Mortierella alpina and application thereof

Abstract

The invention relates to mortierella alpina and application thereof. The strain name is Mortierella alpina RS-01, the preservation number is CGMCC (China General Microbiological Culture Collection Center) No.41944, the preservation time is May 21, 2025, and the preservation location is CGMCC (China General Microbiological Culture Collection Center), No. 3, No.1 yard, West Beichen Road, Chaoyang District, Beijing, China. The mortierella alpina provided by the invention can be used for pathogenic bacteria of ginseng rust rot, pathogenic bacteria of muskmelon fusarium wilt, pathogenic bacteria of pythium aphanidermatum, pathogenic bacteria of apple fruit rot, pathogenic bacteria of northern corn leaf blight, pathogenic bacteria of seedling blight, pathogenic bacteria of walnut canker, pathogenic bacteria of poplar canker, pathogenic bacteria of pepper anthracnose and pathogenic bacteria of blueberry stem canker; the compound has the best antibacterial effect on pathogenic bacteria of apple shell monochromatic alternaria, pathogenic bacteria of tobacco brown spot, and especially pathogenic bacteria of ginseng rust rot.

Description

Technical Field

[0001] The invention belongs to the technical field of microorganisms, and in particular relates to a Mortierella alpina strain and applications thereof. Background Art

[0002] Ginseng, a perennial medicinal plant of the genus Panax in the Araliaceae family, possesses high medicinal value. However, due to long-term continuous cropping and inappropriate use of pesticides, ginseng diseases have become increasingly severe, directly impacting ginseng yield and quality. Ginseng rust, among other diseases, can affect ginseng at all stages of its growth cycle and is the most serious root disease. Its average incidence rate is 15%-30%, and in severe cases, it can exceed 70%, resulting in significant economic losses. Due to their environmental and sustainable nature, microbial agents are increasingly being used to inhibit soil-borne ginseng diseases. In ginseng production and management, the use of microbial agents, replacing chemical fertilizers and pesticides, not only effectively prevents ginseng diseases but also ensures the safety of the medicinal components of the ginseng root. However, selecting the appropriate bacterial strain is crucial to this approach.

[0003] Mortierella, a member of the Zygomycetes, Mucorales, and Mortierellaceae families, is primarily found in soil, plant debris, and animal feces. It is also a key saprophytic fungus in soil and is widely found in forest soils. Mortierella can be used to produce arachidonic acid (ARA), promote plant growth, remediate pollution, and prepare immunomodulators and anti-tumor ingredients. However, there are currently no reports on the use of Mortierella alpina to control the pathogen that causes ginseng rust. Summary of the Invention

[0004] In view of the problems existing in the prior art, the present invention provides a Mortierella alpina strain and its application.

[0005] The technical solution of the present invention to solve the above technical problems is as follows:

[0006] The present invention provides a strain of Mortierella alpina, the strain name is Mortierella alpina (Mortierella alpina) RS-01, the preservation number is CGMCC No.41944, the preservation time is May 21, 2025, and it is deposited in the General Microbiology Center (CGMCC) of the China Culture Collection Administration of Microorganisms, and the preservation location is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, China, Institute of Microbiology, Chinese Academy of Sciences.

[0007] The Mortierella alpina RS-01 provided by the present invention can prevent and control plant pathogens and has the advantages of good prevention and control effect, a broad antibacterial spectrum, and the like.

[0008] The present invention provides a bacterial agent, comprising the above-mentioned Mortierella alpina or the fermentation liquid of the above-mentioned Mortierella alpina.

[0009] The present invention has no special requirements for the dosage form of the microbial agent, and can be a solid preparation, a liquid preparation, or other preparation types. The microbial agent may also include other ingredients, such as excipients, etc. The microbial agent may also include other microorganisms.

[0010] The present invention provides a fermentation method of the Mortierella alpina, comprising the following steps: inoculating the Mortierella alpina into a culture medium for fermentation and culturing.

[0011] For example, the fermentation medium may be PD liquid medium, the culture temperature may be 28° C., and the culture time may be 6 days.

[0012] The fermentation liquid prepared by the above method can prevent and control plant pathogenic bacteria, and has the advantages of good prevention and control effect and a broad antibacterial spectrum.

[0013] The present invention provides a preparation method of the bacterial agent, comprising the following steps: inoculating the above-mentioned Mortierella alpina into a culture medium for fermentation and culturing.

[0014] For example, the fermentation medium may be PD liquid medium, the culture temperature may be 28° C., and the culture time may be 6 days.

[0015] Furthermore, the method further comprises the steps of centrifuging and taking the supernatant.

[0016] The microbial agent prepared by the above method can prevent and control plant pathogenic bacteria and has the advantages of good prevention and control effect and a broad antibacterial spectrum.

[0017] The present invention provides use of the Mortierella alpina or the bacterial agent in preparing a biocontrol preparation.

[0018] The present invention provides use of the above-mentioned Mortierella alpina or the above-mentioned bacterial agent in preventing and controlling plant pathogens.

[0019] The present invention provides a biological control method, comprising the following steps: using the above-mentioned Mortierella alpina or the above-mentioned bacterial agent to control plant pathogens.

[0020] The above-mentioned plant pathogens may include one or more of the pathogens of ginseng rust, melon wilt, melon and fruit rot, apple fruit rot, corn leaf blight, seedling damping-off, walnut canker, poplar canker, pepper anthracnose, blueberry stem canker, apple shell monochromatic septoria, and tobacco brown spot.

[0021] The Mortierella alpina RS-01 provided by the invention has a biological control effect on ginseng rust.

[0022] The present invention isolated fungi from different samples collected in Jilin Province, and conducted an initial screening targeting the pathogen of ginseng rust rot. 12 pathogens that seriously harm agriculture and forestry, such as the pathogen of blueberry stem canker and the pathogen of apple fruit rot, were then rescreened. It was found that the RS-01 strain had a strong antibacterial effect and a broad antibacterial spectrum. Both its live bacteria and fermentation broth had significant inhibitory effects on the 12 test pathogens. Among them, the strain RS-01 had the highest inhibition rate against the pathogen of ginseng rust rot, which was 77.43%. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 This is the confrontation culture of strain RS-01 against the pathogen of ginseng rust rot, among which A1 is the control group and A2 is the confrontation culture of RS-01 against the pathogen of ginseng rust rot.

[0024] Figure 2 The figures show the inhibitory effect of strain RS-01 on different pathogens, where label 1 represents the control group, label 2 represents the experimental group, A represents the pathogen of ginseng rust, B represents the pathogen of melon wilt, C represents the pathogen of citrullus rot, D represents the pathogen of apple fruit rot, E represents the pathogen of corn leaf blight, F represents the pathogen of seedling damping-off, G represents the pathogen of walnut canker, H represents the pathogen of poplar canker, I represents the pathogen of pepper anthracnose, J represents the pathogen of blueberry stem canker, K represents the pathogen of apple shell monochromatic septoria, and L represents the pathogen of tobacco brown spot disease.

[0025] Figure 3 The antibacterial activity of RS-01 fermentation liquid against different pathogens is shown in Figure 1, where label 1 represents the control group, label 2 represents the experimental group, A represents the pathogen of ginseng rust, B represents the pathogen of melon wilt, C represents the pathogen of citrullus rot, D represents the pathogen of apple fruit rot, E represents the pathogen of corn leaf blight, F represents the pathogen of seedling damping-off, G represents the pathogen of walnut canker, H represents the pathogen of poplar canker, I represents the pathogen of pepper anthracnose, J represents the pathogen of blueberry stem canker, K represents the pathogen of apple shell unicolor septoria, and L represents the pathogen of tobacco brown spot disease.

[0026] Figure 4 Phylogenetic analysis of strain RS-01 and related strains. DETAILED DESCRIPTION

[0027] The principles and features of the present invention are described below with reference to the accompanying drawings. The examples given are only used to explain the present invention and are not used to limit the scope of the present invention.

[0028] The present invention isolated 6 fungi from soil and plant tissues under different vegetation in Jilin Province. Through primary screening and fermentation broth rescreening, an antagonistic strain RS-01 capable of effectively inhibiting ginseng rust rot pathogen was obtained. This strain is a highly effective biocontrol strain for ginseng rust rot.

[0029] The present invention uses the live bacteria confrontation method and the poison-containing plate method to determine the antibacterial effect of Mortierella alpina and its fermentation liquid. The taxonomic position of the strain was determined through morphological observation and phylogenetic analysis. The research results show that the strain exhibits inhibitory effects on 12 pathogens that are common and seriously harmful in agricultural and forestry production, and has a broad antibacterial spectrum, especially the inhibitory effect on the pathogens of ginseng rust. In the live bacteria confrontation test, the inhibition rate reached 77.43%, and in the fermentation liquid poison-containing plate method, the inhibition rate was as high as 81.41%. Through morphological characteristics and phylogenetic tree construction analysis based on Bayesian inference, the strain was confirmed to be Mortierella alpina. Strain RS-01 has demonstrated excellent biological control potential and has been used for the control of ginseng rust for the first time, providing a new bacterial species resource for the development of biocontrol agents for ginseng rust.

[0030] The test plant pathogens: Dothiorella gregari, the pathogen of walnut canker, Ilyonectria radicicola, the pathogen of ginseng rust, Botryosphaeria dothidea, the pathogen of poplar canker, Monilinia polystroma, the pathogen of apple fruit rot, and Fusarium oxysporum, the pathogen of seedling damping-off, were isolated and preserved by the Institute of Forestry Resources Protection, Jilin Academy of Forestry Sciences. The pathogen of corn leaf blight Exserohilum turcicum, the pathogen of melon wilt Fusarium oxysporium, the pathogen of pepper anthracnose Colletotrichum capsici, the pathogen of tobacco brown spot Alternaria alternata, and the pathogen of melon and fruit rot Pythium aphanidermatum were donated by the Plant Virology Laboratory of Shenyang Agricultural University. The pathogen of apple shell monochromatic septoria Botryosphaeria stevensii and the pathogen of blueberry stem canker Diaporthe australafricana were provided by Researcher Li Xin of Dalian Customs Technical Center.

[0031] The above plant pathogens are all available to the public for non-commercial purposes only and are used to repeat the examples described in the present invention.

[0032] Test samples: corn soil collected in August 2023 from Eru Township, Qianguo County, Songyuan City, Jilin Province (45°05′03″N, 124°37′15″E), healthy corn stalks from Eru Township, Qianguo County, Songyuan City, Jilin Province (45°05′03″N, 124°37′15″E), wood piles from Eru Township, Qianguo County, Songyuan City, Jilin Province (45°05′03″N, 124°37′15″E), and rice straw from Eru Township, Qianguo County, Songyuan City, Jilin Province (45°05′03″N, 124°37′15″E).

[0033] Test reagents: Ezup column-type fungal genomic DNA extraction kit, purchased from Shanghai Sangon Biotech Co., Ltd.; Taq PCR Master Mix, purchased from Shanghai BBI Bioscience Services Co., Ltd.

[0034] PDA plates were prepared according to the following ratios: 1.0 L potato extract, 20.0 g glucose, 15.0 g agar, and natural pH.

[0035] PD liquid culture medium was prepared according to the following ratio: 1.0 L potato extract and 20.0 g glucose.

[0036] The preparation method of the potato extract comprises: taking 200 g of peeled potatoes, cutting them into small pieces, adding 1.0 L of water and boiling for 30 minutes, filtering out the potato pieces, and filling the filtrate to 1.0 L.

[0037] Instruments: BX53 Olympus optical microscope, Olympus Corporation; S-3400N scanning electron microscope, HITACHI Corporation; PCR instrument, Applied Biosystems.

[0038] Where specific techniques or conditions are not specified in the examples, all methods were performed according to conventional methods, techniques or conditions described in literature in the field, or according to product specifications. Reagents and instruments used, for which the manufacturers are not specified, are conventional products that can be purchased through regular channels.

[0039] In the embodiments, when data analysis is involved, SPSS 25.0 software was used for statistical analysis, and the new multiple range method was used for difference significance analysis.

[0040] The following is an introduction through specific embodiments.

[0041] Example 1 Isolation and purification of antagonistic strains

[0042] The strain screening was carried out by the stepwise dilution and spreading method, which included the following steps: 10 g of soil, straw, corn stalks, and wood pile were baked at 60 °C for 1 h, added to 100 mL of sterile water, and shaken at 100 rpm for 30 min. 1 mL of the suspension was drawn and diluted with sterile water at 10 °C. -3 , 10 -4 and 10 -5 Perform a gradient dilution by pipetting 0.1 mL of each dilution solution at different concentrations onto a PDA plate and evenly spreading the solution. Place the plate upside down in a 28°C incubator and incubate for 3-10 days. Observe and select colonies with distinct morphologies every 24 hours. Purify the colonies 3-5 times before transferring them to a PDA slant and incubating them for 5-7 days. Number the colonies and store them in a 4°C refrigerator until ready for use. Initial screening of strains was performed using the above method.

[0043] After different strains were isolated and purified, live bacteria confrontation experiments were conducted using the pathogen of ginseng rust as the target. The experimental method included the following steps:

[0044] (1) Activation of ginseng rust pathogen: Prepare a PDA plate with a culture medium suitable for bacterial growth, restore the bacterial strain from the preserved state to room temperature, inoculate the preserved bacterial strain onto the PDA plate and culture it at 28°C for 7 days, select the colonies that grow vigorously on the PDA plate and inoculate them onto a new PDA plate, culture it at 28°C for 7 days, and repeat this step 2-3 times to obtain well-grown ginseng rust pathogen colonies.

[0045] (2) The activated ginseng rust pathogen was inoculated on a PDA plate and cultured at 28°C for 7 days for later use.

[0046] (3) Experimental group: All strains isolated from the initial screening were inoculated on PDA plates. When each strain grew vigorously, the pathogen of ginseng rust was used as the target bacteria, and the inhibitory activity of the initial screening strains on the pathogen of ginseng rust was evaluated by plate confrontation culture method. The specific operation included: the pathogen of ginseng rust and the initial screening strain were made into 5mm diameter cakes, which were placed on a 90mm diameter PDA plate. The ginseng rust pathogen and the initial screening strain were located on both sides of the center of the plate, and the center of the ginseng rust pathogen cake, the center of the initial screening strain cake, and the center of the plate were in a straight line. The center of the ginseng rust pathogen cake was 2.5cm away from the center of the plate, and the center of the initial screening strain cake was 2.5cm away from the center of the plate. After the plate was cultured at a constant temperature of 28℃ for 72h, the growth diameter of the ginseng rust pathogen was measured by cross measurement method.

[0047] Control group: Based on the experimental group, the agar cake on the PDA plate (5 mm in diameter) was used instead of the primary screening strain, and the rest were the same as the experimental group.

[0048] Each of the above treatments was repeated three times.

[0049] (4) The antibacterial rate was calculated using the following formula: Antibacterial rate (%) = (colony diameter of the control group - colony diameter of the treated group) / colony diameter of the control group × 100%.

[0050] The experimental results are shown in Table 1. Among the molds isolated and purified in the same batch, 6 strains had inhibition zone diameters of less than 50 mm after confrontation culture with the ginseng rust pathogen. In particular, strain RS-01 showed the best antibacterial effect, with an inhibition zone diameter of only 20.312 mm and an inhibition rate of 77.43%. Figure 1 The results of the confrontation culture experiment of strain RS-01 against the pathogen of ginseng rust rot were shown in Figure 2. Therefore, strain RS-01 was selected for subsequent experiments.

[0051] Table 1 Antibacterial activity of Mortierella alpina against ginseng rust pathogens

[0052]

[0053] Note: The data in the table are mean ± standard deviation.

[0054] Example 2 Determination of antibacterial activity and antibacterial spectrum of strain RS-01

[0055] The RS-01 strain was selected for determination of the antibacterial spectrum.

[0056] The plant pathogens tested were ginseng rust pathogen (I. radicicola), melon wilt pathogen (F. oxysporium), melon fruit rot pathogen (P. aphanidermatum), apple fruit rot pathogen (M. polystroma), corn leaf blight pathogen (E. turcicum.), seedling damping-off pathogen (F. oxysporum), walnut canker pathogen (D. gregari), poplar canker pathogen (B. dothidea), pepper anthracnose pathogen (C. Capsici), blueberry stem canker pathogen (D. australa africana), apple shell monochromatic septum spore pathogen (B. stevensii) and tobacco brown spot pathogen (A. alternata).

[0057] The antibacterial activity of strain RS-01 against the aforementioned plant pathogens was verified. The test pathogens were first activated. The specific process included preparing PDA plates, returning the test pathogens to room temperature from a stored state, inoculating the stored test pathogens onto the PDA plates and incubating them at 28°C for 7 days. Strongly growing colonies on the PDA plates were selected and inoculated onto fresh PDA plates and incubated at 28°C for another 7 days. This process was repeated 2-3 times to obtain well-grown test pathogen colonies.

[0058] A total of 12 treatments were set up, each with an experimental group and a control group. The experimental group was inoculated with both the test pathogen and strain RS-01. In addition to the experimental group, the control group was inoculated with agar cake (5 mm in diameter) on a PDA plate instead of strain RS-01. All other conditions were the same as the experimental group. The experimental method was similar to that in Example 1.

[0059] The results of the antibacterial spectrum determination of strain RS-01 are shown in Table 2 and Figure 2 As shown, the growth of the tested pathogens was inhibited to varying degrees, and strain RS-01 had a broad antibacterial spectrum. Among them, strain RS-01 had the strongest inhibitory effect on the pathogen of ginseng rust. After confrontation culture, the diameter of the pathogen was only 20.31 mm, and the inhibition rate was as high as 77.43%, which was significantly different from the other tested pathogens. It also had a strong inhibitory effect on the pathogen of pepper anthracnose, the pathogen of blueberry stem canker and the pathogen of walnut canker, and the growth of the pathogens was inhibited. When the diameter of the pathogen in the control reached 90 mm, the diameters of the pathogens in confrontation culture of strain RS-01 were 24.10 mm, 26.33 mm and 24.37 mm, respectively, and the inhibition rates were 73.22%, 70.74% and 72.90%, respectively. The inhibitory effect on the pathogen of poplar canker was relatively weak, but when the control filled the dish, the diameter of the pathogen was 45.73 mm, and the inhibition rate could still reach 49.19%.

[0060] Table 2 Inhibitory spectrum of live bacteria of Mortierella alpina RS-01 on toxic plates

[0061] pathogens Diameter of live bacteria colony (mm) Antibacterial rate (%) I. radicicola, the pathogen of ginseng rust 20.31±3.09d 77.43a Melon wilt pathogen F.oxysporium 30.83±3.29c 65.74bc Pythium aphanidermatum 34.34±4.22b 61.84bcd Apple fruit rot pathogen M. polystroma 31.42±3.21bc 65.09c E. turcicum, the pathogen of corn leaf blight 41.47±2.51a 53.93cd F.oxysporum, the pathogen of seedling damping-off disease 33.64±2.32b 62.62c Walnut canker pathogen D.gregari 24.37±1.44cd 72.90ab Poplar canker pathogen B. dothidea 45.73±3.44ab 49.19cd Pepper anthracnose pathogen C. Capsici 24.10±3.14d 73.22ab Blueberry stem canker pathogen D. australafricana 26.33±4.28bcd 70.74b The pathogen of apple shell septoria unicolor, B. stevensii. 30.44±4.45c 66.18b Tobacco brown spot pathogen A. alternata 37.08±1.51ab 53.80d

[0062] Note: Data in the table are mean ± standard deviation. Different letters after the data in the same column indicate significant differences at the P < 0.05 level as tested by Duncan's new multiple range test.

[0063] Example 3 Determination of the antibacterial spectrum of RS-01 fermentation broth

[0064] Set up experimental and control groups.

[0065] (1) Experimental group:

[0066] The plant pathogens tested were ginseng rust pathogen (I. radicicola), melon wilt pathogen (F. oxysporium), melon fruit rot pathogen (P. aphanidermatum), apple fruit rot pathogen (M. polystroma), corn leaf blight pathogen (E. turcicum.), seedling damping-off pathogen (F. oxysporum), walnut canker pathogen (D. gregari), poplar canker pathogen (B. dothidea), pepper anthracnose pathogen (C. Capsici), blueberry stem canker pathogen (D. australa africana), apple shell monochromatic septum spore pathogen (B. stevensii) and tobacco brown spot pathogen (A. alternata).

[0067] Preparation of RS-01 bacterial cakes involves inoculating activated Mortierella alpina RS-01 onto a PDA plate. The specific process involves returning the RS-01 strain from storage to room temperature, inoculating the preserved RS-01 strain onto the PDA plate, and incubating at 28°C for 7 days. Selecting robust colonies grown on the PDA plate, inoculating them onto a new PDA plate, and incubating them at 28°C for another 7 days. Repeat this process 2-3 times to obtain well-grown colonies. Set aside. Use a sterile workbench to create RS-01 bacterial cakes with a diameter of 5 mm using a microporator.

[0068] Add 100 mL of PD liquid medium to a 200 mL Erlenmeyer flask, add six RS-01 bacterial cakes (5 mm in diameter), and incubate at 28°C, 150 rpm, and constant temperature shaking for 6 days. After incubation, centrifuge at 7800 rpm and 25°C for 10 minutes to remove the precipitate. Collect the supernatant as RS-01 fermentation broth for subsequent experiments.

[0069] Add 5 mL of RS-01 fermentation broth prepared using the above method to 50 mL of PDA and shake well to obtain a mixture. This mixture was evenly distributed among three 90 mm diameter Petri dishes to create PDA-containing plates. Next, a 5 mm diameter cake of the test plant pathogen was placed in the center of the plate. Incubate for another 5 days at 25°C.

[0070] (2) Control group: Based on the experimental group, the RS-01 fermentation broth was replaced with an equal amount of PD liquid culture medium, and the rest was the same as the experimental group.

[0071] (3) Each of the above treatments was repeated three times, and the growth diameter of the test plant pathogens was measured using the cross-cross method. The inhibition rate was calculated using the method of Example 1.

[0072] The experimental results are shown in Table 3 and Figure 3As shown, the antibacterial activity of the fermentation broth of the antagonistic strain RS-01 was determined using the toxin-containing plate method. The RS-01 fermentation broth exhibited high antibacterial activity, demonstrating the strongest antibacterial activity against the pathogen of ginseng rust, with a pathogen diameter of only 16.73 mm and an inhibition rate of 81.41%. It also exhibited strong antagonistic activity against the pathogens of blueberry stem canker and walnut canker, with pathogen diameters of 19.88 mm and 22.30 mm, respectively, and inhibition rates of 77.92% and 75.22%, respectively. It also demonstrated strong inhibitory activity against the pathogens of pepper anthracnose, citrus rot, and melon wilt, with pathogen diameters of 26.02 mm, 26.31 mm, and 25.44 mm, respectively, with inhibition rates of 71.09%, 70.77%, and 71.74%, respectively. These results demonstrate that the fermentation broth of this antagonistic strain exhibits excellent inhibitory activity against all tested pathogens.

[0073] Table 3 Antibacterial spectrum of the toxic plate containing Mortierella alpina RS-01 fermentation liquid

[0074] pathogens Diameter of bacterial colonies in fermentation broth (mm) Antibacterial rate (%) I. radicicola, the pathogen of ginseng rust 16.73±2.55d 81.41a Melon wilt pathogen F.oxysporium 25.44±3.57bcd 71.74b Pythium aphanidermatum 26.31±2.41c 70.77bc Apple fruit rot pathogen M. polystroma 27.80±2.95bc 69.12bc E. turcicum, the pathogen of corn leaf blight 31.19±2.29ab 65.35cd F.oxysporum, the pathogen of seedling damping-off disease 30.03±2.47a 66.63d Walnut canker pathogen D. gregari 22.30±2.10cd 75.22ab Poplar canker pathogen B. dothidea 30.47±4.42b 66.14bcd Pepper anthracnose pathogen C. Capsici 26.02±2.28c 71.09b Blueberry stem canker pathogen D. australafricana 19.88±1.42cd 77.92a The pathogen of apple shell septoria unicolor, B. stevensii. 31.66±4.32a 64.83d Tobacco brown spot pathogen A. alternata 28.26±5.28b 68.61c

[0075] Note: Data in the table are mean ± standard deviation. Different letters after the data in the same column indicate significant differences at the P < 0.05 level as tested by Duncan's new multiple range test.

[0076] Example 4 Morphological identification of strain RS-01

[0077] The RS-01 strain was inoculated onto a PDA plate, and a sterilized coverslip was inserted into the culture medium at a 45° angle. After culturing at 28°C for 3-5 days, the coverslip was removed and the morphological characteristics of the strain were observed under an optical microscope. At the same time, strains with good growth status were selected for scanning electron microscopy observation and photographed.

[0078] When strain RS-01 was cultured on a PDA plate at 28°C for 24-48 hours, the colony was white and velvety with dense hyphae. After 3-5 days of culture, the hyphae gradually thickened and formed layers, with sporangiophores growing from the aerial hyphae without microsporangiospores, unbranched sporangia, terminal and spherical, without a spindle, easily broken membrane, and multiple spores. The colony surface was flat, with a light yellow back, no exudate, and no soluble pigment. Electron microscopy revealed that the conidiophores had no patterns on their surface, the phialides were closely arranged, and the spores were accumulated in chains or clusters without accessory structures.

[0079] Example 5 Molecular Biological Identification of Strain RS-01

[0080] The DNA of RS-01 strain was extracted using the Ezup column-based fungal genomic DNA extraction kit.

[0081] Primers ITS1 and ITS4 were used for amplification. Primers ITS1 and ITS4 were synthesized by Kumei.

[0082] ITS1: 5′-TCCGTAGGTGAACCTGCGG-3′ (SEQ ID NO: 1);

[0083] ITS4: 5'-TCCTCCGCTTATTGATATGC-3' (SEQ ID NO: 2).

[0084] The PCR reaction system includes: ITS1 (10 μmol / L) 2 μL, ITS4 (10 μmol / L) 2 μL, Taq PCR MasterMix 25 μL, DNA template 1 μL, ddH2O 20 μL.

[0085] The PCR reaction conditions included: pre-denaturation at 95°C for 5 min; denaturation at 95°C for 30 s, annealing at 55°C for 50 s, and extension at 72°C for 1 min, 39 cycles; and final extension at 72°C for 10 min.

[0086] The amplified products were sent to Changchun Kumei Biotechnology Co., Ltd. for bidirectional sequencing. The sequences were analyzed and manually corrected and spliced ​​using software such as BioEdit7.0.1. The spliced ​​sequences were logged into the BLAST page in NCBI for comparison. Partial sequences of similar species that had been logged in were downloaded from GenBank, and sequence alignment was performed using Clustal W. Multi-gene sequence alignment was performed using the concatenate sequence (CS) method of PhyloSuite software, and the phylogenetic tree was viewed using FigTree v1.4.3 software.

[0087] The comparison method can refer to the following literature:

[0088] Thompson JD,Higgins DG,Gibson TJ.CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice[J]. Nucleic acidsresearch, 1994, 22(22):4673-4680.

[0089] Ronquist F,Teslenko M,van der Mark P,et al.MrBayes 3.2: efficientBayesian phylogenetic inference and model choice across a large model space[J].Systematic Biology,2012,61,539-542.

[0090] Zhang D,Gao FL, I, et al. Phylosuite: an integrated and scalable desktop platform for streamlined molecular sequence data management and evolutionary phylogenetics studies [J]. Molecular Ecology Resources, 2020, 20, 348-355.

[0091] The ITS gene of the RS-01 strain was amplified and sequenced to obtain a fragment sequence as shown in SEQ ID NO: 3.

[0092] (SEQ ID NO: 3).

[0093] Eighteen sequences with high homology to strain RS-01 were selected from NCBI, and phylogenetic analysis of strain RS-01 was performed using I TS. Figure 4 As shown in the figure, strain RS-01 and Mortierella alpina clustered together in a clade with a 99% posterior probability, indicating that they were sister clades. Strain RS-01 and other Mortierella species (M. thaxteri and M. sp.) belonged to separate clades. Therefore, combined with morphological and molecular biological identification, RS-01 was clearly identified as Mortierella alpina.

[0094] The strain RS-01 was deposited in the General Microbiology Center of the China Culture Collection Administration of Microorganisms (CGMCC) on May 21, 2025, at the Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, China, with the deposit number CGMCC No. 41944. The name at the time of deposit was Mortierella alpina RS-01, and the proposed taxonomic name was Mortierella alpina.

[0095] Mortierella alpina can regulate the microbial composition of ginseng roots, stabilize the microbial community structure of ginseng roots, while increasing soil sucrase activity and reducing the abundance of harmful bacteria in the ginseng rhizosphere soil, thereby helping ginseng resist the invasion of pathogens.

[0096] The present invention isolated 6 fungi from different forest soils and host plants in Jilin Province, and conducted an initial screening targeting the pathogen of ginseng rust rot. 12 pathogens that seriously harm agriculture and forestry, such as the pathogen of blueberry stem canker and the pathogen of apple fruit rot, were then rescreened. Among them, the RS-01 strain had a strong antibacterial effect (an inhibition rate of 77.43%) and a broad antibacterial spectrum. Its fermentation broth had a significant inhibitory effect on all 12 test pathogens. Among them, the RS-01 fermentation broth had the strongest antibacterial effect on the pathogen of ginseng rust rot, with an inhibition rate of 81.41%.

[0097] Sequences with high homology to strain RS-01 were selected from NCBI. Phylogenetic analysis of strain RS-01 confirmed that strain RS-01 and the Mortierella alpina strain submitted to GenBank clustered in a clade with a 99% posterior probability. This clade is sister to Mortierella alpina and distinct from other Mortierella species (M. globalpina and M. sp). Therefore, combined morphological and molecular identification confirmed that RS-01 is a Mortierella alpina strain.

[0098] In summary, strain RS-01 has bactericidal activity, and its live bacteria and fermentation liquid both show strong inhibitory effects against ginseng rust pathogens, and have the advantage of a broad antibacterial spectrum.

[0099] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A Mortierella alpina strain, characterized in that The strain is named Mortierella alpina RS-01, and its deposit number is CGMCC No.41944.

2. A bacterial agent, characterized in that The method comprises the Mortierella alpina described in claim 1 or the fermentation liquid of the Mortierella alpina described in claim 1.

3. The fermentation method of Mortierella alpina according to claim 1, wherein The following steps are involved: The Mortierella alpina described in claim 1 is inoculated into a culture medium for fermentation.

4. Use of the Mortierella alpina described in claim 1 in preparing a biocontrol agent.

5. Use of the microbial agent according to claim 2 in the preparation of biocontrol agents.

6. Use of the Mortierella alpina described in claim 1 in preventing and controlling plant pathogens.

7. The application according to claim 6, characterized in that The plant pathogens include one or more of the pathogens of ginseng rust, melon wilt, melon fruit rot, apple fruit rot, corn leaf blight, seedling damping-off, walnut canker, poplar canker, pepper anthracnose, blueberry stem canker, apple shell monochromatic septoria, and tobacco brown spot.

8. Use of the microbial agent according to claim 2 in preventing and controlling plant pathogenic bacteria.

9. The application according to claim 8, characterized in that: The plant pathogens include one or more of the pathogens of ginseng rust, melon wilt, melon fruit rot, apple fruit rot, corn leaf blight, seedling damping-off, walnut canker, poplar canker, pepper anthracnose, blueberry stem canker, apple shell monochromatic septoria, and tobacco brown spot.

10. A biological control method, characterized in that: The method comprises the following steps: using the Mortierella alpina described in claim 1 or the microbial agent described in claim 2 to control plant pathogenic bacteria.

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