Arbuscular mycorrhizal fungus monospore separating, sterilizing and filtering tool and method

Abstract

The invention relates to a tool and a method for separating, sterilizing and filtering arbuscular mycorrhizal fungi monospores. The device comprises a microoperation module and a multi-stage filtering module which work cooperatively. Wherein the microoperation module comprises a microsuction device and a filtering module, the filtering module comprises an upper filtering section and a lower filtering section, the upper filtering section comprises a first cylindrical section, a second cylindrical section and a third cylindrical section which are coaxially arranged and sequentially connected, and a filter screen is arranged at the end of the third cylindrical section; the lower filtering section comprises a fourth filtering section and a fifth filtering section which are coaxially arranged and connected with each other, the fourth filtering section is used for sleeving the third filtering section, and the fourth filtering section and the fifth filtering section jointly form a filter for filtering spores extracted by the micro-suction device. The tool has the innovativeness that the tool can effectively solve the problem of single separation of AMF fungal spores, and loss of the spores is reduced in the sterilization process.

Description

Technical Field

[0001] The invention relates to a tool and method for separating and sterilizing filtration of arbuscular mycorrhizal fungi single spores, and belongs to the technical field of precise separation of microorganisms. Background Art

[0002] Arbuscular mycorrhizal fungi (AMF) form mutually beneficial symbiotic relationships with plant roots (Faghihinia, Jansa et al. 2022). They are found in 80% of plant species in terrestrial ecosystems, including major food crops such as rice, maize, wheat, and sorghum (Wen, Chen et al. 2022, Alotibi, Alotaibi et al. 2023, Duan, Luo et al. 2024, Hu, Chen et al. 2024). AMF significantly enhance crop uptake of nutrients such as nitrogen and phosphorus by increasing root extensibility, while also conferring tolerance to drought, salinity, and heavy metal pollution (Chen, Wang et al. 2023, Wen, Wu et al. 2024). As a key factor in sustainable agriculture, AMF shows great potential in replacing chemical fertilizers, maintaining soil fertility and improving ecosystem stability (Duan, Feng et al. 2024). Research on related symbiotic mechanisms and interactions has become a research hotspot in the field of biology.

[0003] Currently, AMF propagation relies primarily on two techniques: open pot culture using boiling sand as a substrate, and a dual culture system using arbuscular mycorrhizae and carrot hairy roots under sterile conditions (BéCard and Fortin 2006). While the pot culture method is simple to operate, the open environment is prone to microbial contamination, resulting in insufficient spore purity. While the dual culture method can produce sterile spores, it still faces bottlenecks such as low efficiency in isolating original spores and high spore loss during the sterilization process (traditional methods reach up to 20%). The diverse morphology of AMF spores in soil (mostly approximately 40-200 μm in diameter) and their mixed distribution further exacerbate the technical challenges of accurately isolating individual spores. For example, in agricultural applications, low-purity AMF inoculants can lead to fluctuations in symbiotic efficiency. In ecological restoration, screening for target spores in heavy metal-contaminated soils can take weeks. Existing microscopy methods often rely on manual isolation, with a success rate of less than 60%. Furthermore, the sterilization process requires more than five spore transfers, resulting in a 35% loss of viability. Therefore, there is an urgent need to develop an integrated tool to achieve efficient single spore capture, in situ sterilization and zero-loss filtration to support the large-scale application of AMF in crop yield increase, soil remediation and symbiotic mechanism research. Summary of the Invention

[0004] In response to the above technical problems, the present invention provides a tool and method for isolating and sterilizing filtration of single spores of arbuscular mycorrhizal fungi. The tool uses a glass micropipette (inner diameter 0.58 mm, adjustable) combined with three-dimensional micromanipulation technology to achieve high-precision capture of single spores (efficiency >95%), and is equipped with a multi-layer filter sterilization device (including a 400-mesh interception layer) to ensure zero spore loss and sterilization treatment. The tool is particularly suitable for agricultural production increase (preparation of symbiotic microbial agents for crops such as wheat, rice, and corn), ecological restoration (screening of bacterial strains in heavy metal-contaminated soil), and research on microbial interaction mechanisms. It overcomes the technical bottlenecks of high contamination rate of traditional potted propagation and less than 80% spore recovery rate in sterile systems, providing key technical support for sustainable agriculture and precision microbial research.

[0005] To achieve the above object, the present invention adopts the following technical solutions: A tool for separating and sterilizing filtration of single spores of arbuscular mycorrhizal fungi, comprising: A micropipette is a tubular structure with a diameter gradually decreasing from the tail end to the tip. The tip diameter of the micropipette is 50-300 μm. The micropipette is mounted on a microscope and is used for microscopic observation of spores and single spore aspiration operations. The filter comprises an upper filter section and a lower filter section, wherein the upper filter section comprises a first cylindrical section, a second cylindrical section, and a third cylindrical section coaxially arranged and sequentially connected, and a filter screen is provided at the end of the third cylindrical section; the lower filter section comprises a fourth filter section and a fifth filter section coaxially arranged and mutually connected, and the fourth filter section is used to be sleeved on the third cylindrical section. The two together constitute the filter for filtering spores extracted by the micropipette.

[0006] The tool for isolating and sterilizing single spores of arbuscular mycorrhizal fungi, preferably, the micropipette is obtained by the following method: after the glass tube is drawn by a microelectrode drawing instrument, the tip diameter is adjusted by the shoulder scratching method: holding a glass tube of target specification in one hand and an auxiliary tube in the other hand, the shoulder of the auxiliary tube is used to lightly scratch the surface of the glass tube in a direction perpendicular to the glass tube, and pressure is applied to the breakpoint to obtain a predetermined aperture.

[0007] In the arbuscular mycorrhizal fungus single spore separation and sterilization filtration tool, preferably, the pore size of the filter is less than 38 μm, and the first cylindrical section is used to adapt to a syringe and a pipette tip.

[0008] A second aspect of the present invention provides a method for using the above-mentioned arbuscular mycorrhizal fungus single spore separation and sterilization filtration tool, comprising the following steps: Step 1: Mount the micropipette on the glass tube holder of the microinjector, then mount the glass tube holder on a three-dimensional micromanipulator, and then mount the three-dimensional micromanipulator on a microscope; Step 2: Observe the spores under a microscope and use the micropipette to aspirate a single spore; Step 3: Using a three-dimensional micromanipulator, move the micropipette to the location of the spores, rotate the microinjector, and suck the spores into the micropipette. Then, move the three-dimensional micromanipulator and rotate the microinjector in the opposite direction to remove the spores and collect them in a centrifuge tube for later use. Step 4: Soak the spores obtained in step 3 in sterilizing solution A for a certain period of time, rinse with sterile water for a certain number of times, then transfer to sterilizing solution B for a certain period of time, rinse with sterile water for a certain number of times, and set aside; Step 5: Pipette the spores to be used in step 4 into a centrifuge tube, add sterilizing solution A, and ultrasonically vibrate for a certain period of time to remove impurities attached to the surface of the spores. After the vibration is completed, use a pipette tip or a sterile syringe to draw the sterilizing solution A containing the spores from the entrance of the first cylindrical segment into the filter. At this time, the spores will fall onto the filter screen. Use a sterile syringe or a pipette tip to inject sterile water from the entrance of the first cylindrical segment to clean it a certain number of times; invert the filter so that the entrance of the fifth filter segment is on the top. Use a pipette tip or a sterile syringe to draw sterilizing solution B and inject it into the entrance of the fifth filter segment. At this time, the mixture of sterilizing solution B and spores falls into the centrifuge tube again. Repeat the above steps again to sterilize with sterilizing solution B. After the sterilization is completed, rinse the filter with sterile water a certain number of times until the sterilization is completed.

[0009] In the method for using the tool for isolating and sterilizing filtration of single spores of arbuscular mycorrhizal fungi, preferably, in step 1, the tilt angle of the glass tube holder is adjusted to 20-40° to avoid optical interference.

[0010] In the method for using the arbuscular mycorrhizal fungus single spore separation and sterilization filtration tool, preferably, step 3 includes the following specific steps: Use low-power microscopy to initially screen and identify spore-rich areas in the soil suspension; Differential interference contrast imaging was used to distinguish spores from foreign particles using a high-power microscope; The microinjection apparatus was rotated step by step to monitor the spore adsorption status in real time.

[0011] In the method for using the arbuscular mycorrhizal fungus single spore separation and sterilization filtration tool, preferably, sterilization solution A is 2% by mass of chloramine T + 0.01% by mass of Tween 20; and sterilization solution B is a mixture of 200 mg / L streptomycin + 100 mg / L gentamicin.

[0012] The present invention has the following advantages due to the adoption of the above technical solution: 1. The present invention achieves precise and efficient separation of AMF single spores in complex matrices through the coordinated control of a micron-level glass tube micropipette (tip diameter adjustable 50-300μm) and a three-dimensional micromanipulator, with an efficiency of >95%, solving the problem of low efficiency of traditional manual sorting.

[0013] 2. The present invention is based on the design of a multi-layer filter tool, which reduces the spore transfer step to 2 times in the sterilization process. Compared with the existing method with a spore loss rate of ≥20%, the spore retention rate of this method is ≥98%.

[0014] 3. The present invention integrates the chloramine T-antibiotic dual-stage sterilization process, significantly reducing the microbial contamination rate and meeting laboratory needs.

[0015] 4. By improving the geometry of the micropipette tip, spore aspiration efficiency is increased to 50%. The filter employed in this invention achieves a spore capture rate of 95.2% during the filtration and sterilization process. This makes it particularly suitable for the isolation, culture, and sterilization of AMF spores, significantly reducing the risk of contamination while maintaining spore bioactivity. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 A schematic diagram of the process of aspirating and removing spores using a micropipette according to one embodiment of the present invention, wherein Figure 1 is a spore positioning diagram, Figure 2 is a spore aspiration diagram, Figure 3 is a spore removal diagram, and Figure 4 is a spore collection diagram; Figure 2 Figure 1 is a diagram of the process for aspirating and removing spores using a micropipette according to this embodiment of the present invention, wherein Figure 1 is a diagram of spore positioning, Figure 2 is a diagram of spore aspiration, Figure 3 is a diagram of spore removal, and Figure 4 is a diagram of spore collection; Figure 3 A schematic diagram of a micropipette provided in this embodiment of the present invention; Figure 4 Schematic diagram of the spore sterilizing filter provided in this embodiment of the present invention, wherein Figure a is a stereoscopic view and Figure b is a cross-sectional view; Figure 5 Schematic diagram of the spore sterilizing filter provided in this embodiment of the present invention, wherein Figure a is a schematic diagram of the upper filtration section, and Figure b is a schematic diagram of the lower filtration section; Figure 6 Schematic diagram of the combination of a 1 ml sterile syringe and a spore sterilizing filter provided in this embodiment of the present invention; Figure 7 Schematic diagram of the assembly of the pipette tip and the spore sterilizing filter provided in this embodiment of the present invention; Figure 8 A spore germination diagram provided for this embodiment of the present invention; The reference numerals in the figures are as follows: 1-micropipette; 2-filter; 3-upper filter section; 4-lower filter section; 5-first cylindrical section; 6-second cylindrical section; 7-third cylindrical section; 8-fourth filter section; 9-fifth filter section. DETAILED DESCRIPTION

[0017] To make the objectives, technical solutions, and advantages of the present invention more clear, the technical solutions of the present invention are described clearly and completely below. Obviously, the embodiments described are only some of the embodiments of the present invention, not all of them. All other embodiments derived by ordinary persons in this field based on the embodiments of the present invention without creative effort are within the scope of protection of the present invention.

[0018] Unless otherwise defined, the technical or scientific terms used in the present invention shall have the usual meanings understood by persons of ordinary skill in the field to which the present invention belongs. The words "first", "second", "third", "fourth" and similar terms used in the present invention do not indicate any order, quantity or importance, but are only used to distinguish different components. Words such as "include" or "comprise" mean that the elements or objects preceding the word include the elements or objects listed after the word and their equivalents, without excluding other elements or objects. Words such as "connect" or "connected" are not limited to physical or mechanical connections, but may include electrical connections, whether direct or indirect.

[0019] For ease of description, spatially relative terms may be used herein to describe the relationship of one element or feature relative to another element or feature as shown in the figures, such as "inside," "outside," "inner side," "outer side," "lower," "upper," etc. Such spatially relative terms are intended to encompass different orientations of the device in use or operation in addition to the orientation depicted in the figures.

[0020] Currently, AMF propagation relies primarily on two techniques: open pot culture using boiling sand as a substrate, and a dual culture system using arbuscular mycorrhizae and carrot hairy roots under sterile conditions (BéCard and Fortin 2006). While the pot culture method is simple to operate, the open environment is prone to microbial contamination, resulting in insufficient spore purity. While the dual culture method can produce sterile spores, it still faces bottlenecks such as low efficiency in isolating original spores and high spore loss during the sterilization process (traditional methods reach up to 20%). The diverse morphology of AMF spores in soil (mostly approximately 40-200 μm in diameter) and their mixed distribution further exacerbate the technical challenges of accurately isolating individual spores. For example, in agricultural applications, low-purity AMF inoculants can lead to fluctuations in symbiotic efficiency. In ecological restoration, screening for target spores in heavy metal-contaminated soils can take weeks. Existing microscopy methods often rely on manual isolation, with a success rate of less than 60%. Furthermore, the sterilization process requires more than five spore transfers, resulting in a 35% loss of viability. Therefore, there is an urgent need to develop an integrated tool to achieve efficient single spore capture, in situ sterilization and zero-loss filtration to support the large-scale application of AMF in crop yield increase, soil remediation and symbiotic mechanism research.

[0021] To address these technical challenges, the present invention provides a simple and efficient single-spore isolation and sterilization filtration tool for arbuscular mycorrhizal fungi (AMF). This tool effectively addresses the challenge of single-spore isolation of AMF fungi and reduces spore loss during the sterilization process. This novel single-spore isolation method and sterilizing filter achieves single spore isolation and spore retention during sterilization, demonstrating its significant application value.

[0022] like Figure 3 、 Figure 4 、 Figure 5 As shown, the single spore isolation and sterilization filtration tool for arbuscular mycorrhizal fungi of the present invention comprises: a micropipette 1, which is a tubular structure with a diameter gradually decreasing from the tail end to the tip end. The tip end of the micropipette 1 has a diameter of 50-300 μm. The micropipette 1 is mounted on a microscope for microscopic observation of spores and single spore aspiration. The tip end of the present invention refers to the end for aspirating spores, and the other end is the tail end. A filter 2 comprises an upper filter section 3 and a lower filter section 4. The upper filter section 3 comprises a first cylindrical section 5, a second cylindrical section 6, and a third cylindrical section 7, which are coaxially arranged and sequentially connected. A filter screen (a 400-mesh nylon filter screen with a pore size of <38 μm) is provided at the end of the third cylindrical section 7. The lower filter section 4 comprises a fourth filter section 8 and a fifth filter section 9, which are coaxially arranged and interconnected. The fourth filter section 8 is configured to be sleeved on the third cylindrical section 7. Together, the fourth filter section 8 and the fifth filter section 9 constitute the filter 2 for filtering spores extracted by the micropipette 1.

[0023] Specifically, the micropipette 1 of the present invention is a borosilicate glass tube (outer diameter 1.00 mm, inner diameter 0.58 mm, total length 10 cm), prefabricated using a microelectrode puller (P-97) according to the parameters (Table 1). The micropipette 1 is produced by the following method: After the glass tube is pulled using the microelectrode puller, the tip diameter is adjusted using the shoulder scratching method: holding a glass tube of the target specification in one hand and an auxiliary tube in the other, the shoulder of the auxiliary tube is used to lightly scratch the surface of the glass tube in a single stroke perpendicular to the glass tube, applying pressure to the breakpoint to obtain the desired aperture.

[0024] Table 1 Glass tube drawing parameters

[0025] A second aspect of the present invention provides a method for using the above-mentioned arbuscular mycorrhizal fungus single spore separation and sterilization filtration tool, comprising the following steps: Step 1: Mount the micropipette 1 on the glass tube holder (CellTram® Oil, Eppendorf) of the microinjector (CellTram® Oil). The glass tube holder is then mounted on a three-dimensional micromanipulator (M3301L, World Precision Instruments). The three-dimensional micromanipulator is then mounted on a microscope (BX50WI, Olympus).

[0026] Step 2: Observe the spores under a microscope (BX50WI, Olympus, supporting low-magnification positioning (40×), (100×) and high-magnification (400×) precise operation) and use a micropipette to aspirate a single spore.

[0027] Step 3: Use the three-dimensional micromanipulator that can move up and down, left and right, and forward and backward to move the micropipette 1 to the location of the spores, gently rotate the microinjector, and then suck the spores into the micropipette. Then move the three-dimensional micromanipulator and rotate the microinjector in the opposite direction to remove the AMF spores and collect them in a 1.5ml centrifuge tube for use. Figure 1 and Figure 2 shown.

[0028] The single spore isolation operation is as follows: Low-power initial screening: Identify spore-rich areas in soil suspension; High-magnification lock-on: Differential interference contrast (DIC) imaging is used to distinguish AMF spores from foreign particles. Negative pressure control: The microinjector rotates in steps (minimum adjustment volume < 0.02 μL) to monitor the spore adsorption status in real time.

[0029] Furthermore, if Figure 1 、 2As shown, the single spore separation operation is as follows: ① Spore positioning: Displays the spore positioning process under a microscope. Use a low-power microscope to find the spore position, and switch to a high-power microscope for precise operation.

[0030] ② Spore aspiration: The micropipette 1 is moved to the spore position by the three-dimensional micromanipulator, and the microinjector is gently rotated to aspirate the spores into the micropipette 1.

[0031] ③ Spore removal: The aspirated spores are removed into a 1.5 ml centrifuge tube by rotating the microinjector in the reverse direction.

[0032] ④ Spore collection: The spores are successfully removed and collected in the centrifuge tube, ready for the next sterilization operation.

[0033] Step 4: Sterilization Solution A consists of 2% chloramine T (by mass) and 0.01% Tween 20 (by mass) (pH 7.0-7.5). Mix thoroughly and filter through a 0.22 μm filter. Sterilization Solution B consists of a mixture of 200 mg / L streptomycin and 100 mg / L gentamicin sulfate (sterilized through a 0.22 μm filter (store at 4°C for ≤ 48 h)). Soak AMF spores in Sterilization Solution A for 10 minutes, rinse 3-5 times with sterile water, then transfer to Sterilization Solution B for 10 minutes. Rinse 3-5 times with sterile water before use.

[0034] Step 5: Make a sterilizing filter 2. The filter 2 is divided into three parts. The upper part is composed of the first cylindrical section 5, the second cylindrical section 6 and the third cylindrical section 7 which are coaxially arranged and connected in sequence. The middle part is composed of a 400-mesh filter screen cut into a circle. The lower part is composed of the fourth filter section 8 and the fifth filter section 9 which are coaxially arranged and connected to each other. The three parts are bonded to form a filter 2 with a filter screen in the middle and openings on both sides. The two ends of the openings can be matched with a 1ml syringe or a 1ml pipette tip, such as Figure 6 、 Figure 7 shown.

[0035] Step 6: Pipette the spores to be used in step 4 into a new sterilized 1.5ml centrifuge tube, and add sterilizing solution A to the scale line to 1ml. Ultrasonic vibration for 1 minute to remove impurities attached to the surface of the spores. After the vibration is completed, use a 1ml pipette tip or a 1ml sterile syringe to draw the sterilizing solution containing spores to the inlet of the first cylindrical section 5 and inject it into the filter 2. At this time, the spores will fall onto the middle filter screen. Use a sterile syringe or a 1ml pipette tip to inject sterile water from the inlet to clean 3-5 times; turn the filter 2 upside down, at this time the inlet of the fifth filter section 9 is on the top, use a 1ml pipette tip or a 1ml sterile syringe to draw sterilizing solution B and inject it into the inlet of the fifth filter section 9. At this time, the mixture of sterilizing solution B and spores falls into the centrifuge tube again, and repeat again until sterilization is completed. The germination diagram of a single spore is shown in the figure below. Figure 8shown.

[0036] In the method for using the tool for isolating and sterilizing filtration of single spores of arbuscular mycorrhizal fungi, preferably, in step 1, the tilt angle of the glass tube holder is adjusted to 20-40° to avoid optical interference.

[0037] Compared with the prior art, the present invention has the following advantages: Table 2 Comparison of technical advantages

[0038] This invention provides a simple and efficient tool for the isolation and sterilization filtration of single spores of arbuscular mycorrhizal fungi (AMF). This tool effectively addresses the challenge of isolating individual spores of AMF fungi and reduces spore loss during the sterilization process. This novel single-spore isolation method and sterilizing filter achieves single spore isolation and spore retention during sterilization, demonstrating its significant application value.

[0039] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the various embodiments of the present invention.

Claims

1. A tool for separating and sterilizing spores of arbuscular mycorrhizal fungi, characterized in that: include: A micropipette (1) is a tubular structure with a diameter gradually decreasing from the tail end to the tip end, the tip diameter of the micropipette (1) is 50-300 μm, and the micropipette (1) is mounted on a microscope for microscopic observation of spores and single spore aspiration operation; A filter (2), the filter (2) comprising an upper filter section (3) and a lower filter section (4), the upper filter section (3) comprising a first cylindrical section (5), a second cylindrical section (6) and a third cylindrical section (7) coaxially arranged and sequentially connected, a filter screen being provided at the end of the third cylindrical section (7), the lower filter section (4) comprising a fourth filter section (8) and a fifth filter section (9) coaxially arranged and mutually connected, the fourth filter section (8) being used to be sleeved on the third cylindrical section (7), and the two together forming the filter (2) for filtering the spores extracted by the micropipette (1).

2. The arbuscular mycorrhizal fungus single spore separation and sterilization filtration tool according to claim 1, characterized in that: The micropipette (1) is obtained by the following method: after the glass tube is drawn by a microelectrode drawing instrument, the tip diameter is adjusted by a shoulder scratching method: holding a glass tube of target specification in one hand and an auxiliary tube in the other hand, the surface of the glass tube is lightly scratched once with the shoulder of the auxiliary tube in a direction perpendicular to the glass tube, and pressure is applied to the breakpoint to obtain a predetermined aperture.

3. The arbuscular mycorrhizal fungus single spore separation and sterilization filtration tool according to claim 1, characterized in that: The pore size of the filter is less than 38 μm, and the first cylindrical section (5) is used to adapt to a syringe and a pipette tip.

4. A method for using the arbuscular mycorrhizal fungus single spore separation and sterilization filtration tool according to any one of claims 1 to 3, characterized in that: The steps include: Step 1: Mount the micropipette (1) on the glass tube holder of the microinjection instrument, then mount the glass tube holder on a three-dimensional micromanipulator, and then mount the three-dimensional micromanipulator on a microscope; Step 2: Observe the spores under a microscope and use the micropipette (1) to aspirate a single spore; Step 3: Using a three-dimensional micromanipulator, move the micropipette (1) to the location of the spores, rotate the microinjector, and suck the spores into the micropipette (1), then move the three-dimensional micromanipulator, and rotate the microinjector in the opposite direction to remove the spores and collect them in a centrifuge tube for use; Step 4: Soak the spores obtained in step 3 in sterilizing solution A for a certain period of time, rinse with sterile water for a certain number of times, then transfer to sterilizing solution B for a certain period of time, rinse with sterile water for a certain number of times, and set aside; Step 5: Pipette the spores to be used in step 4 into a centrifuge tube, add sterilizing solution A, and ultrasonically vibrate for a certain period of time to remove impurities attached to the surface of the spores. After the vibration is completed, use a pipette tip or a sterile syringe to draw the sterilizing solution A containing the spores from the inlet of the first cylindrical section (5) and inject it into the filter (2). At this time, the spores will fall onto the filter screen. Use a sterile syringe or a pipette tip to inject sterile water from the inlet of the first cylindrical section (5) for cleaning; turn the filter (2) upside down, at this time the inlet of the fifth filter section (9) is on the top, use a pipette tip or a sterile syringe to draw sterilizing solution B and inject it into the inlet of the fifth filter section (9). At this time, the mixture of sterilizing solution B and spores falls into the centrifuge tube again, and repeat the above steps again to sterilize with sterilizing solution B. After the sterilization is completed, rinse the filter with sterile water for a certain number of times until the sterilization is completed.

5. The method for using the tool for separating and sterilizing filtration of single spores of arbuscular mycorrhizal fungi according to claim 4, characterized in that: In step 1, the tilt angle of the glass tube holder was adjusted to 20-40° to avoid optical interference.

6. The method for using the tool for separating and sterilizing filtration of single spores of arbuscular mycorrhizal fungi according to claim 4, characterized in that: Step 3 includes the following specific steps: Use low-power microscopy to initially screen and identify spore-rich areas in the soil suspension; Differential interference contrast imaging was used to distinguish spores from foreign particles using a high-power microscope; The microinjection apparatus was rotated step by step to monitor the spore adsorption status in real time.

7. The method for using the tool for separating and sterilizing filtration of single spores of arbuscular mycorrhizal fungi according to claim 4, characterized in that: Sterilization solution A is a mixture of 2% chloramine T by mass and 0.01% Tween 20 by mass; sterilization solution B is a mixture of 200 mg / L streptomycin and 100 mg / L gentamicin.

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