Application of mortierella alpina agent

Abstract

The invention relates to application of a mortierella alpina agent, which is prepared by the following method: inoculating mortierella alpina into a culture medium for fermentation culture, centrifuging, and taking supernate; the name of the bacterial strain of the mortierella alpina is Mortierella alpina RS-01, and the preservation number of the mortierella alpina is CGMCC (China General Microbiological Culture Collection Center) No.41944. The invention further discloses a preparation method of the mortierella alpina. The mortierella alpina agent provided by the invention can be used for preventing and treating pathogenic bacteria of ginseng rust rot, pathogenic bacteria of muskmelon fusarium wilt, pathogenic bacteria of melon and fruit rot, 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 and pathogenic bacteria of pepper anthracnose; the compound has the best antibacterial effect on pathogenic bacteria of blueberry stem canker, pathogenic bacteria of apple shell monochromatic alternaria, pathogenic bacteria of tobacco brown spot, and especially pathogenic bacteria of ginseng rust rot.

Description

[0001] This invention is a divisional application. The original Chinese invention patent application number was 202510872493.6, the application date was June 26, 2025, and the patent title at the time of application was: A strain of alpine spores and its application. Technical Field

[0002] This invention belongs to the field of microbial technology, and in particular relates to the application of an alpine spore fungicide. Background Technology

[0003] Ginseng, a perennial medicinal plant belonging to the genus *Panax* of the Araliaceae family, possesses high medicinal value. Due to prolonged continuous cropping and improper pesticide use, ginseng diseases have worsened year by year, directly impacting yield and quality. Among these, ginseng rust rot, which can affect all stages of ginseng's growth, is the most serious root disease, with an average incidence rate of 15%-30%, exceeding 70% in severe cases, leading to significant economic losses. Due to its environmentally friendly and sustainable characteristics, microbial agents are increasingly widely used to suppress soil-borne diseases in ginseng. In ginseng production management, using microbial agents to replace chemical fertilizers and pesticides not only effectively prevents ginseng diseases but also ensures the safety of the medicinal components in ginseng roots. However, selecting suitable strains is crucial to this method.

[0004] Morphyra ( Mortierella *Mortierellaceae*, belonging to the class Zygomycetes, order Mucorales, and family Mortierellaceae, is mainly found in soil, plant debris, and animal feces. It is also an important saprophytic fungus in soil, widely distributed in forest soils. *Mortierellaceae* can be used to produce arachidonic acid (ARA), promote plant growth, remediate pollution, and prepare immunomodulators and antitumor components. However, there are currently no reports on the use of *Mortierellaceae* in controlling ginseng rust rot pathogens. Summary of the Invention

[0005] In view of the problems existing in the prior art, the present invention provides an application of alpine spore fungicide.

[0006] The technical solution of the present invention to solve the above-mentioned technical problems is as follows:

[0007] This invention provides a strain of *Morchella alpineensis*, named *Morchella alpineensis* (… Alpine Mortierella RS-01, accession number CGMCC No.41944, deposited on May 21, 2025, is deposited 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, China.

[0008] The alpine spores RS-01 provided by this invention can control plant pathogens and has the advantages of good control effect and broad antibacterial spectrum.

[0009] The present invention provides a microbial agent comprising the above-mentioned *Morchella alpina* or the fermentation broth of the above-mentioned *Morchella alpina*.

[0010] This invention does not impose any special requirements on the dosage form of the microbial agent; it can be a solid dosage form, a liquid dosage form, or other dosage forms. The microbial agent may also include other components, such as excipients. The microbial agent may also include other microorganisms.

[0011] The present invention provides a fermentation method for the above-mentioned alpine mollusc, comprising the following steps: fermenting the above-mentioned alpine mollusc inoculated culture medium.

[0012] For example, the fermentation medium can be PD liquid medium, the culture temperature can be 28°C, and the culture time can be 6 days.

[0013] The fermentation broth prepared by the above method can control plant pathogens and has the advantages of good control effect and broad antibacterial spectrum.

[0014] The present invention provides a method for preparing the above-mentioned microbial agent, comprising the following steps: fermenting the above-mentioned *Morchella alpineensis* inoculation medium.

[0015] For example, the fermentation medium can be PD liquid medium, the culture temperature can be 28°C, and the culture time can be 6 days.

[0016] Furthermore, it also includes the steps of centrifugation and collecting the supernatant.

[0017] The microbial agent prepared by the above method can control plant pathogens and has the advantages of good control effect and broad antibacterial spectrum.

[0018] This invention provides the application of the above-mentioned alpine spores or the above-mentioned fungal agent in the preparation of biocontrol agents.

[0019] This invention provides the application of the above-mentioned alpine spores or the above-mentioned fungal agents in the prevention and control of plant pathogens.

[0020] This invention provides a biological control method, comprising the following steps: using the above-mentioned alpine spores or the above-mentioned fungal agent to control plant pathogens.

[0021] The aforementioned plant pathogens may include one or more of the following: ginseng rust rot pathogen, melon wilt pathogen, fruit rot pathogen, apple fruit rot pathogen, corn leaf spot pathogen, seedling damping-off pathogen, walnut canker pathogen, poplar canker pathogen, pepper anthracnose pathogen, blueberry stem canker pathogen, apple shell monochromatic spore pathogen, and tobacco red spot pathogen.

[0022] The alpine spores RS-01 provided by this invention have a biological control effect on ginseng rust disease.

[0023] This invention isolated fungi from different samples collected in Jilin Province. Initial screening was conducted using the ginseng rust pathogen as the target, followed by secondary screening of 12 other pathogens that cause serious damage to agriculture and forestry, such as the blueberry stem canker pathogen and the apple fruit rot pathogen. 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 showed significant inhibitory effects on the 12 tested pathogens. Among them, the RS-01 strain had the highest inhibition rate against the ginseng rust pathogen, at 77.43%. Attached Figure Description

[0024] Figure 1 The culture was a confrontation culture of strain RS-01 against the pathogen of ginseng rust, where A1 was the control group and A2 was the confrontation culture of RS-01 against the pathogen of ginseng rust.

[0025] Figure 2 The inhibitory effects of strain RS-01 on different pathogens are shown. In the figure, 1 represents the control group and 2 represents the experimental group. A is the pathogen of ginseng rust rot, B is the pathogen of melon wilt, C is the pathogen of fruit rot, D is the pathogen of apple fruit rot, E is the pathogen of corn leaf blight, F is the pathogen of seedling damping-off, G is the pathogen of walnut canker, H is the pathogen of poplar canker, I is the pathogen of pepper anthracnose, J is the pathogen of blueberry stem canker, K is the pathogen of apple shell monochromatic spore disease, and L is the pathogen of tobacco red spot disease.

[0026] Figure 3 The study investigated the antibacterial activity of RS-01 fermentation broth against different pathogens. In the experimental group, group 1 represents the control group, group 2 represents the experimental group, and A represents the pathogen causing ginseng rust rot, B represents the pathogen causing melon wilt, C represents the pathogen causing fruit rot, D represents the pathogen causing apple fruit rot, E represents the pathogen causing corn leaf blight, F represents the pathogen causing seedling damping-off, G represents the pathogen causing walnut canker, H represents the pathogen causing poplar canker, I represents the pathogen causing pepper anthracnose, J represents the pathogen causing blueberry stem canker, K represents the pathogen causing apple shell monochromatic spore disease, and L represents the pathogen causing tobacco red spot disease.

[0027] Figure 4 Phylogenetic analysis of strain RS-01 and related strains. Detailed Implementation

[0028] The principles and features of the present invention are described below with reference to the accompanying drawings. The examples given are only for explaining the present invention and are not intended to limit the scope of the present invention.

[0029] This invention isolated six fungi from soil and plant tissues under different vegetation in Jilin Province. Through primary screening and secondary screening of fermentation broth, one antagonistic strain RS-01 was obtained, which can effectively inhibit the pathogen of ginseng rust rot. This strain is a highly efficient biocontrol strain for the biological control of ginseng rust rot.

[0030] This invention uses the live-cell confrontation method and the toxin-containing plate method to determine the antibacterial effect of *Morchella alpineensis* and its fermentation broth. The taxonomic position of this strain was determined through morphological observation and phylogenetic analysis. The results show that this strain exhibits inhibitory effects against 12 pathogens commonly occurring and causing serious damage in agricultural and forestry production, and possesses a broad spectrum of inhibition, especially against the pathogen causing ginseng rust. In the live-cell confrontation test, the inhibition rate reached 77.43%, and in the toxin-containing plate method of the fermentation broth, the inhibition rate was as high as 81.41%. Through morphological characteristics and phylogenetic tree construction based on Bayesian inference, this strain was confirmed as *Morchella alpineensis*. Alpine Mortierella Strain RS-01 demonstrated excellent potential for biocontrol and was used for the first time in the control of ginseng rust disease, providing a new strain resource for the development of biocontrol agents for ginseng rust disease.

[0031] Tested plant pathogens: *Walnut canker pathogen* Dothiorella gregari Pathogens causing ginseng rust Ilyonectria radicicola Poplar canker pathogen Botryosphaeria dothidea and the pathogen of apple fruit rot Monilinia polystroma Pathogen of seedling damping-off Fusarium oxysporum Isolated and preserved by the Institute of Forestry Resources Conservation, Jilin Provincial Academy of Forestry Sciences. *Maize leaf spot pathogen* Turkish sedge Melon wilt pathogen Fusarium oxysporum Anthracnose pathogen of pepper Colletotrichum capsici Tobacco red spot pathogen Alternaria alternata Pathogens of Pythium spp. in melons and fruits Pythium aphanidermatum The pathogen of apple shell monochromatic septum, donated by the Plant Virus Research Laboratory of Shenyang Agricultural University. Botryosphaeria stevensii Blueberry stem canker pathogen Diaporthe australafricana Provided by Li Xin, researcher at the Dalian Customs Technology Center.

[0032] All of the above-mentioned plant pathogens are available to the public and are used only for non-commercial purposes to repeat the embodiments described in this invention.

[0033] Test samples: In August 2023, the samples were maize soil, healthy maize stalks, wood piles, and rice straw from Eru Township, Qian Gorlos County, Songyuan City, Jilin Province (45°05′03″ N, 124°37′15″ E), collected from Eru Township, Qian Gorlos County, Songyuan City, Jilin Province (45°05′03″ N, 124°37′15″ E).

[0034] Test reagents: Ezup column-based fungal genomic DNA extraction kit, purchased from Sangon Biotech (Shanghai) Co., Ltd.; Taq PCR Master Mix, purchased from BBI Bioscience Services (Shanghai) Co., Ltd.

[0035] PDA plates are prepared in the following proportions: 1.0 L potato extract, 20.0 g glucose, 15.0 g agar, pH at natural.

[0036] PD liquid culture medium was prepared in the following proportions: 1.0 L potato extract and 20.0 g glucose.

[0037] The preparation method of the above potato extract includes: taking 200 g of peeled potatoes, cutting them into small pieces, adding 1.0 L of water and boiling for 30 min, filtering out the potato pieces, and making up the filtrate to 1.0 L.

[0038] Instruments: Olympus BX53 optical microscope, Olympus Corporation; S-3400N scanning electron microscope, HITACHI Corporation; PCR instrument, Applied Biosystems.

[0039] Unless otherwise specified, all methods used in the examples were conventional or performed according to techniques or conditions described in the literature in this field, or in accordance with the product instructions. Reagents and instruments used without specified manufacturers were all conventional products that could be purchased from legitimate channels.

[0040] In the embodiments, if data analysis is involved, SPSS 25.0 software is used for statistical analysis, and the new multiple range method is used for significance analysis of differences.

[0041] The following is a description through specific embodiments.

[0042] Example 1 Isolation and purification of antagonistic strains

[0043] The strains were screened using a stepwise dilution plating method, including the following steps: 10 g of soil, rice straw, corn stalks, and wood pile were baked at 60℃ for 1 h, then added to 100 mL of sterile water. The mixture was shaken at 100 r / min for 30 min. 1 mL of the suspension was then extracted and diluted with sterile water at a ratio of 10:1. -3 10 -4 and 10 -5 Serial dilutions were performed, with 0.1 mL of each dilution added to a PDA plate and spread evenly. The plates were then inverted and incubated at 28°C for 3-10 days. Colonies with different morphologies were observed and selected every 24 hours. After purifying 3-5 times, the colonies were transferred to PDA slants and incubated for 5-7 days. The colonies were then numbered and stored at 4°C for later use. The strains selected for initial screening were obtained using the above method.

[0044] After different strains were isolated and purified, a live bacterial confrontation experiment was conducted using the pathogen causing ginseng rust as a target. The experimental method included the following steps:

[0045] (1) Activation of ginseng rust pathogen: Prepare a suitable culture medium PDA plate for the growth of the strain, restore the strain from the storage state to the room temperature state, inoculate the stored strain onto the PDA plate and incubate at 28℃ for 7 days, select the colonies that grow well on the PDA plate and inoculate them onto a new PDA plate, incubate at 28℃ for 7 days, and repeat this step 2-3 times to obtain well-grown ginseng rust pathogen colonies.

[0046] (2) Inoculate the activated ginseng rust pathogen onto a PDA plate and incubate at 28°C for 7 days for later use.

[0047] (3) Experimental group: All strains isolated from the initial screening were inoculated onto PDA plates. When the growth of each strain was relatively vigorous, the ginseng rust pathogen was used as the target bacterium, and the inhibitory activity of the initial screening strains against the ginseng rust pathogen was evaluated by plate confrontation culture method. The specific operation included: making ginseng rust pathogen and initial screening strain into 5 mm diameter mycelial cakes and placing them on a 90 mm 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 mycelial cake, the center of the initial screening strain mycelial cake, and the center of the plate were on a straight line. The center of the ginseng rust pathogen mycelial cake was 2.5 cm away from the center of the plate, and the center of the initial screening strain mycelial cake was 2.5 cm away from the center of the plate. After the plates were cultured at a constant temperature of 28℃ for 72 h, the growth diameter of the ginseng rust pathogen was determined by cross measurement method.

[0048] Control group: Based on the experimental group, the initial screening strains were replaced with agar cakes (5 mm in diameter) on PDA plates, and all other aspects were the same as the experimental group.

[0049] Each of the above processes is repeated three times.

[0050] (4) The inhibition rate was calculated using the following formula: Inhibition rate (%) = (Coronary diameter of control group - Coronary diameter of treatment group) / Coronary diameter of control group × 100%.

[0051] The experimental results are shown in Table 1. Among the molds isolated and purified in the same batch, the diameter of the inhibition zone of the pathogen was 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 This is the result of the confrontation culture experiment between strain RS-01 and the pathogen causing ginseng rust. Therefore, strain RS-01 was selected for subsequent experiments.

[0052] Table 1. Antibacterial activity of *Morchella alpina* against the pathogen causing ginseng rust.

[0053]

[0054] Note: The data in the table are mean ± standard deviation.

[0055] Example 2: Determination of the antibacterial activity and antibacterial spectrum of strain RS-01

[0056] The RS-01 strain was selected for determination of its antibacterial spectrum.

[0057] The tested plant pathogens were the ginseng rust pathogen ( I. radicicola ), the pathogen of melon wilt ( F. oxysporium ), Pythium spp. pathogen of melons and fruits ( P. aphanidermatum ), the pathogen of apple fruit rot ( M. polystroma ), the pathogen of maize leaf spot ( E. turcicum .), the pathogen of seedling damping-off ( F. oxysporum ), walnut canker pathogen ( D. to gather ), the pathogen of poplar canker ( B. dothidea ), the pathogen of anthracnose in peppers ( C. Capsicum ), blueberry stem canker pathogen ( D. australafricana ), the pathogen of apple shell monochromatic septum disease ( B. Stevens ) and the pathogen of tobacco red spot disease ( A. alternata ).

[0058] Using the aforementioned plant pathogens as test pathogens, the antibacterial activity of strain RS-01 against the test pathogens was verified. First, the test pathogens were activated. The specific procedures included: preparing PDA plates, restoring the test pathogens from their storage state to room temperature, inoculating the stored test pathogens onto PDA plates and incubating them at 28℃ for 7 days, selecting robust colonies from the PDA plates, inoculating them onto new PDA plates, and continuing incubation at 28℃ for another 7 days. This step was repeated 2-3 times to obtain well-grown test pathogen colonies.

[0059] A total of 12 treatments were set up for AL, with an experimental group and a control group in each treatment. The experimental group was inoculated with the test pathogen and strain RS-01 simultaneously. In addition to the experimental group, the control group was inoculated with agar cakes (5 mm in diameter) on PDA plates instead of strain RS-01, and all other aspects were the same as the experimental group. The experimental method is the same as in Example 1.

[0060] The results of the antibacterial spectrum determination of strain RS-01 are shown in Table 2 and Figure 2 As shown, the growth of all tested pathogens was inhibited to varying degrees, with strain RS-01 exhibiting a broad antibacterial spectrum. Among them, strain RS-01 showed the strongest inhibitory effect against the ginseng rust pathogen, with the pathogen diameter after confrontation culture reaching only 20.31 mm and an inhibition rate as high as 77.43%, significantly different from other tested pathogens. It also showed strong inhibitory effects against the pepper anthracnose pathogen, blueberry stem canker pathogen, and walnut canker pathogen, inhibiting pathogen growth. When the diameter of the control pathogen reached 90 mm, the diameters of the pathogens cultured with strain RS-01 were 24.10 mm, 26.33 mm, and 24.37 mm, respectively, with inhibition rates of 73.22%, 70.74%, and 72.90%. The inhibitory effect against the poplar canker pathogen was relatively weak, but when the control reached full coverage, the pathogen diameter was 45.73 mm, and the inhibition rate still reached 49.19%.

[0061] Table 2. Antimicrobial spectrum of live bacteria of *Morchella alpina* RS-01 on plate.

[0062]

[0063] Note: Data in the table are mean ± standard deviation. Different letters after the data in the same column indicate that the results were obtained through Duncan's new multiple range test. P The difference was statistically significant at levels <0.05.

[0064] Example 3: Determination of the antibacterial spectrum of RS-01 fermentation broth

[0065] Experimental and control groups were set up.

[0066] (1) Experimental group:

[0067] The tested plant pathogens were the ginseng rust pathogen ( I.radicicola ), the pathogen of melon wilt ( F. oxysporium ), Pythium spp. pathogen of melons and fruits ( P. aphanidermatum ), the pathogen of apple fruit rot ( M. polystroma ), the pathogen of maize leaf spot ( E. turcicum. ), seedling damping-off pathogen ( F. oxysporum ), walnut canker pathogen ( D. to gather ), the pathogen of poplar canker ( B. dothidea ), the pathogen of anthracnose in peppers ( C. Capsicum ), blueberry stem canker pathogen ( D. australafricana ), the pathogen of apple shell monochromatic septum disease ( B. Stevens ) and the pathogen of tobacco red spot disease ( A. alternata ).

[0068] The preparation of RS-01 mycelial cakes includes the following steps: Activated *Morchella alpineensis* RS-01 is inoculated onto PDA plates. The specific procedures include: restoring the RS-01 culture from its storage state to room temperature; inoculating the stored RS-01 culture onto PDA plates and incubating at 28°C for 7 days; selecting robustly growing colonies on the PDA plates, inoculating them onto new PDA plates, and continuing incubation at 28°C for another 7 days; repeating this step 2-3 times to obtain well-grown colonies. These are then set aside. RS-01 mycelial cakes with a diameter of 5 mm are obtained using a punch in a sterile environment.

[0069] Add 100 mL of PD liquid culture medium to a 200 mL Erlenmeyer flask, add six 5 mm diameter RS-01 bacterial pellets, and incubate at 28℃ and 150 rpm for 6 days with constant temperature shaking. After incubation, centrifuge at 7800 rpm and 25℃ for 10 minutes to remove the precipitate, and collect the supernatant as the RS-01 fermentation broth for subsequent experiments.

[0070] Add 5 mL of RS-01 fermentation broth prepared using the above method to 50 mL of PDA, shake well to obtain a mixture; evenly distribute the mixture into three 90 mm diameter petri dishes to prepare PDA-containing plates. Then, place a 5 mm diameter mycelium of the tested plant pathogen in the center of the plate and continue culturing for 5 days at a culture temperature of 25℃.

[0071] (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.

[0072] (3) Each of the above treatments was repeated three times, and the growth diameter of the pathogenic bacteria in the tested plants was measured using the cross-multiplication method. The inhibition rate was calculated using the method in Example 1.

[0073] The experimental results are shown in Table 3 and Figure 3 As shown, the antibacterial activity of the fermentation broth of the antagonistic strain RS-01 was determined using the toxic plate method. The RS-01 fermentation broth exhibited high antibacterial activity, with the strongest inhibitory effect against the pathogen causing ginseng rust, achieving an inhibition rate of 81.41% against pathogens with a diameter of only 16.73 mm. It showed secondary antagonistic effects against the pathogens causing blueberry stem canker and walnut canker, with inhibition rates of 77.92% and 75.22% for pathogens with diameters of 19.88 mm and 22.30 mm, respectively. It also showed good inhibitory effects against the pathogens causing anthracnose in peppers, pyrophyllosis in melons, and wilt in melons, with inhibition rates of 71.09%, 70.77%, and 71.74% for pathogens with diameters of 26.02 mm, 26.31 mm, and 25.44 mm, respectively. These results indicate that the fermentation broth of this antagonistic bacterium has a good inhibitory effect on all tested pathogens.

[0074] Table 3. Antimicrobial spectrum of toxic plate of *Morchella esculenta* RS-01 fermentation broth.

[0075]

[0076] Note: Data in the table are mean ± standard deviation. Different letters after the data in the same column indicate that the results were obtained through Duncan's new multiple range test. P The difference was statistically significant at levels <0.05.

[0077] Example 4 Morphological identification of strain RS-01

[0078] Inoculate RS-01 strain onto PDA plates and cover with sterile coverslips at 45°C. o After slanting the strains in the culture medium and incubating at 28°C for 3-5 days, remove the coverslips and observe the morphological characteristics of the strains under an optical microscope. At the same time, select strains with good growth status for observation under a scanning electron microscope and take pictures.

[0079] When strain RS-01 was cultured on PDA plates at 28℃ for 24-48 h, the colonies were white and fluffy with dense hyphae. After 3-5 days of culture, the hyphae gradually thickened and formed layers. Sporangiophores grew from the aerial hyphae, without microsporangiospores. The sporangiophores were unbranched, the sporangia were terminal, spherical, without a columella, the membrane was easily broken, and there were many spores. The colony surface was flat, the reverse side was light yellow, there was no exudate, and there was no soluble pigment. Electron microscopy revealed that the surface of the conidiophores was undecorated, the phloems were tightly arranged, and the spores were stacked in chains or clusters without any accessory structures.

[0080] Example 5 Molecular biological identification of strain RS-01

[0081] DNA was extracted from strain RS-01 using the Ezup column-based fungal genomic DNA extraction kit.

[0082] Amplification was performed using primers ITS1 and ITS4. Primers ITS1 and ITS4 were synthesized by Cummins.

[0083] ITS1: 5´-TCCGTAGGTGAACCTGCGG-3´ (SEQ ID NO: 1);

[0084] ITS4: 5´-TCCTCCGCTTATTGATATGC-3´ (SEQ ID NO: 2).

[0085] The PCR reaction system includes: 2 μL ITS1 (10 μmol / L), 2 μL ITS4 (10 μmol / L), 25 μL Taq PCRMaster Mix, 1 μL DNA template, and 20 μL ddH2O.

[0086] 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, extension at 72°C for 1 min, for 39 cycles; and final extension at 72°C for 10 min.

[0087] The amplified products were sent to Changchun Kumei Biotechnology Co., Ltd. for bidirectional sequencing. The sequences were analyzed and manually corrected and assembled using software such as BioEdit 7.0.1. The assembled sequences were then entered into the BLAST page of NCBI for alignment. Partial sequences of each similar species that have been registered in GenBank were downloaded, and sequence alignment was performed using Clustal W. Multi-gene sequence alignment was performed using the concatenate sequence (CS) method of PhyloSuite software. The phylogenetic tree was viewed using FigTree v1.4.3 software.

[0088] The comparison method can be found in the following literature:

[0089] 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 acids research, 1994, 22(22): 4673-4680.

[0090] 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.

[0091] Zhang D, Gao FL, Jakovlić 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.

[0092] The ITS gene of strain RS-01 was amplified and sequenced, and the sequence of the fragment is shown in SEQ ID NO:3.

[0093] (SEQ ID NO:3).

[0094] Eighteen sequences with high homology to strain RS-01 were selected from NCBI, and phylogenetic analysis of the ITS of strain RS-01 was performed. Figure 4 As shown. Strain RS-01 and Alpine Mortierella Clustered in one branch, with a posterior probability of 99%, and Alpine Mortierella As a sister branch, strain RS-01 is associated with other *Monosporium* species (…). M.thaxteri , M. (sp) belong to different branches. Therefore, based on morphological and molecular biological identification, RS-01 was identified as *Morchella alpineensis*.

[0095] Strain RS-01 was deposited at the China General Microbiological Culture Collection Center (CGMCC) on May 21, 2025, at the Institute of Microbiology, Chinese Academy of Sciences, No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, China, with accession number CGMCC No. 41944 and the name at the time of deposit as *Moriophyta alpinea*. Mortierella alpine RS-01, the suggested classification name is Alpine Moniliformis ( Alpine Mortierella ).

[0096] Alpine spores can regulate the composition of ginseng root microorganisms, stabilize the structure of ginseng root microbial communities, increase soil sucrase activity, and reduce the abundance of harmful bacteria in ginseng rhizosphere soil, thereby helping ginseng resist pathogen invasion.

[0097] This invention isolated six fungal strains from different forest soils and host plants in Jilin Province. Initial screening was conducted using *Ginseng rust rot pathogen* as the target, followed by secondary screening against 12 other pathogens causing serious damage to agriculture and forestry, including *Blueberry stem canker pathogen* and *Apple fruit rot pathogen*. The RS-01 strain showed strong antibacterial activity (inhibition rate of 77.43%) and a broad antibacterial spectrum. Its fermentation broth significantly inhibited all 12 tested pathogens, with the RS-01 fermentation broth exhibiting the strongest antibacterial effect against *Ginseng rust rot pathogen*, with an inhibition rate of 81.41%.

[0098] Sequences with high homology to strain RS-01 were selected from NCBI. Phylogenetic analysis of strain RS-01 was performed, comparing RS-01 with *Morodermus alpineus* strain submitted to GenBank. Alpine Mortierella They cluster together on one branch, with a posterior probability of 99%, and... Alpine Mortierella It is a sister branch, and differs from other *Monosporium* genera ( M.globalpina , M (.sp) belong to different branches. Therefore, based on morphological and molecular biological identification, RS-01 was identified as *Morchella alpineensis*.

[0099] In summary, strain RS-01 has bactericidal activity. Both its live bacteria and fermentation broth showed strong inhibitory effects on ginseng rust pathogens and have the advantage of a broad antibacterial spectrum.

[0100] 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 within the protection scope of the present invention.

Claims

1. The application of an alpine spore fungicide, characterized in that, For the preparation of biocontrol agents, the *Morchella alpineensis* agent is prepared by the following method: *Morchella alpineensis* is inoculated into a culture medium for fermentation, centrifuged, and the supernatant is collected; the strain name of *Morchella alpineensis* is *Morchella alpineensis* (…). Mortierella alpina RS-01, accession number CGMCC No. 41944; The biocontrol agent is used to control one or more of the following pathogens: ginseng rust rot pathogen, melon wilt pathogen, fruit rot pathogen, apple fruit rot pathogen, corn leaf blight pathogen, seedling damping-off pathogen, walnut canker pathogen, poplar canker pathogen, pepper anthracnose pathogen, blueberry stem canker pathogen, apple shell monochromatic spore pathogen, and tobacco red spot pathogen.

2. The application according to claim 1, characterized in that, The culture medium is PD liquid culture medium.

3. The application according to claim 1 or 2, characterized in that, The fermentation process was carried out with shaking at 150 rpm, at a fermentation temperature of 28℃, and for 6 days.

4. The application according to claim 1 or 2, characterized in that, The centrifugation speed was 7800 rpm, the centrifugation temperature was 25℃, and the centrifugation time was 10 minutes.

5. The application of an alpine spore fungicide, characterized in that, For the prevention and control of plant pathogens, the *Morchella alpineensis* agent is prepared by the following method: *Morchella alpineensis* is inoculated into a culture medium and fermented, then centrifuged, and the supernatant is collected; the strain name of *Morchella alpineensis* is *Morchella alpineensis* (…). Mortierella alpina RS-01, accession number CGMCC No. 41944; The plant pathogens mentioned include one or more of the following: ginseng rust rot pathogen, melon wilt pathogen, fruit rot pathogen, apple fruit rot pathogen, corn leaf spot pathogen, seedling damping-off pathogen, walnut canker pathogen, poplar canker pathogen, pepper anthracnose pathogen, blueberry stem canker pathogen, apple shell monochromatic spore pathogen, and tobacco red spot pathogen.

6. The application according to claim 5, characterized in that, The culture medium is PD liquid culture medium.

7. The application according to claim 5 or 6, characterized in that, The fermentation process was carried out with shaking at 150 rpm, at a fermentation temperature of 28℃, and for 6 days.

8. The application according to claim 5 or 6, characterized in that, The centrifugation speed was 7800 rpm, the centrifugation temperature was 25℃, and the centrifugation time was 10 minutes.

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