed after 25 days of normal management and cultivation. During the normal cultivation process, the plants were regularly watered with sterile water.

[0109] II. Results

[0110] 1. Flat Plate Standoff Experiment

[0111] After five days of inverted culture, the pathogen almost completely covered the control plate. At this point, it was found that the endophytic strain (Alternaria alternata) had a significant antagonistic effect on the mycelial growth of the pathogen (Alternaria spp.). Figure 15 The inhibitory effect of endophytic bacterial strains on pathogens is shown (the pathogen is in the middle of the figure, and the endophytic bacterial strains are on both sides). Based on the colony diameter calculation in Table 3, the inhibition rate of *Alternaria alternata* LG-EN-N3 against *Alternaria spp.* LG-BB-2-2 reached approximately 83.88%.

[0112] Table 4. Antibacterial activity of *Alternaria alterniflora* LG-EN-N3 against *Alternaria spp.* LG-BB-2-2

[0113] Antibacterial rate (%) = [(90-5) - (18.7-5)] / (90-5) = 83.88%

[0114] Currently, existing studies have reported that *Euphorbia lactea* has inhibitory effects on different pathogens (Table 5), but the *Euphorbia lactea* in this study ( Irpex lacteus LG-EN-N3 has a certain advantage in inhibiting pathogens compared to previously discovered *Alternaria* species, although there are reports indicating that *Alternaria* inhibits *Alternaria* species (…). Alternaria alternata While it exhibits inhibitory activity (inhibition rate 37.82%), research on *Alternaria spp.*, the main pathogen of *Ligusticum liaothomsonii* leaf spot disease, has not been reported. This study demonstrates that *Irpex lacteus* LG-EN-N3 has an inhibition rate of approximately 83.88% against *Alternaria spp.* LG-BB-2-2, providing a new candidate strain for the biocontrol of *Ligusticum liaothomsonii* leaf spot disease. Compared with existing *Irpex lacteus* inhibition rates, the *Irpex lacteus* LG-EN-N3 of this invention demonstrates a higher inhibitory effect, significantly outperforming *Irpex lacteus* LL210 and *Irpex lacteus* CMU-8413 in inhibiting *Alternaria spp.* / pepper leaf spot and *Anthracnose* (Table 5 and...). Figure 16 ).

[0115] Table 5. Different types of *Alternaria alternifolia* ( Irpex lacteus Inhibition rate against four plant pathogens

[0116]

[0117] 2. Potted plant verification experiment

[0118] The results showed that the two pots of Ligusticum striatum exhibited different growth patterns after 25 days (Table 6 and 1). Figure 17 Potted plants irrigated with the pathogen LG-BB-2-3 showed some wilting and curling of leaves, with black diseased leaves appearing. Potted plants irrigated with a 1:1 solution of pathogen LG-BB-2-3 and endophytic fungus LG-EN-N3 are currently growing well, with fewer diseased leaves, and the incidence of disease has been greatly reduced.

[0119] Table 6. Effect of endophytic bacterium LG-EN-N3 on the incidence of leaf spot disease in Ligusticum liaoense.

[0120]

[0121] III. Conclusion

[0122] This study first isolated and purified the pathogen from diseased leaf samples of *Ligusticum liaotiensis* grown in the field. After obtaining suspected strains through tissue isolation, a pathogen causing leaf spot disease in *Ligusticum liaotiensis* was finally isolated and identified as *Alternaria spp.*, named LG-BB-2-3, through a combination of morphological observation (colon morphology, spore morphology and size) and molecular biological identification. This pathogen grew rapidly on PDA medium, producing grayish-black, fluffy colonies with chain-like conidia exhibiting both transverse and longitudinal septa, highly consistent with the morphological and molecular characteristics of known *Alternaria spp.*. Furthermore, pathogenicity testing showed that this strain successfully induced typical leaf spot symptoms in healthy *Ligusticum liaotiensis* leaves, confirming its status as the pathogenic agent.

[0123] Based on the green control concept of "biological control," this study further isolated multiple endophytic fungal strains from healthy *Ligusticum liaoense* leaves to screen for biocontrol strains targeting this pathogen. Multiple endophytic fungal and bacterial strains were obtained through surface disinfection and streak plating. Screening was performed using the plate inhibition method, and one endophytic fungus showed a particularly significant inhibitory effect on *Alternaria spp.* morphological observation (colonies are milky white, dense, and have regular edges) and molecular analysis identified this endophytic fungus as *Alternaria spp.* To further verify the biocontrol potential of *Alternaria spp.*, this study conducted plate inhibition experiments and pot experiments: Indoor plate inhibition experiments showed that *Alternaria spp.* strongly inhibited the mycelial growth and spore germination of *Alternaria spp.*, with an inhibition rate of approximately 85%; in the pot experiment, the incidence of leaf spot disease in *Ligusticum liaoense* plants was significantly reduced after applying a mixed bacterial solution of *Alternaria spp.* and *Alternaria spp.* via irrigation compared to direct application of *Alternaria spp.* bacterial solution.

[0124] In summary, this study isolated and identified an endophytic bacterium (LG-EN-N3) from healthy leaves of *Ligusticum liaothomsonii* that exhibits significant inhibitory activity against the previously isolated pathogen of *Ligusticum liaothomsonii* leaf spot (*Alternaria spp.* LG-BB-2-3*). Indoor plate inhibition tests and pot experiments demonstrated that this strain has the potential to be developed into a green biocontrol agent. This discovery not only provides a new candidate strain for the biological control of *Ligusticum liaothomsonii* leaf spot but also provides important evidence and practical support for reducing the use of chemical pesticides in the cultivation of medicinal plants, ensuring the quality and safety of medicinal materials, and promoting the sustainable development of the ecological environment.

[0125] The above are merely preferred embodiments of the present invention. It should be noted that those skilled in the art can make various improvements and modifications 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. Irpex lacteus LG-EN-N3 with accession number CGMCC No.42470.

2. An inoculum agent comprising Irpex lacteus LG-EN-N3 as described in claim 1.

3. The microbial agent according to claim 2, characterized in that, Dosage forms include granules, liquids, and / or dry powders.

4. The application of at least one of the following (I) to (II) in inhibiting plant pathogens: I) Irpex lacteus LG-EN-N3 as described in claim 1; II) The bacterial agent according to claim 2 or 3; The pathogens are: Alternaria stenozoata, Alternaria alternata, Colletotrichum anthracis, Colletotrichum multiforme, Colletotrichum vetta, Penicillium expansum, Alternaria microphylla, Fusarium oxysporum, Staphylococcus aureus, or Coccidioides anthracis.

5. The application of at least one of the following (I) to (II) in promoting plant growth: I) Irpex lacteus LG-EN-N3 as described in claim 1; II) The bacterial agent according to claim 2 or 3; The plant is a tomato, pepper, potato, or wheat.

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