Device for detecting nutrient flow of hyphae of arbuscular mycorrhizal fungi and use method of device

Abstract

The invention belongs to the technical field of intelligent detection of agricultural microorganisms, and relates to a detection device for nutrient flow of arbuscular mycorrhizal fungi hyphae and a use method thereof, the detection device comprises a sterile culture room, a microfiltration membrane and a fluorescence detection module; the sterile culture room is a transparent hollow box body, and infectious microbes in the sterile culture room are removed through high-pressure steam; the device is divided into a plant chamber and a hypha chamber through a microfiltration membrane, the two chambers are both filled with an MSR culture medium containing plant gel, and the microfiltration membrane can only allow AMF hyphae to pass through but block the root system; the fluorescence detection module is arranged outside a plant room, and a high-sensitivity photodiode is used for detecting the signal intensity of cytoplasm circulation fluorescent dye in a symbiotic area in real time and dynamically analyzing the nutrition transfer process. Accurate sterile co-culture of the plant root system and the single AMF hypha is achieved, the problems of infectious microbe interference, environmental heterogeneity, dynamic monitoring deficiency and the like in traditional screening are solved, and the method is suitable for high-throughput screening of AMF functional strains.

Description

Technical Field

[0001] This invention relates to a device for detecting nutrient flow of arbuscular mycorrhizal fungi hyphae and its method of use, belonging to the field of agricultural microbial detection technology. Background Technology

[0002] Arbuscular mycorrhizal fungi (AMF), obligate symbiotic fungi that live in symbiosis with terrestrial plants, have established symbiotic systems dating back approximately 460 million years, representing a key evolutionary characteristic of plant adaptation to terrestrial environments. AMFs establish transdomain nutrient exchange channels by forming highly specialized symbiotic interface structures, significantly enhancing the host plant's efficiency in acquiring mineral nutrients such as nitrogen and phosphorus; their nitrogen transport flux can reach 3-5 times that of autonomous root uptake. Molecular mechanism studies have shown that AMF hyphae, through H… + -ATPase-driven high-efficiency membrane transport system for selective absorption of ammonium nitrogen (NH4) + -N) and nitrate nitrogen (NO3) - -N), and completes transmembrane transport to the symbiotic interface via a specific AMT / PTR transporter protein family. Regarding trace element transport, AMF hyphae mediate Fe transport by secreting low-molecular-weight siderophores and organic acid anions. 3+ Mn 2+ 、Zn 2+ The chelation and dissolution mechanism of metal ions significantly enhances the bioavailability of insoluble metal oxides.

[0003] After forming a symbiotic relationship with plant roots, arbuscular mycorrhizal fungi (AMF) can continuously transport the nutrients they need to promote plant growth. Their transport mechanism mainly includes cytoplasmic flow. Cytoplasmic flow promotes the diffusion and transport of nutrients within cells through the circulation of cytoplasm within the hyphae. Large amounts of nutrients absorbed by the ends of the hyphae are transported towards the base of the hyphae via cytoplasmic flow, and after being transported to the arbuscular structures within the plant root cortex cells, the nutrients are released at the symbiotic interface and further transported to the plant cells. Cytoplasmic flow ensures efficient nutrient transport by AMFs and maintains the symbiotic relationship with plants. Therefore, detecting the rate and intensity of cytoplasmic flow in AMF hyphae within the symbiotic relationship is crucial for screening AMF hyphae that efficiently absorb and transport nutrients.

[0004] However, existing AMF functional hyphae screening systems have significant technical limitations: phenotypic screening platforms based on traditional pot culture methods are constrained by multiple biotic and abiotic interference factors. Specifically:

[0005] 1. Interference in microbial community interactions: Open culture systems cannot eliminate the competitive effects of native microorganisms (such as Glomus spp.), leading to a functional phenotype assessment bias of >30%;

[0006] 2. Impact of environmental heterogeneity: The spatial heterogeneity of soil aggregate structure leads to uneven distribution of mycelial network, resulting in a coefficient of variation (CV) of 25%-40% for element uptake flux.

[0007] 3. Time limitation: Stable phenotypic data can only be obtained after the entire growth period of the host plant (approximately 120-150 days for C3 crops), resulting in low screening throughput (<100 mycelia / year);

[0008] 4. Lack of dynamic monitoring: The lack of non-invasive real-time monitoring technology (such as microscopic imaging) makes it difficult to analyze the spatiotemporal dynamic characteristics of nutrient transport.

[0009] 5. Deficiencies in the evaluation system: The current nutrient element accumulation index cannot distinguish the contribution of direct mycelial translocation to soil availability improvement, with a quantitative error of >15%. Summary of the Invention

[0010] To address the aforementioned problems, the present invention aims to provide a detection device and method for detecting nutrient flow of arbuscular mycorrhizal fungi hyphae, which can be used for rapid screening and efficient identification of AMF hyphae that promote the absorption of nutrient elements in crops, thus providing technical support for the development of green agriculture.

[0011] To achieve the above objectives, the present invention proposes the following technical solution: a detection device for nutrient flow of arbuscular mycorrhizal fungi hyphae, comprising: a sterile culture chamber, a microporous filter membrane, and a fluorescence detection module; the sterile culture chamber is a transparent hollow box, and high-pressure steam is used to remove contaminants from the sterile culture chamber; the sterile culture chamber is divided into two parts by the microporous filter membrane, one part being a plant chamber and the other part being a hyphae chamber, the microporous filter membrane only allows AMF hyphae to pass through but blocks the roots; the fluorescence detection module is installed outside the plant chamber for real-time detection of the signal intensity of cytoplasmic circulating fluorescent dye.

[0012] Furthermore, the sterile culture chamber includes a U-shaped glass plate and a cover glass, the cover glass being placed on the U-shaped glass plate, sealed with high-altitude silicone grease, and covering the hollow area of ​​the U-shaped glass plate.

[0013] Furthermore, two symmetrical grooves are provided on the inner wall of the "U"-shaped glass plate, and the grooves are located at the center line of the "U"-shaped glass plate to fix the microporous filter membrane.

[0014] Furthermore, the plant chamber and mycelium chamber are placed in MSR solid culture medium to simulate the process of AMF mycelium directional transport of nutrients.

[0015] Furthermore, the microporous filter membrane is a nylon microporous filter membrane, and the AMF hyphae form a symbiotic relationship with the plants in the plant chamber through the nylon microporous filter membrane.

[0016] Furthermore, fluorescent dye is dropped into the mycelium chamber at a preset distance from the symbiotic area, and the plant sample with added fluorescent dye is placed in the area corresponding to the fluorescence detection module, and its fluorescence signal is detected within a preset time.

[0017] Furthermore, the fluorescence detection module includes an excitation light source, a first filter, a second filter, and a photoelectric sensor. The excitation light source and the first filter are placed above the plant chamber, and the second filter and the photoelectric sensor are placed at corresponding positions below the plant chamber. The excitation light source is used to emit laser light, which passes through the plant sample containing the fluorescent dye. The photoelectric sensor is used to receive the laser light passing through the plant sample containing the fluorescent dye.

[0018] Furthermore, the photoelectric sensor includes a power supply filter module, a photodiode, and a transimpedance amplifier. The power signal passes through the power supply filter module, then through the photodiode, and is output. The transimpedance amplifier is connected in parallel with the photodiode.

[0019] Furthermore, the power supply filtering module includes a tantalum capacitor and a bypass capacitor, which are connected in parallel to form a multi-stage filtering network; the transimpedance amplifier includes a first resistor and a first capacitor, which are connected in parallel.

[0020] This invention also discloses a method for using a detection device for nutrient flow of arbuscular mycorrhizal fungi hyphae, characterized in that the detection device for nutrient flow of arbuscular mycorrhizal fungi hyphae as described above includes the following steps: inoculating AMF hyphae and plants in a sterile culture chamber, placing the sterile culture chamber in a constant temperature and light incubator until the hyphae and roots form a symbiotic relationship; selecting a fluorescence detection target area according to the location of the symbiotic relationship, adding fluorescent dye near the target area, and placing the device at the corresponding position of the fluorescence detection module for fluorescence signal detection; obtaining the fluorescence signal change curve over time to determine the maximum value of the AMF hyphae's ability to transport fluorescent dye.

[0021] The technical solution of the present invention has at least the following technical effects or advantages:

[0022] 1. The AMF mycelium-plant symbiotic sterile culture chamber developed in this invention enables precise sterile co-culture of plant roots and single AMF mycelium, completely eliminating interference from other microorganisms, maintaining a long-term stable symbiotic environment, and ensuring the reliability of screening results. The AMF mycelium-plant symbiotic sterile culture chamber eliminates the influence of soil heterogeneity and climate fluctuations on experimental results, ensuring the reproducibility of screening results and the comparability of data from different batches.

[0023] 2. This invention creatively combines fluorescent probe technology with photodiodes to directly detect the real-time quantitative detection of cytoplasmic circulation at the hyphae-root interface in the symbiotic system, thereby realizing the dynamic process analysis of nutrient absorption and transport.

[0024] 3. The present invention uses a photodiode, which has high sensitivity, low noise, and high signal-to-noise ratio. Its bandwidth meets the requirements of AMF mycelium for nutrient absorption, and it has high stability and strong anti-interference, making it suitable for high electromagnetic interference environments. Attached Figure Description

[0025] Figure 1 This is a schematic diagram of a sterile culture room in one embodiment of the present invention. Figure 1 (a) is a model diagram of a sterile culture room; Figure 1 (b) is a schematic diagram of the structure of a sterile culture room;

[0026] Figure 2 This is a schematic diagram illustrating the symbiotic relationship between plants and AMF mycelium in one embodiment of the present invention;

[0027] Figure 3 This is a microscopic image of the symbiotic region between the plant and AMF mycelium in one embodiment of the present invention;

[0028] Figure 4 This is a schematic diagram of a fluorescence detection module in one embodiment of the present invention. Figure 4 (a) is a photodiode. Figure 4 (b) is the first filter; Figure 4 (c) is the second filter; Figure 4 (d) is a physical image of the fluorescence detection module;

[0029] Figure 5 This is a circuit diagram of a photodiode in one embodiment of the present invention;

[0030] Figure 6 This is a frequency response curve of a photodiode in one embodiment of the present invention;

[0031] Figure 7 This is a noise contribution analysis diagram in one embodiment of the present invention;

[0032] Figure 8 This is a graph showing the change of fluorescence signal over time in one embodiment of the present invention. Detailed Implementation

[0033] To enable those skilled in the art to better understand the technical solutions of the present invention, the present invention is described in detail through specific embodiments. However, it should be understood that the specific embodiments are provided only for a better understanding of the present invention and should not be construed as limiting the present invention. In the description of the present invention, it should be understood that the terminology used is for descriptive purposes only and should not be construed as indicating or implying relative importance.

[0034] The existing screening technology for arbuscular mycorrhizal fungi (AMF) strains faces the following key technical bottlenecks:

[0035] (1) The interference of contaminating microorganisms cannot be eliminated, and the purity of the symbiotic system is difficult to guarantee. Traditional screening methods rely on open environment sandy soil or field pot experiments. The soil microbial community is complex and cannot effectively isolate the interference of non-target microorganisms such as competing fungi and bacteria. Contaminating microorganisms may affect the screening results by competing for nutrients, producing inhibitory metabolites, interfering with AMF-plant signaling or symbiotic processes, and changing the physicochemical properties of the rhizosphere microenvironment.

[0036] (2) The detection methods have limitations. Existing technologies mainly rely on indirect detection and endpoint determination to analyze the nutrient content of plant tissues. The defects are: ① It is impossible to track and monitor the dynamic transport process of mineral nutrients in the symbiotic system in real time (such as mycelium → root system); ② There is a lack of means to observe the absorption and transport of mineral nutrients; ③ The detection sensitivity is insufficient, making it difficult to identify subtle functional differences.

[0037] (3) Low experimental efficiency. Traditional screening methods face the following efficiency bottlenecks: ① Field or pot experiments require several months to several years to verify the effectiveness of AMF strains, resulting in a long experimental cycle; ② Manual sampling, cultivation, and chemical analysis are cumbersome, time-consuming, and labor-intensive; ③ The number of strains that can be screened in a single experiment is limited, making it difficult to meet the needs of high-throughput screening; ④ The experiment is significantly affected by environmental factors, resulting in poor reproducibility. Soil physicochemical properties (such as pH and organic matter) and climate fluctuations (temperature and humidity) can mask the true function of AMF strains, leading to poor reproducibility of screening results, low comparability of experimental data from different batches, and difficulty in establishing a standardized evaluation system.

[0038] To address the problems of microbial community interaction interference, environmental heterogeneity, lack of dynamic monitoring, and lack of evaluation systems in existing technologies, this invention provides a detection device and method for nutrient flow of arbuscular mycorrhizal (AMF) hyphae. It integrates microbial co-culture technology, non-destructive in vivo detection technology of plant physiological indicators, and machine learning algorithms. It is primarily used for rapid screening and efficient identification of AMF strains that promote crop nutrient absorption. By constructing a sterile AMF hyphae-plant symbiotic culture chamber, precise co-culture of a single AMF hyphae with plant roots can be achieved. A fluorescence detection module using a precision photodiode detects the cytoplasmic circulation rate and intensity of hyphae in the symbiotic culture chamber labeled with FM4-64 fluorescent dyes, enabling efficient screening of AMF strains for efficient nutrient absorption and transport. This invention can be widely applied in microbial fertilizer development, soil improvement, precision agricultural management, and other fields, involving multiple professional fields such as agricultural microbiology, plant nutrition, and biosymbiosis, providing technical support for the development of green agriculture. The following detailed description of the invention, in conjunction with the accompanying drawings, provides an example to illustrate the invention.

[0039] Example 1

[0040] This embodiment discloses a device for detecting nutrient flow of hyphae in arbuscular mycorrhizal fungi, comprising: a sterile culture chamber, a microporous filter membrane, and a fluorescence detection module;

[0041] like Figure 1 As shown, the aseptic culture chamber is a transparent hollow box. It includes a U-shaped glass plate and a cover glass. The cover glass is placed on the U-shaped glass plate and sealed with high-volume silicone grease, covering the hollow area of ​​the U-shaped glass plate. The aseptic culture chamber is preferably made of high-transmittance quartz glass. In this embodiment, the external dimensions of the aseptic culture chamber are preferably 70mm × 70mm × 5mm (length × width × height), and the internal chamber is preferably 40mm × 40mm × 5mm (length × width × height). The dimensions of the aseptic culture chamber can be set according to actual needs and are not limited to the dimensions disclosed in this embodiment. In this embodiment, two symmetrical grooves are provided on the inner wall of the U-shaped glass plate. The grooves are located at the center line of the U-shaped glass plate and are used to fix the microporous filter membrane. The dimensions of the grooves are preferably 1mm × 1mm × 5mm (length × width × height), but their dimensions can also be set as needed and are not limited to the dimensions disclosed in this embodiment.

[0042] In this embodiment, high-pressure steam is used to remove contaminants from the sterile culture chamber. The ultraviolet light has a wavelength of 254 nm and an intensity ≥50 μW / cm². 2 The high-pressure steam temperature is 121℃, and the high-pressure steam application time is more than 15 minutes to ensure the sterility of the chamber. The co-culture process of plant roots and AMF mycelium is carried out in a sterile incubator. Before cultivation, it is necessary to remove mold again by ultraviolet light to completely eliminate interference from other microorganisms.

[0043] In this embodiment, the sterile culture chamber enables precise sterile co-culture of plant roots and single AMF hyphae, completely eliminating interference from other microorganisms, maintaining a long-term stable symbiotic environment, ensuring the reliability of screening results, eliminating the influence of soil heterogeneity and climate fluctuations on experimental results, and ensuring the reproducibility of screening results and the comparability of data from different batches.

[0044] like Figure 2 As shown, the sterile culture chamber is divided into two parts by a microporous membrane: a plant chamber and a mycelium chamber. The microporous membrane allows only AMF mycelia to pass through. A fluorescence detection module is installed outside the plant chamber to detect the signal intensity of the cytoplasmic circulating fluorescent dye in real time. In this embodiment, the microporous membrane is preferably a nylon microporous membrane with a pore size of 30 μm. AMF mycelia form a symbiotic relationship with the plants in the plant chamber through the nylon microporous membrane. A microscopic image of the AMF mycelial symbiotic region is shown below. Figure 3 As shown. Figure 3 The microstructure shows that AMF hyphae extend to the plant roots.

[0045] Plant room and mycelium room, i.e. Figure 1 MSR medium, containing 3 g / L plant gel, was prepared. MSR medium is a synthetic medium specifically designed for the pure or co-culture of arbuscular mycorrhizal fungi (AMFs, such as Rhizophagus irregularis). It is an optimized version of Suleman's medium and is suitable for laboratory studies of AMF spore germination, mycelial growth, and symbiotic relationships with plant roots. The composition of MSR medium is shown in Table 1.

[0046] Table 1 Composition of MSR medium

[0047] Culture medium components mg / L <![CDATA[Magnesium sulfate heptahydrate MgSO4·7H2O]]> 739.0 <![CDATA[Potassium nitrate KNO3]]> 76.0 Potassium chloride (KCl) 65.0 <![CDATA[Potassium dihydrogen phosphate KH2PO4]]> 4.1 <![CDATA[Calcium nitrate tetrahydrate Ca(NO3)2·4H2O]]> 359.0 Sodium iron EDTA (NaFeEDTA) 8.0 <![CDATA[Ammonium molybdate tetrahydrate (NH4)6Mo7O 24 ·4H2O]]> 0.035 <![CDATA[Manganese sulfate tetrahydrate MnSO4·4H2O]]> 2.45 <![CDATA[Zinc sulfate heptahydrate ZnSO4·7H2O]]> 0.29 <![CDATA[Boric acid H3BO3]]> 1.86 <![CDATA[Copper(II) sulfate pentahydrate CuSO4·5H2O]]> 0.24 <![CDATA[Sodium molybdate dihydrate Na2MoO4·2H2O]]> 0.0024 Calcium Pantothenate 0.9 Thiamine Hydrochloride 1.0 Pyridoxine Hydrochloride 0.9 Nicotinic Acid 1.0 <![CDATA[Vitamin B 12 Vitamin B 12 > 0.4 Biotin 0.0009 Sucrose 10000 pH 5.5

[0048] Fluorescent dye is added near the symbiotic area, and the plant sample with added fluorescent dye is placed in the corresponding area of ​​the fluorescence detection module. Fluorescence signal is detected on the sample within a preset time.

[0049] like Figure 4 As shown, the fluorescence detection module includes an excitation light source, a first filter, a second filter, and a photoelectric sensor. The excitation light source and the first filter are placed above the plant chamber, and the second filter and the photoelectric sensor are placed at corresponding positions below the plant chamber. The excitation light source is used to emit excitation light, which passes through the plant sample with added fluorescent dye. The photoelectric sensor is used to receive the excitation light from the plant sample with added fluorescent dye.

[0050] like Figure 5As shown, biofluorescence signals are typically weak (nA level), low-frequency (Hz to kHz range) current signals, requiring high-gain, low-noise amplification circuits for effective detection. Therefore, the photoelectric sensor in this embodiment includes a power supply filter module, a photodiode, and a transimpedance amplifier. The power signal passes through the power supply filter module, then through the photodiode, and is output. The transimpedance amplifier is connected in parallel with the photodiode. The power supply filter module includes a tantalum capacitor and a bypass capacitor, which are connected in parallel to form a multi-stage filter network. The transimpedance amplifier includes a first resistor and a first capacitor, which are connected in parallel.

[0051] In this embodiment, the photodiode (Hamamatsu S1336-44BQ) used has a light-receiving surface area of ​​3.6 × 3.6 mm. 2 With a reverse bias voltage of 0V, a spectral response range of 190 to 1100nm, a capacitance of Cd = 150pF, a shunt resistor of Rsh = 600MΩ, and a dark current ≤ 50pA, this photodiode is used to convert optical signals into current signals. The photodiode's anode is grounded, and its cathode is connected to the inverting input (-IN) of an AD795. A transimpedance amplifier is connected across the op-amp's output and inverting input to suppress high-frequency noise gain and improve phase margin.

[0052] This embodiment uses a low-noise precision operational amplifier to construct a transimpedance amplifier. The transimpedance amplifier (ADIAD795) has a feedback network consisting of a parallel connection of an Rf = 100MΩ (Vishay VR37) and a Cf = 1.3pF (Murata GQM18), with a gain of 10V / 100nA. Its balanced boost is 100MΩ, enabling the detection of currents in the nA range. Figure 6 The frequency bandwidth shown is 3.26kHz. AMF mycelium absorption of nutrients is a signal with extremely low frequency changes; therefore, the frequency bandwidth in this embodiment fully meets the requirements. The Q value (Quality Factor) is an important indicator for quantifying the "sharpness" and selectivity of the signal curve at the resonant frequency. A higher Q value indicates a sharper signal characteristic, while a lower Q value indicates a smoother signal characteristic. In this embodiment, the Q value is 0.453, indicating that the photodiode damping is moderate and oscillations are avoided; within the range of -40℃ to 85℃, the gain drift is ≤0.01% / ℃. Figure 7 As shown, the peak output signal is 10V, the signal-to-noise ratio (SNR) is 86.6dB, the effective number of bits (ENOB) is 14.1bit, and the total output noise is only 164μVrms. The noise mainly comes from the operational amplifier voltage noise (155μV) and the feedback resistor thermal noise (55.7μV). Performance is optimized through the selection of low-noise components. Among these, It is the standard unit for noise current spectral density, used to quantify the noise characteristics of devices at different frequencies, helping engineers optimize the system signal-to-noise ratio. In this embodiment, the common-mode rejection ratio (CMRR) is ≥120dB, suitable for high electromagnetic interference environments.

[0053] Power supply filtering module: The power supply voltage is ±12.5V to ±18V, supporting a wide power supply range; tantalum capacitors Clant+ / Clant- provide low-frequency decoupling, with a capacitance of 10μF, enhancing power supply stability; bypass capacitors Cbp+ / Cbp- are used for high-frequency noise suppression, with a capacitance of 100nF, employing X7R capacitors. X7R capacitors are temperature-stable ceramic capacitors used in mid-to-high frequency circuits, featuring high capacitance density, stability, and low distortion characteristics. The power supply pins (V+ / V-) are grounded through the bypass capacitors and tantalum capacitors to ensure low-noise power supply. This embodiment uses tantalum capacitors (for low-frequency filtering), significantly reducing the impact of power supply ripple on the signal chain, adapting to wide temperature environments, and extending service life.

[0054] All components in the fluorescence detection module are packaged in 0603 / 6032 packages (resistance tolerance ±0.1%), suitable for high-density PCB layouts, and have dimensions of 45mm x 35mm, making them compatible with portable testing equipment.

[0055] This invention integrates a nutrient element fluorescent labeling and real-time detection module, enabling dynamic monitoring of nutrient element translocation processes in the AMF-plant symbiotic system. It also allows for intelligent assessment and grading of the nutrient absorption capacity of AMF strains, achieving high-throughput and precise screening, significantly improving screening efficiency and accuracy. Furthermore, the detection unit in this invention is characterized by its small size, high precision, and good stability, allowing for high-density integrated design, significantly improving production efficiency, reducing costs, and meeting the needs of scientific research and agricultural fields for high-throughput and high-precision production.

[0056] Example 2

[0057] Based on the same inventive concept, this embodiment discloses a method for using a detection device for nutrient flow of arbuscular mycorrhizal fungi hyphae, which is used for any of the above-mentioned detection devices for nutrient flow of arbuscular mycorrhizal fungi hyphae, and includes the following steps:

[0058] S1 involves inoculating AMF mycelia and plants into a sterile culture chamber, which is then placed in a constant temperature and light incubator until the mycelia and roots form a symbiotic relationship.

[0059] S2 selects the target area for fluorescence detection based on the location of the symbiotic organism, namely the symbiotic area where AMF hyphae and roots are in contact. Fluorescent dye FM4-64 is added near the target area, and the module is placed at the corresponding position for fluorescence signal detection. In the hyphae chamber, fluorescent dye FM4-64 is added at a certain distance from the target symbiotic area (preferably 1 cm in this embodiment, but not limited to this) to ensure that the dye can be absorbed by the hyphae and transported to the target symbiotic area through cytoplasmic circulation.

[0060] S3 acquires the fluorescence signal change curve over time to determine the maximum transport capacity of AMF hyphae for fluorescent dyes.

[0061] The fluorescence signal changes over time as shown in the curve. Figure 8 As shown, the hyphae transport the fluorescent dye FM4-64 from the distal end, i.e., 1 cm from the target area, to the fluorescence detection area via cytoplasmic circulation. Figure 8 As shown, within the time range of 0 to 300 s, the average fluorescence intensity of the symbiotic region continuously increases over time, indicating that the hyphae continuously transport dye to the symbiotic region through cytoplasmic circulation. However, when it reaches approximately 300 s, the fluorescence intensity of the symbiotic region tends to stabilize or reaches its peak, indicating that the AMF hyphae's ability to transport fluorescent dye reaches its maximum. AMF hyphae transport nutrients from distant locations to the symbiotic region through cytoplasmic circulation. This embodiment, by detecting the cytoplasmic circulation transport rate and intensity of hyphae, can screen out strains with strong and efficient nutrient transport capabilities from different AMF strains, providing feasible solutions and technical support for the development of microbial fertilizers.

[0062] In this embodiment, the method combines fluorescent probe technology with photodiodes to directly detect the real-time quantitative detection of cytoplasmic circulation at the hyphae-root interface in the symbiotic system, thereby achieving a technological upgrade from "static endpoint detection" to "dynamic process analysis" in the absorption and transport of nutrients.

[0063] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and not to limit it. Although the present invention has been described in detail with reference to the above embodiments, those skilled in the art should understand that modifications or equivalent substitutions can still be made to the specific embodiments of the present invention. Any modifications or equivalent substitutions that do not depart from the spirit and scope of the present invention should be covered within the protection scope of the claims of the present invention. The above content is only a specific embodiment of the present invention, but the protection scope of the present invention is not limited thereto. Any changes or substitutions that can be easily conceived by those skilled in the art within the technical scope disclosed in the present invention should be covered within the protection scope of the present invention. Therefore, the protection scope of the present invention should be determined by the protection scope of the claims.

Claims

1. A device for detecting nutrient flow of arbuscular mycorrhizal fungi hyphae, characterized in that, include: Sterile culture chamber, microporous filter membrane and fluorescence detection module; The sterile culture chamber is a transparent hollow box, and high-pressure steam is used to remove bacteria from the sterile culture chamber. The sterile culture chamber is divided into two parts by the microporous filter membrane, one part being the plant chamber and the other part being the mycelium chamber. The microporous filter membrane only allows AMF mycelium to pass through but blocks the roots. The fluorescence detection module is installed outside the plant chamber to detect the signal intensity of the cytoplasmic circulating fluorescent dye in real time.

2. The detection device for nutrient flow of arbuscular mycorrhizal fungal hyphae as described in claim 1, characterized in that, The sterile culture chamber includes a U-shaped glass plate and a cover glass. The cover glass is placed on the U-shaped glass plate, sealed with high-altitude silicone grease, and covers the hollow area of ​​the U-shaped glass plate.

3. The detection device for nutrient flow of arbuscular mycorrhizal fungal hyphae as described in claim 2, characterized in that, Two symmetrical grooves are provided on the inner wall of the "U"-shaped glass plate. The grooves are located at the center line of the "U"-shaped glass plate and are used to fix the microporous filter membrane.

4. The detection device for nutrient flow of arbuscular mycorrhizal fungal hyphae as described in claim 2 or 3, characterized in that, The plant chamber and mycelium chamber are placed in MSR solid culture medium to simulate the process of AMF mycelium directional transport of nutrients.

5. The detection device for nutrient flow of arbuscular mycorrhizal fungal hyphae as described in claim 1, characterized in that, The microporous filter membrane is a nylon microporous filter membrane, and the AMF hyphae form a symbiotic relationship with the plants in the plant chamber through the nylon microporous filter membrane.

6. The detection device for nutrient flow of arbuscular mycorrhizal fungal hyphae as described in claim 5, characterized in that, Fluorescent dye is dropped into the mycelium chamber at a preset distance from the symbiotic area. The plant sample with the added fluorescent dye is placed in the area corresponding to the fluorescence detection module, and the fluorescence signal is detected within a preset time.

7. The detection device for arbuscular mycorrhizal fungal hyphae as described in claim 6, characterized in that, The fluorescence detection module includes an excitation light source, a first filter, a second filter, and a photoelectric sensor. The excitation light source and the first filter are placed above the plant chamber, and the second filter and the photoelectric sensor are placed at corresponding positions below the plant chamber. The excitation light source is used to emit laser light, which passes through the plant sample containing the fluorescent dye. The photoelectric sensor is used to receive the laser light passing through the plant sample containing the fluorescent dye.

8. The detection device for arbuscular mycorrhizal fungal hyphae as described in claim 7, characterized in that, The photoelectric sensor includes a power filter module, a photodiode, and a transimpedance amplifier. The power signal passes through the power filter module, then through the photodiode, and is output. The transimpedance amplifier is connected in parallel with the photodiode.

9. The detection device for nutrient flow of arbuscular mycorrhizal fungal hyphae as described in claim 8, characterized in that, The power supply filtering module includes a tantalum capacitor and a bypass capacitor, which are connected in parallel to form a multi-stage filtering network; the transimpedance amplifier includes a first resistor and a first capacitor, which are connected in parallel.

10. A method of using a device for detecting nutrient flow of arbuscular mycorrhizal fungal hyphae, characterized in that, The device for detecting nutrient flow of arbuscular mycorrhizal fungal hyphae as described in any one of claims 1-9 comprises the following steps: AMF mycelium and plants were inoculated in a sterile culture chamber, which was then placed in a constant temperature and light incubator until the mycelium and roots formed a symbiotic relationship. Based on the location of the symbiont, a target area for fluorescence detection is selected, and fluorescent dye is dropped near the target area. The dye is then placed in the corresponding position of the fluorescence detection module to detect the fluorescence signal. The fluorescence signal was obtained as a function of time to determine the maximum value of the AMF hyphae's ability to transport fluorescent dyes.

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