Method for evaluating grassland degradation based on microbial community structure and composition

Abstract

The invention provides a method for evaluating grassland degradation based on microbial community structures and composition, and belongs to the technical field of grassland degradation degree evaluation. The method comprises the following steps: selecting a representative sample plot in a to-be-evaluated grassland degradation area, selecting a sample square in the sample plot, and collecting rhizosphere soil in the sample square; mixing rhizosphere soil of all quadrat belonging to the sample plot to obtain a soil sample; analyzing the OTU number of bacteria and the OTU number of fungi in the soil sample by adopting high-throughput sequencing; calculating a grassland degradation grade evaluation value according to the OTU number of the bacteria and the OTU number of the fungi; according to the grassland degradation grade evaluation value and the grassland degradation grade evaluation standard, grassland degradation evaluation is carried out, and an evaluation result is obtained. The grassland degradation grade evaluation value of the to-be-evaluated area is calculated by taking the microflora structure and composition of the to-be-evaluated area as evaluation indexes, and the grassland degradation grade of the to-be-evaluated area is determined in combination with the grassland degradation grade evaluation standard.

Description

Technical Field

[0001] The present invention belongs to the technical field of grassland degradation degree assessment, and in particular relates to a method for assessing grassland degradation based on microbial community structure and composition. Background Art

[0002] Grasslands are a vital component of terrestrial ecosystems, serving crucial production and ecological functions. They contribute significantly to soil and water conservation, water conservation, climate regulation, and biodiversity protection.

[0003] Grassland degradation is a type of land degradation and a major manifestation of desertification. It not only threatens the balance of ecosystems but also poses a potential risk to regional and even global ecological security. Therefore, to protect grassland ecosystems, research on the restoration and management of degraded grasslands has become a hot topic.

[0004] The grassland degradation assessment index system includes evaluation methods using vegetation and soil as indicators and comprehensive evaluation methods using ecological indicators and auxiliary monitoring projects. However, vegetation and soil indicators are greatly affected by external interference. Summary of the Invention

[0005] The purpose of the present invention is to provide a method for evaluating grassland degradation based on the structure and composition of microbial communities. The method of the present invention is a method that is less affected by external interference.

[0006] The present invention provides a method for assessing grassland degradation based on microbial community structure and composition, comprising:

[0007] In the grassland degradation area to be assessed, a representative sample plot is selected, a quadrat is selected from the sample plot, and rhizosphere soil is collected from the quadrat; the rhizosphere soils of all quadrat belonging to the sample plot are mixed to obtain a soil sample;

[0008] High-throughput sequencing is used to analyze the number of bacterial OTUs and the number of fungal OTUs in the soil sample; and a grassland degradation grade assessment value is calculated based on the number of bacterial OTUs and the number of fungal OTUs;

[0009] Conducting grassland degradation assessment according to the grassland degradation grade assessment value and grassland degradation grade assessment standard to obtain an assessment result;

[0010] The representative sample plots cover different terrains, vegetation types and soil types in the grassland degradation area to be assessed;

[0011] The calculation formula of the grassland degradation grade assessment value is shown in Formula 1:

[0012] P = X / Y (Equation 1); in Equation 1, P represents the grassland degradation grade assessment value, X represents the number of bacterial OTUs in the soil sample, and Y represents the number of fungal OTUs in the soil sample;

[0013] The grassland degradation grade assessment standards include:

[0014] If P < 3, the grassland degradation level is non-degraded;

[0015] If 3≤P<4, the grassland degradation level is slightly degraded;

[0016] If 4≤P<5, the grassland degradation level is moderate;

[0017] If P>5, the grassland degradation level is severe degradation.

[0018] Preferably, the grassland degradation area to be assessed includes desert steppe or alpine meadow.

[0019] Preferably, the number of sample plots is 9 to 15; the number of sample plots in each sample plot is 3 to 5; the soil sample of each sample plot is collected by the five-point sampling method; the rhizosphere soil of all sample plots in the sample plot is mixed to obtain the soil sample of each sample plot; the number of bacterial OTUs and the number of fungal OTUs in the soil sample of each sample plot are analyzed by high-throughput sequencing; the grassland degradation grade assessment value of each sample plot is calculated based on the number of bacterial OTUs and the number of fungal OTUs; if the average level of degradation status of the entire grassland degradation area to be assessed is assessed, the median of the grassland degradation grade assessment value is taken; if the most serious area in the grassland degradation area to be assessed is assessed, the maximum value of the grassland degradation grade assessment value is taken.

[0020] Preferably, the distance between adjacent sample plots is 10 to 50 m.

[0021] Preferably, the soil sample is collected at a depth of 0 to 20 cm; and the soil includes rhizosphere soil.

[0022] Preferably, after collecting the rhizosphere soil sample, the method further includes removing surface litter from the soil sample, mixing all the soil samples to obtain a mixed soil sample, and drying the mixed soil sample to remove litter and root fragments.

[0023] Preferably, after removing fallen leaves and root fragments, the method further comprises sieving the soil to 2 mm.

[0024] Preferably, the method for determining the number of OTUs of the bacteria comprises 16S rRNA gene sequencing.

[0025] Preferably, the method for determining the number of OTUs of the fungus includes ITS sequencing; the primer pair used in the ITS sequencing includes an ITS1 region primer pair and an ITS2 region primer pair.

[0026] Preferably, after obtaining the evaluation results, it also includes using drone hyperspectral remote sensing to verify the evaluation results.

[0027] The present invention provides a method for assessing grassland degradation based on microbial community structure and composition, comprising: selecting a representative sample plot in a degraded grassland area to be assessed, selecting a quadrat within the plot, and collecting rhizosphere soil within the quadrat; mixing the rhizosphere soil from all quadrat samples within the plot to obtain a soil sample; analyzing the number of bacterial and fungal OTUs in the soil sample using high-throughput sequencing; calculating a grassland degradation grade based on the number of bacterial and fungal OTUs; and assessing grassland degradation based on the grassland degradation grade and grassland degradation grade criteria to obtain an assessment result. Soil microorganisms respond differently to grassland degradation. Compared with bacteria, fungi are more sensitive to degradation and their numbers decrease relatively sharply, while bacteria increase relatively. Grassland degradation leads to a shift from fungal-dominated to bacterial-dominated grassland. However, when external environmental factors cause the death of some microorganisms, some coexisting microorganisms with different taxonomic status can perform the same metabolic functions. Even if the number of microbial OTUs changes in the short term, the P value remains stable. The present invention uses the structure and composition of microbial communities in the area to be assessed as evaluation indicators, calculates a comprehensive assessment value of grassland degradation in the area to be assessed, and combines it with the grassland degradation grade assessment standard to determine the grassland degradation grade of the area to be assessed. DETAILED DESCRIPTION

[0028] The present invention provides a method for assessing grassland degradation based on microbial community structure and composition, comprising:

[0029] In the grassland degradation area to be assessed, a representative sample plot is selected, a quadrat is selected from the sample plot, and rhizosphere soil is collected from the quadrat; the rhizosphere soils of all quadrat belonging to the sample plot are mixed to obtain a soil sample;

[0030] High-throughput sequencing is used to analyze the number of bacterial OTUs and the number of fungal OTUs in the soil sample; and a grassland degradation grade assessment value is calculated based on the number of bacterial OTUs and the number of fungal OTUs;

[0031] Conducting grassland degradation assessment according to the grassland degradation grade assessment value and grassland degradation grade assessment standard to obtain an assessment result;

[0032] The representative sample plots cover different terrains, vegetation types and soil types in the grassland degradation area to be assessed;

[0033] The calculation formula of the grassland degradation grade assessment value is shown in Formula 1:

[0034] P = X / Y (Equation 1); in Equation 1, P represents the grassland degradation grade assessment value, X represents the number of bacterial OTUs in the soil sample, and Y represents the number of fungal OTUs in the soil sample;

[0035] The grassland degradation grade assessment standards include:

[0036] If P < 3, the grassland degradation level is non-degraded;

[0037] If 3≤P<4, the grassland degradation level is slightly degraded;

[0038] If 4≤P<5, the grassland degradation level is moderate;

[0039] If P>5, the grassland degradation level is severe degradation.

[0040] The present invention first selects a representative sample plot in a grassland degradation area to be assessed, selects a quadrat in the sample plot, collects rhizosphere soil in the quadrat; and mixes the rhizosphere soil of all quadrat in the sample plot to obtain a soil sample.

[0041] As an embodiment, the microbial community structure and composition include the taxonomic composition and quantitative distribution of bacteria and fungi.

[0042] The present invention selects representative sample plots, and the representative sample plots cover different terrains, vegetation types and soil types in the grassland degradation area to be assessed, so as to ensure the representativeness of the sample plots. Different grassland types have different degradation characteristics.

[0043] As an embodiment, the grassland degradation area to be assessed includes desert steppe or alpine meadow.

[0044] As an implementation mode, the number of the sample plots is 9 to 15; the number of quadrats in each sample plot is 3 to 5; and the soil sample of each quadrat is collected using a five-point sampling method.

[0045] As an embodiment, the present invention mixes the rhizosphere soil of all the quadrats belonging to the sample plot to obtain a soil sample of each sample plot; uses high-throughput sequencing to analyze the number of bacterial OTUs and the number of fungal OTUs in the soil sample of each sample plot; calculates the grassland degradation grade assessment value of each sample plot based on the number of bacterial OTUs and the number of fungal OTUs; if the average level of degradation of the entire grassland degradation area to be assessed is assessed (overall grading is performed), the median of the grassland degradation grade assessment value is taken; if the most serious area (degraded core area) in the grassland degradation area to be assessed is assessed, the maximum value of the grassland degradation grade assessment value is taken. In the present invention, the overall grading reflects the average level or intermediate level of grassland degradation in the entire study area from a macro perspective; the assessment of the degradation core area reflects the area with the most serious grassland degradation in the study area, which helps to take targeted control measures.

[0046] As an implementation method, the spacing between adjacent sample plots is 10 to 50 meters, and further 10, 20, and 50 meters, to avoid excessively dense sampling points leading to excessive correlation between samples, maintain relatively consistent environmental conditions within the sample plot, and ensure comparability of data within the sample plot.

[0047] As an embodiment, the soil sample is collected at a depth of 0 to 20 cm; the soil includes rhizosphere soil.

[0048] As an embodiment, the soil sample is collected using a soil drill with a diameter of 3.5 cm, which is simple to collect and highly operable.

[0049] In one embodiment, after collecting the rhizosphere soil sample, the method further includes removing surface litter from the soil sample, mixing all the soil samples to obtain a mixed soil sample, and drying the mixed soil sample to remove litter and root debris. In one embodiment, the drying includes air drying at room temperature.

[0050] As an embodiment, after removing litter and root debris, the soil is further sieved to 2 mm to standardize the particle size and prevent coarse particles from interfering with chemical extraction or instrumental analysis.

[0051] After obtaining the soil sample, the present invention uses high-throughput sequencing to analyze the number of bacterial OTUs and the number of fungal OTUs in the soil sample.

[0052] The present invention uses high-throughput sequencing to quickly obtain the structure and composition of microbial communities and quickly determine the degree of grassland degradation.

[0053] In one embodiment, the method for determining the number of bacterial OTUs includes 16S rRNA gene sequencing. In one embodiment, the 16S rRNA gene sequencing includes: adding a known copy number of spike-in synthetic sequence to sample DNA, then constructing and sequencing a 16S amplicon library of the sample, then plotting a standard curve based on the number of 16S amplicon sequences (reads) and their copy number of the spike-in, and calculating the 16S rRNA gene copy number of the species corresponding to the OTU representative sequence in the sample based on the number of OTU sequences (reads); adding the 16S spike-in to the sample template and performing high-throughput sequencing of the 16S rRNA gene amplicon to obtain species composition information in the sample.

[0054] As an embodiment, the method for determining the number of OTUs of the fungus includes ITS sequencing; the primer pairs used in the ITS sequencing include an ITS1 region primer pair and an ITS2 region primer pair; as an embodiment, the ITS sequencing includes: extracting DNA from a soil sample using a DNA extraction kit; using the DNA of the soil sample as a template, amplifying using ITS1 and ITS2 region primers, and sequencing the amplified products after agarose gel electrophoresis detection.

[0055] After obtaining the number of bacterial OTUs and the number of fungal OTUs, the present invention calculates the grassland degradation grade assessment value based on the number of bacterial OTUs and the number of fungal OTUs;

[0056] After calculating the grassland degradation grade assessment value, the present invention performs grassland degradation assessment according to the grassland degradation grade assessment value and grassland degradation grade assessment standards to obtain an assessment result.

[0057] The present invention assesses grassland degradation based on microbial composition and realizes dynamic monitoring. It can reduce ecological restoration investment through early warning, effectively reduce economic losses caused by grassland degradation, and improve grassland restoration efficiency.

[0058] As an implementation method, after obtaining the assessment results, it also includes using drone hyperspectral remote sensing to verify the assessment results, which helps to build a more comprehensive grassland degradation assessment system.

[0059] The present invention uses drone hyperspectral remote sensing to verify the assessment results and identify technical errors. If the verification results are inconsistent with the assessment results, the present invention rechecks the bacterial and fungal OTU counts (OTU clustering parameters), reanalyzes the original data, or resamples. Furthermore, due to the lag in responses between microorganisms and vegetation, if the verification results are inconsistent with the assessment results, the present invention can also retest indicators; these retest indicators include vegetation cover and / or soil organic matter. If the retested indicators meet the degradation level classification of the "Classification Indicators for Natural Grassland Degradation, Desertification, and Salinization" (GB 19377-2003), the national standard takes precedence.

[0060] To further illustrate the present invention, a method for assessing grassland degradation based on microbial community structure and composition provided by the present invention is described in detail below in conjunction with examples, but they should not be construed as limiting the scope of protection of the present invention.

[0061] Example 1

[0062] The area to be assessed is located in the Sanjiangyuan region in the heart of the Qinghai-Tibet Plateau. Surveys and statistics were conducted on various indicators of natural grassland soil and vegetation. Combined with the overall situation of the grassland in the region, the natural grasslands in the area to be assessed were divided into three types according to the "Classification Standard for Natural Grassland Degradation, Desertification and Salinization" (GB 19377-2003): non-degraded grassland, moderately degraded grassland, and severely degraded grassland.

[0063] Four 10m × 10m plots were selected in each degraded grassland type, for a total of 12 plots;

[0064] Three 1 × 1 m quadrats were randomly set up in each plot, and rhizosphere soils with a depth of 0–10 cm from the three quadrats were collected and mixed as one sample; the rhizosphere soils from the three quadrats in each plot were mixed;

[0065] The obtained samples were sieved to 2 mm, stored in liquid nitrogen containers, and then subjected to microbial sequencing to obtain the number of bacterial OTUs and the number of fungal OTUs;

[0066] The grassland degradation grade assessment value is calculated based on the bacterial OTU number and the fungal OTU number;

[0067] Conducting grassland degradation assessment according to the grassland degradation grade assessment value and grassland degradation grade assessment standard to obtain an assessment result;

[0068] The calculation formula of the grassland degradation grade assessment value is shown in Formula 1:

[0069] P = X / Y (Equation 1); in Equation 1, P represents the grassland degradation grade assessment value, X represents the number of bacterial OTUs in the soil sample, and Y represents the number of fungal OTUs in the soil sample;

[0070] The grassland degradation grade assessment standards include:

[0071] If P < 3, the grassland degradation level is non-degraded;

[0072] If 3≤P<4, the grassland degradation level is slightly degraded;

[0073] If 4≤P<5, the grassland degradation level is moderate;

[0074] If P>5, the grassland degradation level is severe degradation.

[0075] Among them, the ratio of the number of bacterial and fungal OTUs in the sampling quadrats of the non-degraded area was P = 2.77, that is, P < 3, which was consistent with the judgment of the non-degraded level of grassland.

[0076] Example 2

[0077] The area to be assessed is located in Luqu County, Gannan Tibetan Autonomous Prefecture, Gansu Province. Based on surveys and statistics of soil and vegetation indicators of natural grasslands and the overall grassland conditions in the area, the natural grasslands within the area were classified into three types: slightly degraded grassland, moderately degraded grassland, and severely degraded grassland, in accordance with the "Classification Standard for Natural Grassland Degradation, Desertification, and Salinization" (GB19377-2003).

[0078] Five 10m × 10m plots were selected in each degraded grassland type, and within each plot, a 1 × 1m quadrat was selected using the five-point method;

[0079] Rhizosphere soil from each plot with a depth of 0 to 20 cm was collected and mixed into one sample. The rhizosphere soil from each plot was mixed, sterile-sealed, and transported to the laboratory at 4°C for high-throughput sequencing to obtain the number of bacterial OTUs and fungal OTUs.

[0080] The grassland degradation grade assessment value is calculated based on the bacterial OTU number and the fungal OTU number;

[0081] Conducting grassland degradation assessment according to the grassland degradation grade assessment value and grassland degradation grade assessment standard to obtain an assessment result;

[0082] The calculation formula of the grassland degradation grade assessment value is shown in Formula 1:

[0083] P = X / Y (Equation 1); in Equation 1, P represents the grassland degradation grade assessment value, X represents the number of bacterial OTUs in the soil sample, and Y represents the number of fungal OTUs in the soil sample;

[0084] The grassland degradation grade assessment standards include:

[0085] If P < 3, the grassland degradation level is non-degraded;

[0086] If 3≤P<4, the grassland degradation level is slightly degraded;

[0087] If 4≤P<5, the grassland degradation level is moderate;

[0088] If P>5, the grassland degradation level is severe degradation.

[0089] Among them, the ratio of the number of bacterial and fungal OTUs in the sampling quadrats in the slightly degraded area was P = 3.86, that is, 3≤P<4, which is consistent with the judgment of the grassland being slightly degraded.

[0090] Example 3

[0091] The area to be assessed is located in Maqin County, Golog Tibetan Autonomous Prefecture, Qinghai Province. Surveys and statistics on soil and vegetation indicators of natural grasslands were conducted. Based on the overall grassland conditions in the area, the natural grasslands within the area were classified into three types: slightly degraded grassland, moderately degraded grassland, and severely degraded grassland, in accordance with the "Classification Standard for Degradation, Desertification, and Salinization of Natural Grasslands" (GB19377-2003).

[0092] Four 10m × 10m plots were randomly selected in each degraded grassland type, each plot contained four 1m × 1m quadrats, and the distance between adjacent quadrats was 50m;

[0093] The five-point sampling method was used in four quadrats in each plot, and the rhizosphere soil of each quadrats with a depth of 0 to 20 cm was mixed as one sample; the rhizosphere soil of the quadrats in each plot was mixed;

[0094] 10 g of soil was taken from each sample and stored in a −80°C refrigerator for DNA extraction and high-throughput sequencing to obtain the number of bacterial OTUs and the number of fungal OTUs;

[0095] The grassland degradation grade assessment value is calculated based on the bacterial OTU number and the fungal OTU number;

[0096] Conducting grassland degradation assessment according to the grassland degradation grade assessment value and grassland degradation grade assessment standard to obtain an assessment result;

[0097] The calculation formula of the grassland degradation grade assessment value is shown in Formula 1:

[0098] P = X / Y (Equation 1); in Equation 1, P represents the grassland degradation grade assessment value, X represents the number of bacterial OTUs in the soil sample, and Y represents the number of fungal OTUs in the soil sample;

[0099] The grassland degradation grade assessment standards include:

[0100] If P < 3, the grassland degradation level is non-degraded;

[0101] If 3≤P<4, the grassland degradation level is slightly degraded;

[0102] If 4≤P<5, the grassland degradation level is moderate;

[0103] If P>5, the grassland degradation level is severe degradation;

[0104] Among them, the ratio of the number of bacterial and fungal OTUs in the sampling quadrats in the moderately degraded area was P = 4.58, that is, 4≤P<5, which is consistent with the judgment of moderate grassland degradation level.

[0105] Example 4

[0106] The area to be assessed is located in the Songnen Plain. Based on surveys and statistics of various indicators of natural grassland soil and vegetation, and considering the overall grassland situation in the region, the natural grasslands in the area are divided into three types: slightly degraded grassland, moderately degraded grassland, and severely degraded grassland in accordance with the "Classification Standard for Natural Grassland Degradation, Desertification, and Salinization" (GB 19377-2003).

[0107] Three 10m×10m plots were randomly selected in each degraded grassland type, each plot contained three 5m×5m quadrats, and the spacing between adjacent quadrats was 10m.

[0108] A five-point sampling method was used in four plots in each sample site. Rhizosphere soil from each plot was collected at a depth of 0 to 20 cm and mixed into one sample. The rhizosphere soil from each plot was mixed, placed in a refrigerator, and transported to the laboratory. It was stored at -80°C for high-throughput sequencing to obtain the number of bacterial and fungal OTUs.

[0109] The grassland degradation grade assessment value is calculated based on the bacterial OTU number and the fungal OTU number;

[0110] Conducting grassland degradation assessment according to the grassland degradation grade assessment value and grassland degradation grade assessment standard to obtain an assessment result;

[0111] The calculation formula of the grassland degradation grade assessment value is shown in Formula 1:

[0112] P = X / Y (Equation 1); in Equation 1, P represents the grassland degradation grade assessment value, X represents the number of bacterial OTUs in the soil sample, and Y represents the number of fungal OTUs in the soil sample;

[0113] The grassland degradation grade assessment standards include:

[0114] If P < 3, the grassland degradation level is non-degraded;

[0115] If 3≤P<4, the grassland degradation level is slightly degraded;

[0116] If 4≤P<5, the grassland degradation level is moderate;

[0117] If P>5, the grassland degradation level is severe degradation.

[0118] Among them, the ratio of the number of bacterial and fungal OTUs in the sampling quadrats in the severely degraded area was P = 10.32, or P > 5, which was consistent with the judgment of the severe degradation level of grassland.

[0119] In summary, the present invention uses the microbial composition in the area to be assessed as an evaluation indicator, and can quickly and accurately calculate the assessment result of grassland degradation in the area to be assessed.

[0120] Although the above embodiment provides a detailed description of the present invention, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the scope of protection of the present invention.

Claims

1. A method for assessing grassland degradation based on microbial community structure and composition, characterized in that: include: In the grassland degradation area to be assessed, a representative sample plot is selected, a quadrat is selected from the sample plot, and rhizosphere soil is collected from the quadrat; the rhizosphere soil of all quadrat belonging to the sample plot is mixed to obtain a soil sample; High-throughput sequencing is used to analyze the number of bacterial OTUs and the number of fungal OTUs in the soil sample; and a grassland degradation grade assessment value is calculated based on the number of bacterial OTUs and the number of fungal OTUs; Conducting grassland degradation assessment according to the grassland degradation grade assessment value and grassland degradation grade assessment standard to obtain an assessment result; The representative sample plots cover different terrains, vegetation types and soil types in the grassland degradation area to be assessed; The calculation formula of the grassland degradation grade assessment value is shown in Formula 1: P = X / Y (Equation 1); in Equation 1, P represents the grassland degradation grade assessment value, X represents the number of bacterial OTUs in the soil sample, and Y represents the number of fungal OTUs in the soil sample; The grassland degradation grade assessment standards include: If P < 3, the grassland degradation level is non-degraded; If 3≤P<4, the grassland degradation level is slightly degraded; If 4≤P<5, the grassland degradation level is moderate; If P>5, the grassland degradation level is severe degradation.

2. The method according to claim 1, characterized in that The grassland degradation areas to be assessed include desert steppes or alpine meadows.

3. The method according to claim 1, characterized in that The number of sample plots is 9 to 15; the number of quadrats in each sample plot is 3 to 5; a soil sample is collected from each sample plot using a five-point sampling method; the rhizosphere soil of all the quadrats in the sample plot is mixed to obtain a soil sample for each sample plot; the number of bacterial OTUs and the number of fungal OTUs in the soil sample of each sample plot are analyzed using high-throughput sequencing; and a grassland degradation grade assessment value for each sample plot is calculated based on the number of bacterial OTUs and the number of fungal OTUs. If the average level of degradation in the entire grassland degradation area to be assessed is to be evaluated, the median of the grassland degradation grade assessment values ​​shall be taken; if the most severely degraded area in the grassland degradation area to be assessed is to be assessed, the maximum value of the grassland degradation grade assessment values ​​shall be taken.

4. The method according to claim 3, characterized in that The distance between adjacent sample plots is 10 to 50 m.

5. The method according to claim 1 or 3, characterized in that The soil sample is collected at a depth of 0 to 20 cm; the soil includes rhizosphere soil.

6. The method according to claim 3, characterized in that After collecting the rhizosphere soil samples, the process also includes removing the surface litter from the soil samples, mixing all the soil samples to obtain mixed soil samples, and drying the mixed soil samples to remove litter and root debris.

7. The method according to claim 6, characterized in that This also includes sieving the soil to 2 mm after removing litter and root debris.

8. The method according to claim 1, characterized in that The method for determining the number of OTUs of the bacteria includes 16SrRNA gene sequencing.

9. The method according to claim 1, characterized in that The method for determining the number of OTUs of the fungus includes ITS sequencing; the primer pair used in the ITS sequencing includes an ITS1 region primer pair and an ITS2 region primer pair.

10. The method according to claim 1, characterized in that After obtaining the assessment results, the assessment results are also verified using drone hyperspectral remote sensing.