Mortierella alpina GRB-1, bacterial agent and its application

Abstract

The present invention discloses a strain of Mortierella alpina GRB-1, a microbial agent and its application, belonging to the field of agricultural microbiology. The present invention is the first to isolate a strain of Mortierella alpina ( Alpine Mortierella ) GRB‑1, a strain that simultaneously produces proteases, siderophores, phosphate solubilization, ammonia, and auxin, can inhibit Fusarium spores and has the potential to alleviate continuous cropping problems. Combining it with crop straw to create a product has shown excellent results in alleviating continuous cropping problems with apples. It can promote the growth of both above- and below-ground parts of continuously cropped apple trees, increase the relative abundance of bacterial communities and nutrient content in soils containing continuously cropped apples, and enhance apple root vitality, making it suitable for preventing and treating continuous cropping problems.

Description

Technical Field

[0001] The present invention relates to the technical field of agricultural microorganisms, and in particular to a Mortierella alpina GRB-1, a bacterial agent and applications thereof. Background Art

[0002] Apple continuous cropping disorder is an important problem faced in the process of replanting and rebuilding apple orchards. At present, it is generally believed that the imbalance of soil microbial community structure, the decline of plant's own resistance, the disorder of soil nutrient structure and self-toxic substances are the main causes of apple continuous cropping disorder.

[0003] Fusarium is currently known to be the primary pathogen causing apple cropping problems. Crop rotation, chemical fumigation, and biological control are currently the primary preventive measures for apple cropping problems. Pre-planting soil fumigation with methyl bromide is recognized internationally and internationally as the best method for preventing apple cropping problems. However, methyl bromide fumigation reduces soil microbial populations. Directly available nutrients in the soil, aside from those added manually, primarily come from microbial decomposition. This reduction in soil microbial activity and decomposition capacity leads to a decrease in nutrient content and serious environmental pollution. Consequently, methyl bromide fumigation is gradually being phased out. Crop rotation is also time-consuming and difficult to promote in production. Biological control, on the other hand, is a green and healthy prevention and control method currently being widely researched. It utilizes beneficial microorganisms to suppress pathogen populations in the soil or interfere with plant pathogen infection. Therefore, with increasing environmental awareness and the commercialization and mechanization of orchard management, crop rotation and chemical fumigation are being replaced by biological control to save time and minimize environmental pollution.

[0004] Mortierella alpina is a saprophytic fungus found widely in soil. It produces large quantities of unsaturated fatty acids and is currently the primary fungus for industrial production of unsaturated fatty acids. Unsaturated fatty acids have been shown to play a key role in plant signaling, plant-microbe communication, and fungal virulence. Furthermore, Mortierella alpina can promote fungal parasitism by secreting proteases. Its strong adaptability to environmental factors suggests significant potential for green agriculture development, but its effectiveness in preventing and controlling continuous cropping of apple has yet to be reported. Summary of the Invention

[0005] In view of the above-mentioned prior art, the present invention aims to provide a strain of Mortierella alpina GRB-1, a fungal agent and its application. After long-term technical and practical exploration, the inventors isolated a strain of Mortierella alpina ( Mortierella alpina ) GRB-1, and used it in combination with cow dung and crop straw to prepare a product with excellent effect in alleviating apple continuous cropping obstacles, which can be used for the prevention and control of apple continuous cropping obstacles.

[0006] To achieve the above object, the present invention adopts the following technical solutions:

[0007] In a first aspect, the present invention provides a strain of Mortierella alpina ( Mortierella alpina ) GRB-1, the Mortierella alpina ( Mortierella alpina ) GRB-1 was deposited on March 28, 2025 in the General Microbiology Center of China Culture Collection Administration (CGMCC, address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing), and its deposit number is CGMCC No: 41869.

[0008] In a second aspect of the present invention, a bacterial agent is provided, wherein the bacterial agent contains the above-mentioned Mortierella alpina ( Mortierella alpina )GRB-1.

[0009] In the bacterial agent, Mortierella alpina ( Mortierella alpina ) GRB-1 exists in the form of live bacterial suspension, spore suspension or fermentation broth.

[0010] The spore suspension is prepared by the following method:

[0011] Mortierella alpina ( Mortierella alpina ) GRB-1 was cultured on PDA medium at 28°C, and the spores were washed with sterile water to prepare a spore suspension.

[0012] The Mortierella alpina ( Mortierella alpina ) The fermentation broth of GRB-1 was prepared by the following method:

[0013] Mortierella alpina ( Mortierella alpina ) GRB-1 spore suspension was inoculated into fermentation medium 1 and cultured in a shaking incubator at 34°C and 180 rpm for 72 h to prepare Mortierella alpina ( Mortierella alpina ) GRB-1 fermentation broth.

[0014] In a third aspect, the present invention provides the above-mentioned Mortierella alpina ( Mortierella alpina ) GRB-1 or containing Mortierella alpina ( Mortierella alpina ) Use of the GRB-1 bacterial agent in any of the following (1)-(3):

[0015] (1) Producing siderophores, dissolving phosphate and producing ammonia;

[0016] (2) Inhibit the pathogen Fusarium solani;

[0017] (3) Prepare products to alleviate the obstacles of continuous cropping of apples.

[0018] The method of reducing the obstacle of continuous apple cropping is specifically manifested in: increasing the relative abundance of bacterial communities in the soil of continuous apple cropping, increasing the nutrient content in the soil of continuous apple cropping, increasing the vitality of apple root systems, and promoting the growth of apple plants.

[0019] In a fourth aspect, the present invention provides a solid bacterial fertilizer for alleviating the obstacle of continuous cropping of apples, wherein the solid bacterial fertilizer is prepared from Mortierella alpina ( Mortierella alpina ) GRB-1 is the active ingredient.

[0020] The solid bacterial fertilizer also includes cow dung and corn straw.

[0021] The preparation method of the solid bacterial fertilizer comprises the following steps:

[0022] (i) preparing a carrier by mixing cow dung and crop straw in a mass ratio of 3:1, and sterilizing the mixture;

[0023] (ii) Mortierella alpina ( Mortierella alpina ) GRB-1 spore suspension was inoculated into fermentation medium 2 at an inoculum rate of 1 mL: (8-12) g, and fermented at 30-34°C for 10-14 days to obtain a solid bacterial fertilizer that alleviates the obstacles of continuous cropping of apples.

[0024] The solid bacterial fertilizer contains Mortierella alpina ( Mortierella alpina ) The amount of viable GRB-1 bacteria is (2-3) × 10 9 cfu g -1 .

[0025] Preferably, the solid bacterial fertilizer contains Mortierella alpina ( Mortierella alpina )The amount of viable GRB-1 bacteria is 2×10 9 cfu·g -1 .

[0026] Preferably, the Mortierella alpina solid fertilizer is applied at a ratio of 1:1000 (w / w) to soil weight.

[0027] Beneficial effects of the present invention:

[0028] The present invention is the first to isolate a strain of Mortierella alpina ( Mortierella alpina ) GRB-1, a strain that simultaneously produces proteases, siderophores, phosphate, ammonia, and growth hormones, can inhibit the pathogen Fusarium solani and has the potential to alleviate continuous cropping problems. Combining it with crop straw to create a product has shown excellent results in alleviating the problem of continuous cropping of apple trees. It can promote the growth of both above-ground and below-ground parts of continuously cropped apple trees, increase the relative abundance of bacterial communities and nutrient content in soils used for continuous cropping, and enhance apple root vitality, making it suitable for preventing and treating continuous cropping problems. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 is the morphological diagram of GRB-1 strain; among them, Figure 1 A in the middle is the colony morphology of PDA culture medium. Figure 1 Middle B is the phylogenetic tree of Mortierella alpina GRB-1, Figure 1 C in the middle is the hyphae morphology, Figure 1 D in the middle is the spore form.

[0030] Figure 2 This is a plate inhibition experiment of GRB-1 against Fusarium solani.

[0031] Figure 3 This is a seedling infection experiment to test the inhibitory effect of GRB-1 on Fusarium solani.

[0032] Figure 4 This study aimed to investigate the effects of GRB-1 bacterial fertilizer treatment on root activity of Camellia sinensis seedlings grown in pots under continuous apple cropping.

[0033] Figure 5 The effects of GRB-1 bacterial fertilizer treatment on the physical and chemical properties of soil in continuous apple cropping; Figure 5 a in the middle is the detection of ammonium nitrate content, Figure 5 b in the middle is the nitrate nitrogen content test, Figure 5 c in the middle is the effective potassium content test, Figure 5 In the middle, d is the detection of organic matter content. Figure 5 The "e" in the figure is the available phosphorus content.

[0034] Figure 6 The effect of GRB-1 bacterial fertilizer treatment on culturable microorganisms in soil; Figure 6 A in the middle is the detection of bacterial count in the soil, Figure 6 B in the middle is the detection of fungal quantity in the soil. DETAILED DESCRIPTION

[0035] It should be noted that the following detailed descriptions are illustrative and intended to provide further explanation of the present application. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which the present application belongs.

[0036] As mentioned in the background section, continuous cropping of apples can lead to reduced yield and quality, increased pests and diseases, weakened trees, and even death, causing severe economic losses to fruit growers. Currently, there are no reports on the use of Mortierella alpina as a biocontrol agent for controlling continuous cropping of apples.

[0037] Based on this, the present invention isolated a fungus GRB-1 from the rhizosphere of healthy fruit trees in a continuously cropped apple orchard. According to morphological, physiological and biochemical characteristics detection and ITS-based phylogenetic analysis, the fungus was identified as Mortierella alpina. Different from the existing reported Mortierella alpina, the Mortierella alpina isolated by screening in the present invention is a new type of biocontrol fungus with great development potential. It has the ability to produce proteases, siderophores, phosphates, ammonia, and growth hormones, and can inhibit the pathogen Fusarium solani. It integrates multiple functions in one and has the application potential to alleviate continuous cropping obstacles. The product prepared by combining it with crop straw has excellent effects in alleviating apple continuous cropping obstacles, can promote the growth of the aboveground and underground parts of continuously cropped apple trees, increase the relative abundance of bacterial communities and the nutrient content in the soil of continuously cropped apples, improve the vitality of apple roots, and is used for the prevention and treatment of apple continuous cropping obstacles.

[0038] In order to enable those skilled in the art to more clearly understand the technical solution of the present application, the technical solution of the present application will be described in detail below with reference to specific examples. If the specific experimental conditions are not specified in the examples, they are usually based on conventional conditions or the conditions recommended by the reagent company; the reagents, consumables, etc. used in the following examples can be obtained through commercial channels unless otherwise specified. Among them:

[0039] PDA medium: 200.0 g peeled potatoes, 20.0 g glucose, 20.0 g agar, 1000 mL distilled water.

[0040] LB medium: peptone 10.0 g, yeast extract powder 5.0 g, sodium chloride 10.0 g, distilled water 1000 mL.

[0041] Fermentation medium 1: 40 g / L corn flour medium supplemented with 10 g / L glucose and inorganic salts (nitrate (2 g / L KNO3), phosphate (1 g / L NaH2PO4) and magnesium ion (0.3 g / L MgSO4•7H2O), and 1000 mL distilled water.

[0042] The composition of fermentation medium 2 is: glucose 50.00 g, (NH4)2SO4 2.00 g, KH2PO4 0.30 g, NaH2PO4 0.08 g, MgSO4‧7H2O 0.30 g, ZnSO4‧7H2O 0.08 g, CaCO3 10.00 g (CaCO3 is wrapped in tin foil and sterilized separately and then added to fermentation medium 2 to balance the pH), and distilled water 1000 mL.

[0043] The primer synthesis and amplified fragment sequencing used in the examples were completed by Shanghai Sangon Co., Ltd.

[0044] The Fusarium solani used in the examples of the present invention is recorded in "Analysis of fungal community structure in continuous cropping soil around Bohai Sea and study on alleviating continuous cropping obstacles of apples by mixed cropping of onions".

[0045] Example 1: Isolation and identification of strains

[0046] 1. Isolation and purification of strains:

[0047] The rhizosphere soil of healthy apple trees was collected from the continuous cropping orchard in Wangshen Village, Beixin Township, Linyi County, Yuncheng City, Shanxi Province by gradient dilution coating method and brought back to the laboratory for immediate screening of rhizosphere fungi.

[0048] 10g of fresh sample was placed in 90mL of sterile water and shaken for 30min before diluting to 10 -4 100 μl of the diluted solution was added to each plate of PDA culture medium containing 0.4% penicillin-streptomycin (100×), and after plating, rhizospheric fungi were isolated. The plates were then kept at 28°C for one week. Mycelium from a single colony was then transferred to a new PDA plate without antibiotics and cultured. A total of over 80 rhizospheric fungi were isolated.

[0049] 2. Screening of strains:

[0050] Using a dilution plate spread method of root abrasive solution, over 30 different endophytic fungi were isolated and identified. A potted experiment using Pingyi sweet tea (apple seedling) seedlings (using rhizosphere soil from a perennial (over 25 years old) orchard with continuous cropping problems) identified a strain with the best growth-promoting effect on Pingyi sweet tea seedlings, designated GRB-1. This strain grows rapidly, with hyphae covering the entire culture plate in just 4-5 days. Its metabolites significantly inhibit the disease-causing effects of the pathogen Fusarium solani (ARD) on plants. These characteristics facilitate its colonization and the prevention and control of ARD, leading to the decision to further study GRB-1.

[0051] 3. Identification of strains:

[0052] (1) Morphological identification: When grown on PDA solid culture medium, the colonies are white and layered. They grow rapidly, and their hyphae can cover the entire culture plate in 4-5 days. When observed under a 60x ordinary optical microscope, the spores are oval. The sporangium stalks grow from the aerial hyphae, without microspores, the sporangium stalks are unbranched, and the sporangia are terminal and spherical ( Figure 1 ).

[0053] (2) Sequence analysis and phylogenetic analysis:

[0054] Amplification of fungal ITS (Internal Transcribed Spacer) fragments refers to the use of PCR (polymerase chain reaction) technology to specifically replicate the non-coding region sequences located between ribosomal RNA (rRNA) genes in the fungal genome. This operation is a core step in fungal molecular identification and diversity research.

[0055] The DNA of the GRB-1 strain was extracted using a fungal genomic DNA extraction kit (Solarbio Cat#D2300), and the primer sequences shown in SEQ ID No. 1 to SEQ ID No. 2 were used to amplify the ITS fragment. The amplification process was as follows: pre-denaturation at 94°C for 1 min, denaturation at 94°C for 1 min, annealing at 50°C for 1 min, extension at 74°C for 90 s, 30 cycles, and a final extension at 74°C for 7 min;

[0056] ITS1: 5'-TCCGTAGGTGAACCTGCGG-3' (SEQ ID No. 1),

[0057] ITS2: 5'-GCTGCGTCTTCATCGATGC-3' (SEQ ID No. 2).

[0058] Sequencing results were compared with the National Center for Biotechnology Information (NCBI) nucleotide database using BLAST. Sequences of the isolated strains were analyzed and a phylogenetic tree constructed using the neighbor-joining (NJ) cluster analysis method using MEGA 7 software, with a bootstrap value of 1000 replicates. The rDNA-ITS sequence of the strain was 595 bp long. Blast analysis revealed that the sequence matched that of Mortierella alpina ( Mortierella alpina , MT365986) ITS sequence homology reached 100%. MEGA7.0 software was used to compare with the parts registered in the GenBank database Mortierella The phylogenetic tree constructed from genus sequences revealed that strain GRB-1 was closely related to Mortierella alpina ( Mortierella alpina ) had the highest homology and clustered into one branch. Combined with the results of morphological analysis, it was shown that the GRB-1 strain was a strain of Mortierella alpina ( Mortierella alpina ).

[0059] Based on the morphological and functional analyses of GRB-1 and the results of ITS homology analysis, the isolated GRB-1 strain was identified as Mortierella alpina. This strain has been deposited as a biological deposit, and the deposit information is as follows:

[0060] Species name: Mortierella alpina

[0061] Latin name: Mortierella alpina

[0062] Strain ID: GRB-1

[0063] Depository: General Microbiology Center of China Culture Collection Administration

[0064] Abbreviation of depository institution: CGMCC

[0065] Address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing

[0066] Deposit date: March 28, 2025

[0067] The registration number of the CGMCC Collection Center is: CGMCC No.41869.

[0068] Example 2: Experiment on the inhibition of GRB-1 strain on pathogens causing apple continuous cropping problems

[0069] (1) Plate confrontation experiment: A confrontation experiment was conducted using Fusarium solani as the test pathogen. Fusarium solani was isolated and preserved in our laboratory. The inhibitory effect of GRB-1 was verified by co-culturing Fusarium solani and Mortierella alpina GRB-1 on PDA solid culture medium.

[0070] The experimental results showed that compared with the control group (CK), when Fusarium solani and Mortierella alpina GRB-1 were co-cultured for 5 days and 7 days, Mortierella alpina showed a certain inhibitory effect on Fusarium solani, the pathogen causing apple continuous cropping problems ( Figure 2 ).

[0071] (2) Seedling infection experiment: 9 Pingyi sweet tea seedlings with 4-5 leaves were taken and divided into a blank control group, a Fusarium solani group, and a Mortierella alpina group, with 3 plants in each group. The treatments of the three groups were as follows:

[0072] Blank control group: The roots of Pingyi sweet tea seedlings with 4-5 leaves were washed and placed in a sterile medium;

[0073] Fusarium solani group: The roots of Pingyi sweet tea seedlings with 4-5 leaves were washed and placed in a sterile substrate. After growing for seven days, 2×10 7 cfu·g -1 5mL of pathogenic Fusarium solani;

[0074] Mortierella alpina group: The roots of 4-5 leaves of Pingyi sweet tea seedlings were washed and placed in 2×10 7 cfu·g -1 After the roots were soaked in Mortierella alpina liquid for 30 minutes, they were planted in sterile substrates and inoculated with 2×10 7 cfu·g-1 5mL of pathogenic Fusarium solani.

[0075] After each group was treated, the survival status of the seedlings was observed after 7 days.

[0076] The experimental results showed that compared with the blank control group, the seedlings in the Fusarium solani group wilted, the plants were short, and the leaves turned yellow, while the Mortierella alpina group was able to significantly alleviate the growth inhibition of the seedlings caused by the inoculation of Fusarium solani and restore the healthy growth of the seedlings. The test results showed that Mortierella alpina had a significant inhibitory effect on the disease of plants infected by the pathogen Fusarium solani ( Figure 3 ).

[0077] Example 3: Mortierella alpina ( Mortierella alpina ) Functional identification of GRB-1

[0078] The protease, ammonia, auxin, and siderophore production capacities of strain GRB-1 were determined according to the method of Dong and Cai (2001). The specific testing steps are as follows:

[0079] Protease production: Conduct a milk peptone test using skim milk culture medium to observe whether the milk becomes clear;

[0080] Ammonia production test: Use urea agar (containing phenol red indicator) to observe whether the culture medium turns red;

[0081] Production of elongin: According to the indole test, Mortierella alpina is co-cultured with tryptophan and then reacted with Kovac's reagent to observe whether a red color is formed.

[0082] Iron carrier production: blue chromium method, whether the color of the culture medium can change from blue to orange.

[0083] Decomposition of inorganic phosphorus: solid NBRIP medium, inoculate GRB-1 and observe whether a transparent hydrolysis zone can be formed on the solid medium.

[0084] Potassium hydrolysis: Use a potassium-selective medium (in this example, Alexander Roth medium) to observe whether a transparent hydrolysis zone can be formed on the solid culture medium.

[0085] Cellulase production: Congo red staining method (CMC-Na plate method) to observe whether the enzymatic hydrolysis area can show a transparent hydrolysis zone.

[0086] The experimental results showed that Mortierella alpina (GRB-1) has the ability to produce protease, siderophore, phosphate, ammonia, and auxin (Table 1).

[0087] Table 1: Detection of biological characteristics of Mortierella alpina

[0088]

[0089] Example 4: Preparation of a product containing Mortierella alpina GRB-1

[0090] 1. Preparation of Mortierella alpina GRB-1 fermentation broth

[0091] A spore suspension of Mortierella alpina GRB-1 was inoculated into fermentation medium 1 at an inoculum rate of 1%, and the culture was carried out at 34°C and 180 rpm for 72 h.

[0092] 2. Preparation of products to alleviate apple continuous cropping problems

[0093] (1) Cow dung and corn straw (corn straw was purchased from Dezhou Chuangdi Microbial Resources Co., Ltd. and was a fully decomposed product) were mixed in a mass ratio of 3:1 to prepare a carrier, which was sterilized for later use;

[0094] (2) A spore suspension of Mortierella alpina GRB-1 was inoculated into fermentation medium 2 at a 1% inoculum rate and cultured in a shaker at 180 rpm at 34°C for 48 h to obtain a fermentation broth. The fermentation broth was mixed with the sterilized carrier at a ratio of 1 mL:8 g and cultured at 30°C for 10 days.

[0095] Dezhou Chuangdi Microbial Resources Co., Ltd. prepared a product (Mortellini alpina GRB-1 solid bacterial fertilizer) to alleviate the obstacles of apple continuous cropping according to the above preparation method. The amount of live bacteria of Mortellini alpina GRB-1 in the bacterial fertilizer was 2.00×10 9 cfu g -1 , effective nitrogen content 0.51 mg·g -1 , available phosphorus content 1.35 mg·g -1 , effective potassium content 1.03mg·g -1 .

[0096] Example 5: Preparation of a composition for alleviating apple continuous cropping obstacles

[0097] (1) Cow dung and corn straw were mixed in a mass ratio of 3:1 to prepare a carrier, which was sterilized for later use;

[0098] (2) A spore suspension of Mortierella alpina GRB-1 was inoculated into fermentation medium 2 at a 1% inoculum rate and cultured in a shaking incubator at 34°C and 180 rpm for 48 h to obtain a fermentation broth. The fermentation broth was then mixed with a sterilized carrier at a ratio of 1 mL:12 g and cultured at 34°C for 14 days to produce a product that alleviates the problem of continuous cropping of apples.

[0099] Example 6: Potted plant test

[0100] 1. Experimental Design

[0101] The soil used in the potted experiment was taken from a 30-year-old apple orchard in Xintai, Tai'an City, Shandong Province. Soil was randomly collected from multiple points at a depth of 10 to 40 cm, 80 cm from the tree trunk, and mixed evenly. The soil type was sandy loam.

[0102] The pot experiment was conducted at the National Apple Engineering Experimental Center, South Campus, Shandong Agricultural University from March to October 2024. The test plants were Pingyi sweet tea ( Malus hupehensis Rehd . ) seedlings. Pingyi sweet tea seeds were stratified at 4°C for 30 days. Once the seeds appeared white, they were sown in pots filled with seedling medium for seedling cultivation. When the seedlings had 5–6 true leaves, plants with uniform growth and no pests or diseases were selected and transplanted in early May into clay pots (25 cm top diameter, 17 cm bottom diameter, 18 cm height) filled with 6.5 kg of soil from different treatments. Three treatments were set up in the experiment: a continuous cropping soil treatment group (CK, negative control), a methyl bromide fumigation continuous cropping soil treatment group (CK1, positive control), and a Mortierella alpina GRB-1 bacterial fertilizer treatment group (H1). The treatments for each group were as follows:

[0103] Continuous cropping soil treatment group (CK): 7.5 kg of 30-year-old apple orchard rhizosphere soil per pot

[0104] Methyl bromide fumigated continuous cropping soil treatment group (CK1): 7.5 kg of rhizosphere soil from a 30-year-old apple orchard per pot was fumigated with methyl bromide for 14 days.

[0105] Mortierella alpina GRB-1 bacterial fertilizer treatment group (H1): 75 g of Mortierella alpina GRB-1 solid bacterial fertilizer prepared in Example 4 was added to 7.5 kg of rhizosphere soil from a 30-year-old apple orchard per pot, mixed well, and then placed in the pot.

[0106] Each treatment group was set up with 20 pots, and one seedling was planted in each pot. Fertilizer and water management was uniform. Plant biomass was measured after 60 days of cultivation. Continued cultivation continued until 90 days. Rhizosphere soil and plant samples from each treatment were uniformly sampled to measure soil microorganisms, soil physical and chemical properties, and root activity, and the average values ​​were calculated. The determination method is as follows:

[0107] Determination of soil microorganisms: The number of culturable bacteria and fungi in the soil was determined using LB and PDA culture media, respectively, using the plate dilution coating method.

[0108] Determination of soil physicochemical properties: Fresh soil from the rhizosphere of Pingyi sweet tea seedlings was taken, dried, and passed through a 2mm sieve. The organic matter content was measured using the organic matter-potassium dichromate organic volume method, and ammonium nitrogen and nitrate nitrogen (NH4 + -N and NO3— The ammonium and nitrate nitrogen contents were measured by the flow injection analyzer method with CaCl2 extraction; the available phosphorus content was measured by the effective phosphorus (P2O5)-antimony-molybdenum colorimetric method, and the available potassium (K2O)-flame spectrophotometric method.

[0109] Biomass determination: Plant height and ground diameter were measured with a ruler and a vernier caliper, respectively. When measuring fresh mass, the plants were first washed with tap water, dried, and measured with an electronic balance. The plants were then dried at 80°C to a constant weight before measuring the mass.

[0110] Root activity assay: The root activity of seedlings was assayed using the 2,3,5-triphenyltetrazolium (TTC) method.

[0111] The measured data were calculated and plotted using Microsoft Excel 2010 and Graphpad Prism. SPSS 23.0 was used for comparison of means and one-way ANOVA. Duncan's new multiple range method was used for comparison of significant differences (a = 0.05).

[0112] 2. Experimental Results

[0113] (1) Growth-promoting effect of GRB-1 bacterial fertilizer on Pingyi sweet tea seedlings

[0114] Table 2 shows that, compared to the continuous cropping control (CK), the Mortierella alpina fertilizer treatment (H1) promoted biomass growth in Pingyi sweet tea seedlings under potted conditions. Plant height and ground diameter increased by 29.82% and 52.95%, respectively, in July, with similar trends in July and August. Although the growth of Pingyi sweet tea seedlings treated with GRB-1 fertilizer (H1) failed to surpass that of the methyl bromide fumigation treatment (CK1), the Mortierella alpina fertilizer treatment (H1) showed significant differences compared to the continuous cropping control (CK). These results suggest that applying Mortierella alpina GRB-1 to continuously cropped soil can alleviate apple continuous cropping problems to a certain extent.

[0115] Table 2: Effects of GRB-1 bacterial fertilizer on the growth of Camellia sinensis seedlings in continuous cropping soil

[0116]

[0117] (2) Effects of GRB-1 bacterial fertilizer treatment on root activity of Pinellia truncatula seedlings grown in pots under continuous apple cropping

[0118] Depend on Figure 3 It can be seen that compared with the continuous cropping control (CK) and methyl bromide fumigation treatment (CK1), the root activity after applying Mortierella alpina fertilizer (H1) showed a significant increase compared with the continuous cropping soil and methyl bromide fumigation treatment ( Figure 4 ).

[0119] (3) Effects of GRB-1 bacterial fertilizer treatment on the physical and chemical properties of soil under continuous apple cropping

[0120] The inoculation of Mortierella alpina had a significant effect on the nutrients in the continuously cropped soil. The nitrate nitrogen content in the soil increased significantly after inoculation with Mortierella alpina, indicating that Mortierella alpina can effectively help convert nitrogen sources in the soil, converting ammonium nitrogen that is difficult for plants to use into nitrate nitrogen that can be directly used. Compared with the continuously cropped soil, the nitrate nitrogen content in the soil increased by 45.37% after inoculation with Mortierella alpina. In addition to the significant effect on nitrogen sources, the inoculation with Mortierella alpina showed a significant increase in the content of soil organic matter and available phosphorus, which increased by 27.20% and 22.06% respectively compared with the continuously cropped soil. The effective potassium content in the soil increased by 15.39% after inoculation with Mortierella alpina compared with the continuously cropped treatment ( Figure 5 ).

[0121] (4) Effects of GRB-1 bacterial fertilizer treatment on culturable microorganisms in soil

[0122] like Figure 6 As shown in the figure, the fungi and bacteria in the soil after three months of treatment with Mortierella alpina fertilizer showed that compared with the continuous cropping control (CK), the GRB-1 fertilizer treatment (H1) significantly increased the number of culturable bacteria in the soil to a certain extent. However, it did not have a significant effect on the number of culturable fungi in the soil.

[0123] The above description is only a preferred embodiment of the present application and is not intended to limit the present application. For those skilled in the art, the present application may have various modifications and variations. Any modification made within the spirit and principles of the present application shall be

[0124] Equivalent replacements, improvements, etc. should all be included in the protection scope of this application.

Claims

1. A strain of Mortierella alpina ( Mortierella alpina ) GRB-1, characterized in that The Mortierella alpina ( Mortierella alpina ) GRB-1 was deposited in the General Microbiology Center of China Culture Collection Administration on March 28, 2025, and its deposit number is CGMCC No: 41869.

2. A bacterial agent, characterized in that The bacterial agent contains the Mortierella alpina described in claim 1 ( Mortierella alpina )GRB-1.

3. The microbial agent according to claim 2, characterized in that In the bacterial agent, Mortierella alpina ( Mortierella alpina ) GRB-1 exists in the form of live bacterial suspension, spore suspension or fermentation broth.

4. The microbial agent according to claim 3, characterized in that The spore suspension is prepared by the following method: Mortierella alpina ( Mortierella alpina ) GRB-1 was cultured on PDA medium at 28°C, and the spores were washed with sterile water to prepare a spore suspension.

5. the Mortierella alpina described in claim 1 ( Mortierella alpina ) Use of GRB-1 or the bacterial agent according to claim 2 in any of the following (1) to (3): (1) Producing siderophores, dissolving phosphate and producing ammonia; (2) Inhibit pathogenic Fusarium solani; (3) Prepare products to alleviate the obstacles of continuous cropping of apples.

6. The use according to claim 5, characterized in that The method of reducing the obstacle of continuous apple cropping is specifically manifested in: increasing the relative abundance of bacterial communities in the soil of continuous apple cropping, increasing the nutrient content in the soil of continuous apple cropping, increasing the vitality of apple root systems, and promoting the growth of apple plants.

7. A solid bacterial fertilizer for alleviating the obstacle of continuous cropping of apples, characterized in that: The solid bacterial fertilizer is made of the Mortierella alpina ( Mortierella alpina ) GRB-1 is the active ingredient.

8. The solid bacterial fertilizer according to claim 7, characterized in that The solid bacterial fertilizer also includes cow dung and corn straw.

9. The solid bacterial fertilizer according to claim 7, characterized in that The preparation method of the solid bacterial fertilizer comprises the following steps: (i) preparing a carrier by mixing cow dung and corn straw in a mass ratio of 3:1, and sterilizing the mixture; (ii) Mortierella alpina ( Mortierella alpina ) GRB-1 spore suspension was inoculated into fermentation medium 2 at an inoculum rate of 1 mL: (8-12) g, and fermented at 30-34°C for 10-14 days to obtain a solid bacterial fertilizer that alleviates the obstacles of continuous cropping of apples.

10. The solid bacterial fertilizer according to claim 7, characterized in that The solid bacterial fertilizer contains Mortierella alpina ( Mortierella alpina ) The amount of viable GRB-1 bacteria is (2-3) × 10 9 cfu g -1 .

Images