CN111788991A - Method and device for mutual reciprocal symbiosis of isotope C-N labeled endophytic fungi and host plants - Google Patents
Method and device for mutual reciprocal symbiosis of isotope C-N labeled endophytic fungi and host plants Download PDFInfo
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01G—HORTICULTURE; CULTIVATION OF VEGETABLES, FLOWERS, RICE, FRUIT, VINES, HOPS OR SEAWEED; FORESTRY; WATERING
- A01G18/00—Cultivation of mushrooms
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- A—HUMAN NECESSITIES
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Abstract
The invention discloses an isotope13C‑15N-labeled endophytic fungi and host plants are symbiotic and reciprocal. The invention relates to a method and a device for researching symbiotic relationship of plants and microorganisms, belonging to a method for quantitatively analyzing microorganisms to promote mineral elements absorbed by a host through an isotope labeling test. The invention uses an integrated device assembled by nylon nets and organic glass plates with different apertures as a tool to mark isotopes13C‑15And N is used for quantitatively analyzing the reciprocal symbiotic relationship between the endophytic fungi and the host plants. The method fully utilizes the characteristics of rapidness, accuracy and strong controllability of the integrated device, simultaneously marks various isotopes in the same tree body, and rapidly reveals the interaction between the endophytic fungi and the host plant. The invention realizes the integrated marking device for researching the reciprocal symbiotic relationship between the endophytic fungi and the host plants, solves the problem of quantitatively analyzing the functional characteristics of the endophytic fungi, and greatly improves the isotope marking efficiency and the marking precision.
Description
Technical Field
The invention belongs to the technical field of scientific research of symbiotic relationship of plants and microorganisms, and particularly relates to a method for researching reciprocal symbiosis of endophytic fungi and host plants, and also relates to a device for the reciprocal symbiosis of isotopic-labeled endophytic fungi and host plants.
Background
Mineral nutrients are the basis of plant growth and development, however, in traditional agriculture, growers excessively pursue yield, unreasonable large-amount application of chemical fertilizers changes the soil microbial community structure, destroys the positive effect of beneficial microbial communities in the natural environment on host plants, and limits research on the reciprocal symbiotic relationship between root microbial communities and host plants. Therefore, designing isotopes13C-15The N-labeled endophytic fungi and host plants are mutually and reciprocally symbiotically integrated, and the N-labeled endophytic fungi and host plants have important ecological significance for developing and utilizing beneficial microbial communities of plants and healthy cultivation of agriculture and forestry.
CO2As a main raw material for photosynthesis of plants, plants absorb CO through photosynthesis2And assimilates the organic matters to transport the organic matters to each organ of the plant body, thereby providing energy for the growth and development of the plant. Meanwhile, after the root system microorganisms infect the root system of the host plant, the host plant provides energy substances (such as carbon sources) and a breeding place for the growth of the root system microorganisms, and the beneficial root system microorganisms promote the host plant to absorb nutrients,Water and increase the biotic and abiotic stress capacity of the host plant. Current research is primarily focused on the use of isotopes13C marks the influence of distribution and transportation of photosynthetic products on the growth and development of plants.
With the maturity and development of isotope labeling technology, isotope labeling devices are applied to the research of plant-microorganism symbiosis relationship with the advantages of high efficiency, low cost and easy control, and beneficial microbial communities and synthetic microbial communities are applied to the cultivation mode of agriculture and forestry health with the advantages of ecological environmental protection and sustainability. Thus, the isotope is improved13C-15The N marking efficiency and accuracy are used for disclosing the reciprocal symbiotic relationship between the root microorganisms and the host plants, and the technical problem to be solved urgently is solved.
Disclosure of Invention
The invention provides an isotope label for rapidly revealing the reciprocal symbiotic relationship between plants and microorganisms, which is characterized in that the transportation and distribution of plant leaf photosynthetic products and the strength of the absorption and transportation capacity of root system nutrients supplement each other, when the reciprocal symbiotic relationship between plants and microorganisms is explored and the transportation and distribution of plant photosynthetic products or mineral elements in different seedlings are singly marked by isotopes, the differences of infection of root system microorganisms on hosts can cause different host growth vigors, so that the quantitative analysis of isotope label results can be influenced by the differences among different seedlings, and the infection among different seedlings has the defects of large workload, high cost and the like13C-15N-labelled endophytic fungi and host plants, and isotopes of the invention13C-15And N is used for marking the same seedling, so that the isotope marking result can be conveniently and quantitatively analyzed, the symbiotic difference caused by the growth difference of different seedlings can be reduced, and the reliability and the accuracy of the result can be ensured.
Another object of the present invention is to provide an isotope13C-15N mark endophytic fungi and host plant intergrowth device that reciprocal, the device can carry out simultaneous marker analysis to plant-microorganism interaction. The device is small and exquisite, has simple structure, and makesThe portable integrated device is convenient to use, has a light source, is less influenced by the weather environment, uses nylon nets with different apertures to separate root systems and fungal hyphae in the device, and is a portable integrated device for researching the functional characteristics of endophytic fungi and the interaction between the endophytic fungi and host plants.
In order to achieve the purpose, the invention adopts the following technical scheme:
the purpose of the invention is realized as follows:
isotope element13C-15An N-labeled endophytic fungi and host plant reciprocal symbiotic device comprising:
the device comprises a box body 12, wherein an N mark chamber, a middle part and an N mark chamber are sequentially arranged in the box body 12 from left to right; the middle part is provided with a fungus chamber I9 with marks and a plant growth chamber (from front to back)13C marker chamber) 11, fungus chamber II10 with markers; for placing13C-15N isotope, endophytic fungi with a mark, plants to be marked and a marking device;
the isolation layer is arranged on the contact surfaces of the plant growth chamber 11, the N mark chamber, the fungus chamber I, II with the mark and the fungus chamber I, II with the mark and is used for separating the N mark chamber, the fungus chamber with the mark and the plant growth chamber;
the isolation layer includes: an organic glass plate, a 2mm nylon net or a 20 μm nylon net; the organic glass plate is arranged on the isolation layer with the height of more than 15cm at the bottom of the box body, and the 2mm nylon net and the 20 mu m nylon net are arranged on the isolation layer with the height of less than 15cm at the bottom of the box body;
the organic glass plate also uniformly divides the two N marking chambers into 4N marking chambers respectively, namely NⅠ,NⅡ,NⅢ,NⅣ，NⅠ′,NⅡ′,NⅢ′,NⅣ′For analysing the differences in uptake of endophytic fungi to host plants15Influence of N form and host plant pair differentiation15N form preference, all the joints of the organic glass plates are sealed by waterproof glue;
13c gas reaction generator in the plant growth chamber for generatingBecome into13C-CO2Gas is carried out13C is a label of13The C gas reaction generating device comprises: a plug-attached waste liquid discharge pipe 15 for discharging reaction waste liquid;13C-CO2a reaction cup 16 arranged in the plant growth chamber for accommodating13C-Na2CO3A solution; a three-way valve burette 20 with scales, which is arranged above the reaction cup 16, wherein the three-way valve in the three-way valve burette 20 with scales is arranged at the top of the box body, and HCl solution and the three-way valve are injected into the reaction cup 1613C-Na2CO3Solution reaction formation13C-CO2Gas is carried out13C, marking;
a gas sensor 18 disposed in the plant growth chamber for monitoring13CO before and after C-labelling and during labelling2And O2Concentration, CO2And O2The gas is used as raw material and product of plant photosynthesis, and the change of its concentration can real-time represent the growth condition of seedling to preliminarily determine isotope13C labeling efficiency;
the fluorescent lamp 19 is arranged in the plant growing room and fixed at the top of the box body by long screws, the electric wire of the fluorescent lamp is punched out from the top end of the box body, and waterproof glue is used for sealing the joint so as to supplement light in insufficient illumination or rainy days, thereby avoiding the influence of the rainy days on the marking test;
a wide-mouth bottle component with a rubber plug, which is arranged in the plant growing chamber and is used for absorbing13CO before and after C labelling2A gas; the wide-mouth bottle component with the rubber plug comprises:
a hook 22 arranged at the top of the plant growing chamber, a rubber plug is fixed on the hook and can move up and down to open or close the wide-mouth bottle,13c mark front lifting hook to open wide-mouth bottle to fully absorb CO in plant growth chamber2Gas, after the mark begins, the hook moves down to plug the wide-mouth bottle, so as to prevent the wide-mouth bottle from being plugged13C-CO2The gas is absorbed by alkaline solution to influence the marking efficiency, and after the marking is finished, the hook is lifted up to open the wide-mouth bottle to fully absorb the residual gas in the plant growth chamber13C-CO2Gas, prevention of CO2Air pollution by gas;
a wide-mouth bottle with a rubber plug for containing alkaline solution to fully absorb CO in the plant growth chamber2Gas, thereby improving marking efficiency and preventing environmental pollution;
the hygrothermograph 23 is arranged in the plant growth chamber and used for monitoring the temperature and the humidity in the plant growth chamber and preventing the inappropriate temperature and humidity from influencing the normal growth of the plant, because the temperature and the humidity are important environmental factors influencing the photosynthesis of the plant, the temperature and the humidity in the plant growth chamber are monitored and adjusted in real time, which is favorable for improving the photosynthetic efficiency of the plant, thereby improving the isotope13C labeling efficiency;
the temperature control assembly can be communicated with the interior of the box body and is used for adjusting the temperature and the humidity in the plant growing chamber; the temperature control assembly includes: the cooling pipe 24 is arranged in the plant growth chamber and used for adjusting the temperature and the humidity of the plant growth chamber; a cold water generator 26 arranged outside the box body and used for refrigerating and controlling the temperature; and the air pump 25 is arranged on the outer side of the box body, is connected with the cooling pipe 24 and the cold water generator 26 through rubber hoses, and is used for pumping the temperature-controlled water in the cold water generator into the cooling pipe.
Furthermore, the box is made by organic glass, the organic glass board at box top only seals the plant growth room, N mark room, the fungus room I that has the mark, the fungus room II that has the mark are uncovered or ventilative sealless.
The right side of the top of the plant growth chamber is provided with a box door 21 with a handle, the box door can be opened and closed at any time, the box door is sealed by a rubber gasket, and air leakage cannot occur during closing.
The 2mm nylon net divided N-mark chamber (N)Ⅲ,NⅣ,NⅠ′,NⅡ′) The marked fungus chamber II can pass through fungus hypha and plant fine root, so that the isotope in the N marked chamber divided by 2mm nylon net15N can be absorbed by endophytic fungi and plant fine roots; but 20 μm nylon mesh divided N-labeled chambers (N)Ⅰ,NⅡ,NⅢ′,NⅣ′) The marked fungus chamber I can only pass through fungus hypha, so the isotope in the N marked chamber divided by the 20 mu m nylon net15N can only be absorbed by endophytic fungi.
The above-mentioned13C gas reaction generating device, the13C-CO2A reaction cup 16 is placed at the left side of the plant growth chamber 11, the reaction cup13C-CO2A waste liquid discharge pipe 15 with a rubber plug is arranged at the bottom of the reaction cup 16 and passes through NⅢPasses through the wall of the box body to the outside of the box body.
The box wall and the penetrating positions of all the guide pipes are sealed by waterproof glue so as to ensure that the box body is strictly sealed.
The length, width and height of the box body can be 40, 40 and 50 cm; the size of the N marking chamber is 10 x 40cm, and N isⅠ、NⅡ、NⅢ、NⅣ，NⅠ′、NⅡ′、NⅢ′、NⅣ′The marking chambers were each 10X 10cm in size. The size of the marked fungus chamber I and the marked fungus chamber II is 20 multiplied by 10 multiplied by 50cm, and the size of the plant growth chamber is 20 multiplied by 50 cm.
The invention provides a method for researching isotope by using the device13C-15The N-labeled endophytic fungus and host plant reciprocal symbiosis method includes the following steps:
(1) preparing a substrate: weighing the substrate which is continuously sterilized for 3 times at high temperature, and putting the substrate into a clean plant growth chamber, an N mark chamber and a fungus chamber with marks; the thickness of the substrate of each chamber is 20-25cm, and the thickness of the substrate exceeds the height of the nylon mesh isolation layer by 5-10cm, so that each compartment is relatively sealed and airtight;
(2) transplanting seedlings to be marked: selecting seedling seedlings with the seedling age of 20-30d, cutting off main roots, transplanting the seedling seedlings into plant growth chambers, wherein each plant growth chamber is provided with one seedling, placing the device in a greenhouse or a natural environment, irrigating sterile water in the plant growth chambers, an N marking chamber and a marked fungus chamber after transplanting to ensure that the water content of a matrix is 60 +/-5%, irrigating 1/2Long-Ashton medium nutrient solution in the N marking chamber every 7d after the seedlings survive, replacing 1/2Long-Ashton medium nutrient solution with N-deficient 1/2Long-Ashton medium nutrient solution at 21d, keeping other elements unchanged, and irrigating for 7d for once until the isotope labeling test is finished; nutrient solution is not poured into the plant growth chamber and the marked fungus chamber to promote the root system or hyphae to absorb nutrients through the nylon net;
(3) establishing a symbiotic system: equal amounts (e.g.: 1X 10) of the labelled endophytic fungi are added7cfu/ml, 10ml) is inoculated in a sterile marked endophytic fungi chamber I and a sterile marked endophytic fungi chamber II which are separated by a nylon net with the diameter of 20 mu m and a nylon net with the diameter of 2mm, whether the seedling root system has the marked endophytic fungi is detected after a plurality of days, the colonization part and the infection rate of the marked endophytic fungi in the plant root system are observed, and after the root system is successfully colonized, the isotope is started13C-15Marking by N;
(4) isotope of carbon monoxide15Marking by N: in that15Before N marking, the seedlings to be marked are subjected to N starvation treatment for 1 week, the seedlings are normally irrigated by improved 1/2Long-Ashton medium nutrient solution lacking N, and 4N marking chambers N on the left side of the box body are used for one week laterⅠ,NⅡ,NⅢ,NⅣAdding the same amount of isotope15N(K15NO3，15Abundance of N10.2%), 4 Nitrogen labeling chambers N on the right side of the cabinetⅠ′,NⅡ′,NⅢ′,NⅣ′Adding the same amount of isotope15N((15NH4)2S04，15Abundance of N10.2%) begin to label, isotope in N label chamber15The concentration of N is 1mM, the 1/2Long-Ashton medium nutrient solution lacking N is normally irrigated after the marking is started, other elements are unchanged, the irrigation time is still 7d, the nutrient solution is irrigated until the isotope marking test is finished, and the isotope marking is carried out after the isotope marking is continuously carried out for 7-10d15Detecting N abundance, adjusting the water content of the matrix to be 60 +/-5% by using sterile water during sampling,15measuring the N abundance by adopting an isotope mass spectrometer, and then calculating the percentage content of nitrate nitrogen (or ammonium nitrogen) in the marked plants; the calculation formula is as follows:
nitrate nitrogen (or ammonium nitrogen) (% of measurement sample)15N abundance (%) -Nature15N abundance (%)]10.2% of the natural world15The N abundance is about 0.336%; wherein the endophytic fungi assist the host in uptake15NO3 -Is an amount of NⅠAnd NⅡAmount of spent marker compartment, uptake by host root system15NO3 -Is an amount of NⅢAnd NⅣTotal amount of marker chamber consumed minus NⅠAnd NⅡTotal amount of marker chamber consumed; endophytic fungi help host to absorb15NH4Is an amount of NⅢ′And NⅣ′Amount of spent marker compartment, uptake by host root system15NH4Is an amount of NⅠ′And NⅡ′Total amount of marker chamber consumed minus NⅢ′And NⅣ′Total amount of marker chamber consumed;
(5) isotope of carbon monoxide13C, marking: time of day was marked from 09 am: 00 to 17 pm: 00 end, sufficiently absorbing CO in the plant growth chamber by using alkaline solution in a wide-mouth bottle before marking2Gas and carbon starving treatment of the nursery stock to be marked for one day so as to increase isotope13C labeling efficiency; before marking, by13C gas reaction generating device13C-Na2CO3Reaction of the solution with HCl solution to form13C-CO2Gas is carried out13C mark, during which the observation in the plant growth chamber is carried out by means of a gas sensor13C-CO2(0.03%) and O2(21%) concentration; observing temperature and humidity change in the plant growth chamber by a hygrothermograph, continuously marking for 7-10 days, and analyzing isotope of the substrate in the plant growth chamber, the N marking chamber and the marked fungus chamber by using an isotope mass spectrometer13C abundance, and isotopes of plant material (roots, stems, leaves) and labeled fungi13C abundance, after each marking, using alkaline solution in the wide-mouth bottle to fully absorb the C in the plant growth chamber13C-CO2Gas, prevent air pollution.
In the step 1) of the method, the high-temperature continuous sterilization is performed for 20min at 121 ℃. The matrix is prepared from fine sand and vermiculite according to the volume ratio of 1:1 in a certain proportion.
In the step 2) of the method, the greenhouse conditions are as follows: day/night: 25 +/-2/18 +/-2 ℃; the illumination is 12h, 400 +/-50 mu mol-2.s-1(ii) a Relative humidity: 65 plus or minus 5 percent.
In the step 3) of the method, considering that different strains have different infection times on different plants, when the seedling is a walnut seedling and the endophytic fungus is a phiocephala fortinii fungus, the step 3) detects whether the seedling root system has the endophytic fungus carrying the marker after 30-40 days.
In step 4), the N labeling chamber NⅠ,NⅡ,NⅢ,NⅣIsotope of (1)15N is represented by K15NO3The method comprises the steps of providing a material,15the abundance of N is 10.2%; the N labeling chamber NⅠ′,NⅡ′,NⅢ′,NⅣ′Isotope of (1)15N is represented by15NH4)2S04The method comprises the steps of providing a material,15the abundance of N was 10.2%.
In step 5) of the above method, in the plant growth chamber during the marking13C-CO2Is maintained at a concentration of 0.03% and O2The concentration was 21%. If it is13C-CO2The gas concentration is lower than 0.03%, and HCl solution is required to be added dropwise or supplemented in time13C-Na2CO3Solution reaction enhancement13C-CO2The gas concentration.
In the step 5) of the method, the temperature in the plant growth chamber during the marking period is not more than 30 ℃, and the humidity is about 60 percent; when the temperature exceeds 30 ℃, the temperature is adjusted to the proper temperature range of the plants by using the temperature control component.
The plant body includes plant roots, stems and leaves.
The endophytic fungus having a label in the above method may be an endophytic fungus having a fluorescent label.
The endophytic fungus with a label can be an endophytic fungus with a fluorescent protein label.
The endophytic fungi labeled with the fluorescent protein can be endophytic fungi which are introduced with genes for coding the marker protein in the endophytic fungi so as to express the marker protein. The marker protein is a protein that can be observed or observed.
Illustratively, the marker protein can be a fluorescent protein, such as a red fluorescent protein, a green fluorescent protein, a yellow fluorescent protein, and the like. Illustratively, the marker protein may be a GFP protein.
The Long-Ashton medium nutrient solution comprises the following components:
2mM NH4NO3，0.6mM KH2PO4，0.042mM K2HPO4，1mM K2SO4，0.9mM CaCl2·2H2O，0.9mMMgSO4·7H2O，10.4uM MnSO4·4H2O，69uM H3BO3，1.2uM ZnSO4·7H2O，1.7uM CuSO4·5H2O，0.13uM NaMoO4·2H2O，0.05uM CoSO4·7H2o, 10uM Fe-EDTA. The 1/2Long-Ashton medium nutrient solution used in the invention reduces the concentration of each component by half; the improved N-deficient 1/2Long-Ashton medium nutrient solution used in the invention is obtained by removing NH from 1/2Long-Ashton medium nutrient solution4NO3The components and other nutrient components are kept unchanged.
Compared with the prior art, the invention has the following advantages:
(1) the integrated marking device realizes the research of the reciprocal symbiotic relationship between the endophytic fungi and the host plants:
the traditional method adopts a way of carrying out single isotope labeling among different trees for researching the symbiotic relationship between plants and microorganisms, and has the disadvantages of complex work and high cost. The invention separates the nursery stock to be marked, the endophytic fungi and the element to be marked into an integrated device through nylon nets with different apertures, simultaneously marks a plurality of isotopes in the same tree body, and rapidly reveals the interaction between the endophytic fungi and the host plant, which is particularly important for the development and utilization of beneficial microbial communities and synthetic microbial communities in soil.
(2) The problem of quantitative analysis of the functional characteristics of endophytic fungi is solved:
the invention adopts 2mm and 20 mu m nylon nets to separate the N marking chamber, the GFP fungus chamber and the plant growth chamber, and is more convenient to research the functional characteristics of endophytic fungi and host planting compared with the traditional tieback infection method (the root system and the fungus hyphae are in the same environment)Effect of organisms on growth of endophytic fungi by analyzing isotopes in N-labeled chambers separated by 2mm and 20 μm Nylon mesh15N concentration difference, thereby quantitatively revealing the influence of the endophytic fungi on the mineral element absorption of a host;
(3) greatly improve the isotope13C-15N labeling efficiency:
the invention adopts the integrated marking device, compared with the traditional marking method (single isotope marking among different trees), the operation is simple and quick, and the controllability of the marking gas, elements and temperature and humidity in the device is strong, thus the method for simultaneously marking in the integrated device inevitably and greatly improves the marking efficiency and the marking accuracy.
Drawings
FIG. 1 shows an isotope of the present invention13C-15Schematic diagram of N-labeled endophytic fungi and host plant reciprocal symbiotic device.
1-N labeling Chamber NⅠ2-N labeling Chamber NⅡ3-N labeling Chamber NⅢ4-N labeling Chamber NⅣ5-N labeling Chamber NⅠ′6-N labeling Chamber NⅡ′7-N labeling Chamber NⅢ′8-N labeling Chamber NⅣ′9-GFP fungus chamber I, 10-GFP fungus chamber II, 11-plant growth chamber, 12-box body, 13-20 micrometer nylon net, 14-2 mm nylon net, 15-waste liquor discharge pipe with rubber plug, 16-reaction cup, 17-plant to be marked, 18-gas sensor, 19-fluorescent lamp, 20-three-way valve burette with scale, 21-box door with handle, 22-hook, 23-hygrothermograph, 24-cooling pipe, 25-air pump, 26-cold water generator, 27-wide-mouth bottle with plug for containing alkaline solution, 28-K15NO3、29—(15NH4)2S0430-plant fine roots, 31-hypha of fungi carrying GFP labels, 32-plant growth chamber: (13C label chamber).
Detailed Description
The present invention is described below with reference to the following embodiments and the accompanying drawings, but the present invention is not limited thereto, and any modification, equivalent replacement, and improvement made within the spirit and principle of the present invention should be included in the scope of the present invention.
The experimental procedures used in the following examples are all conventional procedures unless otherwise specified.
Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.
The strain Dialocephala fortinii is a walnut root system endophytic fungus resource which is separated from a walnut root system in a laboratory and is preserved for a long time. The plasmid pCT74 has the GFP gene and the hygromycin resistance gene and the ampicillin resistance gene. pCT74 plasmid: wuhan vast Ling Biotech, Inc.
200ml of PDB culture medium is added into a 500ml triangular flask, and 5ml of Dialocephala fortini spore liquid (2X 10)5cfu/ml), cultured at 200rpm at 28 ℃ for 36 h. Filtering with sterile filter paper, collecting mycelium, washing with sterile 0.7M NaCl solution for 3-4 times, transferring into sterile 50ml centrifuge tube, adding lyase solution, and performing enzymolysis at 28 deg.C and 200rpm for 2 h. Filtering the zymolyte with sterile filter paper in 0.7M NaCl solution (5 times/2 min), centrifuging the collected filtrate at 4 deg.C and 4000rpm for 15min, discarding the supernatant, adding 5ml STC solution, gently shaking, centrifuging at 4 deg.C and 4000rpm for 15min, and collecting the white precipitate as protoplast (1 × 10)6cfu/ml)。
Mixing the above prepared protoplast (5ml) and pCT74 plasmid (50 μ L), adding into 10ml centrifuge tube, gently shaking for 1-2 times, ice-cooling for 15min, adding 2ml PEG4000 solution (60%), gently shaking for 1-2 times, ice-cooling for 15min, adding 5ml PDB culture solution, mixing, culturing at 28 deg.C for 12-24h, collecting 100ml culture solution, coating on the surface containing coating containing 8 mg.L-1A transformant was obtained by culturing on a hygromycin (Hyg) resistant PDA plate at 28 ℃ for 7-10 days in the dark, inoculated into a PDA medium for 3-5 days, and subcultured 5 times in succession, and then examined by using an LSM 510 laser confocal microscope (Carl Zeiss, Oberkochen, Germany) for the presence of a fluorescent tag in the transformant.
FIG. 1 shows an isotope13C-15Schematic diagram of N-labeled Dialocephala fortini and host plant reciprocal symbiotic device.
As shown in fig. 1, the integrated apparatus of the present invention includes: a box body made of organic glass and comprising an N-mark chamber, a GFP fungus chamber and a plant growth chamber (13A C marking chamber) for placing the plant to be marked, the Phialocephalafitinii fungus with GFP label, the isotope to be marked and the marking equipment; n marking chamber for placing the object to be marked15N isotope and mineral nutrients required by plant growth; a GFP fungus chamber for culturing a Phialocephala fortinii fungus carrying a GFP tag; a plant growth chamber for placing the plant to be marked and sealing13C-CO2Isotope labeled gas and labeled material; 2mm and 20 mu m nylon nets for separating the N marking chamber, the GFP fungus chamber and the plant growing chamber, so as to be convenient for quantitatively researching the symbiotic relationship between plants and microorganisms;13c gas reaction generating means for releasing13C-CO2Isotope labeled gas, which comprises a waste liquid discharge pipe with a rubber plug and is used for discharging reaction waste liquid; a reaction cup arranged in the plant growth chamber for accommodating13C-Na2CO3A solution; a three-way burette with scales, which is arranged in the plant growth chamber and is filled with HCl solution through a three-way valve at the top of the box body13C-Na2CO3Solution reaction formation13C-CO2Gas is carried out13C, marking; a wide-mouth bottle with a rubber plug for containing alkaline solution (strong alkaline solution such as sodium hydroxide or potassium hydroxide or calcium hydroxide, the concentration is 2mol/L) before and after absorption and marking and in the process of controlling marking13C-CO2Concentration; the fluorescent lamp is used for supplementing light; the hygrothermograph is used for monitoring the temperature and humidity change in the plant growth chamber in real time; gas sensor for real-time monitoring of CO in a plant growth chamber2And O2A change in concentration; and the temperature control assembly is used for adjusting the temperature and the humidity of the plant growing chamber.
Specifically, the integrated device of the present invention is shown in fig. 1, the box body is made of organic glass, the length x width x height of the box body is 40 x 50cm, the box body is strictly sealed and airtight, and the box body is divided by an organic glass plate and nylon nets with the length x width x height of the box body and the height x 40 x 50cm to form 8N-marked chambers, 2 GFP fungus chambers and 1 plant growth chamber. The organic glass plate on the top of the box body only seals the plant growth chamber, the N marking chamber and the GThe FP fungus room is open or air-permeable and airtight, thus being convenient for watering and applying nutrient solution. The N-mark chamber on the left side of the box body is divided into N parts by an organic glass plateⅠ、NⅡ、NⅢAnd NⅣThe N marking chamber on the right side of the box body is divided into N by an organic glass plateⅠ′、NⅡ′、NⅢ′And NⅣ′The size of the 8 marking chambers uniformly divided was 10X 50cm (length X width X height, the same applies hereinafter), NⅠ、NⅡSeparated from GFP fungus chamber I using a 20 μm nylon mesh (0-15cm height) and plexiglass plates (15-50 cm); n is a radical ofⅠ′、NⅡ′Separated from GFP fungus chamber I by 2mm nylon net (0-15cm) and plexiglass plate (15-50 cm); n is a radical ofⅢ、NⅣIs separated from GFP fungus room II by 2mm nylon net (0-15cm) and plexiglass plate (15-50 cm); n is a radical ofⅢ′、NⅣ′Separated from GFP fungus chamber II by a 20 μm nylon mesh (0-15cm height) and plexiglass plate (15-50 cm); the GFP fungus chamber I and the plant growth chamber are separated by a 20-micron nylon net (0-15cm) and an organic glass plate (15-50 cm); GFP fungus room II and plant growth room are separated by 2mm nylon net (0-15cm) and plexiglass plate (15-50cm), the size of GFP fungus room I and GFP fungus room II is 20X 10X 50cm, and the size of plant growth room is 20X 50 cm. The right side of the top of the plant growth chamber is provided with a box door 21 with a handle, the box door can be opened and closed at any time, the box door is sealed by a rubber gasket, and air leakage cannot occur during closing. Moist (60 + -5% of saturated water content) sterile substrate (fine sand: vermiculite: 1, v/v) continuously sterilized (121 ℃, 15min)3 times was loaded into N-mark chamber, GFP fungus chamber and plant growth chamber, the substrate thickness was 20-25cm (the specific thickness was determined depending on the seedling size). Placed at the left side of plant growth chamber 1113C-CO2 A reaction cup 16, a waste liquid discharge pipe 15 with a rubber plug is arranged at the bottom of the reaction cup 16 and passes through NⅢThe reaction waste liquid is discharged through the box wall to the outside of the box, and is connected into a three-way burette 20 with scales through the top of the box and arranged above the reaction cup 16; the top of the three-way valve burette with the scale is controlled by a three-way valve, and the joint of the three-way valve and the top of the box is sealed by waterproof glue. A wide-mouth bottle 27 with a plug for containing alkaline solution is arranged at the right side of the plant growth chamber, and a rubber plug of the wide-mouth bottle is usedThe hook is connected with the top of the box and can move up and down to open or close the jar,13c mark front lifting hook to open wide-mouth bottle to fully absorb CO in plant growth chamber2Gas, after the mark begins, the hook moves down to plug the wide-mouth bottle, so as to prevent the wide-mouth bottle from being plugged13C-CO2The gas is absorbed by alkaline solution to influence the marking efficiency, and after the marking is finished, the hook is lifted up to open the wide-mouth bottle to fully absorb the residual gas in the plant growth chamber13C-CO2Gas, preventing surplus13C-CO2The gas pollutes the environment. The plant growth chamber is suspended with a gas sensor 18 and a thermo-hygrometer 23, sealed with a waterproof glue at the top of the box, for real-time detection of the gas Concentration (CO) in the plant growth chamber2And O2) And temperature and humidity variations. A60-watt fluorescent lamp 19 is connected into the plant growing room, a power line penetrates through the top of the box to be connected with a power switch outside the box, light is supplemented, and an isotope labeling test can be normally carried out in rainy days. A cooling pipe 24 is arranged at the right side of the plant growth chamber and is connected with a rubber conduit through NⅢ′And the air pump 25 and the cold water generator 26 which are arranged outside the box body are connected through the right box wall and are used for adjusting the temperature and humidity change of the plant growth chamber. The penetration parts of the box wall and all the guide pipes are sealed by waterproof glue to ensure the strict sealing of the box body and improve the isotope13C-CO2Marking efficiency.
The isotope provided by the invention13C-15The N-labeled endophytic fungus and host plant reciprocal symbiosis method includes the following steps:
(1) the integrated device is installed as described above and placed in a greenhouse or natural environment. In this embodiment, isotope labeling research is performed on the symbiotic relationship between the walnut root system Phialocephala fortini fungus and the host thereof, and the isotope labeling research can also be used for symbiotic relationship research between root microorganisms and the host of other economic forest trees or perennial woody plants or mineral element transportation and distribution relationship research.
Preparing a substrate: 5-10kg of a substrate (fine sand: vermiculite: 1, volume ratio) which was continuously sterilized at a high temperature (121 ℃, 20min) for 3 times was weighed and charged into a clean plant growth chamber (length × width × height ═ 20 × 20 × 50cm), an N-mark chamber (length × width × height ═ 10 × 40 × 50cm) and a GFP fungus chamber (length × width × height ═ 20 × 10 × 50cm), the thickness of the substrate was 20-25cm, and the thickness of the substrate exceeded the thickness of the nylon mesh barrier layer by 5-10cm, so that the above 3 compartments were relatively sealed and airtight.
In the research, the walnut seedlings of 20-30 days are transplanted to a plant growth room after main roots (convenient for lateral roots/fibrous roots to grow) are cut off, the Dialocephala fortini fungi carrying GFP labels are cultured to a GFP fungus room after the transplanted seedlings survive, and the GFP fungus room is placed in a greenhouse (day/night: 25 +/-2/18 +/-2 ℃, illumination is 12 hours, 400 +/-50 mu mol m-2.s-1(ii) a Relative humidity: 65 +/-5 percent of the total nutrient solution is cultured (the box door with a handle at the top of the plant growth chamber is opened before isotope labeling), sterile water is poured into the plant growth chamber, the N labeling chamber and the fungus chamber with the label after transplanting to ensure that the water content of the substrate is 60 +/-5 percent, 1/2Long-Ashton medium nutrient solution is poured into the N labeling chamber every 7 days after the seedlings survive, and 1/2Long-Ashton medium nutrient solution is replaced by N-deficient 1/2Long-Ashton medium nutrient solution at 21 days (the N element starvation treatment is carried out for 7 days to improve the isotope labeling)15N marking efficiency), keeping other elements unchanged, and pouring for 7d once, pouring nutrient solution until the isotope marking test is finished, detecting whether the walnut seedling root system is infected by Dialochela fortini fungi carrying GFP labels or not after 30d, using the successfully infected seedlings for the isotope marking test, and simultaneously using an organic glass cover at the top of a waterproof glue sealing device.
In particular, to isotopes15When marking N, before marking, carrying out N starvation treatment on walnut seedlings for 1 week, normally irrigating by using improved 1/2Long-Ashton medium nutrient solution lacking N, and after one week, placing 4 walnut seedlings on the left side of the box body15N labeling Chamber (N)Ⅰ,NⅡ,NⅢ,NⅣ) Adding equal amounts of isotopes15N(K15NO3，15Abundance of N10.2%), 4 on the right side of the tank15N labeling Chamber (N)Ⅰ′,NⅡ′,NⅢ′,NⅣ′) Adding equal amounts of isotopes15N((15NH4)2S04，15Abundance of N10.2%) begin to label, eight N labels isotopes in the chamber15The N concentration is 1mMWherein N is separated by a nylon mesh with a pore diameter of 20 mu mⅠ、NⅡ、NⅢ′And NⅣ′The marking chamber only has N separated by a nylon net with 2mm aperture and allowing fungal hypha to pass throughⅢ、NⅣ、NⅠ′And NⅡ′The marker chamber may pass through the fine roots of the plant and fungal hyphae, with a 2mm nylon mesh being chosen because the endophytic fungi predominantly infest the root line below this pore size (about 1-2 mm). And after the marking is started, the walnut seedlings are normally irrigated with 1/2Long-Ashton medium nutrient solution lacking N, other elements are unchanged, the irrigation time is still 7d, and the nutrient solution is irrigated until the isotope marking test is finished. After continuously marking for 7-10 days, carrying out isotope labeling15And (4) detecting the abundance of the N,15the N abundance is measured by an isotope mass spectrometer in nature15The N abundance was about 0.336%, and was calculated as: nitrate nitrogen (or ammonium nitrogen) (% of measurement sample)15N abundance (%) -Nature15N abundance (%)]10.2 percent. Wherein the endophytic fungi assist the host in uptake15NO3 -Is an amount of NⅠAnd NⅡAmount of spent marker compartment, uptake by host root system15NO3 -Is an amount of NⅢAnd NⅣTotal amount of marker chamber consumed minus NⅠAnd NⅡThe total amount of the marking chamber consumed,15NH4 +the same is true.
(2) Reacting hydrochloric acid with gas by gas reaction generator13C-sodium carbonate solution reaction release13C-CO2Gas, carrying out isotope production13C mark, in the process of injecting hydrochloric acid solution, slowly dripping hydrochloric acid until the reaction is too violent in order to prevent the reaction from being too violent until13C-CO2Stopping the injection when the gas concentration is 0.03%, and facilitating the observation in the reaction cup13C-consumption of sodium carbonate solution, before adding hydrochloric acid, dripping 2-3 drops of phenolphthalein indicator, when the solution in the reaction bottle turns colorless, indicating that13The C-sodium carbonate solution is exhausted, the reaction waste liquid is emptied through a waste liquid discharge pipe 15, and the reaction liquid is added. In the marking process, the humiture in the plant growth chamber is observed in real time through a hygrothermograph, and when the temperature exceeds 30 ℃, the temperature is adjusted by using a temperature control assemblyThe temperature is in a range suitable for plants; real-time observation of plant growth chamber by gas sensor 1813C-CO2And O2Gas concentration, thereby increasing isotope concentration13C labeling efficiency.
In particular, to isotopes13C, in the case of marking, 09 a.m.: 00 to 17 pm: 00 and before marking, using alkaline solution in the wide-mouth bottle 27 to fully absorb CO in the plant growth chamber2Gas, then performing carbon starvation treatment on the walnut seedlings to be marked, namely fully absorbing CO in the plant growth chamber through a wide-mouth bottle 27 (strong alkaline solution such as sodium hydroxide or potassium hydroxide or calcium hydroxide with the concentration of 2mol/L) containing alkaline solution2Gas, reducing CO in the plant growth chamber2Gas, to place the plant at carbon starvation level and reduce the original CO2Gas pair13C-CO2Influence of gas, thereby increasing isotope content13C labeling efficiency. Before marking, by13C gas reaction generating device13C-Na2CO3Reaction of the solution with HCl solution to form13C-CO2Gas is carried out13And C, marking. During the marking, observing in the plant growth chamber by means of a gas sensor13C-CO2(0.03%) and O2(21%) concentration of13C-CO2The gas concentration is lower than 0.03 percent, and the reaction is improved by adding hydrochloric acid solution dropwise in time13C-CO2Gas concentration if O2The concentration exceeds 21 percent, which indicates that the photosynthesis capability of the plant is stronger; in addition, the walnut root system is a temperature sensitive plant, and the life activity process of the plant root system stops when the soil temperature exceeds 30 ℃, so that the device is provided with a hygrothermograph 23 and a temperature control assembly (a cooling pipe 24, an air pump 25 and a cold water generator 26), the humiture change in the plant growth chamber is observed through the hygrothermograph (the temperature does not exceed 30 ℃ during marking and the humidity is about 60 percent), if the temperature exceeds 30 ℃, the temperature control assembly is opened in time to regulate and control the temperature of the plant growth chamber, and meanwhile, the cooling pipe in the temperature control assembly can regulate the humidity in the plant growth chamber; after the continuous labeling for 7-10 days, the plant growth chamber, the N-labeled chamber and the GFP fungus chamber were analyzed for the medium by an isotope mass spectrometerIsotopes of matter13C abundance, and isotopes of plant material (roots, stems, leaves) and GFP-tagged Phyacocephala fortinii fungi13C abundance, after the end of marking, using alkaline solution in the wide-mouth bottle to fully absorb the C in the plant growth chamber13C-CO2Gas, preventing surplus13C-CO2The gas contaminates the air.
The time for infecting walnut roots by the Phialocephala fortini fungus selected in the research needs about 30 days, and the isotope is13C-15The marking is finished after the N marking lasts for 7-10 days, and the marking time is determined according to the time for infecting the host by the target fungi and the time for absorbing, transporting and assimilating mineral elements by the host for other plants.
The above is only a preferred example of the present invention, and is not intended to limit the present invention, and the skilled worker can modify the present invention according to the actual needs.
Claims (10)
1. Isotope element13C-15An N-labeled endophytic fungi and host plant reciprocal symbiotic device comprising:
the box body is internally provided with an N marking chamber, a middle part and an N marking chamber from left to right in sequence; the middle part is sequentially provided with a fungus chamber I with a mark, a plant growth chamber and a fungus chamber II with a mark from front to back; the two N marking chambers are respectively and uniformly divided into 4N marking chambers, namely NⅠ,NⅡ,NⅢ,NⅣ，NⅠ′,NⅡ′,NⅢ′,NⅣ′；
The isolation layer is arranged on the contact surfaces of the plant growth chamber and the N-marked chamber, the marked fungus chamber I, II and the marked fungus chamber I, II, and is used for separating the N-marked chamber, the marked fungus chamber and the plant growth chamber;
the isolation layer includes: an organic glass plate, a 2mm nylon net or a 20 μm nylon net; the organic glass plate is arranged on the isolation layer with the height of more than 15cm at the bottom of the box body, and the 2mm nylon net and the 20 mu m nylon net are arranged on the isolation layer with the height of less than 15cm at the bottom of the box body;
13c gas reaction generating device arranged in the plant growth chamber and used for generating13C-CO2Gas is carried out13C, marking; the above-mentioned13The C gas reaction generating device comprises: the waste liquid discharge pipe with the rubber plug is used for discharging reaction waste liquid;13C-CO2reaction cup for containing13C-Na2CO3A solution; a three-way burette with scales arranged on13C-CO2Above the reaction cup 16, a three-way valve in the graduated three-way valve burette is arranged at the top of the box body and is arranged in the direction of the three-way valve13C-CO2Injecting HCl solution into the reaction cup13C-Na2CO3Solution reaction formation13C-CO2Gas is carried out13C, marking;
a gas sensor disposed in the plant growth chamber for monitoring13CO before and after C-labelling and during labelling2And O2Concentration;
the fluorescent lamp is arranged in the plant growing chamber;
a wide-mouth bottle component with a rubber plug, which is arranged in the plant growing chamber and is used for absorbing13CO before and after C labelling2A gas; the wide-mouth bottle component with the rubber plug comprises: a wide-mouth bottle with a rubber plug for containing alkaline solution to fully absorb CO in the plant growth chamber2A gas; the hook is arranged at the top of the plant growing chamber and used for fixing the rubber plug in the rubber plug wide-mouth bottle;
the hygrothermograph is arranged in the plant growing chamber and is used for monitoring the temperature and the humidity in the plant growing chamber;
the temperature control assembly is communicated with the interior of the box body and is used for adjusting the temperature and the humidity in the plant growing chamber; the temperature control assembly includes: the cooling pipe is arranged in the plant growth chamber and used for adjusting the temperature and the humidity of the plant growth chamber; the cold water generator is arranged outside the box body and used for refrigerating and controlling the temperature; and the air pump is arranged on the outer side of the box body, is connected with the cooling pipe and the cold water generator through a rubber hose and is used for pumping the temperature-controlled water in the cold water generator into the cooling pipe.
2. The apparatus of claim 1, wherein: the box body is made of organic glass, the organic glass plate on the top of the box body only seals the plant growth chamber, and the N mark chamber, the fungus chamber I with the mark and the fungus chamber II with the mark are open or air-permeable and airtight;
a box door with a handle is arranged on the right side of the top of the plant growing chamber; the door with the handle is sealed by a rubber gasket, so that air leakage cannot occur when the door is closed.
3. The apparatus of claim 1 or 2, wherein: the above-mentioned13C gas reaction generating device, the13C-CO2The reaction cup is arranged at the left side of the plant growth chamber, the reaction cup13C-CO2A waste liquid discharge pipe with a rubber plug is arranged at the bottom of the reaction cup and passes through NⅡPenetrating through the wall of the box body to the outside of the box body;
the box wall of the box body and the penetrating positions of all the guide pipes are sealed by waterproof glue so as to ensure that the box body is strictly sealed.
4. The apparatus of any one of claims 1-3, wherein: the length, the width and the height of the box body are 40, 40 and 50 cm; the size of the N marking chamber is 10 x 40cm, and N isⅠ、NⅡ、NⅢ、NⅣ，NⅠ′、NⅡ′、NⅢ′、NⅣ′The sizes of the marking chambers are all 10 multiplied by 10 cm; the size of the marked fungus chamber I and the marked fungus chamber II is 20 multiplied by 10 multiplied by 50cm, and the size of the plant growth chamber is 20 multiplied by 50 cm.
5. Investigation of isotopes using the device of any of claims 1 to 413C-15The N-labeled endophytic fungus and host plant reciprocal symbiosis method includes the following steps:
(1) preparing a substrate: weighing the substrate which is continuously sterilized for 3 times at high temperature, and putting the substrate into a clean plant growth chamber, an N mark chamber and a fungus chamber with marks; the thickness of the substrate of each chamber is 20-25cm, and the thickness of the substrate exceeds the height of the nylon mesh isolation layer by 5-10cm, so that each compartment is relatively sealed and airtight;
(2) transplanting seedlings to be marked: selecting seedling seedlings with the seedling age of 20d to 30d, cutting off main roots, transplanting the seedling seedlings into plant growth chambers, wherein each plant growth chamber is provided with one seedling, placing the device in a solar energy greenhouse or a natural environment according to any one of claims 1 to 4, irrigating sterile water in the plant growth chambers, the N marking chamber and the marked fungus chamber after transplanting to ensure that the water content of a substrate is 60 +/-5%, irrigating 1/2Long-Ashton medium nutrient solution in the N marking chamber every 7d after the seedling survives, replacing 1/2Long-Ashton medium nutrient solution with N-deficient 1/2Long-Ashton medium nutrient solution at 21d, keeping other elements unchanged, and irrigating for 7d until the isotope labeling test is finished; nutrient solution is not poured into the plant growth chamber and the marked fungus chamber to promote the root system or hyphae to absorb nutrients through the nylon net;
(3) establishing a symbiotic system: the marked endophytic fungi are equivalently inoculated in a sterile marked endophytic fungi chamber I and a sterile marked endophytic fungi chamber II which are separated by a nylon net with the diameter of 20 mu m and a nylon net with the diameter of 2mm, whether the seedling root system has the marked endophytic fungi or not is detected after a plurality of days, the colonization part and the infection rate of the marked endophytic fungi in the plant root system are observed, and after the root system is successfully colonized, the isotope is started13C-15Marking by N;
(4) isotope of carbon monoxide15Marking by N: in that15Before N marking, the seedlings to be marked are subjected to N starvation treatment for 1 week, the seedlings are normally irrigated by improved 1/2Long-Ashton medium nutrient solution lacking N, and 4N marking chambers N on the left side of the box body are used for one week laterⅠ,NⅡ,NⅢ,NⅣAdding the same amount of isotope15N, 4 nitrogen marker chambers N on the right side of the box bodyⅠ′,NⅡ′,NⅢ′,NⅣ′Adding the same amount of isotope15N-start labeling, isotopes in N-label chamber15The concentration of N is 1mM, 1/2 Long-Ashtonmedia nutrient solution lacking N is normally irrigated after marking is started, other elements are unchanged, the irrigation time is 7d, isotope is continuously labeled for 7-10d15Detecting N abundance, and sampling with sterileAdjusting the water content of the substrate to 60 +/-5 percent by water,15measuring the N abundance by adopting an isotope mass spectrometer, and then calculating the percentage content of nitrate nitrogen (or ammonium nitrogen) in the marked plants; the calculation formula is as follows:
nitrate nitrogen (or ammonium nitrogen) (% of measurement sample)15N abundance (%) -Nature15N abundance (%)]10.2% of the natural world15The N abundance is about 0.336%; wherein the endophytic fungi assist the host in uptake15NO3 -Is an amount of NⅠAnd NⅡAmount of spent marker compartment, uptake by host root system15NO3 -Is an amount of NⅢAnd NⅣTotal amount of marker chamber consumed minus NⅠAnd NⅡTotal amount of marker chamber consumed; endophytic fungi help host to absorb15NH4Is an amount of NⅢ′And NⅣ′Amount of spent marker compartment, uptake by host root system15NH4Is an amount of NⅠ′And NⅡ′Total amount of marker chamber consumed minus NⅢ′And NⅣ′Total amount of marker chamber consumed;
(5) isotope of carbon monoxide13C, marking: time of day was marked from 09 am: 00 to 17 pm: 00 end, sufficiently absorbing CO in the plant growth chamber by using alkaline solution in a wide-mouth bottle before marking2Gas and carbon starving treatment of the nursery stock to be marked for one day so as to increase isotope13C labeling efficiency; before marking, by13C gas reaction generating device13C-sodium carbonate solution reacts with hydrochloric acid to generate13C-CO2Gas is carried out13C mark, during which the observation in the plant growth chamber is carried out by means of a gas sensor13C-CO2And O2Concentration; observing temperature and humidity change in the plant growth chamber by a hygrothermograph, continuously marking for 7-10 days, and analyzing isotope of the substrate in the plant growth chamber, the N marking chamber and the marked fungus chamber by using an isotope mass spectrometer13Abundance of C, and isotopes of plant material and labeled fungi13C abundance, after each marking, using alkaline solution in the wide-mouth bottle to fully absorb the C in the plant growth chamber13C-CO2Gas, prevent air pollution.
6. The method of claim 5, wherein: in the step 1), the high-temperature continuous sterilization condition is sterilization at 121 ℃ for 20 min; the matrix is prepared from fine sand and vermiculite according to the volume ratio of 1:1 in a certain proportion.
7. The method according to claim 5 or 6, characterized in that: in the step 2), the greenhouse conditions are as follows: day/night: 25 +/-2/18 +/-2 ℃; the illumination is 12h, 400 +/-50 mu mol-2.s-1(ii) a Relative humidity: 65 plus or minus 5 percent.
8. The method according to any one of claims 5-7, wherein:
in the step 2), the seedlings are walnut seedlings, the endophytic fungi are Phialocephala fortini fungi, and whether the seedling roots have the endophytic fungi carrying the markers is detected after 30-40 days in the step 3);
in the step 4), the N marks a chamber NⅠ,NⅡ,NⅢ,NⅣIsotope of (1)15N is represented by K15NO3The method comprises the steps of providing a material,15the abundance of N is 10.2%; the N labeling chamber NⅠ′,NⅡ′,NⅢ′,NⅣ′Isotope of (1)15N is represented by15NH4)2S04The method comprises the steps of providing a material,15the abundance of N was 10.2%.
9. The method according to any one of claims 5-8, wherein: in said step 5), in the plant growth chamber during marking13C-CO2Is maintained at a concentration of 0.03% and O2The concentration is 21%;
if it is13C-CO2The gas concentration is lower than 0.03 percent, and the reaction is improved by adding hydrochloric acid solution dropwise in time13C-CO2The gas concentration;
in the step 5), the temperature in the plant growth chamber during marking is not more than 30 ℃, and the humidity is about 60%; when the temperature exceeds 30 ℃, the temperature is adjusted to the proper temperature range of the plants by using the temperature control component.
The plant body includes plant roots, stems and leaves.
10. The method according to any one of claims 5-9, wherein: the endophytic fungi with the label are endophytic fungi with a fluorescent label;
preferably, the endophyte with the label is an endophyte with a fluorescent protein label;
the endophytic fungi with the fluorescent protein marker is the endophytic fungi which introduces a gene coding the marker protein into the endophytic fungi so as to express the marker protein; the marker protein is a protein that can be observed or observed.
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