Estimation of the contribution of ectomycorrhizal fungi to nitrogen acquisition in Abies minjiangensis and Betula sclerophylla

Abstract

The present application discloses a method for estimating the contribution of ectomycorrhizal fungi to nitrogen acquisition by Abies minjiangensis and Betula sclerosa, which relates to the technical field of estimating the contribution of plants to nitrogen acquisition. The method comprises collecting leaf, stem and root samples of ectomycorrhizal trees in a target area and soil samples directly below the ectomycorrhizal trees; measuring the leaf, stem and root samples to determine the nitrogen stable isotope values ​​and nitrogen contents of the leaf, stem and root samples; measuring the soil sample to determine the nitrogen stable isotope value of the total dissolved nitrogen of the soil sample; and calculating the contribution rate of ectomycorrhizal fungi to nitrogen acquisition by ectomycorrhizal trees using an isotope mass balance model and an isotope mixing effect model based on the nitrogen stable isotope values ​​and nitrogen contents of the leaf, stem and root samples and the nitrogen stable isotope value of the total dissolved nitrogen of the soil sample. The present application can improve the accuracy of estimating the contribution of ectomycorrhizal fungi to nitrogen acquisition by Abies minjiangensis and Betula sclerosa.

Description

Technical Field

[0001] The present application relates to the technical field of estimating the contribution of plants to nitrogen acquisition, and in particular to a method for estimating the contribution of ectomycorrhizal fungi to nitrogen acquisition of Abies minjiangensis and Betula sclerotiorum. Background Art

[0002] Nitrogen availability is an important factor in determining forest productivity and regulating forest community structure. Nitrogen availability is declining in many unfertilized forest ecosystems worldwide, that is, the nitrogen supply is declining relative to the nitrogen demand of plants. In order to meet the nitrogen demand of plants, in addition to absorbing inorganic nitrogen sources such as ammonium nitrogen (NH4 + ) and nitrate nitrogen (NO3 - ), and also absorb soluble organic nitrogen (DON) in the soil. In addition, plants also rely on mycorrhizal fungi for nitrogen acquisition, especially ectomycorrhizal fungi, which play an important role in plant nitrogen nutrition. The ectomycorrhizal plants distributed in the eastern edge of the Qinghai-Tibet Plateau are mainly Minjiang Abies and rough-barked Betula. The nitrogen of the above ectomycorrhizal plants mainly comes from the direct absorption of soil NH by the plants through fine roots (i.e., the root pathway). 4+ 、NO 3- and DON, and soil nitrogen is obtained through the mycelium of ectomycorrhizal fungi (i.e., mycelial pathway) and then transferred to fine roots. However, in natural ecosystems, ectomycorrhizal plants such as Abies chinensis and Betula rugosa absorb soil NH through the root pathway. 4+ 、NO 3- Estimates of the relative contributions of nitrogen uptake by Abies minjiangensis and Betula ostreae to DON and nitrogen acquired through the mycelial pathway and transferred to fine roots are highly uncertain. Elucidating the underlying mechanisms by which plants maintain efficient nitrogen uptake is crucial for developing optimized management strategies in forest ecosystems dominated by Abies minjiangensis and Betula ostreae.

[0003] Currently, existing methods for estimating plant nitrogen acquisition contributions are primarily based on laboratory incubation experiments combined with isotope labeling. However, due to the complexity of natural environmental conditions, laboratory incubation experiments cannot fully represent the nitrogen acquisition processes of Abies chinensis and Betula serrata trees growing under natural conditions, resulting in inaccurate estimates of nitrogen acquisition contributions. Furthermore, isotope labeling involves artificial nitrogen addition, which inevitably interferes with soil nitrogen pools, microbial turnover, and plant nitrogen uptake, further inaccurately estimating nitrogen acquisition contributions. Summary of the Invention

[0004] The purpose of this application is to provide a method for estimating the contribution of ectomycorrhizal fungi to nitrogen acquisition of Abies minjiangensis and Betula ostreae, which can improve the accuracy of estimating the contribution of ectomycorrhizal fungi to nitrogen acquisition of Abies minjiangensis and Betula ostreae.

[0005] To achieve the above objectives, this application provides the following solutions.

[0006] A method for estimating the contribution of ectomycorrhizal fungi to nitrogen acquisition of Abies minjiangensis and Betula sclerotiorum includes the following steps.

[0007] Leaf, stem and root samples of ectomycorrhizal trees in the target area and soil samples directly below the ectomycorrhizal trees were collected; the ectomycorrhizal trees were Abies minjiangensis or Betula rough-barked.

[0008] The leaf, stem and root samples are measured to determine the nitrogen stable isotope values ​​and nitrogen contents of the leaf, stem and root samples.

[0009] The soil sample is measured to determine the nitrogen stable isotope value of the total dissolved nitrogen in the soil sample.

[0010] Based on the nitrogen stable isotope values ​​and nitrogen contents of the leaf, stem and root samples and the nitrogen stable isotope values ​​of the total dissolved nitrogen of the soil sample, the contribution rate of ectomycorrhizal fungi to nitrogen acquisition of ectomycorrhizal trees was calculated using the isotope mass balance model and the isotope mixing effect model.

[0011] Optionally, collecting leaf, stem and root samples of ectomycorrhizal trees in the target area and soil samples directly below the ectomycorrhizal trees specifically includes the following steps.

[0012] Measure the diameter at breast height, tree height, and rooting depth of ectomycorrhizal trees in the target area.

[0013] According to the diameter at breast height and the tree height of the ectomycorrhizal tree, leaves, stems and roots of the ectomycorrhizal tree are collected to obtain leaf, stem and root samples.

[0014] According to the rooting depth of the ectomycorrhizal tree, soil at a corresponding depth directly below the ectomycorrhizal tree is collected to obtain the soil sample.

[0015] Optionally, measuring the leaf, stem and root samples to determine the nitrogen stable isotope values ​​and nitrogen contents of the leaf, stem and root samples specifically includes the following steps.

[0016] The leaf, stem and root samples are dried respectively to obtain dried leaf, stem and root samples.

[0017] The dried leaf, stem and root samples were ground into powder using a ball mill to obtain ground leaf, stem and root samples.

[0018] The nitrogen contents of the ground leaf, stem and root samples were measured using an elemental analyzer, respectively, as the nitrogen contents of the leaf, stem and root samples.

[0019] The nitrogen stable isotope values ​​of the ground leaf, stem and root samples were measured respectively by an isotope ratio mass spectrometer as the nitrogen stable isotope values ​​of the leaf, stem and root samples.

[0020] Optionally, measuring the soil sample to determine the nitrogen stable isotope value of the total dissolved nitrogen in the soil sample specifically includes the following steps.

[0021] The soil sample and potassium chloride solution are mixed in a preset ratio to obtain a soil extract.

[0022] The total dissolved nitrogen concentration in the soil extract was measured using a fully automatic chemical analyzer.

[0023] According to the total dissolved nitrogen concentration in the soil extract, the nitrogen stable isotope value of the total dissolved nitrogen in the soil sample is measured by an isotope ratio mass spectrometer and a gas analyzer using a denitrifying bacteria method.

[0024] Optionally, based on the nitrogen stable isotope values ​​and nitrogen contents of the leaf, stem and root samples and the nitrogen stable isotope values ​​of the total dissolved nitrogen of the soil sample, an isotope mass balance model and an isotope mixing effect model are used to calculate the contribution rate of ectomycorrhizal fungi to the nitrogen acquisition of ectomycorrhizal trees, which specifically includes the following steps.

[0025] Based on the leaf, stem and root samples, the biomass of the leaves, stems and roots in the leaf, stem and root samples were calculated respectively using the allometric equation.

[0026] The nitrogen content of the whole ectomycorrhizal tree was calculated based on the biomass of the leaves, stems and roots in the leaf, stem and root samples and the nitrogen content of the leaf, stem and root samples using an isotope mass balance model.

[0027] The nitrogen stable isotope value of the whole ectomycorrhizal tree is calculated using an isotope mass balance model based on the nitrogen content of the whole ectomycorrhizal tree, the biomass of the leaves, stems and roots in the leaf, stem and root samples, and the nitrogen stable isotope value and nitrogen content of the leaf, stem and root samples.

[0028] According to the nitrogen stable isotope value of the whole ectomycorrhizal tree and the nitrogen stable isotope value of the total dissolved nitrogen in the soil sample, the contribution rate of ectomycorrhizal fungi to the nitrogen acquisition of the ectomycorrhizal tree was calculated using the isotope mixing effect model and the fractionation coefficient model.

[0029] Alternatively, the nitrogen content of the whole ectomycorrhizal tree was calculated using the following formula.

[0030] N plant =N leaf ×F leaf +N stem ×Fstem +N root ×F root ;

[0031] Among them, N plant Indicates the nitrogen content of the whole ectomycorrhizal tree, N leaf 、N stem and N root represents the nitrogen concentration in leaves, stems and roots of ectomycorrhizal trees; F leaf 、F stem and F root Represent the percentage of biomass of leaves, stems and roots in the whole plant of ectomycorrhizal trees, respectively.

[0032] Alternatively, the nitrogen stable isotope value of the whole ectomycorrhizal tree was calculated using the following formula.

[0033] δ 15 N plant =δ 15 N leaf × (N leaf ×F leaf / N plant )+δ 15 N stem × (N stem ×F stem / N plant )+δ 15 N root × (N root ×F root / N plant );

[0034] Among them, δ 15 N plant represents the nitrogen stable isotope value of the whole ectomycorrhizal tree, δ 15 N leaf represents the nitrogen stable isotope values ​​of leaves of ectomycorrhizal trees, δ 15 N stem represents the nitrogen stable isotope value of the stems of ectomycorrhizal trees, δ 15 N root The nitrogen stable isotope values ​​of the roots of ectomycorrhizal trees, N plant Indicates the nitrogen content of the whole ectomycorrhizal tree, N leaf 、N stem and N root represents the nitrogen concentration in leaves, stems and roots of ectomycorrhizal trees; F leaf 、F stem and F root Represent the percentage of biomass of leaves, stems and roots in the whole plant of ectomycorrhizal trees, respectively.

[0035] Optionally, the expression of the isotope mixing effect model is as follows.

[0036] δ 15 N plant =δ 15 N transfer × f fungi +δ 15 N available N × (1- f fungi );

[0037] Among them, δ 15 N plant represents the nitrogen stable isotope value of the whole ectomycorrhizal tree, δ 15 N transfer represents the nitrogen stable isotope value corresponding to the nitrogen transferred from ectomycorrhizal fungi to ectomycorrhizal trees; δ 15 N available N represents the nitrogen stable isotope value corresponding to the nitrogen available to ectomycorrhizal trees in the soil sample; f fungi represents the contribution rate of ectomycorrhizal fungi to nitrogen acquisition of ectomycorrhizal trees, 1- f fungi It represents the contribution of nitrogen in soil samples absorbed by ectomycorrhizal trees through the root pathway to the nitrogen acquisition of ectomycorrhizal trees.

[0038] Optionally, the expression of the fractionation coefficient model is as follows.

[0039] Δ f =(δ 15 N TDN -δ 15 N transfer ) / (1+δ 15 N transfer );

[0040] Among them, Δ f This indicates that mycelium is involved in the transfer of nitrogenous compounds to ectomycorrhizal trees. 15 The fractionation coefficient corresponding to the N fractionation effect, δ 15 N TDN represents the nitrogen stable isotope value of the total dissolved nitrogen in the soil sample, δ 15 N transfer Nitrogen stable isotope values ​​representing nitrogen transferred from ectomycorrhizal fungi to ectomycorrhizal trees.

[0041] Alternatively, the following formula is used to calculate the contribution of ectomycorrhizal fungi to nitrogen acquisition by ectomycorrhizal trees.

[0042] f fungi =[(δ15 N plant -δ 15 N TDN )×(Δ f +1)] / [-(δ 15 N TDN +1)×Δ f ];

[0043] in, f fungi represents the contribution rate of ectomycorrhizal fungi to nitrogen acquisition of ectomycorrhizal trees, δ 15 N TDN represents the nitrogen stable isotope value of the total dissolved nitrogen in the soil sample, δ 15 N plant represents the nitrogen stable isotope value of the whole ectomycorrhizal tree, Δ f This indicates that mycelium is involved in the transfer of nitrogenous compounds to ectomycorrhizal trees. 15 N is the fractionation coefficient corresponding to the fractionation effect.

[0044] According to the specific embodiments provided in this application, this application has the following technical effects.

[0045] The present application provides a method for estimating the contribution of ectomycorrhizal fungi to nitrogen acquisition of Abies minjiangensis and Betula serrata. From the perspective of ectomycorrhizal fungi, the application aims to explore the contribution of ectomycorrhizal fungi to the nitrogen acquisition process of Abies minjiangensis and Betula serrata. Leaf, stem and root samples and soil samples of ectomycorrhizal trees are collected in the target area, and the nitrogen stable isotope values ​​and nitrogen content of the leaf, stem and root samples and the nitrogen stable isotope values ​​of the total dissolved nitrogen of the soil samples are measured. Based on the above data, the isotope mass balance model and the isotope mixing effect model are used to calculate the contribution rate of ectomycorrhizal fungi to nitrogen acquisition of Abies minjiangensis and Betula serrata. This application collects real leaf, stem and root samples and soil samples without artificial nitrogen addition, and truly characterizes the nitrogen acquisition process of Abies chinensis and Betula serrata growing under natural environmental conditions, and uses the isotope mass balance model and the isotope mixing effect model for calculation. Since the isotope mass balance model and the isotope mixing effect model are derived from the nitrogen isotope natural abundance method, they can make full use of the characteristics of the nitrogen isotope natural abundance method that does not interfere with natural environmental conditions and can reflect the relative contributions of the root pathway and the mycelium pathway to the nitrogen acquisition of Abies chinensis and Betula serrata, and thus can accurately calculate the contribution rate of ectomycorrhizal fungi to the nitrogen acquisition of Abies chinensis and Betula serrata. BRIEF DESCRIPTION OF THE DRAWINGS

[0046] In order to more clearly illustrate the embodiments of the present application or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments. Obviously, the drawings described below are only some embodiments of the present application. For ordinary technicians in this field, other drawings can be obtained based on these drawings without creative work.

[0047] Figure 1 This is a flow chart of a method for estimating the contribution of ectomycorrhizal fungi to nitrogen acquisition by Abies minjiangensis and Betula ostreae, provided in one embodiment of the present application. DETAILED DESCRIPTION

[0048] The following will be combined with the drawings in the embodiments of this application to clearly and completely describe the technical solutions in the embodiments of this application. Obviously, the embodiments described are only part of the embodiments of this application, not all of the embodiments. Based on the embodiments in this application, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of this application.

[0049] Currently, existing methods for estimating plant nitrogen acquisition contributions are primarily based on laboratory incubation experiments combined with isotope labeling. However, due to the complexity of natural environmental conditions, laboratory incubation experiments cannot fully represent the nitrogen acquisition processes of trees growing under natural conditions. Furthermore, while isotope labeling techniques can track and quantify the relative contributions of ectomycorrhizal fungi to nitrogen acquisition in Abies chinensis and Betula ostreae, this anthropogenic nitrogen addition inevitably interferes with soil nitrogen pools, microbial turnover, and plant nitrogen uptake, thus hindering our understanding of tree nitrogen acquisition strategies in natural ecosystems. Furthermore, nitrogen isotope natural abundance has been applied to gain deeper insights into tree nitrogen acquisition strategies. Compared to other measurement methods, a major advantage of nitrogen isotope natural abundance is that it does not interfere with natural environmental conditions. Current experimental studies and theoretical models generally consider the root system and mycorrhizal fungal mycelium as a whole, lacking further differentiation between root and mycelial pathways in nitrogen acquisition in Abies chinensis and Betula ostreae. The nitrogen isotope natural abundance method can reflect the relative contributions of the root pathway and mycelium pathway to nitrogen acquisition by Abies minjiangensis and Betula serrata in natural ecosystems.

[0050] The purpose of this application is to provide a method for estimating the contribution of ectomycorrhizal fungi to nitrogen acquisition of Abies minjiangensis and Betula serrata, and to utilize the above advantages of the nitrogen isotope natural abundance method to improve the accuracy of the estimation of the contribution of ectomycorrhizal fungi to nitrogen acquisition of Abies minjiangensis and Betula serrata, so as to solve the problem of low accuracy of nitrogen acquisition contribution estimation results caused by the existing methods based on laboratory cultivation experiments and isotope labeling due to the inability to characterize the nitrogen acquisition process of trees growing under natural environmental conditions and the artificial nitrogen addition.

[0051] In order to make the above-mentioned purposes, features and advantages of the present application more obvious and easy to understand, the present application is further described in detail below with reference to the accompanying drawings and specific implementation methods.

[0052] like Figure 1 As shown, this embodiment proposes a method for estimating the contribution of ectomycorrhizal fungi to nitrogen acquisition of Abies minjiangensis and Betula ostreatus, which specifically includes the following steps.

[0053] Step S1: collecting leaf, stem and root samples of ectomycorrhizal trees in a target area and soil samples directly below the ectomycorrhizal trees; wherein the ectomycorrhizal trees are Abies minjiangensis or Betula serrata.

[0054] In this embodiment, step S1 collects leaf, stem and root samples of ectomycorrhizal trees in the target area and soil samples directly below the ectomycorrhizal trees, which specifically includes the following steps.

[0055] Step S11: measuring basic data such as the diameter at breast height, tree height, and rooting depth of ectomycorrhizal trees in the target area.

[0056] Step S12: collecting leaves, stems and roots of the ectomycorrhizal trees according to the diameter at breast height and the tree height of the ectomycorrhizal trees to obtain leaf, stem and root samples.

[0057] Step S13: According to the rooting depth of the ectomycorrhizal tree, soil at a corresponding depth directly below the ectomycorrhizal tree is collected to obtain the soil sample.

[0058] This embodiment adopts a field monitoring approach, with the eastern edge of the Qinghai-Tibet Plateau as the target area, and collects leaf, stem, and root samples and soil samples from ectomycorrhizal trees at various growth stages on site. Based on these samples, the contribution of ectomycorrhizal fungi to nitrogen acquisition by ectomycorrhizal trees is calculated. Compared with the laboratory culture test method, this embodiment can completely and truly characterize the nitrogen acquisition process of ectomycorrhizal trees growing under natural environmental conditions, thereby ensuring the accuracy and reliability of the calculation results.

[0059] Step S2: measuring the leaf, stem and root samples to determine the nitrogen stable isotopes (δ 15 N) value and nitrogen content.

[0060] In this embodiment, step S2 measures the leaf, stem and root samples to determine the nitrogen stable isotope values ​​and nitrogen contents of the leaf, stem and root samples, which specifically includes the following steps.

[0061] Step S21: Dry the leaf, stem and root samples respectively to obtain dried leaf, stem and root samples.

[0062] Step S22: using a ball mill to grind the dried leaf, stem and root samples into powder, respectively, to obtain ground leaf, stem and root samples.

[0063] Step S23: Using an elemental analyzer, respectively measure the nitrogen content in the ground leaf, stem, and root samples as the nitrogen content of the leaf, stem, and root samples.

[0064] Step S24: using an isotope ratio mass spectrometer to measure the nitrogen stable isotope values ​​of the ground leaf, stem and root samples, respectively, as the nitrogen stable isotope values ​​of the leaf, stem and root samples.

[0065] Step S3: measuring the soil sample to determine the nitrogen stable isotope value of the total dissolved nitrogen (TDN) of the soil sample.

[0066] In this embodiment, step S3 measures the soil sample to determine the nitrogen stable isotope value of the total dissolved nitrogen in the soil sample, which specifically includes the following steps.

[0067] Step S31: Mix the soil sample with potassium chloride (KCl) solution in a preset ratio to obtain a soil extract.

[0068] Step S32: Using a fully automatic chemical analyzer, measure the total dissolved nitrogen concentration in the soil extract.

[0069] Step S33: Based on the total dissolved nitrogen concentration in the soil extract, an isotope ratio mass spectrometer and a gas analyzer are used to measure the nitrogen stable isotope value of the total dissolved nitrogen in the soil sample using a denitrifying bacteria method.

[0070] Step S4: Based on the nitrogen stable isotope values ​​and nitrogen contents of the leaf, stem and root samples and the nitrogen stable isotope value of the total dissolved nitrogen of the soil sample, the contribution rate of ectomycorrhizal fungi to nitrogen acquisition of ectomycorrhizal trees is calculated using an isotope mass balance model and an isotope mixing effect model.

[0071] In this embodiment, step S4 calculates the contribution rate of ectomycorrhizal fungi to nitrogen acquisition of ectomycorrhizal trees based on the nitrogen stable isotope values ​​and nitrogen contents of the leaf, stem and root samples and the nitrogen stable isotope value of the total dissolved nitrogen of the soil sample using the isotope mass balance model and the isotope mixing effect model, which specifically includes the following steps.

[0072] Step S41: Based on the leaf, stem and root samples, the biomass of the leaves, stems and roots in the leaf, stem and root samples are calculated using the Allometric Growth Equation.

[0073] Step S42: Calculate the nitrogen content of the entire ectomycorrhizal tree using an isotope mass balance model based on the biomass of the leaves, stems and roots in the leaf, stem and root samples and the nitrogen content of the leaf, stem and root samples.

[0074] Step S43: Calculate the nitrogen stable isotope value of the whole ectomycorrhizal tree using an isotope mass balance model based on the nitrogen content of the whole ectomycorrhizal tree, the biomass of the leaves, stems, and roots in the leaf, stem, and root samples, and the nitrogen stable isotope value and nitrogen content of the leaf, stem, and root samples.

[0075] Step S44: Based on the nitrogen stable isotope value of the whole ectomycorrhizal tree and the nitrogen stable isotope value of the total dissolved nitrogen in the soil sample, an isotope mixing effect model and a fractionation coefficient model are used to consider the occurrence of nitrogen-containing compounds transferred from mycelium to the ectomycorrhizal tree during the generation of nitrogen-containing compounds. 15 N fractionation occurs during the transfer of nitrogenous compounds from the mycelium to the ectomycorrhizal trees. 15 The fractionation coefficient corresponding to the N fractionation effect is used, and the nitrogen stable isotope value of the total dissolved nitrogen in the soil sample is used as the nitrogen stable isotope value corresponding to the nitrogen available to the ectomycorrhizal trees in the soil sample, and the contribution rate of ectomycorrhizal fungi to the nitrogen acquisition of ectomycorrhizal trees is calculated.

[0076] In order to make the technical solution of this embodiment clearer, the specific implementation process of the technical solution of this embodiment is described in detail below in the form of examples, including the following contents.

[0077] (1) Collect and measure leaf, stem, root and soil samples.

[0078] In this example, leaf, stem, and root samples, as well as soil samples, were collected and measured using field monitoring. The diameter at breast height (DBH) and height of ectomycorrhizal trees were measured. Leaves, stems, and roots of the ectomycorrhizal trees were collected for determination of nitrogen stable isotopes and nitrogen content. For coniferous trees, biennial leaves were collected. Soil samples were collected at a depth corresponding to the rooting depth of the ectomycorrhizal trees. Each soil sample was sieved through a 2 mm sieve, and any visible living plant material and rocks were manually removed from the sieved soil samples.

[0079] The leaves, stems, and roots of the collected ectomycorrhizal trees were then dried in an oven to constant weight (70°C, 72 h) and then ground into powder using a MM200 ball mill (Retsch GmbH, Haan, Germany). The δ 15 N and nitrogen content. δ 15 The calculation formula of N is formula (1).

[0080] δ 15 N=[(R sample / R standard )-1]×1000‰ (1).

[0081] Among them, δ 15 N represents a stable isotope of nitrogen, R sample Indicates leaf, stem, and root samples 15 N / 14 The molar ratio of N, R standard Indicates the standard substance (N2) 15 N / 14 The molar ratio of N.

[0082] In this example, 10 g of fresh soil sample without any treatment was extracted with 2 M potassium chloride solution at a soil:potassium chloride solution ratio of 1:5. Before use, potassium chloride was heated to 450°C and maintained for 48 hours to reduce nitrogen blank. The mixture was shaken on a shaker for 1 hour, the extract was filtered and stored frozen at −20°C to obtain a soil extract. The total dissolved nitrogen concentration in the soil extract was then measured using an automatic chemical analyzer (Smartchem300, AMS, Italy). Subsequently, the total dissolved nitrogen in the soil extract was converted into nitrate (NO3 - ), NO3 - δ 15 N value (δ 15 N NO3- ) is measured using the denitrifying bacteria method, which uses denitrifying bacteria that lack N2O reductase to convert NO3 - or NO2 - Converted into nitrous oxide (N2O). The δ 15Nitrogen was determined by an isotope ratio mass spectrometer (IsoPrime 100; IsoPrime Limited, Stockport, UK) coupled with a gas analyzer (TraceGas, IsoPrime Limited).

[0083] (2) The contribution of ectomycorrhizal fungi to nitrogen acquisition by ectomycorrhizal trees was calculated using the nitrogen isotope natural abundance method based on the isotope mass balance model, isotope mixing effect model and fractionation coefficient model.

[0084] This example first uses the allometric growth equation to calculate the biomass percentage of leaves, stems, and roots of ectomycorrhizal trees in the whole plant, including F in formula (2) leaf 、F stem and F root The allometric equation is a mathematical model that describes the relationship between the size or mass of different parts of an organism. Its form is Y=aX b , where Y and X represent the mass, length, or other measurements of two different parts of an organism, respectively; a is the proportionality coefficient, representing the value of Y when X = 1; and b is the allometric exponent, reflecting the rate and pattern of change of Y with X. For woody plants without specific allometric equations, the allometric equations of the same genus or phylogenetically related species, or the mixed species equation, can also be used to obtain the percentage of leaf, stem, and root biomass in the entire plant for the ectomycorrhizal trees.

[0085] In this embodiment, after calculating the biomass percentages of leaves, stems, and roots of the ectomycorrhizal tree in the whole plant through the allometric growth equation, the isotope mass balance model is used. The isotope mass balance model is a mathematical model based on the isotope mass balance equation. The isotope mass balance equation is used to calculate the nitrogen content of the whole ectomycorrhizal tree. The calculation formula is formula (2).

[0086] N plant =N leaf ×F leaf +N stem ×F stem +N root ×F root (2).

[0087] Among them, N plant Indicates the nitrogen content of the whole ectomycorrhizal tree, N leaf 、N stem and N root represents the nitrogen concentration in leaves, stems and roots of ectomycorrhizal trees; F leaf 、F stem and F root Represent the percentage of biomass of leaves, stems and roots in the whole plant of ectomycorrhizal trees, respectively.

[0088] Then, this example uses the isotope mass balance equation to calculate the nitrogen stable isotope value of the entire ectomycorrhizal tree, which is expressed as formula (3).

[0089] δ 15 N plant =δ 15 N leaf × (N leaf ×F leaf / N plant )+δ 15 N stem × (N stem ×F stem / N plant )+δ 15 N root × (N root ×F root / N plant ) (3).

[0090] Among them, δ 15 N plant represents the nitrogen stable isotope value of the whole ectomycorrhizal tree, δ 15 N leaf represents the nitrogen stable isotope values ​​of leaves of ectomycorrhizal trees, δ 15 N stem represents the nitrogen stable isotope value of the stems of ectomycorrhizal trees, δ 15 N root The nitrogen stable isotope values ​​of the roots of ectomycorrhizal trees, N plant Indicates the nitrogen content of the whole ectomycorrhizal tree, N leaf 、N stem and N root represents the nitrogen concentration in leaves, stems and roots of ectomycorrhizal trees; F leaf 、F stem and F root Respectively represent the biomass percentage of leaves, stems and roots of ectomycorrhizal trees in the whole plant. leaf +F stem +F root =1.

[0091] This example considers that the nitrogen in the fine roots of ectomycorrhizal trees comes from direct absorption of soil nitrogen by the trees through their fine roots, or from soil nitrogen that is transferred to the fine roots by the mycelium of ectomycorrhizal fungi. Based on this, this example uses the isotope mixing effect model, a mathematical model based on differences in stable isotope ratios that quantifies the contribution of different sources to a mixture. The isotope mixing effect model is based on the mass conservation law of stable isotope ratios and is expressed as Equation (4).

[0092] δ M = f 1×δ1+ f 2×δ2+ f n ×δ n (4).

[0093] Among them, δ M is the stable isotope value of the mixture; δ i is the stable isotope value of the i-th source, f i is the contribution ratio of the i-th source, i=1, 2,…, n; δ n is the stable isotope value of the nth source; f n is the contribution ratio of the nth source.

[0094] In this embodiment, the isotope mixing effect model is expressed as formula (5).

[0095] δ 15 N plant =δ 15 N transfer × f fungi +δ 15 N available N × (1- f fungi ) (5).

[0096] Among them, δ 15 N plant represents the nitrogen stable isotope value of the whole ectomycorrhizal tree, δ 15 N transfer represents the nitrogen stable isotope value corresponding to the nitrogen transferred from ectomycorrhizal fungi to ectomycorrhizal trees; δ 15 N available N represents the nitrogen stable isotope value corresponding to the nitrogen available to ectomycorrhizal trees in the soil sample; f fungi represents the contribution rate of ectomycorrhizal fungi to nitrogen acquisition of ectomycorrhizal trees, 1- f fungi It represents the contribution of nitrogen in soil samples absorbed by ectomycorrhizal trees through the root pathway to the nitrogen acquisition of ectomycorrhizal trees.

[0097] Plants have been shown to absorb and utilize a variety of dissolved organic nitrogen forms, ranging from simple low-molecular-weight compounds (e.g., amino acids, oligopeptides, nucleotides, and urea) to more complex polymers (e.g., proteins). The soil organic nitrogen sources that can directly affect plant nitrogen nutrition or be directly absorbed and utilized by plants are primarily composed of the soluble fraction of soil organic nitrogen nutrients. Therefore, in this example, the nitrogen stable isotope value of the total dissolved nitrogen in the soil sample is used as the nitrogen stable isotope value corresponding to the nitrogen available to the ectomycorrhizal trees in the soil sample, that is, δ 15 N TDN As δ 15 N available N , substituted into the isotope mixing effect model in formula (5).

[0098] In this embodiment, the fractionation coefficient model is a model that takes into account the mycelium's production and transfer of nitrogenous compounds to trees. 15 The mathematical model of N fractionation uses Δ f This indicates that mycelium is producing nitrogenous compounds that are transferred to the tree. 15 The fractionation coefficient corresponding to the N fractionation effect, the expression of the fractionation coefficient model is formula (6).

[0099] Δ f =(δ 15 N TDN -δ 15 N transfer ) / (1+δ 15 N transfer ) (6).

[0100] Among them, Δ f This indicates that mycelium is involved in the transfer of nitrogenous compounds to ectomycorrhizal trees. 15 The fractionation coefficient corresponding to the N fractionation effect, δ 15 N TDN represents the nitrogen stable isotope value of the total dissolved nitrogen in the soil sample, δ 15 N transfer Nitrogen stable isotope values ​​representing nitrogen transferred from ectomycorrhizal fungi to ectomycorrhizal trees.

[0101] In this embodiment, formula (5) and formula (6) are combined to obtain formula (7). It can be seen that according to the nitrogen stable isotope value δ of the total dissolved nitrogen in the soil sample, 15 N TDN , nitrogen stable isotope values ​​of whole ectomycorrhizal trees δ 15 N plant and mycelium during the production and transfer of nitrogenous compounds to ectomycorrhizal trees 15 Fractionation coefficient Δ corresponding to N fractionation f, substituted into formula (7), the contribution rate of ectomycorrhizal fungi to nitrogen acquisition of ectomycorrhizal trees can be calculated.

[0102] f fungi =[(δ 15 N plant -δ 15 N TDN )×(Δ f +1)] / [-(δ 15 N TDN +1)×Δ f ] (7).

[0103] in, f fungi represents the contribution rate of ectomycorrhizal fungi to nitrogen acquisition of ectomycorrhizal trees, δ 15 N TDN represents the nitrogen stable isotope value of the total dissolved nitrogen in the soil sample, δ 15 N plant represents the nitrogen stable isotope value of the whole ectomycorrhizal tree, Δ f This indicates that mycelium is involved in the transfer of nitrogenous compounds to ectomycorrhizal trees. 15 The fractionation coefficient corresponding to the N fractionation effect is, in this embodiment, Δ f The value is 9‰.

[0104] This example uses data from field studies and laboratory culture studies on ectomycorrhizal trees to derive Δ in formulas (6) and (7). f The value of is 8~10‰, in this embodiment Δ f The value is 9‰. It is worth noting that Δ f The value of was developed from multiple well-studied high-latitude tundra and forest ecosystems where plant productivity is primarily limited by nitrogen and may not be applicable to the high nitrogen conditions typical of many tropical forests. The target area of ​​this example is located in the eastern Qinghai-Tibet Plateau, which is characterized by limited nitrogen mineralization and low soil nitrogen availability. Therefore, 9‰ was used as the Δ f The contribution rate of ectomycorrhizal fungi to nitrogen acquisition of ectomycorrhizal trees can be calculated by combining formula (5) and formula (6) to obtain formula (7). f fungi This is the final result of the method for estimating the contribution of ectomycorrhizal fungi to nitrogen acquisition of Abies minjiangensis and Betula sclerotiorum in this example, and 1- f fungi It is the contribution rate of nitrogen in soil samples absorbed by ectomycorrhizal trees through roots to the nitrogen acquisition of ectomycorrhizal trees, thus completing the complete process of estimating the contribution of ectomycorrhizal fungi to nitrogen acquisition of Abies minjiangensis and Betula roughy.

[0105] This example conducted an experiment on Abies chinensis to calculate the contribution of ectomycorrhizal fungi to nitrogen acquisition in the plant and explore the feasibility of this technical solution. Fifteen Abies chinensis trees were selected for the experiment, numbered 1 to 15. Root, stem, leaf, and soil data were measured for each tree. The measured data for the Abies chinensis trees are shown in Table 1.

[0106] Table 1 Measurement data of Abies chinensis

[0107]

[0108] This example uses the allometric growth equation of Abies chinensis from the Forest Biomass Model Manual, combined with formulas (1) to (7) of this example, Δ f The value was taken as 9‰ to calculate the contribution rate of ectomycorrhizal fungi to nitrogen acquisition of Abies minjiangensis. The final experimental results of Abies minjiangensis are shown in Table 2.

[0109] Table 2 Experimental results of Abies minjiangensis

[0110]

[0111] It can be seen from Tables 1 and 2 that according to the technical solution of this embodiment, by collecting real leaf, stem and root samples and soil samples, the contribution rate of the ectomycorrhizal fungi corresponding to each Minjiang fir tree to the nitrogen acquisition of Minjiang fir can be accurately calculated, and an accurate and reliable contribution rate value can be obtained.

[0112] This example conducted an experiment on Betula serrata to calculate the contribution of ectomycorrhizal fungi to nitrogen acquisition in Betula serrata and to explore the feasibility of this technical solution. This example selected 15 Betula serrata trees for the experiment, numbered 16 to 30 (following the experimental numbering used for Abies minjiangensis). Root, stem, leaf, and soil data were measured for each Betula serrata tree. The measured data for Betula serrata are shown in Table 3.

[0113] Table 3 Measurement data of Betula serrata

[0114]

[0115] This example uses the allometric growth equation of Betula serrata from the Forest Biomass Model Manual, combined with formulas (1) to (7) of this example, Δ f The value of 9‰ was taken to calculate the contribution rate of ectomycorrhizal fungi to nitrogen acquisition of Betula serrata. The final experimental results of Betula serrata are shown in Table 4.

[0116] Table 4 Experimental results of Betula serrata

[0117]

[0118] As can be seen from Tables 3 and 4, according to the technical solution of this embodiment, by collecting real leaf, stem and root samples and soil samples, the contribution rate of the ectomycorrhizal fungi corresponding to each Betula serrata plant to the nitrogen acquisition of Betula serrata can be accurately calculated, and an accurate and reliable contribution rate value can be obtained.

[0119] This example starts from the perspective of ectomycorrhizal fungi and aims to explore the contribution of ectomycorrhizal fungi to the nitrogen acquisition process of Abies minjiangensis and Betula serrata. The ectomycorrhizal trees are Abies minjiangensis or Betula serrata. Leaf, stem and root samples and soil samples of ectomycorrhizal trees are collected in the target area, and the nitrogen stable isotope values ​​and nitrogen content of the leaf, stem and root samples and the nitrogen stable isotope value of the total dissolved nitrogen of the soil samples are measured. Based on the above data, the isotope mass balance model and the isotope mixing effect model are used to calculate the contribution rate of ectomycorrhizal fungi to the nitrogen acquisition of the ectomycorrhizal trees. By collecting real leaf, stem, root and soil samples, without artificial nitrogen addition, the nitrogen acquisition process of ectomycorrhizal trees growing under natural environmental conditions is truly characterized. The isotope mass balance model and isotope mixing effect model in the nitrogen isotope natural abundance method are used to fully utilize the characteristics of the nitrogen isotope natural abundance method, which does not interfere with natural environmental conditions and can reflect the relative contributions of root and mycelium pathways to the nitrogen acquisition of ectomycorrhizal trees. In turn, the contribution rate of ectomycorrhizal fungi to the nitrogen acquisition of ectomycorrhizal trees can be accurately calculated.

[0120] The technical features of the above embodiments can be combined arbitrarily. To make the description concise, not all possible combinations of the technical features in the above embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0121] This document uses specific examples to illustrate the principles and implementation methods of this application. The description of the above examples is only intended to help understand the method and core concept of this application. At the same time, for those skilled in the art, based on the concept of this application, there may be changes in the specific implementation methods and application scope. In summary, the content of this specification should not be understood as limiting this application.

Claims

1. A method for estimating the contribution of ectomycorrhizal fungi to nitrogen acquisition in Abies minjiangensis and Betula ostreatus, characterized in that: include: Collecting leaf, stem and root samples of ectomycorrhizal trees in the target area and soil samples directly below the ectomycorrhizal trees; wherein the ectomycorrhizal trees are Abies minjiangensis or Betula serrata; measuring the leaf, stem and root samples to determine the nitrogen stable isotope values ​​and nitrogen contents of the leaf, stem and root samples; measuring the soil sample to determine the nitrogen stable isotope value of the total dissolved nitrogen in the soil sample; Based on the nitrogen stable isotope values ​​and nitrogen contents of the leaf, stem and root samples and the nitrogen stable isotope value of the total dissolved nitrogen of the soil sample, the contribution rate of ectomycorrhizal fungi to nitrogen acquisition of ectomycorrhizal trees was calculated using an isotope mass balance model and an isotope mixing effect model, specifically including: Based on the leaf, stem and root samples, the biomass of the leaves, stems and roots in the leaf, stem and root samples are calculated using allometric growth equations; Calculating the nitrogen content of the entire ectomycorrhizal tree using an isotope mass balance model based on the biomass of the leaves, stems and roots in the leaf, stem and root samples and the nitrogen content of the leaf, stem and root samples; Calculating the nitrogen stable isotope value of the whole ectomycorrhizal tree using an isotope mass balance model based on the nitrogen content of the whole ectomycorrhizal tree, the biomass of the leaves, stems, and roots in the leaf, stem, and root samples, and the nitrogen stable isotope value and nitrogen content of the leaf, stem, and root samples; Based on the nitrogen stable isotope value of the whole ectomycorrhizal tree and the nitrogen stable isotope value of the total dissolved nitrogen in the soil sample, the contribution rate of ectomycorrhizal fungi to the nitrogen acquisition of the ectomycorrhizal tree is calculated using an isotope mixing effect model and a fractionation coefficient model; The nitrogen content of the whole ectomycorrhizal tree was calculated using the following formula: Among them, N plant Indicates the nitrogen content of the whole ectomycorrhizal tree, N leaf 、N stem and N root represents the nitrogen concentration in leaves, stems and roots of ectomycorrhizal trees; F leaf 、F stem and F root represent the percentage of biomass of leaves, stems, and roots in the whole plant of ectomycorrhizal trees, respectively; The nitrogen stable isotope value of the whole ectomycorrhizal tree was calculated using the following formula: Among them, δ 15 N plant represents the nitrogen stable isotope value of the whole ectomycorrhizal tree, δ 15 N leaf represents the nitrogen stable isotope values ​​of leaves of ectomycorrhizal trees, δ 15 N stem represents the nitrogen stable isotope value of the stems of ectomycorrhizal trees, δ 15 N root represents the nitrogen stable isotope values ​​of roots of ectomycorrhizal trees; The expression of the isotope mixing effect model is: Among them, δ 15 N transfer represents the nitrogen stable isotope value corresponding to the nitrogen transferred from ectomycorrhizal fungi to ectomycorrhizal trees; δ 15 N available N represents the nitrogen stable isotope value corresponding to the nitrogen available to ectomycorrhizal trees in the soil sample; f fungi represents the contribution rate of ectomycorrhizal fungi to nitrogen acquisition of ectomycorrhizal trees, 1- f fungi represents the contribution of nitrogen in soil samples absorbed by ectomycorrhizal trees through the root pathway to nitrogen acquisition by ectomycorrhizal trees; The expression of the fractionation coefficient model is: Among them, Δ f This indicates that mycelium is involved in the transfer of nitrogenous compounds to ectomycorrhizal trees. 15 The fractionation coefficient corresponding to the N fractionation effect, δ 15 N TDN represents the nitrogen stable isotope value of the total dissolved nitrogen in the soil sample; The contribution of ectomycorrhizal fungi to nitrogen acquisition of ectomycorrhizal trees was calculated using the following formula:

2. The method for estimating the contribution of ectomycorrhizal fungi to nitrogen acquisition in Abies minjiangensis and Betula ostreae according to claim 1, characterized in that: Collect leaf, stem, and root samples from ectomycorrhizal trees in the target area, as well as soil samples directly beneath the trees, including: Measure the diameter at breast height, tree height, and rooting depth of ectomycorrhizal trees in the target area; collecting leaves, stems and roots of the ectomycorrhizal trees according to the diameter at breast height and the tree height of the ectomycorrhizal trees to obtain leaf, stem and root samples; According to the rooting depth of the ectomycorrhizal tree, soil at a corresponding depth directly below the ectomycorrhizal tree is collected to obtain the soil sample.

3. The method for estimating the contribution of ectomycorrhizal fungi to nitrogen acquisition in Abies minjiangensis and Betula ostreae according to claim 1, characterized in that: Measuring the leaf, stem, and root samples to determine the nitrogen stable isotope values ​​and nitrogen contents of the leaf, stem, and root samples, specifically comprising: Drying the leaf, stem and root samples respectively to obtain dried leaf, stem and root samples; Grinding the dried leaf, stem and root samples into powder using a ball mill to obtain ground leaf, stem and root samples; Using an elemental analyzer, measuring the nitrogen content in the ground leaf, stem and root samples, respectively, as the nitrogen content of the leaf, stem and root samples; The nitrogen stable isotope values ​​of the ground leaf, stem and root samples were measured respectively by an isotope ratio mass spectrometer as the nitrogen stable isotope values ​​of the leaf, stem and root samples.

4. The method for estimating the contribution of ectomycorrhizal fungi to nitrogen acquisition in Abies minjiangensis and Betula ostreae according to claim 1, characterized in that: Measuring the soil sample to determine the nitrogen stable isotope value of the total dissolved nitrogen in the soil sample specifically includes: Mixing the soil sample with potassium chloride solution in a preset ratio to obtain a soil extract; Using a fully automatic chemical analyzer, measuring the total dissolved nitrogen concentration in the soil extract; According to the total dissolved nitrogen concentration in the soil extract, the nitrogen stable isotope value of the total dissolved nitrogen in the soil sample is measured by an isotope ratio mass spectrometer and a gas analyzer using a denitrifying bacteria method.

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