A non-destructive sampling method for studying the interaction between AM fungi and hyphal bacteria

Abstract

This invention discloses a non-destructive sampling method for studying the interaction between AM fungi and mycelial bacteria. Step 1: Sterilized culture medium is filled into the root chamber and the mycelial growth chamber, and the outer end of the mycelial growth chamber is sealed; the inner end of the mycelial growth chamber is connected to the root chamber. Step 2: Corn seeds are planted in the culture medium in the root chamber, and AM fungal spores are added to the root chamber while simultaneously cultivating them using nutrient solution and water. Step 3: Collection is performed, consisting of collecting pure mycelia and collecting fresh soil affected by pure AM fungal mycelia. This invention uses non-destructive sampling under pot conditions to collect pure, uncontaminated AM fungal mycelia and mycelial soil affected by AM fungi for scientific research on mycelial interactions.

Description

Technical Field

[0001] This invention belongs to the field of container cultivation technology, specifically a method for non-destructive sampling to study the interaction between AM fungi and mycelial bacteria. Background Technology

[0002] Amphipathic fungi (AM) are filamentous microorganisms widely found in soil. Their hyphae are approximately 10 micrometers in diameter, and their spores are approximately 100 micrometers in diameter. They can form symbiotic relationships with 80% of terrestrial plants on Earth and are widely distributed in agricultural ecosystems. This symbiotic relationship between AM fungi and plants makes a significant contribution to the acquisition of soil nutrients. Under suitable conditions, AM fungi can provide plants with over 90% of their phosphorus, over 50% of their nitrogen, as well as potassium, manganese, magnesium, sulfate, and water. Therefore, AM fungi have significant research value for both plant growth and agricultural production.

[0003] The extra-root hyphae of AM fungi are an important component for plants to benefit from. One end of the AM fungal hyphae can enter the internal cortical cells of the plant root to form arbuscular mycelia and exchange nutrients with the host plant; while the other end of the extra-root hyphae can penetrate out of the rhizosphere, extending the range of nutrient absorption from the root surface to more than 12 cm away, extending into the soil to form a huge hyphal network, greatly expanding the area on which the host plant can absorb nutrients.

[0004] The hyphal region is an ecological zone composed of the extra-root hyphae of AM fungi and other soil microorganisms. It is an important gateway for nutrients, water and other substances to enter the plant through AM fungal hyphae.

[0005] However, since AM fungi live in symbiosis with roots and have hyphae diameters of about 10 micrometers and spore diameters of about 100 micrometers, it is necessary to distinguish AM fungi from roots when studying the hyphae. Therefore, it is difficult to directly collect pure AM fungal cells and their surface hyphae microorganisms from the soil. Some studies have attempted to use membrane enrichment to collect AM fungi onto membranes, but this method collects a small amount of hyphae and is difficult to operate in practice. Summary of the Invention

[0006] To address the problems existing in the background technology, and in order to establish an ectopic mycelial growth system of AM fungi, this invention provides a non-destructive sampling method for studying the interaction between AM fungi and mycelial bacteria. This method ensures, on the one hand, the collection of pure, uncontaminated AM fungal hyphae and the mycelial soil affected by AM fungi; on the other hand, it ensures non-destructive collection, enabling continuous dynamic monitoring of mycelial interactions for scientific research. The technical solution includes:

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

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

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

[0010] When it is necessary to collect pure mycelium, open the outer end of the mycelium growth chamber and collect the pure mycelium inside the mycelium growth chamber;

[0011] When collecting fresh soil affected by pure AM fungal mycelia, first open the outer end of the mycelial growth chamber to check the growth of the outermost mycelia. If the mycelial growth outside the mycelial growth chamber is good, fill the mycelial chamber with fresh soil, then seal the mycelial growth chamber and install it outside the mycelial growth chamber. After cultivating for more than 4 weeks, remove the mycelial growth chamber and collect the fresh soil inside the mycelial growth chamber. If the mycelial growth outside the mycelial growth chamber does not meet the requirements, continue to seal the outer end of the mycelial growth chamber and continue cultivation.

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

[0013] The number of mycelial growth chambers is at least one.

[0014] The mycelial growth chamber has a pore size of 4.5 cm and an axial length of 3.5 cm.

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

[0016] The method for sealing the outer end of the mycelial growth chamber is to tighten the mycelial growth cover by threading it into the mycelial growth chamber.

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

[0018] The culture medium filling the root chamber is a mixture of loose filler, which is one or more of vermiculite, coconut coir and sawdust, with a volume ratio of 2:1 between the culture medium and the loose filler.

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

[0020] Step 101: Inoculate fresh carrot hairy roots transfected with Ri plasmids with AM fungal spores on MSR solid medium and culture at 28℃ for 4 months to obtain plates of mature AM fungal spores; prepare 0.1M citric acid solution as solution A: weigh 5.25g of citric acid and dilute to 250mL with sterile water; prepare 0.1M trisodium citrate solution as 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 then dilute to 500mL; the volume ratio of buffer solution to solid medium is 2:1 to 5:1;

[0021] Step 102: Dissolving solid culture medium: Open the petri dish and transfer it to a 1L beaker using a glass rod. Add 2 to 5 times the volume of citrate buffer according to the quantity and specifications of the solid culture medium and stir thoroughly until no agar lumps are visible to the naked eye.

[0022] Step 103, Spore isolation and extraction: Transfer the dissolved buffer solution to a crusher or juicer, crush for 10 seconds, pour into 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 solution, and transfer the spores on the sieve to a beaker or reagent bottle using a wash bottle;

[0023] Step 104, Spore Count Calculation: After thoroughly mixing the spore suspension, use a 200μL pipette tip to take 10μL of the spore suspension onto a glass slide and observe the spore count using a stereomicroscope; calculate at least 10 times to estimate the concentration of the spore suspension.

[0024] The cultivation in step 2 includes:

[0025] Step 201: Cover the root chamber with a plastic bag to keep it warm. After 7 days, the seeds will germinate and the seedlings will be thinned out, leaving only one corn seedling in each root chamber. Prepare Hogland nutrient solution, adjusting the P to 20-500μM.

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

[0027] In step 203, at week 6, the second mycelial growth tube was removed. During the culture period, the mycelial growth tube connected to the root chamber was removed to collect mycelium after 4 weeks.

[0028] The beneficial effects of this invention are: non-destructive sampling is carried out under potted conditions to collect pure and uncontaminated AM fungal hyphae and the mycelial soil affected by AM fungi for scientific research on mycelial interactions. Attached Figure Description

[0029] Figure 1 This is a flowchart illustrating an embodiment of a non-destructive sampling method for studying the interaction between AM fungi and hyphal bacteria according to the present invention.

[0030] Figure 2 This is a schematic diagram of the cultivation process using a cultivation device in an embodiment of the present invention;

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

[0032] Figure 4 A comparative diagram of the soil bacterial community structure in the mycelial area of ​​AM fungi at three time points during non-destructive continuous sampling (where blue represents the soil bacterial community structure 2 weeks after AM fungal hyphae entered the mycelial area, red represents 4 weeks, and green represents 6 weeks).

[0033] Figure 5 This is a partial exploded structural diagram of the culture device used in an embodiment of the present invention.

[0034] In the diagram, 100 is the root chamber, 110 is the spiral section, 120 is the 30μm membrane, 200 is the mycelial growth chamber, 300 is the mycelial compartment, and 400 is the mycelial growth cap. Detailed Implementation

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

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

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

[0038] Step 2: Plant corn seeds in the culture medium of the root chamber, and add AM fungal spores to the root chamber while cultivating them using nutrient solution (specifically Hogland nutrient solution) and water (specifically deionized water);

[0039] Step 3, collection, is divided into collecting 500g of pure mycelium and collecting fresh soil affected by pure AM fungal mycelium:

[0040] When it is necessary to collect pure mycelium, open the outer end of the mycelium growth chamber and collect the pure mycelium inside the mycelium growth chamber;

[0041] When collecting fresh soil affected by pure AM fungal mycelium, first open the outer end of the mycelial growth chamber to check the growth of the outermost mycelium. If the mycelial growth outside the mycelial growth chamber is good, fill the mycelial chamber with fresh soil, then seal the mycelial growth chamber and install it outside the mycelial growth chamber (replacing the mycelial growth cover). After cultivating for more than 4 weeks, remove the mycelial growth chamber and collect the fresh soil inside the mycelial growth chamber. If the mycelial growth outside the mycelial growth chamber does not meet the requirements, continue to seal the outer end of the mycelial growth chamber and continue cultivation.

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

[0043] In this embodiment, the mycelial growth chamber has a pore size of 4.5 cm and an axial length of 3.5 cm.

[0044] In this embodiment, the outer end of the mycelial growth chamber is sealed by tightening the mycelial growth cap in the mycelial growth chamber via a threaded connection; the inner thread diameter of the mycelial cap is 4.8 cm, and the outer diameter is 5.5 cm.

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

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

[0047] like Figure 2 and Figure 5 The culture device shown includes: a root chamber 100, a mycelial growth chamber 200, a mycelial compartment 300, and a mycelial growth cover 400. The mycelial growth chamber is installed outside the central root chamber. The mycelial compartment or the mycelial growth cover is installed outside the mycelial growth chamber by threads. The threaded part 110 extending from the inner side of the mycelial growth chamber and the side wall of the root chamber is tightened by thread engagement to facilitate disassembly for mycelial collection.

[0048] The number of mycelial growth chambers is at least one. In this embodiment, it specifically includes four mycelial growth chambers and mycelial compartments. At different time points, only one mycelial growth chamber and mycelial compartment can be removed, while the other three mycelial growth chambers and mycelial compartments are retained without affecting plant growth. The second mycelial growth chamber and mycelial compartment can be removed at the next time point until the sample is harvested.

[0049] The culture medium includes a mixture of perlite and sand, or a mixture of expanded clay and sand, in a volume ratio of 1:1. The culture medium filled into the root chamber can also be mixed with a loose filler, which is one or more of vermiculite, coconut coir, and sawdust, in a volume ratio of 2:1.

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

[0051] Step 101: Inoculate fresh carrot rootlets transfected with the Ri plasmid with spores of the AM fungus *Rhizophagus irregularis* MUCL 43194 onto MSR solid medium. After culturing at 28°C for 4 months, plates of mature AM fungal spores are obtained. Prepare 0.1M citric acid solution (Solution A): Weigh 5.25g of citric acid and dilute to 250mL with sterile water. Prepare 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 then dilute to 500mL. The buffer solution is determined based on the number of MSR plates dissolved; the volume ratio of buffer solution to solid medium is typically 2:1 to 5:1.

[0052] Step 102: Dissolving Solid Culture Medium. Sterilize beakers, glass rods, tweezers, and sieves with alcohol beforehand, and begin the procedure after the alcohol has evaporated. Open the petri dish and transfer the medium to a 1L beaker using a glass rod. Add 2-5 times the volume of citrate buffer, depending on the quantity and specifications of the solid culture medium, and stir thoroughly until no agar lumps are visible to the naked eye. A 150mm plate typically contains 100mL of MSR solid culture medium and requires 200-500mL of buffer for dissolution; a 90mm plate and double-septated plates typically contain 25mL of MSR solid culture medium and require 50-125mL of buffer for dissolution.

[0053] Step 103: Spore isolation and extraction. The culture medium dissolved in the buffer solution contains roots; whether to remove them depends on the situation: the roots can be retained because spores are also present inside; or the roots can be removed if necessary for the experiment. Transfer the dissolved buffer solution to a crusher or juicer, crush for 10 seconds, pour through a 400-mesh stainless steel sieve, discard the filtrate, and retain the spores on the sieve. Rinse the bottle twice with sterile water to remove the buffer solution, and transfer the spores on the sieve to a beaker or reagent bottle using a wash bottle.

[0054] Step 104: Spore Count Calculation. Thoroughly mix the spore suspension. Immediately use a 200μL pipette tip to take 10μL of the spore suspension onto a glass slide and observe the spore count using a stereomicroscope. Calculate at least 10 times to estimate the spore suspension concentration (250 spores / mL). Label and store at 4℃.

[0055] The corn seeds (Zhengdan 958) used in this embodiment were soaked in a 2.5% sodium hypochlorite solution for 10 minutes, then surface-sterilized with 75% alcohol for 1 minute, and rinsed clean with sterile water 6-7 times. The seeds were then placed on a petri dish and cultured for 24 hours. After germination, 3 corn seeds were sown into the root chamber of the device and then covered with 200ml of culture medium.

[0056] The cultivation in step 2 includes:

[0057] Step 201: Cover the root chamber with a plastic bag to keep it warm. After 7 days, the seeds will germinate and the seedlings will be thinned out, leaving only one corn seedling in each root chamber. Prepare Hogland nutrient solution, adjusting the P to 20-500μM.

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

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

[0060] Under this culture system, plants and AM fungi can establish a very stable symbiotic relationship, and a large number of visible AM ​​fungal hyphae exist in the culture medium. The infection rate of AM fungi in maize roots reaches more than 80%. The length of pure AM fungal hyphae harvested in the mycelial growth chamber can even reach more than 6 cm.

[0061] Example 2 utilizes the method of Example 1 to perform non-destructive continuous sampling to study the interaction dynamics between AM fungal hyphae and mycelial bacteria. The preparation of the AM fungal spore inoculum and the corn disinfection method are the same as in Example 1.

[0062] The experiment was conducted on June 3, 2024, with sowing and cultivation of a symbiotic system. Two months after irrigation with the improved culture medium, on August 3, 2024, 50g of fresh soil (brown soil from Tai'an City, Shandong Province) was added to the mycelial chamber device. The caps on the outside of the mycelial growth tubes were removed, and the tubes were carefully screwed into the mycelial chamber device containing fresh soil, followed by normal cultivation. On August 21, 2024, one of the mycelial growth chambers and the mycelial chamber were removed from the device as the first sample.

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

[0064] Experimental Results: Non-destructive continuous sampling of mycelial soil revealed that, compared to fresh soil without access to the device, the growth of AM fungi in the soil significantly affected the soil bacterial community within the mycelial chamber, such as... Figure 3 As shown, there are significant differences in bacterial community composition between the two. Furthermore, the results of non-destructive continuous sampling experiments at three time points revealed that the impact of AM fungi on the soil bacterial community exhibits temporal dynamic changes. The bacterial community composition of the samples at the three time points is as follows: Figure 4 As shown, there were significant differences in phosphatase activity across the range of studies.

Claims

1. A method of non-destructively sampling the interaction of AM fungi with mycelial-intercelled bacteria, characterized in that, The application comprises the following steps: Step 1, filling the sterilized culture medium into the root chamber and the mycelium growth chamber, and sealing the outer end of the mycelium growth chamber; the inner end of the mycelium growth chamber is communicated with the root chamber; Step 2, planting corn seeds in the culture medium of the root chamber, and adding AM fungal spores into the root chamber while using nutrient solution and water for cultivation; Step 3, collecting and separating into collecting pure mycelium and collecting fresh soil affected by the pure AM fungal mycelium: When the pure mycelium needs to be collected, the outer end of the mycelium growth chamber is opened, and the pure mycelium in the mycelium growth chamber is collected; When the fresh soil affected by the pure AM fungal mycelium needs to be collected, the outer end of the mycelium growth chamber is first opened to check the growth of the mycelium at the outermost side, if the mycelium at the outer end of the mycelium growth chamber grows well, the fresh soil is filled into the mycelium growth chamber, then the mycelium growth chamber is sealed and installed outside the mycelium growth chamber, and after more than 4 weeks of cultivation, the mycelium growth chamber is removed, and the fresh soil in the mycelium growth chamber is collected; if the mycelium at the outer end of the mycelium growth chamber does not grow well, the outer end of the mycelium growth chamber is continuously sealed for further cultivation.

2. The method for non-destructive sampling study of AM fungal-bacteria interactions according to claim 1, wherein, The axis of the mycelium growth chamber is directed to the center of the corn root system.

3. The method for non-destructive sampling study of AM fungal-bacteria interactions according to claim 1, wherein, The number of the mycelium growth chamber is at least one.

4. The method for non-destructive sampling study of AM fungal-bacteria interactions according to claim 1, wherein, The pore diameter of the mycelium growth chamber is 4.5 cm, and the axial length is 3.5 cm.

5. A method for non-destructive sampling studies of AM fungal-bacteria interactions according to one of claims 1 to 4, characterized in that, A 30 μm film is arranged between the root chamber and the mycelium growth chamber.

6. The method of non-destructive sampling of AM fungal-bacteria interactions with mycelium according to claim 1, wherein, The outer end of the mycelium growth chamber is sealed by screwing the mycelium growth cover to the mycelium growth chamber through screw connection.

7. The method of non-destructive sampling of AM fungal-bacteria interactions with mycelium according to claim 1, wherein, The culture medium comprises a mixture of perlite and sand, or a mixture of ceramic granules and sand, and the volume ratio is 1:

1.

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

1.

9. The method of non-destructive sampling study of AM fungal-bacteria interactions with mycelium according to claim 1, characterized in that, The culture process of the AM fungal spores comprises the following steps: Step 101, inoculating AM fungal spores on the MSR solid culture medium with fresh Ri plasmid transformed carrot hair roots, and obtaining a mature AM fungal spore plate after 4 months of cultivation at 28°C; preparing 0.1M citric acid solution as A liquid: weighing 5.25 g of citric acid and dissolving in 250 mL of sterile water; preparing 0.1M trisodium citrate solution as B liquid: weighing 7.35 g of trisodium citrate dihydrate and dissolving in 250 mL of sterile water; mixing 9.5 mL of A liquid with 40.5 mL of B liquid, diluting to 480 mL, and adjusting the pH to 6.0 with 0.1M citric acid or sodium hydroxide, and then dissolving in 500 mL; the volume ratio of the buffer solution and the solid culture medium is 2:1-5:1; Step 102, dissolving the solid culture medium: opening the culture dish and transferring to a 1L beaker with a glass rod, adding 2-5 times the volume of citric acid buffer solution according to the number and size of the solid culture medium, and stirring to dissolve until no agar block is visible to the naked eye; Step 103, separation and extraction of spores: transferring the dissolved buffer solution to a crusher or juicer, crushing for 10S, pouring into a 400 mesh stainless steel sieve, discarding the filtrate, and leaving the spores on the sieve; washing the bottle with sterile water for 2 times to remove the buffer solution, and transferring the spores on the sieve to a beaker or reagent bottle with a bottle washer. Step 104, spore quantity calculation: the spore suspension is fully mixed, and 10 μL of the spore suspension is taken from the 200 μL gun head to the slide in time, and the spore quantity is observed by using a body microscope; at least 10 times are calculated to estimate the concentration of the spore suspension.

10. The method of non-destructive sampling of AM fungal-bacteria interactions with mycelium according to claim 1, wherein, The incubation in step 2 comprises: Step 201, the fresh-keeping bag is used to keep warm on the root chamber, and after 7 days, the seedlings are emerged and thinned, only one corn seedling is reserved in each root chamber; the Hoggland nutrient solution is prepared, and the P in the Hoggland nutrient solution is adjusted to 20-500 μM; Step 202, 2 weeks after the thinning, 50 mL of 1 / 4 nutrient solution is added to each corn root chamber every 2 days; from the third week to the fourth week, 1 / 2 nutrient solution is replaced; from the fourth week, full nutrient solution is used, and the corn is cultured for another 4 weeks; during the period, watering and pest control are paid attention to, and after 2-3 months of cumulative culture, the corn is harvested; Step 203, the second mycelium growth tube is removed at 6 weeks, and the mycelium growth tube connected with the root chamber is removed during the culture period to collect the mycelium for 4 weeks.

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