A method for obtaining extrarhizomatous hyphae of arbuscular mycorrhizal fungi and its application

Abstract

This invention discloses a method for obtaining arbuscular mycorrhizal fungal extrarhizomes and its application. The method includes the following steps: S1. Thoroughly soaking the plant roots and soil within the root growth range of the plant symbiotic with arbuscular mycorrhiza (AM) in an soaking solution, collecting the soaking material, and then mixing the soaking material, solid particles, and soaking solution evenly to obtain a mixture; the soaking solution is an ice-water mixture containing Tween 20; S2. Performing solid-liquid separation on the mixture obtained in step S1, filtering the resulting slurry with a sieve, and collecting the separated material that has not passed through the sieve; S3. Thoroughly soaking the separated material in ice water, collecting the flocculent material entangled with solid particles, thus obtaining the final product. This method can achieve rapid separation and collection of AM fungal extrarhizomes under ordinary laboratory conditions without the need for a microscope. It is simple to operate, highly efficient, and the obtained AM fungal extrarhizomes have high infectivity. It avoids the waste of live mycorrhizae in traditional methods for collecting fungal spores, saving resources and demonstrating high feasibility.

Description

Technical Field

[0001] This invention relates to the field of fungal hyphae isolation technology, specifically to a method for obtaining extrarhizomatous hyphae of arbuscular mycorrhizal fungi and its application. Background Technology

[0002] Arbuscular mycorrhizal (AM) fungi can form symbiotic relationships with over 72% of terrestrial plants. AM fungi not only form arbuscular structures and exist within the root cortex, but also distribute abundant extra-root hyphae. These extra-root hyphae absorb nutrients from the soil and transport them into the root system, thereby increasing nutrient uptake by the plant. Furthermore, most AM fungal extra-root hyphae grow rapidly and can survive for up to 5 months ("Lifespan and functionality of mycorrhizal fungal mycelium are uncoupled from host plant lifespan"), serving as propagules for AM fungi ("Colonization of roots by arbuscular mycorrhizal fungi using different sources of inoculum"), allowing AM fungi to colonize new host plant roots and form new arbuscular mycorrhizal symbionts. Isolating and collecting live extra-root hyphae of AM fungi from the soil facilitates the diversification and expansion of arbuscular mycorrhizal fungal propagules.

[0003] In mycorrhizal research, those skilled in the art need to use large quantities of AM fungal propagules when exploring the intrinsic mechanisms of arbuscular mycorrhizal symbiosis and conducting field application trials of arbuscular mycorrhizal fungi. Current techniques typically involve directly inoculating plants with AM fungal spores for propagation, obtaining a large number of AM fungal spores as AM fungal propagules. Examples include pot culture, glass bead compartment culture, atomized culture, and aseptic double culture. These methods require breaking up plant roots or rhizosphere soil, first using a wet sieve to decanter the spores, then using a gradient centrifugation with a sucrose solution to separate plump spores from the plant roots and soil. This process is time-consuming, and the spores are easily inactivated in sucrose. Simultaneously, a large amount of viable mycelium in the soil is easily lost during spore separation, resulting in a waste of AM fungal propagule resources. Furthermore, since the spore production rate of AM fungi is much lower than the viable mycelium production rate, directly isolating viable AM ​​fungal mycelium is more conducive to the rapid propagation of AM fungi. However, traditional methods for isolating live hyphae of AM fungi are rather crude, requiring repeated stirring, centrifugation, and filtration processes ("External hyphal production of vesicular-arbuscular mycorrhizal fungi pasture and tallgrass prairie communities"). Moreover, to obtain complete live hyphae, hyphae need to be picked out in an ice-water bath under a stereomicroscope, which is time-consuming and laborious. Summary of the Invention

[0004] To address the lack of a simple, efficient, and visually-based method for separating and collecting arbuscular mycorrhizal (AM) fungal extraradical hyphae from soil, this invention provides a method for obtaining arbuscular mycorrhizal fungal extraradical hyphae and its application.

[0005] The first objective of this invention is to provide a method for obtaining arbuscular mycorrhizal fungal extrarhizomes.

[0006] A second objective of this invention is to provide the application of the method in the propagation of arbuscular mycorrhizal fungi.

[0007] A third objective of this invention is to provide the application of the method in the preparation of arbuscular mycorrhizal fungal symbionts.

[0008] To achieve the above objectives, the present invention is implemented through the following solution:

[0009] This invention discovers that living ectodermal hyphae can form large, continuous mycelial masses on the outer surface of the host plant's roots. During stirring in water, these mycelia intertwine and attach to small, irregularly shaped solid particles. A sieve's mesh traps these mycelial particles. The trapped material is then poured onto a petri dish and immersed in ice water. Due to buoyancy, the mycelia and their entangled carriers appear as visible, cotton-like mycelial clumps in the ice water. Even slight water ripples can move these clumps, allowing the ectodermal hyphae of AM fungi to be collected directly with tweezers or a pipette without the need for a microscope.

[0010] A method for obtaining arbuscular mycorrhizal fungal extrarhizomes includes the following steps:

[0011] S1. Soak the plant roots and the soil within the root growth range thoroughly with the soaking solution, collect the soaking material, and then mix the soaking material, solid particles and soaking solution evenly to obtain a mixture;

[0012] The plant and arbuscular mycorrhizal fungi are in arbuscular mycorrhizal symbiosis; the soaking solution is an ice-water mixture containing Tween 20; the surface of the solid particles is rough and the density is less than that of water;

[0013] S2. Perform solid-liquid separation on the mixture obtained in step S1 to obtain slurry and precipitate; filter the obtained slurry with a sieve and collect the unfiltered slurry; the aperture of the sieve is equal to the maximum diameter of the solid particles;

[0014] S3. Soak the isolate obtained in step S2 in ice water, collect the hyphae, and obtain the arbuscular mycorrhizal fungal extrarhizomycelium.

[0015] Preferably, step S2 includes the following steps:

[0016] S21. The mixture obtained in step S1 is subjected to solid-liquid separation to obtain mud 1 and precipitate 1. The mud 1 is filtered with the screen and the unfiltered precipitate 1 is collected.

[0017] S22. Mix the soaking solution and the precipitate 1 obtained in step S21 evenly, separate the solid and liquid to obtain mud 2 and precipitate 2, filter the mud 2 with the sieve, and collect the unfiltered precipitate 2.

[0018] More preferably, step S2 further includes the following step: S23. Repeat step S22 3 to 4 times.

[0019] More preferably, step S2 further includes the following step: S23. Repeat step S22 3 times.

[0020] Preferably, the solid particles are expanded vermiculite.

[0021] Preferably, the maximum diameter of the solid particles is 0.4 to 0.5 mm.

[0022] More preferably, the maximum diameter of the solid particles is 0.5 mm.

[0023] Preferably, the ice-water mixture consists of ice and water in a mass ratio of (1-2):(2-6).

[0024] More preferably, the ice-water mixture consists of ice and water in a mass ratio of 1:1.

[0025] Preferably, the concentration of Tween 20 in the soaking solution is 0.05 μL / mL to 0.1 μL / mL.

[0026] More preferably, the concentration of Tween 20 in the soaking solution is 0.1 μL / mL.

[0027] Preferably, the arbuscular mycorrhizal fungus is *Rhizophagus irregularis*.

[0028] Preferably, the plant is planted in a cultivation pot. In step S1, the cultivation pot is placed in the soaking solution and soaked, with the water level below the rim of the cultivation pot and the soil surface not submerged.

[0029] The application of any of the methods described in the propagation of arbuscular mycorrhizal fungi should also be within the scope of protection of this invention.

[0030] The application of any of the methods described in the preparation of arbuscular mycorrhizal fungal symbionts should also be within the scope of protection of this invention.

[0031] Preferably, the arbuscular mycorrhizal fungal extrarhizomes obtained by any of the methods are used as propagation bodies.

[0032] Compared with the prior art, the present invention has the following beneficial effects:

[0033] The method provided by this invention firstly designs a low-temperature condition created by ice water, which is less likely to inactivate the extrarhizomes of arbuscular mycorrhizal fungi. Secondly, while designing the low-temperature condition, this invention uses a soil temperature of 20°C as a detergent, which does not affect the protein activity of the live hyphae and can wash away soil particles adhering to the surface of the hyphae, thereby maximizing the suspension of the hyphae in the ice water, which in turn facilitates the entanglement of the hyphae with the added expanded vermiculite particles. Finally, taking advantage of the characteristic that the hyphae entangled with vermiculite particles are easily identified under the condition of water agitation, they can be collected directly with the naked eye without the aid of microscopic magnification equipment, thus enabling the rapid separation and collection of extrarhizomes of arbuscular mycorrhizal fungi under ordinary laboratory conditions.

[0034] This invention offers simple and efficient operation, resulting in more active extra-root hyphae that enhance infection of host plants. It also improves the integrity of extra-root hyphae during the isolation process, providing a more reliable sample source for subsequent physiological or molecular detection studies of arbuscular mycorrhizal fungi extra-root hyphae. Furthermore, it avoids the waste of live hyphae in traditional methods for collecting arbuscular mycorrhizal fungi spores, thus saving resources, reducing costs, and demonstrating high feasibility. Attached Figure Description

[0035] Figure 1 This is a schematic diagram illustrating the process of soaking a potted plant-AM fungal symbiosis using an ice-water mixture containing Tween 20.

[0036] Figure 2 A schematic diagram illustrating the operation of mixing soil and ice water containing Tween 20.

[0037] Figure 3 This is a schematic diagram of the operation of filtering mud using a sieve.

[0038] Figure 4 This is a photograph of the extrarhizomatous hyphae of AM fungi suspended in water.

[0039] Figure 5 Microscopic images of the extrarhizomatous hyphae of AM fungi suspended in water.

[0040] Figure 6 Electrophoretic images of RNA extracted from dermal hyphae of AM fungi obtained by different methods. Detailed Implementation

[0041] The present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments. These embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods; the materials and reagents used, unless otherwise specified, are commercially available. The symbiotic culture method of AM fungi and plants in the following embodiments is a conventional technique, implemented with reference to the prior art "Inoculation with mycorrhizal fungi increases the yield of green peppers in a high P soil".

[0042] Example 1: A method for obtaining dermal hyphae of AM fungi

[0043] This embodiment uses *Rhizophagus irregularis* as an example to provide a method for obtaining live extracellular hyphae of AM fungi. The specific steps are as follows:

[0044] 1. A potted paper mulberry (Broussonetia papyrifera) that has been co-grown with Rhizophagus irregularis for 6 months, such as... Figure 1 As shown, the pot (10cm long * 10cm wide * 10cm high) is placed in a large square pot (30cm long * 30cm wide * 20cm high). Soaking solution is added to the pot. The soaking solution is an ice-water mixture containing 0.1μL / mL Tween 20 (the mass ratio of ice to water is 1:2). Soak for 1 hour to loosen the soil of the mulberry potted plant (the liquid level is below the rim of the pot, and the soil surface is not submerged), and reduce the adhesion between the roots and mycelium and the soil.

[0045] 2. Transfer all the roots and soil from the pot to a 2000mL beaker. Add 1000mL (more than 50 times the total volume of soil and roots) of fresh soaking solution and 10mL of sterile expanded vermiculite with a diameter of 0.4-0.5mm. Figure 2 As shown, stir in a circular motion with a glass rod for 5 minutes. The upper layer in the beaker is a uniform slurry with vermiculite suspended in it, and the lower layer is a sediment of vermiculite, roots, mud and sand.

[0046] 3. After that, as Figure 3 As shown, the mud in the beaker was filtered using a sieve with a 0.5 mm aperture. After filtration, the mixture of mycelium and mud remaining on the sieve was collected.

[0047] 4. Add 1000mL of new soaking solution to the original beaker to soak the precipitate from step 2, and continue to stir with a glass rod for 5 minutes. After that, filter the mud in the beaker using a 0.5mm mesh sieve. After filtration, collect the mixture of mycelium and mud on the sieve. Repeat this step 3 times.

[0048] 5. Place the mixture of mycelium and sediment collected from the sieve into a 15cm diameter glass petri dish, add 40mL of ice water, and it will float in the ice water as if... Figure 4 The cotton-like material wrapped around the vermiculite particles shown is the extracellular hyphae of AM fungi, as seen under a microscope. Figure 5 As shown;

[0049] 6. Use sterilized tweezers to pick up live extraroot hyphae in time and place them in a 1.5mL centrifuge tube that has been pre-cooled on ice for temporary storage. They can be used directly for inoculation of arbuscular mycorrhizal host plants, or frozen at -80℃ for physiological or molecular detection experiments of live extraroot hyphae of AM fungi.

[0050] Example 2: A method for obtaining AM fungal ectomycetes

[0051] This embodiment uses *Rhizophagus irregularis* as an example to provide a method for obtaining ectomycorrhizae of AM fungi. The specific steps are as follows:

[0052] 1. Place the potted paper mulberry (Broussonetia papyrifera) that has been co-grown with Rhizophagus irregularis for 8 months, along with its culture pot (10cm long * 10cm wide * 10cm high), into a large square pot (30cm long * 30cm wide * 20cm high). Add a soaking solution to the pot, which is an ice-water mixture containing 0.05μL / mL Tween 20 (ice to water mass ratio of 1:1). Soak for 3 hours to loosen the soil of the paper mulberry pot and reduce the adhesion between the roots and mycelium and the soil.

[0053] 2. Transfer the roots of the mulberry bonsai and the soil in the pot to a 2000mL beaker. Add 1000mL (more than 50 times the total volume of soil and roots) of fresh soaking solution and 5mL of sterile expanded vermiculite with a diameter of 0.4-0.5mm. Stir in a circular motion with a glass rod for 3 minutes. The upper layer in the beaker is a uniform slurry with vermiculite suspended in it, and the lower layer is a sediment of vermiculite, roots, mud and stones.

[0054] 3. Next, filter the mud in the beaker using a sieve with a 0.5mm aperture. After filtration, collect the mixture of mycelium and vermiculite that remains on the sieve.

[0055] 4. Add 600mL of fresh soaking solution to the original beaker to soak the precipitate from step 2, and continue to stir with a glass rod for 3 minutes. After that, filter the mud in the beaker using a 0.5mm mesh sieve. After filtration, collect the mixture of mycelium and vermiculite on the sieve. Repeat this step 4 times.

[0056] 5. Collect the mixture of mycelium and vermiculite from the sieve and place it in a 15cm diameter glass petri dish. Add 50mL of ice water. The cotton-like material floating in the ice water and wrapped around the vermiculite particles is the ectodermal mycelium of AM fungi.

[0057] 6. Use sterilized tweezers to pick up live mycelia from outside the roots and place them in a 1.5 mL centrifuge tube placed on ice at 4℃ for temporary storage. They can be used directly for inoculation of AM host plants, or frozen at -80℃ for physiological or molecular detection experiments of live mycelia from outside the roots of AM fungi.

[0058] Comparative Example 1: A method for obtaining ectopic hyphae of AM fungi

[0059] This comparative example uses *Rhizophagus irregularis* as an example to provide a method for obtaining ectomycorrhizae of AM fungi. The specific steps are as follows:

[0060] 1. Place the potted paper mulberry (Broussonetia papyrifera) that has been co-grown with Rhizophagus irregularis for 6 months, along with its cultivation pot (10cm long * 10cm wide * 10cm high), into a large square pot (30cm long * 30cm wide * 20cm high). Add room temperature tap water to the pot and soak for 1 hour to loosen the soil of the paper mulberry pot (the water level should be below the rim of the pot, and the soil surface should not be submerged).

[0061] 2. Transfer the roots of the bonsai and the soil in the pot to a 2000mL beaker. Add 1000mL (more than 50 times the total volume of soil and roots) of fresh tap water at room temperature and 10mL of sterile expanded vermiculite with a diameter of 0.4-0.5mm. Stir in a circular motion with a glass rod for 5 minutes. The upper layer of the beaker is a uniform slurry, and the lower layer is the sediment of roots, soil and stones.

[0062] 3. Next, filter the mud in the beaker using a 0.5mm mesh screen. After filtration, collect the mixture of mycelium and mud remaining on the screen.

[0063] 4. Add 1000mL of room temperature tap water to the original beaker to soak the precipitate from step 2, and continue to stir with a glass rod for 5 minutes. After that, filter the mud in the beaker using a 0.5mm mesh sieve. After filtration, collect the mixture of mycelium and mud on the sieve. Repeat this step 3 times.

[0064] 5. Collect the mixture of mycelium and sediment from the sieve and place it in a 15cm diameter glass petri dish. Add 50mL of room temperature tap water. The cotton-like clumps floating in the water are the mycelial clumps of AM fungi.

[0065] 6. Use sterilized tweezers to pick up live mycelia from outside the roots and place them in a 1.5 mL centrifuge tube placed on ice at 4°C for temporary storage. They can be used directly for inoculation of AM host plants, or frozen at -80°C for physiological or molecular experimental studies of live mycelia from outside the roots of AM fungi.

[0066] Comparative Example 2: A method for obtaining ectopic hyphae of AM fungi

[0067] This comparative example uses *Rhizophagus irregularis* as an example to provide a method for obtaining ectomycorrhizae of AM fungi. The specific steps are as follows:

[0068] 1. Place the potted paper mulberry (Rhizophagus irregularis) that has been co-grown with it for 6 months into a large square pot (30cm long, 30cm wide, and 20cm high), along with its cultivation pot (10cm long * 10cm ground diameter * 10cm high). Add a soaking solution to the pot, which is an ice-water mixture containing 0.1μL / mL Tween 20 (ice to water mass ratio of 1:1). Soak for 2 hours to loosen the soil of the potted paper mulberry (the liquid level should be below the rim of the pot, and the soil surface should not be submerged).

[0069] 2. Transfer the roots of the bonsai and the soil in the pot to a 2000mL beaker, add 1000mL (more than 50 times the total volume of soil and roots) of fresh soaking solution, and stir in a circular motion with a glass rod for 5 minutes. The upper layer of the beaker is a uniform mud slurry, and the lower layer is the sediment of roots, mud and stones.

[0070] 3. Next, filter the mud in the beaker using a 0.5mm mesh screen. After filtration, the filaments and mud that did not pass through the screen remain on the screen. Collect the mixture of mycelium and mud on the screen.

[0071] 4. Add 1000mL of new soaking solution to the original beaker to soak the precipitate from step 2, and continue to stir with a glass rod for 5 minutes. After that, filter the mud in the beaker using a 0.5mm mesh sieve. After filtration, collect the mixture of mycelium and mud remaining on the sieve. Repeat this step 3 times.

[0072] 5. Collect the mixture of mycelium and sediment from the sieve and place it in a 15cm diameter glass petri dish. Add 50mL of ice water. The cotton-like clumps floating in the ice water are mycelial clumps of AM fungi.

[0073] 6. Use sterilized tweezers to pick up live extraroot hyphae in time and place them in a 1.5mL centrifuge tube pre-cooled on ice at 4℃ for temporary storage. They can be used directly for inoculation of arbuscular mycorrhizal host plants, or frozen at -80℃ for physiological or molecular experimental studies of live extraroot hyphae of AM fungi.

[0074] Comparative Example 3: A method for obtaining AM fungal spores

[0075] Referring to existing techniques such as "wet sieving and decanting to separate AM fungal spores (Gerdemann and Nicolson (1963) Spores of mycorrhizal endogone species extracted from soil by wet sieving and decanting; Leonard and Vanderwoude (1976) Isolation of plasma membranes from corn roots by sucrose density gradient centrifugation: annomalous effect of ficoll)," the spores of *Rhizophagus irregularis* obtained using traditional methods mainly include the following steps:

[0076] 1. Allow the potted paper mulberry tree that has been coexisting with Rhizophagus irregularis for 6 months to dry naturally. Place the paper mulberry root system and rhizosphere soil into a 2000mL beaker, add 500mL of room temperature tap water, and stir in a circular motion with a glass rod for 5 minutes. Then, use a regular soymilk maker to grind the mixture of paper mulberry root system and rhizosphere soil (maximum speed 1 minute, 3 intervals). Then, pour the ground mixture of root system and rhizosphere soil back into the original beaker.

[0077] 2. Pass the mixture of broken roots and rhizosphere soil from step 1 through 100-mesh and 320-mesh sieves in turn. After passing through the sieves, rinse the sieves repeatedly with room temperature tap water 5 times.

[0078] 3. Collect the spores and sediment trapped on the 320-mesh sieve in step 2 into a 50 mL centrifuge tube. Use a 50% (m / v) sucrose solution to perform gradient centrifugation on the spores and sediment in the centrifuge tube (3000 r / min, 10 min). Finally, collect the floating matter on the surface of the 50% (m / v) sucrose solution, which is the plump spores of the arbuscular mycorrhizal fungus R. irregularis.

[0079] 4. Rinse the spores collected in step 3 five times with room temperature tap water, place them in centrifuge tubes and store them at 4°C for later use in inoculation of AM host plants.

[0080] Application Example 1: Evaluation of the infectivity of AM fungi

[0081] 1. Experimental Methods

[0082] Prepare sterile tissue culture seedlings of Broussonetia papyrifera (4 months old, 10 cm tall), and randomly divide them into 5 groups of 10 seedlings each. Use sterile quartz sand as the substrate, and inoculate them with ectomycorrhizal mycelia obtained according to the methods of Examples 1-2, ectomycorrhizal mycelia obtained according to the methods of Comparative Examples 1-2, and spores obtained according to Comparative Example 3. The inoculation amount of AM fungal ectomycorrhizal mycelia is 200 cm² / plant, and the inoculation amount of spores is 200 spores / plant.

[0083] After inoculation, the seedlings were watered (to maintain adequate moisture content) and nutrient managed (with a low-phosphorus Hoagland nutrient solution applied once a week) in the glass greenhouse.

[0084] Forty-five days after inoculation, the infection rate of Rhizocystis heterophylla in the roots of each group of Broussonetia papyrifera was determined.

[0085] 2. Experimental Results

[0086] Table 1. Effects of different types of inoculum on the colonization rate of AM fungi in the roots of Broussonetia papyrifera seedlings.

[0087]

[0088] Note: Different letters indicate significant differences between groups (P<0.05, one-way ANOVA).

[0089] As shown in Table 1, the infection rates of *Broussonetia papyrifera* seedlings inoculated with the ectodermal hyphae of *AM* fungi from Examples 1-2 and Comparative Example 2 were significantly higher than those inoculated with the ectodermal hyphae of Comparative Example 1 and the spores of Comparative Example 3. Conversely, the infection rate of *Broussonetia papyrifera* seedlings inoculated with the ectodermal hyphae of Comparative Example 1 was significantly lower than that inoculated with the spores of Comparative Example 3. These results indicate that the ice-water separation method can better preserve the activity of *AM* fungal ectodermal hyphae, and the isolated ectodermal hyphae have a better infection effect on the host plant.

[0090] Application Example 2: Evaluation of the efficiency of obtaining ectopic mycelia of AM fungi

[0091] 1. Determination of hyphae length

[0092] Referring to existing technologies, “Jakobsen I, Abbott LK, Robson AD (1992) External hyphae of vesicular-arbuscular mycorrhizal fungi associated with Trifolium subterranean L.1. Spread of hyphae and phosphorus inflow into roots.” and “Tennant D (1975) A test of a modified line intersect method of estimating root length.”, the length of extrarhiophageal hyphae of AM fungi collected according to the methods of Examples 1-2 and Comparative Examples 1-2 was determined. The main steps included:

[0093] (1) Add 0.1 mL of quartz sand to a 1.5 mL centrifuge tube containing AM fungal dermal hyphae, then add 1 mL of sterile water, and vortex for 3 times, 1 min each time, so as to break the continuous hyphae by friction between the quartz sand.

[0094] (2) Pour the liquid containing broken hyphae from the previous step into a 50mL centrifuge tube, and make up to 50mL with sterile water. After shaking well, use a pipette to take 5mL and filter the 5mL solution through a microporous membrane with a pore size of 0.45μm marked with a grid (each grid square has a side length of 3mm).

[0095] (3) The filter membrane containing hyphae in the previous step was stained with 0.05% trypan blue and rinsed with deionized water.

[0096] (4) Observe the hyphal density under a stereomicroscope at 70x magnification and calculate using the grid line intersection method (count all intersections of hyphae with the grid on the microporous filter membrane).

[0097] Table 2. Amount of AM fungal ectomycelium harvested in the examples and comparative examples.

[0098]

[0099] Note: Different letters indicate significant differences between groups (P<0.05, one-way ANOVA).

[0100] As shown in Table 2, the length of the AM fungal ectodermal hyphae collected in Examples 1 and 2 was significantly longer than that collected in Comparative Examples 1 and 2. Meanwhile, analysis of variance showed that the hyphae collected in Comparative Example 2 (without vermiculite) and Comparative Example 1 (without soil temperature 20) were significantly shorter than those in Examples 1 and 2. The results indicate that the methods employed in this invention—using an ice-water mixture to retain hyphae activity, adding expanded vermiculite to wrap the hyphae, and using soil temperature 20 to remove sand from the hyphae—improved the integrity of the isolated ectodermal hyphae and resulted in better visualization of the isolated live fungal ectodermal hyphae.

[0101] 2. RNA extraction and determination from mycelia

[0102] RNA was extracted from the extrarhizomes obtained in Examples 1-2 and Comparative Examples 1-2, which were stored at -80°C, using the Omega Fungal RNA Extraction Kit (EZNAFungal RNA Kit). The RNA was then detected by gel electrophoresis and its concentration was determined.

[0103] Electrophoresis results as follows Figure 6 As shown, the RNA from the extra-root hyphae obtained according to the methods of Examples 1 and 2 exhibits two relatively clear rRNA bands (28S and 18S), with RNA concentrations of 18 ng / μL and 21 ng / μL, respectively. In contrast, the RNA extracted from the hyphae collected in Comparative Example 1 showed no obvious electrophoretic bands, with an RNA concentration of only 1 ng / μL. The RNA extracted from the hyphae collected in Comparative Example 2 showed only faintly visible electrophoretic bands, with an RNA concentration of only 7 ng / μL. These results indicate that the method of separating AM fungal extra-root hyphae using a mixture of ice and water, vermiculite, and a soil temperature of 20°C can better protect the mycelial biological samples and effectively retain more nucleic acid samples, providing a more authentic and reliable sample source for subsequent physiological or molecular detection studies of AM fungal extra-root live hyphae.

[0104] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit the scope of protection of the present invention. For those skilled in the art, other variations or modifications can be made based on the above description and ideas, and it is neither necessary nor possible to exhaustively describe all implementation methods here. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the scope of protection of the claims of the present invention.

Claims

1. A method for obtaining arbuscular mycorrhizal fungal extrarhizomes, characterized in that, Includes the following steps: S1. Soak the plant roots and the soil within the root growth range thoroughly with the soaking solution, collect the soaking material, and then mix the soaking material, solid particles and soaking solution evenly to obtain a mixture; The plant and arbuscular mycorrhizal fungi are in arbuscular mycorrhizal symbiosis; the soaking solution is an ice-water mixture containing Tween 20; the surface of the solid particles is rough and the density is less than that of water; S2. Perform solid-liquid separation on the mixture obtained in step S1 to obtain slurry and precipitate; filter the obtained slurry with a sieve and collect the unfiltered slurry; the aperture of the sieve is equal to the maximum diameter of the solid particles; S3. Soak the isolate obtained in step S2 in ice water, collect the hyphae, and obtain the arbuscular mycorrhizal fungal extrarhizomycelium.

2. The method according to claim 1, characterized in that, Step S2 includes the following steps: S21. The mixture obtained in step S1 is subjected to solid-liquid separation to obtain mud 1 and precipitate 1. The mud 1 is filtered with the screen and the unfiltered precipitate 1 is collected. S22. Mix the soaking solution and the precipitate 1 obtained in step S21 evenly, separate the solid and liquid to obtain mud 2 and precipitate 2, filter the mud 2 with the sieve, and collect the unfiltered precipitate 2.

3. The method according to claim 2, characterized in that, Step S2 also includes the following steps: S23. Repeat step S22 3 to 4 times.

4. The method according to claim 1, characterized in that, The ice-water mixture consists of ice and water in a mass ratio of (1-2):(2-6).

5. The method according to claim 1, characterized in that, The concentration of Tween 20 in the soaking solution is 0.05 μL / mL to 0.1 μL / mL.

6. The method according to claim 1, characterized in that, The maximum diameter of the solid particles is 0.4 mm to 0.5 mm.

7. The method according to claim 1, characterized in that, The solid particles are expanded vermiculite.

8. The method according to claim 1, characterized in that, The arbuscular mycorrhizal fungi include Rhizophagus irregularis.

9. The application of the method according to any one of claims 1 to 8 in the propagation of arbuscular mycorrhizal fungi.

10. The use of the method according to any one of claims 1 to 8 in the preparation of arbuscular mycorrhizal fungal symbionts.

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