A method for collecting arbuscular mycorrhizal fungal hyphae using nylon membrane

Abstract

The present invention provides a method for collecting arbuscular mycorrhizal fungal hyphae using a nylon membrane, and belongs to the field of microbial technology. The method for collecting arbuscular mycorrhizae using a nylon membrane provided by the present invention comprises preparing a hyphae collection device that allows the passage of arbuscular mycorrhizal hyphae while the roots of the host plant cannot pass through; planting the host plant; burying the hyphae collection device; and collecting and separating the arbuscular mycorrhizal hyphae. The method of the present invention is simple to operate, low in cost, convenient and quick, and does not require hyphae separation under a stereoscope, thereby reducing the increased experimental risk caused by interference from other factors. At the same time, the present invention solves the problem of mycelium being unable to be separated due to drying of the nylon membrane after long-term exposure to air, and can maximize the collection of arbuscular mycorrhizal hyphae, avoiding waste of arbuscular mycorrhizal hyphae.

Description

Technical Field

[0001] The invention relates to the technical field of microorganisms, and in particular to a method for collecting arbuscular mycorrhizal fungal hyphae by utilizing a nylon membrane. Background Art

[0002] Arbuscular mycorrhizal fungi (AMF) produce a vast network of hyphae in the soil, called mycelium. This vast network of AMF hyphae allows host plants to access water and nutrients beyond the rhizosphere. The surface of these hyphae in the soil is a "hotspot" of bacterial activity, where a series of complex fungal-bacterial interactions occur. Therefore, the most challenging problem is how to collect and isolate AMF hyphae as completely as possible. Currently, a commonly used method is to exploit the fact that hyphae cannot pass through the micropores of nylon membranes.

[0003] Nylon membrane is a commonly used microporous filtration membrane. Its material is nylon, so it is also called polyamide microporous filtration membrane. As a filtration membrane, its most important function is to be able to filter impurities with particle sizes larger than the pore size of its filter pores and retain them on the liquid inlet side of the nylon membrane to achieve the filtration effect. Currently, after collecting mycelia using nylon membranes, the common method of separating mycelia is to pick them up under a microscope. This separation method is not only labor-intensive but also requires a lot of time to identify the arbuscular mycorrhizal mycelia to confirm that the separated mycelia are arbuscular mycorrhizal mycelia. Therefore, a method that can simply and quickly collect and separate arbuscular mycorrhizal mycelia is urgently needed. Summary of the Invention

[0004] In view of this, the object of the present invention is to provide a method for collecting arbuscular mycorrhizal fungal hyphae using nylon membrane. The method provided by the present invention can simply and effectively collect arbuscular mycorrhizal hyphae, thereby reducing soil bacterial contamination and interference with plant roots.

[0005] In order to achieve the above object, the present invention provides the following technical solutions:

[0006] A method for collecting arbuscular mycorrhizal fungal hyphae using a nylon membrane comprises the following steps:

[0007] (1) Making a hyphae collection device: Take a tubular material with two open ends, place three layers of the first nylon membrane at equal distances inside the tubular material and perpendicular to the tubular wall, and seal both openings with a second nylon membrane; the host plant roots cannot pass through the second nylon membrane, but the arbuscular mycorrhizal hyphae can pass through; the first nylon membrane allows the arbuscular mycorrhizal hyphae to grow on the membrane;

[0008] (2) Planting the host plant: The incubator is divided into a root chamber and a mycelium chamber by two partition materials, wherein the mycelium chamber is located in the middle of the incubator and the root chambers are located on both sides of the mycelium chamber; the host plant is planted in the root chamber; wherein the central areas of the two partition materials are respectively provided with corresponding holes; and after the holes are punched, the partition materials are adhered on both sides and covered with a second nylon membrane;

[0009] (3) burying the mycelium collecting device horizontally in the mycelium chamber, with the mycelium collecting device located in the center of the mycelium chamber;

[0010] (4) Carry out routine planting and management, and remove the mycelium collection device after 30 to 40 days;

[0011] (5) Collecting arbuscular mycorrhizal hyphae: Use a vortex oscillator to separate the arbuscular mycorrhizal hyphae attached to the nylon membrane.

[0012] In certain embodiments, the tubular material is preferably a PVC tube.

[0013] In certain embodiments, the pore size of the first nylon membrane in step (1) is preferably 0.45 μm;

[0014] In certain embodiments, the diameter of the first nylon membrane is slightly smaller than the diameter of the tubular material, so that the hyphae can be collected and the hyphae can be allowed to grow through to the next first nylon membrane.

[0015] In certain embodiments, the pore size of the second nylon membrane in step (1) is preferably 38 μm.

[0016] In certain embodiments, the tubular material in step (1) preferably has a diameter of 5 cm and a length of 8 cm.

[0017] In certain embodiments, the length×width×height of the culture box is preferably 34 cm×22 cm×18 cm; the width of the mycelium chamber is preferably 10 cm, and the width of the root chamber is preferably 12 cm.

[0018] In certain embodiments, the separator material is preferably a PVC board; the length × width of the PVC board is preferably 22 cm × 18 cm; the corresponding holes in step (2) are preferably 6 groups; the holes are all located in the center of the separator material, distributed in three layers, two holes per layer, and spaced uniformly. In certain embodiments, the diameter of the holes is preferably 3 cm, and the distance between the holes on each piece of separator material is preferably 1.5 cm.

[0019] In certain embodiments, the host plant in step (2) includes but is not limited to tillering onion, tomato, corn, and clover. The present invention provides favorable conditions for infection by arbuscular mycorrhizal fungi by selecting herbaceous plants with well-developed root systems, thereby deriving a large amount of hyphae.

[0020] In certain embodiments, the host plant seedling in step (2) is when the host plant has grown two true leaves.

[0021] In certain embodiments, the planting method of the host plant in step (2) includes but is not limited to intercropping and monocropping.

[0022] In certain embodiments, the number of hyphae collecting devices in step (3) is preferably four, arranged in parallel in two layers, with two hyphae collecting devices in each layer. If there are too many hyphae collecting devices, the PVC tube may be crowded, and the excess PVC tube may be exposed to the air; if there are too few hyphae collecting devices, the space may not be fully occupied, and there may be excess space around the hole.

[0023] In certain embodiments, after burying the mycelium collecting device in step (3), the soil should be filled so that the soil height of the mycelium chamber is consistent with the soil height of the root chamber. This ensures that the mycelium collecting device will not be exposed to the outside and damage the internal soil structure to collect mycelia.

[0024] In certain embodiments, the arbuscular mycorrhizal hyphae in step (5) include but are not limited to hyphae of arbuscular mycorrhizal fungi Rhizophagus intraradices, Diversispora epigaea, and Rhizophagus irregularis.

[0025] In certain embodiments, the step (5) of separating the arbuscular mycorrhizal hyphae attached to the nylon membrane comprises the following steps:

[0026] S1. Place the first nylon membrane in a centrifuge tube, add sterile water, and vortex mixer at 3000 rpm for 7-10 min. Remove the first nylon membrane, let the centrifuge tube stand, and then centrifuge. Aspirate the supernatant for later use.

[0027] S2. Place the second nylon membrane in the funnel, pour the supernatant obtained in S1 into the funnel for filtration, repeat the filtration 2 to 3 times, and then filter once with filter paper. The filtrate obtained is the pure arbuscular mycorrhizal hyphae.

[0028] In certain embodiments, the centrifugation in S1 is preferably performed at 7000 rpm to 8000 rpm for 50 s to 70 s, more preferably at 7500 rpm for 60 s.

[0029] In certain embodiments, in S2, the second nylon membrane is placed in the funnel. Preferably, the second nylon membrane is cut into circular sheets, and then folded according to a conventional filter paper folding method and placed in the funnel.

[0030] In certain embodiments, the pore size of the filter paper used in the filter paper filtration in S2 is preferably such that pore size allows passage of arbuscular mycorrhizal hyphae but not plant roots, such as filter paper model 103. The present invention utilizes a first nylon membrane to collect arbuscular mycorrhizal hyphae, then separates the hyphae collected on the first nylon membrane to produce a supernatant, which is then filtered through a second nylon membrane and filter paper to produce a mycelial suspension. This avoids the introduction of other factors while maximizing the collection of arbuscular mycorrhizal hyphae.

[0031] Beneficial Technical Effects: The present invention provides a method for collecting arbuscular mycorrhizal fungal hyphae using a nylon membrane, comprising preparing a hyphae collection device that allows the passage of arbuscular mycorrhizal hyphae while preventing the host plant's roots from passing through; planting the host plant; embedding the hyphae collection device; and collecting and separating the arbuscular mycorrhizal hyphae. This method is simple to operate, low-cost, and convenient. It eliminates the need for stereomicroscope-based mycelial separation, reducing experimental risks associated with interference from other factors. Furthermore, the present invention addresses the issue of mycelial separation being unable to be separated due to drying of the nylon membrane after prolonged exposure to air. This method maximizes the collection of arbuscular mycorrhizal hyphae and avoids waste. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 This is a schematic diagram of the tillering onion and tomato planting pattern;

[0033] Figure 2 This is a schematic diagram of the mycelium collection device;

[0034] Figure 3 Schematic diagram of hyphae staining under a microscope. DETAILED DESCRIPTION

[0035] For a better understanding of the present invention, the following examples are provided to further illustrate the present invention, but the present invention is not limited to the following examples. The materials, reagents, etc. used in the examples and test examples of the present invention, unless otherwise specified, can be obtained from commercial sources; the methods used in the examples and test examples of the present invention, unless otherwise specified, are conventional methods.

[0036] There were two treatments: 38μm-tillering onion accompanied by tomato and 38μm-tomato monoculture, in which the root separation method was 38μm (no root communication, but root exudates and AMF hyphae can communicate).

[0037] Example 1

[0038] (1) Preparation of a hyphae collection device: Take a PVC tube with a diameter of 5 cm and a length of 8 cm, and place three layers of nylon membranes with a pore size of 0.45 μm at equal intervals and vertically inside the PVC tube to allow AM fungal hyphae to grow on the membranes; seal both sides with nylon membranes with a pore size of 38 μm;

[0039] (2) Incubator: Take an incubator with a length × width × height of 34 cm × 22 cm × 18 cm, cut the length and width of the PVC board (22 cm × 18 cm) to the same size as the inner diameter of the incubator, cut six holes of the same size with a diameter of 3 cm in the middle of the PVC, and the distance between the holes is 1.5 cm. Glue a second nylon membrane on both sides of the PVC board; use the cut PVC board to divide the incubator into two root chambers and one mycelium chamber. The cut PVC board can prevent the plant roots from entering the mycelium chamber, allowing the mycelium to pass through and reach the tubular collector for the purpose of collecting mycelium ( Figure 1 ).

[0040] (3) The incubator was filled with 3.5 kg of low-phosphorus soil (obtained from the open-field low-phosphorus soil at the Xiangyang Research Base of Northeast Agricultural University in Harbin). Tomatoes (variety: Dongnong "708") and tillering onions (variety: Suihua "M27") were planted in the root chamber on one side of the incubator when they had two true leaves. Tillering onions were directly sown in the root chamber on the other side. The tomato and tillering onion intercropping ratio was 1:3. Each treatment was replicated three times, with six pots per replicate, and conventional seedling cultivation and management were performed.

[0041] (4) Four mycelium collecting devices are buried in the mycelium chamber. They are distributed in parallel in the upper and lower layers, with two devices in each layer. The four mycelium collecting devices are placed in the middle of the mycelium chamber as a whole structure, and the ports on both sides of the mycelium collecting devices are close to the holes in the middle of the plate.

[0042] (5) Sampling was performed 35 days after planting. The PVC tube in the mycelium chamber was removed.

[0043] (6) Isolation of arbuscular mycorrhizal hyphae on the nylon membrane: Remove the nylon membrane from the PVC tube. Place the 0.45 μm nylon membrane in a 50 mL centrifuge tube, add an appropriate amount of sterile water (higher than the nylon membrane), and vortex mixer at 3000 rpm for 7 to 10 minutes. Remove the nylon membrane with tweezers, let the centrifuge tube stand for 3 minutes, and then centrifuge at 7500 rpm for 1 minute. Use a pipette to aspirate the supernatant after centrifugation and transfer it to a new 50 mL centrifuge tube.

[0044] Prepare a new centrifuge tube and use sterilized tweezers to place the cut 38μm nylon membrane into the funnel. Pour the supernatant into the filter and repeat 2 to 3 times. Filter the supernatant in the centrifuge tube again through filter paper to remove impurities in the supernatant. The final collected filtrate is stored to make a mycelium suspension to achieve the purpose of isolating mycelium. Soak the collected mycelium in sterile water and store it in a 4℃ refrigerator for later use. The storage time should not exceed one week. If it is stored for more than one week, there is a risk of contamination by foreign bacteria, which will affect the accuracy of the experimental data.

[0045] Example 2

[0046] The difference between this embodiment and embodiment 1 is that in step (3), tomatoes are planted alone, and the specific operation is to plant tomatoes in the root chambers on both sides at a ratio of 1:1.

[0047] Test Example 1

[0048] The collected mycelial suspension can be stained with trypan blue to detect mycelia, and the collected arbuscular mycorrhizal mycelia can be detected under a microscope.

[0049] Trypan blue staining was used to identify the mycelial suspension of Example 1. 1 mL was aspirated and dropped onto a glass slide. The mycelial suspension was stained with trypan blue dye and the collection of mycelia in the mycelial suspension was observed under a microscope. The experimental results are shown in FIG. Figure 3 .Depend on Figure 3 It can be seen that the present invention can maximize the collection of arbuscular mycorrhizal hyphae, and the collected arbuscular mycorrhizal hyphae are relatively pure with less interference.

[0050] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as within the scope of protection of the present invention.

Claims

1. A method for collecting arbuscular mycorrhizal fungal hyphae using a nylon membrane, characterized in that: The following steps are involved: (1) Making a hyphae collection device: taking a tubular material with two ends open, placing three layers of a first nylon membrane at equal distances and perpendicular to the tube wall inside the tubular material, and sealing the two openings with a second nylon membrane; wherein the roots of the host plant cannot pass through the second nylon membrane, but the arbuscular mycorrhizal hyphae can pass through; the first nylon membrane can allow the arbuscular mycorrhizal hyphae to grow on the membrane; the pore size of the first nylon membrane in step (1) is 0.45 μm; the pore size of the second nylon membrane in step (1) is 38 μm; (2) Planting the host plant: The incubator is divided into a root chamber and a mycelium chamber by two partition materials, wherein the mycelium chamber is located in the middle of the incubator and the root chambers are located on both sides of the mycelium chamber; the host plant is planted in the root chamber; wherein the central areas of the two partition materials are respectively provided with corresponding holes; and after the holes are punched, the partition materials are adhered on both sides and covered with a second nylon membrane; (3) burying the mycelium collecting device horizontally in the mycelium chamber, with the mycelium collecting device located in the center of the mycelium chamber; (4) Carry out routine planting and management, and remove the mycelium collection device after 30 to 40 days; (5) Collecting arbuscular mycorrhizal hyphae: Use a vortex shaker to separate the arbuscular mycorrhizal hyphae attached to the nylon membrane; The step (5) of separating the arbuscular mycorrhizal hyphae attached to the nylon membrane using a vortex oscillator comprises the following steps: S1. Place the first nylon membrane in a centrifuge tube, add sterile water, and vortex at 3000 rpm for 7 to 10 minutes. Remove the first nylon membrane, let the centrifuge tube stand, and then centrifuge it. The supernatant is aspirated for standby use. The centrifugation in S1 is performed at 7000 rpm to 8000 rpm for 50 to 70 seconds. S2. Place the second nylon membrane in the funnel, pour the supernatant obtained in S1 into the funnel for filtration, repeat the filtration 2 to 3 times, and then filter once with filter paper. The filtrate obtained is the pure arbuscular mycorrhizal hyphae.

2. The method according to claim 1, characterized in that The host plants in step (2) include tillering onion, tomato, corn, and clover.

3. The method according to claim 1, characterized in that The host plant planting method in step (2) includes intercropping and monocropping.

4. The method according to claim 1, wherein The arbuscular mycorrhizal hyphae in step (5) include hyphae of arbuscular mycorrhizal fungi Rhizophagus intraradices, Diversispora epigaea, and Rhizophagus irregularis.

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