Method for obtaining extracellular hyphae of arbuscular mycorrhizal fungi and application of extracellular hyphae

Abstract

The invention discloses a method for obtaining extracellular hyphae of arbuscular mycorrhizal fungi and application of the extracellular hyphae, and the method comprises the following steps: S1, fully soaking a plant root system with arbuscular mycorrhiza (AM) symbiosis and soil within the growth range of the root system in a soaking solution, collecting a soaked material, and then uniformly mixing the soaked material, solid particles and the soaking solution to obtain a mixture; the soaking solution is an ice-water mixture containing Tween 20; s2, carrying out solid-liquid separation on the mixture obtained in the step S1, filtering the obtained slurry by using a screen, and collecting a separated substance which does not pass through the screen; and S3, fully soaking the obtained isolate with ice water, and collecting floccules wound with solid particles. According to the method, rapid separation and collection of the AM fungus ectomycelium can be achieved without the help of a microscope under the condition of a common laboratory, operation is easy, efficiency is high, the obtained AM fungus ectomycelium has high infection activity, the situation that living mycelium is wasted when fungal spores are collected in a traditional method is avoided, resources are saved, and feasibility is high.

Description

Technical Field

[0001] The invention relates to the technical field of fungal hyphae separation, and in particular to a method for obtaining extraradical hyphae of arbuscular mycorrhizal fungi and an application thereof. Background Art

[0002] Arbuscular mycorrhiza (AM) fungi can interact symbiotically with more than 72% of plants on land. AM fungi can not only form arbuscular structures and have intraradical hyphae in the cortex of plant roots, but also distribute a large number of extraradical hyphae outside the roots. Extraradical hyphae absorb nutrients from the soil and transport them to the root system, thereby increasing the nutrient absorption of plants. In addition, most of the extraradical hyphae of AM fungi grow rapidly and can survive for 5 months ("Lifespan and functionality of mycorrhizal fungal mycelium are uncoupled from host plant lifespan"), which can serve as AM fungi propagules ("Colonization of roots by arbuscularmycorrhizal fungi using different sources of inoculum"), thereby enabling AM fungi to colonize on the roots of new host plants and form new arbuscular mycorrhizal symbionts. Isolating and collecting extraradical living hyphae of AM fungi from the soil is conducive to the diversification and expansion of the propagules of arbuscular mycorrhizal fungi.

[0003] In mycorrhizal research, technicians in this field need to use a large number of AM fungal propagules when exploring the intrinsic mechanism of arbuscular mycorrhizal symbiosis and conducting field application tests of arbuscular mycorrhizal fungi. In the prior art, AM fungal spores are usually directly inoculated on plants for propagation, so as to obtain a large number of AM fungal spores as AM fungal propagules, such as potted culture method, glass bead compartment culture method, atomization culture method, aseptic dual culture method, etc. These methods all require breaking the plant root system or rhizosphere soil, first sieving out the spores by wet sieving decantation, and then using sucrose solution to separate the full spores by gradient centrifugation to obtain spores in the plant root system and soil. The process is time-consuming, and the spores are easily inactivated in sucrose. At the same time, in the process of separating spores, a large number of live hyphae in the soil are easily lost, resulting in a waste of AM fungal propagule resources. In addition, since the output rate of AM fungal spores is much lower than the output rate of live hyphae, directly separating the live hyphae of AM fungi is more conducive to the rapid propagation of AM fungi. However, traditional methods for isolating living hyphae of AM fungi are relatively crude, requiring repeated stirring, centrifugation, and filtration processes (“External hyphal production of vesicular-arbuscular mycorrhizal fungi in pasture and tallgrass prairie communities”). In addition, in order to obtain complete living hyphae, the hyphae need to be picked in an ice water bath under a stereo microscope, which is time-consuming and laborious. Summary of the invention

[0004] In order to solve the problem that there is a lack of a simple, efficient and visual method for separating and collecting extraradical living hyphae of AM fungi in soil in the prior art, the present invention provides a method for obtaining extraradical hyphae of arbuscular mycorrhizal fungi and its application.

[0005] The first object of the present invention is to provide a method for obtaining extraradical hyphae of arbuscular mycorrhizal fungi.

[0006] The second object of the present invention is to provide application of the method in the propagation of arbuscular mycorrhizal fungi.

[0007] The third object of the present invention is to provide application of the method in preparing arbuscular mycorrhizal fungal symbionts.

[0008] In order to achieve the above object, the present invention is implemented by the following scheme:

[0009] The present invention finds that living extraradical hyphae can form larger continuous mycelia outside the root of the host plant, which are entangled with each other during stirring in water, and entangled with small irregularly shaped solid particles added, and then the solid particles entangled with the mycelia are trapped on the sieve by using the mesh barrier effect of the sieve. The intercepted material on the sieve is poured onto a culture dish and soaked in ice water. Due to the buoyancy of water, the mycelia and the carrier entangled with it can present a cotton-like mycelial mass visible to the naked eye in the ice water, and the mass can be driven by slight fluctuations in the water. Under the condition of not using a microscope for magnification operation, the extraradical hyphae of AM fungi can be directly collected with tweezers or a straw.

[0010] A method for obtaining extraradical hyphae of arbuscular mycorrhizal fungi comprises the following steps:

[0011] S1. Soak the roots of the plants and the soil within the root growth range with the soaking liquid, collect the soaked material, and then mix the soaked material, solid particles and the soaking liquid to obtain a mixture;

[0012] The plant and the arbuscular mycorrhizal fungi exist in arbuscular mycorrhizal symbiosis; the soaking liquid 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. performing solid-liquid separation on the mixture obtained in step S1 to obtain mud and precipitate; filtering the obtained mud with a sieve to collect the separated matter that has not passed through the sieve; the aperture of the sieve is equal to the maximum diameter of the solid particles;

[0014] S3. Soak the isolate obtained in step S2 fully in ice water and collect the hyphae to obtain the extraradical hyphae of the arbuscular mycorrhizal fungus.

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

[0016] S21. The mixture obtained in step S1 is subjected to solid-liquid separation to obtain mud 1 and precipitate 1, and the mud 1 obtained is filtered through the sieve to collect the separated matter 1 that has not passed through the sieve;

[0017] S22. The soaking liquid and the precipitate 1 obtained in step S21 are mixed evenly, and the solid and liquid are separated to obtain mud 2 and precipitate 2. The obtained mud 2 is filtered through the sieve, and the separated substance 2 that has not passed through the sieve is collected.

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

[0019] Further preferably, step S2 also includes the following steps: S23. Repeat step S22 three times.

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

[0021] Preferably, the maximum diameter of the solid particles is 0.4-0.5 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 culture pot, and in step S1, the culture pot is placed in the soaking liquid and soaked, the water level is lower than the pot mouth of the culture pot, and the soil surface is not immersed.

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

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

[0031] Preferably, the extraradical hyphae of the arbuscular mycorrhizal fungi obtained by any of the methods are used as propagules.

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

[0033] The method provided by the present invention firstly designs a low temperature condition created by ice water, which is not easy to inactivate the extraradical hyphae of arbuscular mycorrhizal fungi; secondly, while designing the low temperature condition, the present invention adopts soil temperature 20 as a detergent, which neither affects the protein activity of the living hyphae nor washes away the soil particles adhering to the surface of the hyphae, thereby suspending the hyphae in the ice water to the greatest extent, and further facilitating the entanglement of the mycelium and the added expanded vermiculite particles. Finally, the mycelium entangled with the vermiculite particles is easy to be identified under the fluctuating conditions in the water, and can be directly collected by naked eyes without the aid of a microscopic magnification device, so that the extraradical hyphae of arbuscular mycorrhizal fungi can be quickly separated and collected under ordinary laboratory conditions.

[0034] The invention is simple in various operations and high in efficiency. The obtained extraradical mycelium maintains stronger activity and has better infection effect on host plants. The integrity of the extraradical mycelium during the separation process is improved, and a more authentic and reliable sample source is provided for subsequent physiological or molecular detection research of extraradical living mycelium of arbuscular mycorrhizal fungi. The invention also avoids the situation in which living mycelium is wasted for collecting arbuscular mycorrhizal fungal spores in traditional methods, thereby saving resources, reducing costs, and having high feasibility. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 Schematic diagram of the operation of soaking the plant-AM fungus symbiosis pot in an ice-water mixture containing Tween 20.

[0036] Figure 2 Schematic diagram of the operation of stirring a mixture of soil and ice water containing Tween 20.

[0037] Figure 3 Schematic diagram of the operation of filtering mud with a screen.

[0038] Figure 4 A photograph of extraradical hyphae of AM fungi suspended in water.

[0039] Figure 5 These are microscopic photographs of AM fungal extraradical hyphae suspended in water.

[0040] Figure 6 Electrophoresis diagrams of RNA extracted from extraradical hyphae of AM fungi obtained by different methods. DETAILED DESCRIPTION

[0041] The present invention is further described in detail below in conjunction with the accompanying drawings and specific examples of the specification. The examples are only used to explain the present invention and are not used to limit the scope of the present invention. The test methods used in the following examples are conventional methods unless otherwise specified; the materials, reagents, etc. used are reagents and materials that can be obtained from commercial channels unless otherwise specified. The symbiotic cultivation method of AM fungi and plants in the following examples is a conventional technical means, and is 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 extraradical hyphae of AM fungi

[0043] This example takes Rhizophagus irregularis as an example to provide a method for obtaining extra-root living hyphae of AM fungi, and the specific steps are as follows:

[0044] 1. Plant Broussonetia papyrifera in a pot that has been symbiotically grown with Rhizophagus irregularis for 6 months. Figure 1 As shown, the culture pot (10 cm long*10 cm wide*10 cm high) is placed in a large square pot (30 cm long*30 cm wide*20 cm high), and a soaking liquid is added to the pot, wherein the soaking liquid is an ice-water mixture containing 0.1 μL / mL Tween 20 (the mass ratio of ice to water is 1:2), and the soil of the paper mulberry pot is softened (the liquid level is lower than the pot mouth, and the soil surface is not immersed) for 1 hour to reduce the adhesion between the roots and hyphae and the soil;

[0045] 2. Transfer all the roots of the paper mulberry potted plant and the soil in the culture pot to a 2000mL beaker, add 1000mL (50 times the total volume of the soil and roots) of new soaking solution and 10mL of sterile expanded vermiculite with a diameter of 0.4-0.5mm, such as Figure 2 As shown, stir in circles with a glass rod for 5 minutes. The upper layer in the beaker is a uniform mud with suspended vermiculite, and the lower layer is vermiculite, roots, mud and other sediments.

[0046] 3. Afterwards, if Figure 3 As shown, a 0.5 mm pore size sieve is used to filter the slurry in the beaker. After filtering, the mixture of mycelium and mud remaining on the sieve that has not passed through the sieve is collected;

[0047] 4. Add 1000 mL of new soaking solution to the original beaker to soak the sediment in step 2, and continue to stir with a glass rod for 5 minutes; then, use a 0.5 mm pore size sieve to filter the mud in the beaker. After filtering, 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 15 cm diameter glass petri dish and add 40 mL of ice water. Figure 4 The cotton-like substance wrapped around the vermiculite particles is the extra-root living hyphae of AM fungi. Its state under a microscope is as follows: Figure 5 As shown;

[0049] 6. Use sterilized tweezers to promptly pick up the extra-root living hyphae and place them in a 1.5 mL centrifuge tube pre-cooled on ice for temporary storage. They can be directly used for inoculation of arbuscular mycorrhizal host plants, or frozen at -80°C for physiological or molecular detection experiments of AM fungi extra-root living hyphae.

[0050] Example 2 A method for obtaining extraradical hyphae of AM fungi

[0051] This example takes Rhizophagus irregularis as an example to provide a method for obtaining extraradical hyphae of AM fungi, and the specific steps are as follows:

[0052] 1. Put the potted Broussonetia papyrifera that has been symbiotic with Rhizophagus irregularis for 8 months into a large square pot (30 cm long * 30 cm wide * 20 cm high) together with the culture pot (10 cm long * 10 cm wide * 10 cm high), add soaking liquid into the pot, the soaking liquid is an ice-water mixture containing 0.05 μL / mL Tween 20 (the mass ratio of ice to water is 1:1), soak for 3 hours to soften the soil of the Broussonetia papyrifera potted plant and reduce the adhesion between the roots and hyphae and the soil;

[0053] 2. Transfer the roots of the paper mulberry potted plant and the soil in the culture pot to a 2000mL beaker, add 1000mL (50 times the total volume of the soil and roots) of new soaking solution and 5mL of sterile expanded vermiculite with a diameter of 0.4-0.5mm, and stir in circles with a glass rod for 3 minutes. The upper layer in the beaker is a uniform slurry with suspended vermiculite, and the lower layer is vermiculite, roots, mud and stone sediments;

[0054] 3. Then, filter the slurry in the beaker with a 0.5 mm pore size sieve. After filtering, collect the mixture of mycelium and vermiculite remaining on the sieve;

[0055] 4. Add 600 mL of new soaking solution to the original beaker to soak the sediment in step 2, and continue to stir with a glass rod for 3 minutes; then, use a 0.5 mm pore size sieve to filter the mud in the beaker. After filtering, 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, put it into a glass culture dish with a diameter of 15 cm, add 50 mL of ice water, and the cotton-like material floating on the vermiculite particles in the ice water is the extraradical living mycelium of AM fungi;

[0057] 6. Use sterilized tweezers to promptly pick up the extra-root living hyphae and place them in a 1.5 mL centrifuge tube placed on ice at 4°C for temporary storage. They can be directly used for inoculation of AM host plants, or frozen at -80°C for physiological or molecular detection experiments of AM fungal extra-root living hyphae.

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

[0059] This comparative example takes Rhizophagus irregularis as an example to provide a method for obtaining extraradical hyphae of AM fungi, and the specific steps are as follows:

[0060] 1. Put the potted Broussonetia papyrifera that has been symbiotic with Rhizophagus irregularis for 6 months into a large square pot (30 cm long, 30 cm wide, and 20 cm high) together with the culture pot (10 cm long, 10 cm wide, and 10 cm high), add normal temperature tap water to the pot, and soak for 1 hour to soften the soil of the Broussonetia papyrifera pot (the liquid level is lower than the pot mouth, and the soil surface is not immersed);

[0061] 2. Transfer the roots of the paper mulberry potted plant and the soil in the culture pot to a 2000mL beaker, add 1000mL (50 times the total volume of the soil and roots) of new room temperature tap water and 10mL of sterile expanded vermiculite with a diameter of 0.4-0.5mm, and stir in circles with a glass rod for 5 minutes. The upper layer in the beaker is uniform mud, and the lower layer is roots, mud and stones and other sediments;

[0062] 3. Then, filter the slurry in the beaker with a 0.5 mm pore size sieve. After filtering, collect the mixture of mycelium and mud that has not passed through the sieve;

[0063] 4. Add 1000 mL of room temperature tap water to the original beaker to soak the sediment in step 2, and continue to stir with a glass rod for 5 minutes; then, use a 0.5 mm pore size sieve to filter the mud in the beaker. After filtering, collect the mixture of mycelium and mud on the sieve; repeat this step 3 times;

[0064] 5. Collect the mixture of hyphae and sediment from the sieve, put it into a glass culture dish with a diameter of 15 cm, add 50 mL of room temperature tap water, and there are cotton-like masses floating in the water, which are the hyphae of AM fungi extraradical hyphae;

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

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

[0067] This comparative example takes Rhizophagus irregularis as an example to provide a method for obtaining extraradical hyphae of AM fungi, and the specific steps are as follows:

[0068] 1. Put the potted paper mulberry that has been symbiotic with Rhizophagus irregularis for 6 months into a large square pot (30 cm long * 30 cm wide * 20 cm high) together with the culture pot (10 cm long * 10 cm ground diameter * 10 cm high), add soaking liquid into the pot, the soaking liquid is an ice-water mixture containing 0.1 μL / mL Tween 20 (the mass ratio of ice to water is 1:1), soak for 2 hours to make the soil of the paper mulberry pot soft (the liquid level is lower than the pot mouth, and the soil surface is not immersed);

[0069] 2. Transfer the roots of the paper mulberry potted plant and the soil in the culture pot to a 2000mL beaker, add 1000mL (50 times the total volume of the soil and roots) of new soaking solution, and stir in circles with a glass rod for 5 minutes. The upper layer in the beaker is uniform mud, and the lower layer is roots, mud and stones and other sediments;

[0070] 3. Afterwards, the slurry in the beaker was filtered through a 0.5 mm pore size sieve. After filtering, hyphae and sediment that did not pass through the sieve remained on the sieve. The mixture of hyphae and sediment on the sieve was collected.

[0071] 4. Add 1000 mL of new soaking solution to the original beaker to soak the sediment in step 2, and continue to stir with a glass rod for 5 minutes; then, use a 0.5 mm pore size sieve to filter the mud in the beaker. After filtering, collect the mixture of mycelium and mud remaining on the sieve; repeat this step 3 times;

[0072] 5. Collect the mixture of hyphae and mud from the sieve, put it into a glass culture dish with a diameter of 15 cm, add 50 mL of ice water, and there are cotton-like masses floating in the ice water, which are the mycelial masses of the extra-root living hyphae of AM fungi;

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

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

[0075] Referring to the prior art "separation of AM fungal spores by wet sieving and decanting and sucrose gradient centrifugation (Gerdemann and Nicolson (1963) Spores of mycorrhizal endogone species extracted from soil by wet sieving and decanting. Leonard and Vanderwoude (1976) Isolation of plasmamembranes from corn roots by sucrose density gradient centrifugation: anomalous effect of ficoll.)", the spores of Rhizophagus irregularis obtained by the traditional method mainly include:

[0076] 1. The potted paper mulberry that had been symbiotic with Rhizophagus irregularis for 6 months was naturally dried to death, the paper mulberry root system and rhizosphere soil were placed in a 2000 mL beaker, 500 mL of room temperature tap water was added, and the mixture of mud and water of the paper mulberry root system and rhizosphere soil was stirred in circles with a glass rod for 5 minutes, and then the mud and water mixture of the paper mulberry root system and rhizosphere soil was broken up with a conventional soybean milk machine (maximum speed 1 minute, intermittent 3 times), and then the broken mud and water mixture of the root system and rhizosphere soil was poured back into the original beaker;

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

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

[0079] 4. Rinse the spores collected in step 3 with room temperature tap water for 5 times, place them in a centrifuge tube and store them at 4°C for future use in inoculation of AM host plants.

[0080] Application Example 1 Evaluation of the infection activity of AM fungi

[0081] 1. Experimental methods

[0082] Sterile tissue culture seedlings of paper mulberry (seedling age 4 months, plant height 10 cm) were prepared, and randomly divided into 5 groups according to 10 plants / group. Sterile quartz sand was used as the matrix, and the extraradical mycelium obtained according to the method of Examples 1 to 2, the extraradical mycelium obtained according to the method of Comparative Examples 1 to 2, and the spores obtained in Comparative Example 3 were inoculated respectively. The inoculation amount of the extraradical mycelium of AM fungi was 200 cm / each plant, and the inoculation amount of the spores was 200 / each plant.

[0083] After inoculation, the seedlings were routinely watered (maintaining appropriate moisture content) and nutrient managed (irrigated with low-phosphorus Hoagland nutrient solution once a week) in the glasshouse.

[0084] 45 days after inoculation, the infection rate of Rhizosporium heteromorphum in the root systems of each group of paper mulberry 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 among the groups (P<0.05, one-way ANOVA).

[0089] As shown in Table 1, the infection rates of the paper mulberry seedlings inoculated with the extraradical mycelia of AM fungi in Examples 1 to 2 and Comparative Example 2 were significantly higher than those of the paper mulberry seedlings inoculated with the extraradical mycelia of Comparative Example 1 and the spores of Comparative Example 3, while the infection rate of the paper mulberry seedlings inoculated with the extraradical mycelia of Comparative Example 1 was significantly lower than that of the paper mulberry seedlings inoculated with the spores of Comparative Example 3. The results show that the ice water separation method can retain the activity of the AM fungal extraradical mycelia to a greater extent, and the separated extraradical mycelia have a better infection effect on the host plant.

[0090] Application Example 2 Evaluation of the efficiency of obtaining extraradical hyphae of AM fungi

[0091] 1. Determination of hyphae length

[0092] With reference to the prior art "Jakobsen I, Abbott LK, Robson AD (1992) External hyphae of vesicular-arbuscular mycorrhizal fungi associated with Trifolium subterraneu mL.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 AM fungi outside the roots collected according to the methods of Examples 1 to 2 and Comparative Examples 1 to 2 was measured, and the main steps included:

[0093] (1) Add 0.1 mL of quartz sand to the 1.5 mL centrifuge tube containing the AM fungus extraradical hyphae, then add 1 mL of sterile water, and vortex for 3 times, each time for 1 min, so that the continuous hyphae can be broken by the friction between the quartz sands;

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

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

[0096] (4) The mycelial density was observed at 70 times magnification under a stereo microscope and calculated using the grid intersection method (counting the intersection points of all mycelial particles with the grid on the microporous filter membrane).

[0097] Table 2 AM fungi extraradical hyphae harvested in the examples and comparative examples

[0098]

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

[0100] As shown in Table 2, the length of the AM fungus extraradical hyphae collected in Examples 1 and 2 is significantly longer than that collected in Comparative Examples 1 and 2. At the same time, variance analysis shows that the length of hyphae collected in Comparative Example 2 without adding vermiculite and Comparative Example 1 without adding Soil Temperature 20 is significantly lower than that in Examples 1 and 2. The results show that the scheme of retaining the activity of hyphae by ice-water mixture, adding expanded vermiculite to entangle the hyphae, and using Soil Temperature 20 to remove the sand on the hyphae in the present invention can improve the integrity of the separated extraradical hyphae, and has a better effect on visually separating the living extraradical hyphae of fungi.

[0101] 2. Extraction and determination of RNA from hyphae

[0102] For the extraradical mycelia obtained from Examples 1 and 2 and Comparative Examples 1 and 2 stored at -80°C, RNA was extracted using an Omega Fungal RNA Extraction Kit (EZNA Fungal RNA Kit), followed by gel electrophoresis detection and RNA concentration determination.

[0103] The electrophoresis results are as follows Figure 6 As shown, the RNA of the extraradical mycelium obtained by the methods of Examples 1 to 2 showed two relatively clear rRNA bands (28S and 18S), and the RNA concentrations were 18ng / μL and 21ng / μL, respectively; while the RNA extracted from the mycelium collected in Comparative Example 1 had no obvious electrophoresis bands, and the RNA concentration was only 1ng / μL, and the RNA extracted from the mycelium collected in Comparative Example 2 had only faintly visible electrophoresis bands, and the RNA concentration was only 7ng / μL. The results show that the method of the present invention for separating the extraradical mycelium of AM fungi by combining ice-water mixture, vermiculite and soil temperature 20 can better protect the mycelium biological samples, effectively retain more nucleic acid samples, and provide a more authentic and reliable sample source for subsequent physiological or molecular detection research of the extraradical mycelium of AM fungi.

[0104] 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 the protection scope of the present invention. For ordinary technicians in this field, other different forms of changes or modifications can be made based on the above descriptions and ideas. It is not necessary and impossible to list all the implementation methods here. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the protection scope of the claims of the present invention.

Claims

1. A method for obtaining extraradical hyphae of arbuscular mycorrhizal fungi, characterized in that: The following steps are involved: S1. Soak the roots of the plants and the soil within the root growth range with the soaking liquid, collect the soaked material, and then mix the soaked material, solid particles and the soaking liquid to obtain a mixture; The plant and the arbuscular mycorrhizal fungi exist in arbuscular mycorrhizal symbiosis; the soaking liquid 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. performing solid-liquid separation on the mixture obtained in step S1 to obtain mud and precipitate; filtering the obtained mud with a sieve to collect the separated matter that has not passed through the sieve; the aperture of the sieve is equal to the maximum diameter of the solid particles; S3. Soak the isolate obtained in step S2 fully in ice water and collect the hyphae to obtain the extraradical hyphae of the arbuscular mycorrhizal fungus.

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, and the mud 1 obtained is filtered through the sieve to collect the separated matter 1 that has not passed through the sieve; S22. The soaking liquid and the precipitate 1 obtained in step S21 are mixed evenly, and the solid and liquid are separated to obtain mud 2 and precipitate 2. The obtained mud 2 is filtered through the sieve, and the separated substance 2 that has not passed through the sieve is collected.

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

10. Use of the method according to any one of claims 1 to 8 in preparing arbuscular mycorrhizal fungal symbionts.

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