Method for researching interaction of AM fungi and interhypha bacteria through non-destructive sampling

Abstract

The invention discloses a method for researching interaction of AM fungi and interhypha bacteria through non-destructive sampling. The method comprises the following steps: step 1, filling a root chamber and a hypha growth chamber with a sterilized culture medium, and sealing the outer end of the hypha growth chamber; the inner end of the hypha growth chamber is communicated with the root chamber; 2, corn seeds are planted in the culture medium of the root chamber, AM fungal spores are added into the root chamber, and meanwhile a nutrient solution and water are used for cultivation; and step 3, collecting, namely collecting pure hyphae and collecting fresh soil influenced by the pure AM fungal hyphae. According to the method, non-destructive sampling is carried out under the potting condition, pure and pollution-free AM fungus hyphae are collected, and interhypha soil influenced by AM fungi is used for scientific research of interhypha interaction.

Description

Technical Field

[0001] The present invention belongs to the technical field of cultivation in containers, and in particular relates to a method for non-destructive sampling research on the interaction between AM fungi and hyphae bacteria. Background Art

[0002] AM fungi are widespread filamentous microorganisms in the soil, with hyphae measuring approximately 10 microns in diameter and spores approximately 100 microns in diameter. They coexist with 80% of Earth's terrestrial plants and are widely distributed in agricultural ecosystems. This symbiotic relationship with AM fungi also significantly contributes to plant nutrient availability in the soil. Under favorable conditions, AM fungi can provide plants with over 90% of phosphorus, over 50% of nitrogen, as well as potassium, manganese, magnesium, sulfate, and water. Therefore, AM fungi hold significant research value for both plant growth and agricultural production.

[0003] The extraradical hyphae of AM fungi are a crucial component of plant health. One end of the hyphae penetrates the inner cortical cells of plant roots, forming arbuscules to exchange nutrients with the host plant. Meanwhile, the other end of the extraradical hyphae can penetrate the rhizosphere, extending the range of nutrient absorption from the root surface to 12 cm beyond, extending into the soil to form a vast mycelial network, greatly expanding the area over which the host plant can absorb nutrients.

[0004] The mycelium is an ecological zone composed of AM fungal extraradical hyphae and other soil microorganisms. It is an important gateway for nutrients, water and other substances to enter the plant body through AM fungal hyphae.

[0005] However, since AM fungi coexist with roots, with hyphae diameters of approximately 10 microns and spore diameters of approximately 100 microns, studying the mycelium requires distinguishing AM fungi from the root system. Therefore, it is difficult to directly collect pure AM fungal bodies and the mycelial microorganisms on their surface in the soil. Some studies have attempted to use thin film enrichment to enrich AM fungi onto thin films for collection. However, this method collects a small amount of mycelium and is difficult to operate, making it difficult to perform in practice. Summary of the Invention

[0006] To address the problems in the prior art and establish an extraradical mycelial growth system for AM fungi, the present invention provides a non-destructive sampling method for studying the interactions between AM fungi and mycelial bacteria. This method ensures the collection of pure, uncontaminated AM fungal mycelia and mycelial soil affected by AM fungi, while also ensuring non-destructive sampling to enable continuous dynamic monitoring of mycelial interactions. The technical solution includes:

[0007] Step 1: Fill the sterilized culture medium into the root chamber and the mycelial growth chamber, seal the outer end of the mycelial growth chamber, and connect the inner end of the mycelial growth chamber to the root chamber;

[0008] Step 2: Plant corn seeds in the culture medium of the root chamber, and add AM fungal spores into the root chamber while cultivating with nutrient solution and water;

[0009] Step 3: Collection, which is divided into collecting pure mycelium and collecting fresh soil affected by pure AM fungal mycelium:

[0010] When pure mycelia need to be collected, the outer end of the mycelia growth chamber is opened to collect the pure mycelia in the mycelia growth chamber;

[0011] When it is necessary to collect fresh soil affected by pure AM fungal hyphae, the outer end of the mycelial growth chamber must first be opened to check the growth of the outermost mycelium. If the mycelial growth at the outer end of the mycelial growth chamber is in good condition, fresh soil is placed in the mycelial chamber, and then the mycelial chamber is sealed and installed outside the mycelial chamber. After incubation for more than 4 weeks, the mycelial chamber is removed and the fresh soil in the mycelial growth chamber is collected. If the mycelial growth at the outer end of the mycelial growth chamber does not meet the requirements, the outer end of the mycelial growth chamber is continued to be closed and incubation continues.

[0012] The axis of the mycelial growth chamber was oriented toward the center of the corn root system.

[0013] The number of the mycelium growth chamber is at least one.

[0014] The mycelium growth chamber has an aperture of 4.5 cm and an axial length of 3.5 cm.

[0015] A 30 μm membrane was placed between the root chamber and the mycelial growth chamber.

[0016] The outer end of the mycelium growth chamber is sealed by tightening a mycelium growth cover in the mycelium growth chamber by means of a threaded connection.

[0017] The culture medium includes: a mixture of perlite and sand, or a mixture of ceramsite and sand, with a volume ratio of 1:1.

[0018] The culture medium filled into the root chamber is mixed with a fluffy filler, the fluffy filler is one or more of vermiculite, coconut husk and sawdust, and the volume ratio of the culture medium to the fluffy filler is 2:1.

[0019] The process of culturing AM fungal spores includes:

[0020] Step 101: Inoculate AM fungal spores with fresh carrot hairy roots transformed with the Ri plasmid on MSR solid culture medium, and culture at 28° C. for 4 months to obtain a plate of mature AM fungal spores; prepare a 0.1M citric acid solution as solution A: weigh 5.25 g of citric acid and dilute to 250 mL with sterile water; prepare a 0.1M trisodium citrate solution as solution B: weigh 7.35 g of trisodium citrate dihydrate and dilute to 250 mL with sterile water; take 9.5 mL of solution A and mix with 40.5 mL of solution B, dilute to 480 mL, adjust the pH to 6.0 with 0.1M citric acid or sodium hydroxide, and dilute to 500 mL after adjusting the pH; the volume ratio of buffer solution to solid culture medium is 2:1 to 5:1;

[0021] Step 102, dissolving the solid culture medium: open the culture dish and transfer it to a 1 L beaker using a glass rod, add 2 to 5 times the volume of citric acid buffer according to the amount and specifications of the solid culture medium, and stir thoroughly to dissolve until no agar lumps are visible to the naked eye;

[0022] Step 103, separation and extraction of spores: transfer the dissolved buffer to a crusher or juicer, crush for 10 seconds, pour through a 400-mesh stainless steel sieve, discard the filtrate, and leave the spores on the sieve; rinse the bottle twice with sterile water to remove the buffer, and transfer the spores on the sieve to a beaker or reagent bottle using the wash bottle;

[0023] Step 104, Calculate the number of spores: Mix the spore suspension thoroughly, and promptly take 10 μL of the spore suspension onto a glass slide using a 200 μL pipette tip. Observe the number of spores using a stereo microscope; count at least 10 times to estimate the concentration of the spore suspension.

[0024] Cultivation in step 2 includes:

[0025] Step 201: Cover the root chamber with a fresh-keeping bag to keep it warm. After 7 days, the seeds emerge and are thinned out, leaving only one corn seedling in each root chamber. Prepare Hoagland nutrient solution, in which the phosphorus content is adjusted to 20-500 μM.

[0026] Step 202: 2 weeks after thinning, add 50 mL of 1 / 4 nutrient solution to the root chamber of each corn pot every 2 days; replace with 1 / 2 nutrient solution from the 3rd to the 4th week; and start using the full nutrient solution from the 4th week onwards, and cultivate for another 4 weeks; during this period, pay attention to watering and pest control, and harvest after a total of 2-3 months of cultivation.

[0027] Step 203: Remove the second mycelium growth tube at 6 weeks. During the culture period, remove the mycelium growth tube connected to the root chamber to collect mycelium for 4 weeks.

[0028] The beneficial effects of the present invention are: non-destructive sampling is performed under potting conditions, and pure and uncontaminated AM fungal hyphae and mycelial soil affected by AM fungi are collected for scientific research on mycelial interactions. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 This is a flow chart of an embodiment of a method for non-destructive sampling to study the interaction between AM fungi and hyphae bacteria according to the present invention;

[0030] Figure 2 Schematic diagram of a cultivation process using a cultivation device according to an embodiment of the present invention;

[0031] Figure 3 A comparison of soil bacterial communities without AM fungi growth and with AM fungi growth (red represents the soil bacterial community structure without AM fungi growth; blue represents the soil bacterial community structure after AM fungi hyphae growth and entry);

[0032] Figure 4 Comparison of the bacterial community structure in the AM fungal mycelium at three time points during non-destructive continuous sampling (blue represents the soil bacterial community structure 2 weeks after AM fungal hyphae entered the mycelium, red represents 4 weeks, and green represents 6 weeks);

[0033] Figure 5 Schematic diagram of the partial explosion structure of the culture device used in the embodiment of the present invention.

[0034] In the figure, 100-root chamber, 110-thread part, 120-30μm membrane, 200-hyphae growth chamber, 300-hyphae inter-chamber, 400-hyphae growth cover. DETAILED DESCRIPTION

[0035] The present invention will be further described in detail below with reference to the accompanying drawings.

[0036] like Figure 1 The embodiment 1 of the present invention shown includes:

[0037] Step 1: Fill the sterilized culture medium into the root chamber and the mycelial growth chamber, seal the outer end of the mycelial growth chamber, and connect the inner end of the mycelial growth chamber to the root chamber;

[0038] Step 2: Plant corn seeds in a culture medium in a root chamber, and add AM fungal spores into the root chamber while cultivating them with a nutrient solution (specifically, Hoagland's nutrient solution) and water (specifically, deionized water);

[0039] Step 3: Collection, which includes collecting pure mycelium 500 and collecting fresh soil affected by pure AM fungal mycelium:

[0040] When pure mycelia need to be collected, the outer end of the mycelia growth chamber is opened to collect the pure mycelia in the mycelia growth chamber;

[0041] When it is necessary to collect fresh soil affected by pure AM fungal hyphae, first open the outer end of the mycelial growth chamber to check the mycelial growth condition on the outermost side. If the mycelial growth condition at the outer end of the mycelial growth chamber is good, put fresh soil into the mycelial chamber, and then seal the mycelial chamber and install it outside the mycelial chamber (replace the mycelial growth cover). After incubation for more than 4 weeks, remove the mycelial chamber and collect the fresh soil inside the mycelial growth chamber. If the mycelial growth condition at the outer end of the mycelial growth chamber does not meet the requirements, continue to close the outer end of the mycelial growth chamber and continue incubation.

[0042] In this embodiment, a 30 μm membrane 120 ( Figure 3 ); the outer dimensions of the root chamber are 12cm*12cm*15cm in length*width*height; the volume of the culture medium stored in the root chamber is usually 800mL.

[0043] In this embodiment, the mycelium growth chamber has an aperture of 4.5 cm and an axial length of 3.5 cm.

[0044] In this embodiment, the outer end of the mycelium growth chamber is sealed by screwing a mycelium growth cover into the mycelium growth chamber through a threaded connection; the mycelium cover has an inner thread diameter of 4.8 cm and an outer diameter of 5.5 cm.

[0045] In this embodiment, the mycelial chamber is filled with 50 g of fresh soil having a moisture content of 10-18% in a clumping state. The mycelial chamber has an inner diameter of 4.4 cm, an outer diameter of 4.8 cm, and an axial length of 2.2 cm.

[0046] In this embodiment, the sterilization method is gamma ray sterilization.

[0047] like Figure 2 and Figure 5 The culture device shown includes: a root chamber 100, a mycelium growth chamber 200, a mycelium chamber 300 and a mycelium growth cover 400. The mycelium growth chamber is installed outside the central root chamber, and the mycelium chamber or mycelium growth cover is installed outside the mycelium growth chamber by screwing. The inner side of the mycelium growth chamber is screwed with a threaded portion 110 extending from the side wall of the root chamber to facilitate disassembly for collecting mycelia.

[0048] The number of mycelial growth chambers is at least one. In this embodiment, four mycelial growth chambers and mycosporium chambers are included. At different times, only one mycelial growth chamber and mycosporium chamber can be removed, while the other three mycelial growth chambers and mycosporium chambers remain, without affecting plant growth. The second mycelial growth chamber and mycosporium chamber can be removed at a later time until the sample is harvested.

[0049] The culture matrix includes: a mixture of perlite and sand, or a mixture of ceramsite and sand, both in a volume ratio of 1:1; the culture matrix filled into the root chamber can also be mixed with fluffy fillers, which are one or more of vermiculite, coconut coir and sawdust, and the volume ratio of the culture matrix and the fluffy filler is 2:1.

[0050] AM fungal spores are obtained from a mature arbuscular mycorrhizal fungus (AM) system cultured in Modified Strullu-Romand Medium (MSR). The main principle is to dissolve the MSR medium in 0.01 M citric acid-sodium citrate buffer (pH 6.0) and calculate the spore concentration in preparation for subsequent experiments. The specific process includes:

[0051] Step 101: Inoculate spores of the AM fungus Rhizophagus irregularis MUCL 43194 on MSR solid medium using fresh carrot hairy roots transfected with the Ri plasmid. After culturing at 28°C for 4 months, obtain a plate containing mature AM fungal spores. Prepare a 0.1M citric acid solution (Solution A): Weigh 5.25g of citric acid and dilute to 250mL with sterile water. Prepare a 0.1M trisodium citrate solution (Solution B): Weigh 7.35g of trisodium citrate dihydrate and dilute to 250mL with sterile water. Mix 9.5mL of Solution A with 40.5mL of Solution B, dilute to 480mL, adjust the pH to 6.0 with 0.1M citric acid or sodium hydroxide, and dilute to 500mL after pH adjustment. The buffer solution is determined by the number of MSR plates to be dissolved; the volume ratio of buffer to solid medium is typically 2:1 to 5:1.

[0052] Step 102: Dissolve the solid culture medium. Disinfect tools such as beakers, glass rods, tweezers, and sieves with alcohol in advance. Start the operation after the alcohol evaporates. Open the culture dish and transfer it to a 1L beaker with a glass rod. Add 2-5 times the volume of citric acid buffer according to the quantity and specifications of the solid culture medium and stir thoroughly to dissolve until the agar lumps are no longer visible to the naked eye. A 150mm large plate usually contains 100mL of MSR solid culture medium and requires 200-500mL of buffer to dissolve. A 90mm small plate and double partition usually contain 25mL of MSR solid culture medium and require 50-125mL of buffer to dissolve.

[0053] Step 103: Isolation and extraction of spores. If roots are present in the culture medium dissolved in buffer, removal is optional. The roots can be retained, as they also contain spores; or they can be removed if necessary for the experiment. Transfer the dissolved buffer to a crusher or juicer. After crushing for 10 seconds, pour through a 400-mesh stainless steel sieve. Discard the filtrate, leaving the spores on the sieve. Rinse the bottle twice with sterile water to remove the buffer. Use the bottle to transfer the spores on the sieve to a beaker or reagent bottle.

[0054] Step 104: Count the spores. Mix the spore suspension thoroughly and promptly transfer 10 μL of the suspension to a glass slide using a 200 μL pipette tip. Observe the spore count using a stereomicroscope. Count the spores at least 10 times to estimate the concentration of the spore suspension (250 spores / mL). Label the suspension and store at 4°C.

[0055] The corn seeds (Zhengdan 958) used in this example were soaked in 2.5% sodium hypochlorite solution for 10 min, then surface-sterilized with 75% alcohol for 1 min, rinsed 6-7 times with sterile water, and then placed on a culture dish and cultured for 24 h. After germination, three corn seeds were sown in the root chamber of the device; then covered with 200 ml of culture medium.

[0056] Cultivation in step 2 includes:

[0057] Step 201: Cover the root chamber with a fresh-keeping bag to keep it warm. After 7 days, the seeds emerge and are thinned out, leaving only one corn seedling in each root chamber. Prepare Hoagland nutrient solution, in which the P content is adjusted to 20-500 μM.

[0058] Step 202: 2 weeks after thinning, add 50 mL of 1 / 4 nutrient solution to the root chamber of each corn pot every 2 days; replace with 1 / 2 nutrient solution from the 3rd to the 4th week; and start using full nutrient solution from the 4th week onwards, and cultivate for another 4 weeks; pay attention to watering and pest control during this period, and harvest after a total of 2-3 months of cultivation.

[0059] Step 203: During the culture period, one of the mycelium growth tubes connected to the root chamber can be removed to collect mycelium for 4 weeks. The second mycelium growth tube can be removed at 6 weeks. Therefore, pure AM fungal mycelium can be collected in the mycelium growth tube to study the growth status and physiological and biochemical indicators of AM fungi.

[0060] Under this culture system, plants and AM fungi establish a remarkably stable symbiotic relationship, with abundant, visible AM fungal hyphae within the culture medium. AM fungi infect over 80% of corn roots. Pure AM fungal hyphae harvested from the mycelium growth chamber can reach lengths exceeding 6 cm.

[0061] Example 2 utilizes the method of Example 1 to conduct non-destructive continuous sampling to study the dynamic interactions between AM fungal hyphae and mycelial bacteria. The AM fungal spore inoculant is prepared as in Example 1, and the corn disinfection method is also the same as in Example 1.

[0062] The experiment involved sowing seeds on June 3, 2024, and establishing a symbiotic system. Two months after watering with the modified culture medium, 50 g of fresh soil, sourced from brown soil in Tai'an City, Shandong Province, was added to the mycelial chamber on August 3, 2024. The cap on the outer side of the mycelial growth tube was removed and carefully screwed into the mycelial chamber containing fresh soil, followed by normal incubation. On August 21, 2024, one of the mycelial growth chambers and the mycelial chamber were removed from the apparatus for the first sample.

[0063] On September 5, 2024, another hyphal growth chamber and interhyphae chamber were removed from the device as a second sample; on September 19, 2024, another hyphal growth chamber and interhyphae chamber were removed as a third sample. Soil bacterial DNA was extracted from the soil sample in the mycelial chamber and sent to Paisonno Biotech Co., Ltd. for 16S rDNA high-throughput sequencing. In addition, pristine soil without AM fungi was also subjected to high-throughput sequencing as a control.

[0064] Test results: The results of the non-destructive continuous sampling mycelial soil test found that compared with fresh soil without access to the device, the growth of AM fungi in the soil can significantly affect the soil bacterial community in the mycelial chamber, such as Figure 3 As shown in Figure 2, there are significant differences in bacterial community composition between the two. The results of non-destructive continuous sampling experiments at three time points found that the effect of AM fungi on soil bacterial communities is dynamic over time. The bacterial community composition of samples at three time points is as follows: Figure 4 As shown, there were significant differences in phosphatase activities.

Claims

1. A method for non-destructive sampling to study the interaction between AM fungi and mycelial bacteria, characterized in that: include: Step 1: Fill the sterilized culture medium into the root chamber and the mycelial growth chamber, seal the outer end of the mycelial growth chamber, and connect the inner end of the mycelial growth chamber to the root chamber; Step 2: Plant corn seeds in the culture medium of the root chamber, and add AM fungal spores into the root chamber while cultivating with nutrient solution and water; Step 3: Collection, which is divided into collecting pure mycelium and collecting fresh soil affected by pure AM fungal mycelium: When pure mycelia need to be collected, the outer end of the mycelia growth chamber is opened to collect the pure mycelia in the mycelia growth chamber; When it is necessary to collect fresh soil affected by pure AM fungal hyphae, the outer end of the mycelial growth chamber must first be opened to check the growth of the outermost mycelium. If the mycelial growth at the outer end of the mycelial growth chamber is in good condition, fresh soil is placed in the mycelial chamber, and then the mycelial chamber is sealed and installed outside the mycelial chamber. After incubation for more than 4 weeks, the mycelial chamber is removed and the fresh soil in the mycelial growth chamber is collected. If the mycelial growth at the outer end of the mycelial growth chamber does not meet the requirements, the outer end of the mycelial growth chamber is continued to be closed and incubation continues.

2. A non-destructive sampling method for studying the interaction between AM fungi and mycelial bacteria according to claim 1, characterized in that: The axis of the mycelial growth chamber was oriented toward the center of the corn root system.

3. The method for non-destructive sampling to study the interaction between AM fungi and mycelial bacteria according to claim 1, characterized in that: The number of the mycelium growth chamber is at least one.

4. The method for non-destructive sampling to study the interaction between AM fungi and mycelial bacteria according to claim 1, characterized in that: The mycelium growth chamber has an aperture of 4.5 cm and an axial length of 3.5 cm.

5. A non-destructive sampling method for studying the interaction between AM fungi and hyphae bacteria according to any one of claims 1 to 4, characterized in that: A 30 μm membrane was placed between the root chamber and the mycelial growth chamber.

6. The method for non-destructive sampling to study the interaction between AM fungi and hyphae bacteria according to claim 1, characterized in that: The outer end of the mycelium growth chamber is sealed by tightening a mycelium growth cover in the mycelium growth chamber by means of a threaded connection.

7. The method for non-destructive sampling to study the interaction between AM fungi and mycelial bacteria according to claim 1, characterized in that: The culture medium includes: a mixture of perlite and sand, or a mixture of ceramsite and sand, with a volume ratio of 1:

1.

8. The method for non-destructive sampling to study the interaction between AM fungi and mycelial bacteria according to claim 7, characterized in that: The culture medium filled into the root chamber is mixed with a fluffy filler, wherein the fluffy filler is one or more of vermiculite, coconut husk and sawdust; the volume ratio of the culture medium to the fluffy filler is 2:

1.

9. The method for non-destructive sampling to study the interaction between AM fungi and hyphae bacteria according to claim 1, characterized in that: The process of culturing AM fungal spores includes: Step 101: Inoculate AM fungal spores with fresh carrot hairy roots transformed with the Ri plasmid on MSR solid culture medium, and culture at 28° C. for 4 months to obtain a plate of mature AM fungal spores; prepare a 0.1M citric acid solution as solution A: weigh 5.25 g of citric acid and dilute to 250 mL with sterile water; prepare a 0.1M trisodium citrate solution as solution B: weigh 7.35 g of trisodium citrate dihydrate and dilute to 250 mL with sterile water; take 9.5 mL of solution A and mix with 40.5 mL of solution B, dilute to 480 mL, adjust the pH to 6.0 with 0.1M citric acid or sodium hydroxide, and dilute to 500 mL after adjusting the pH; the volume ratio of buffer solution to solid culture medium is 2:1 to 5:1; Step 102, dissolving the solid culture medium: open the culture dish and transfer it to a 1 L beaker using a glass rod, add 2 to 5 times the volume of citric acid buffer according to the amount and specifications of the solid culture medium, and stir thoroughly to dissolve until no agar lumps are visible to the naked eye; Step 103, separation and extraction of spores: transfer the dissolved buffer to a crusher or juicer, crush for 10 seconds, pour through a 400-mesh stainless steel sieve, discard the filtrate, and leave the spores on the sieve; rinse the bottle twice with sterile water to remove the buffer, and transfer the spores on the sieve to a beaker or reagent bottle using the wash bottle; Step 104, Calculate the number of spores: Mix the spore suspension thoroughly, and promptly take 10 μL of the spore suspension onto a glass slide using a 200 μL pipette tip. Observe the number of spores using a stereo microscope; count at least 10 times to estimate the concentration of the spore suspension.

10. The method for non-destructive sampling to study the interaction between AM fungi and mycelial bacteria according to claim 1, characterized in that: Cultivation in step 2 includes: Step 201: Cover the root chamber with a fresh-keeping bag to keep it warm. After 7 days, the seeds emerge and are thinned out, leaving only one corn seedling in each root chamber. Prepare Hoagland nutrient solution, in which the phosphorus content is adjusted to 20-500 μM. Step 202: 2 weeks after thinning, add 50 mL of 1 / 4 nutrient solution to the root chamber of each corn pot every 2 days; replace with 1 / 2 nutrient solution from the 3rd to the 4th week; and start using the full nutrient solution from the 4th week onwards, and cultivate for another 4 weeks; during this period, pay attention to watering and pest control, and harvest after a total of 2-3 months of cultivation. Step 203: Remove the second mycelium growth tube at 6 weeks. During the culture period, remove the mycelium growth tube connected to the root chamber to collect mycelium for 4 weeks.

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