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CN-121992063-A - Method for screening arbuscular mycorrhizal fungi in soil

CN121992063ACN 121992063 ACN121992063 ACN 121992063ACN-121992063-A

Abstract

The application belongs to the technical field of microorganism detection and soil biology, in particular to a method for screening arbuscular mycorrhizal fungi in soil, which comprises the steps of preparing a soil propagule suspension, utilizing a microecological bit array to realize single individual separation and physical isolation of propagules, co-culturing with a sterile host root system under a specific abiotic stress condition, and directly obtaining a stress-resistant functional strain through microscopic monitoring, positive symbiont confirmation and molecular identification. Through the technical scheme, the method can avoid competitive bias, efficiently screen the mycorrhizal fungi purified strain with low phosphorus, drought or salt and alkali tolerance, and remarkably improve the objectivity and functional targeting of screening.

Inventors

  • TAN YAN
  • LU YANHUA
  • LI GUANG
  • WANG CHENGXU

Assignees

  • 甘肃农业大学

Dates

Publication Date
20260508
Application Date
20260319

Claims (10)

  1. 1. A method for screening arbuscular mycorrhizal fungi in soil, which is characterized by comprising the following steps: Preparing a soil propagule suspension, processing a soil sample to be detected to obtain a suspension containing arbuscular mycorrhizal fungi propagules (30), and adjusting the concentration of the suspension to a preset value; Providing a microecological array and inoculating, wherein the microecological array comprises a base plate (10) with a plurality of micropores (11) and a top cover plate (20) with a main culture medium accommodating bin (21) and a root system guiding channel (22), and a microporous filter membrane (23) capable of realizing molecular permeation and microbial isolation is arranged at the port of the root system guiding channel (22); Step three, a stress type root system culture system is established and integrated, a stress type MSR culture medium (40) containing specific abiotic stress factors is prepared in a main culture medium accommodating bin (21) of the top cover plate (20), and a sterile host plant root system, namely carrot hairy roots (50), is inoculated on the culture medium, and then the top cover plate (20) and the base plate (10) are assembled, so that each root system guide channel (22) is aligned with one micropore (11), and an array system consisting of a plurality of independent micro-ecological culture units is constructed; step four, co-culturing and monitoring, namely culturing the array system under a proper condition, periodically and non-invasively monitoring germination of propagules (30) and growth conditions of hyphae (31) in micropores (11), and identifying positive candidate units of the hyphae (31) successfully passing through the microporous filter membrane (23); Step five, confirming and harvesting positive strains, carrying out tissue transparentization and staining treatment on host plant root systems corresponding to the positive candidate units, confirming whether a cluster or vesicle structure exists in the roots under a microscope to confirm that symbiotic relationship is successfully established, and harvesting all contents in micropores (11) confirmed to be successful as purified strains screened by specific stress; And step six, molecular identification of the strain, wherein the step aims at accurately identifying the species level of the screened functional strain.
  2. 2. The method for screening arbuscular mycorrhizal fungi in soil according to claim 1, wherein the microecological array provided in the second step is characterized in that the micro holes (11) arranged in a matrix are arranged on the upper surface of the base plate (10), the root guiding channels (22) arranged in the same matrix are arranged on the bottom surface of the top cover plate (20) corresponding to the matrix positions of the micro holes (11), and the base plate (10) and the top cover plate (20) are assembled in an accurate alignment manner through a positioning structure so as to ensure that the lower port of each root guiding channel (22) and the upper space of one micro hole (11) form a one-to-one corresponding and independent functional culture unit.
  3. 3. The method for screening arbuscular mycorrhizal fungi in soil according to claim 2, characterized in that the base plate (10) and the top cover plate (20) are made of transparent polycarbonate materials which can be subjected to high-temperature high-pressure steam sterilization treatment, the bottoms of the micropores (11) are optical-grade transparent planes, the thickness and the flatness of the micropores are suitable for high-magnification differential interference contrast observation through an inverted microscope, and the root system guide channel (22) is of a hollow circular tube structure, and the lower port of the root system guide channel is covered by the polycarbonate microporous filter membrane (23).
  4. 4. A method according to claim 3, wherein the polycarbonate microporous membrane (23) provided in the second step has a pore size of 0.45 μm, which allows water, inorganic salts, saccharides and other small molecule nutrients in the culture medium and host root system secreted signal molecules to pass through the microporous membrane (23), but physically prevents bacteria and other fungal hyphae from crossing, thereby creating a molecular permeable but microbial barrier between the main culture medium holding compartment (21) and the micropores (11).
  5. 5. The method according to claim 4, wherein the stress type root system culture system established in the third step comprises specific abiotic stress factors introduced by precisely adjusting chemical components of a modified Shi Telv Riemann (MSR) medium, wherein the abiotic stress factors are at least one selected from the group consisting of low-phosphorus stress, drought stress and heavy-metal stress.
  6. 6. The method according to claim 5, wherein when the abiotic stress factor is low-phosphorus stress, the method is performed by decreasing the concentration of potassium dihydrogen phosphate, which is the only phosphorus source in the stress type MSR medium (40), to 10% of the concentration of potassium dihydrogen phosphate in the standard MSR medium, i.e., adjusting the concentration of potassium dihydrogen phosphate to 7.4 mg/L medium, thereby constructing an extremely phosphorus-deficient culture environment for screening strains resistant to low-phosphorus stress.
  7. 7. The method for screening arbuscular mycorrhizal fungi in soil according to claim 6, wherein when the abiotic stress factor is drought stress, it is achieved by adding polyethylene glycol having an average molecular weight of 6000 daltons as an impermeable osmotic regulator on the basis of a standard MSR medium and adjusting the final concentration thereof to 200 g/l so that the stress type MSR medium (40) generates a water potential of-0.5 megapascal to simulate physical drought conditions to screen drought stress resistant strains.
  8. 8. The method according to claim 7, wherein when the abiotic stress factor is heavy metal stress, the method is realized by adding a cadmium chloride stock solution into a standard MSR culture medium to precisely control the final concentration of the cadmium chloride stock solution to 100 micromoles/liter, thereby constructing a culture environment with obvious toxicity but non-lethal cadmium ion concentration, and screening strains with a heavy metal cadmium tolerance mechanism.
  9. 9. The method according to claim 8, wherein the preparation of the soil propagule suspension comprises air drying the soil sample to be tested and sieving with a sieve of 2mm in diameter, weighing the sieved soil sample, adding into sterile deionized water containing nonionic surfactant, magnetically stirring and dispersing, standing, collecting supernatant, wet-sieving sequentially with nylon screens of 500 micron, 250 micron, 100 micron and 38 micron in diameter, collecting and washing the material trapped on the 38 micron screen, centrifuging and resuspending in citrate buffer solution, counting propagule units in the resuspension with a hemocytometer plate, and diluting the suspension to a final concentration of 10 propagule units per milliliter according to the counting result.
  10. 10. The method according to claim 8, wherein the step five of confirming and harvesting positive strains comprises cutting carrot hairy root (50) root segments corresponding to the micropores marked as positive candidate units in the step four, sequentially performing high-temperature transparentizing treatment on potassium hydroxide solution, washing and staining with acid fuchsin or trypan blue, then observing the stained root segments under an optical microscope to see whether the arbuscular or vesicle structures are observed in the cortex cells of the root segments as final confirmation indexes for successful establishment of symbiotic relationship, and sucking newly generated spores, residual hypha network and root segments symbiotic with the interior of the micropores confirmed as positive micropores by using a pipette, and transferring the newly generated spores, residual hypha network and residual root segments into fresh culture medium as purified strains for amplification culture.

Description

Method for screening arbuscular mycorrhizal fungi in soil Technical Field The invention belongs to the technical field of microorganism detection and soil biology, and particularly relates to a method for screening arbuscular mycorrhizal fungi in soil. Background The arbuscular mycorrhizal fungi (ArbuscularMycorrhizalFungi, AMF) is used as a key functional microorganism of a land ecological system, can form a symbiotic system with most of higher plant root systems, can obviously expand the root system absorption range, improve the nutrition and moisture absorption efficiency of phosphorus, nitrogen and the like, and can also enhance the tolerance of plants to drought, salt and alkali, heavy metals and pathogens. The method has great application potential in the aspects of improving crop yield, improving soil structure, repairing degenerated ecological system and the like, so the arbuscular mycorrhizal fungus strain with specific excellent properties is efficiently and accurately screened from soil microbial communities, and the arbuscular mycorrhizal fungus strain becomes a core research direction and a technical bottleneck of modern agricultural biotechnology and ecological restoration engineering. The existing arbuscular mycorrhizal fungi screening mainly adopts a host plant trapping culture technology, utilizes the characteristic that the special living nutrition is needed to depend on the root system of living plants to complete the life cycle, takes the soil to be detected as a matrix, plants host plants with strong affinities such as clover, corn and the like, cultures for several months in a greenhouse controllable environment, enables arbuscular mycorrhizal fungi in the soil to be infected and propagated under the induction of the root system to form dormant spores, and then obtains spore mixtures through wet screening, decantation, sucrose centrifugal gradient and other methods for separation. The technology solves the difficult problem of enrichment acquisition caused by incapability of artificial pure culture of arbuscular mycorrhizal fungi, and has been applied for decades. The prior art has the principle limitation that trapping culture is competitive screening under the artificial limited environment, and has 'enrichment paradox'. The strain with quick propagation and strong infectivity is easy to occupy competitive advantage and is dominant in spore library, while the strain with strong stress resistance and special function is low in spore yield due to conservative growth and propagation in primary environment, and is inferior in short-term competition, and the population is not enriched but can be inhibited. The screened community structure deviates from the original soil, the diversity is reduced, the genetic information is lost, the subsequent function screening result is not representative enough, and the high-value stress adaptability strain is easy to miss, so that the method becomes a key bottleneck of accurate screening. For this purpose, the invention provides a method for screening arbuscular mycorrhizal fungi in soil. Disclosure of Invention In order to overcome the deficiencies of the prior art, at least one technical problem presented in the background art is solved. The technical scheme adopted for solving the technical problems is that the method for screening arbuscular mycorrhizal fungi in soil comprises the following steps: Step one, preparing a soil propagule suspension. This step aims at separating and preparing a uniform concentration of propagule suspension containing arbuscular mycorrhizal fungi spores and hyphal fragments from the original soil sample. The method specifically comprises the steps of taking a soil sample to be tested, carrying out air drying treatment for 72 hours at room temperature, and then sieving through a sieve with the aperture of 2 mm to remove large-particle impurities such as plant residues, gravel and the like. 100 g of the sieved soil sample was precisely weighed, placed in a beaker containing 1 liter of sterile deionized water, and polysorbate twenty having a concentration of 0.05% (by volume) was added as a wetting agent, and stirred at 300rpm for 30 minutes using a magnetic stirrer to sufficiently disperse the soil particles and the fungus propagules. After stirring, standing for 5 minutes, and taking supernatant after settling the large-particle soil. The supernatant was wet screened sequentially through a series of nylon screens with pore sizes of 500 microns, 250 microns, 100 microns and 38 microns. The material trapped on the 38 micron screen was collected and repeatedly rinsed with sterile citrate buffer (ph 6.0) and collected into a sterile centrifuge tube. The collection solution was centrifuged at 3000g for 10 minutes at 4 degrees celsius, the supernatant discarded, and the pellet resuspended in 10ml of the citrate buffer. The arbuscular mycorrhizal fungus spores in the re-suspension are counted using a hemocytometer, and based on