A method for extracting and assaying gut microbes of soil nematodes

Abstract

The application discloses a kind of extraction determination methods of soil nematode digestive tract microorganism, belong to the field of microbial detection technology. Including the following steps: (1) separation extraction nematode: shallow dish method is used to separate nematode in soil, in turn through washing and screening, shallow dish standing, starvation treatment, concentration and preservation operation;(2) digestive tract microorganism extraction: after species identification to nematode, select the nematode individual of specific nutrition group, after surface microorganism elution treatment, extract digestive tract microorganism DNA;(3) DNA amplification: using primer to the V3-V4 region of digestive tract bacteria 16S rRNA gene and the ITS region of fungal ITS gene is carried out PCR amplification;(4) sequencing and data analysis: after purification and quantification to PCR amplification product, using Illumina MiSeq platform carries out double-end sequencing, original data is split, quality filtering, sequence splicing, chimeric sequence removal, ASV clustering and species annotation processing.

Description

Technical Field

[0001] This invention relates to the field of microbial detection technology, and in particular to a method for extracting and determining microorganisms from the digestive tract of soil nematodes. Background Technology

[0002] Soil nematodes, as a large and diverse group of invertebrates in the soil ecosystem, play a crucial role in material cycling, energy flow, and soil fertility regulation. The microbial community colonizing their digestive tract is closely related to the nematodes' nutritional metabolism, environmental adaptability, and ecological functions, and also indirectly reflects the health status and stability of the soil micro-ecosystem. Therefore, accurately extracting and measuring the digestive tract microorganisms of soil nematodes is of great significance for elucidating soil ecological processes, assessing soil quality, and exploring functional microbial resources.

[0003] However, existing techniques for extracting and analyzing microorganisms from the digestive tract of soil nematodes still face many bottlenecks, hindering further research in this area: Nematode isolation and purification is challenging: the soil matrix is ​​complex, and the nematodes are tiny (mostly 0.1-1 mm). Traditional separation methods (such as the Bellman funnel method and sucrose density gradient centrifugation) suffer from low separation efficiency and insufficient purity, and are prone to leaving soil particles, other microorganisms and other impurities, which can interfere with the subsequent extraction of digestive tract microorganisms. Furthermore, excessive mechanical force during some separation processes can easily damage the nematode body, causing leakage of digestive tract microorganisms or contamination of the body surface microorganisms.

[0004] Microbial contamination on the body surface is difficult to completely remove: nematodes easily adsorb bacteria, fungi and other microorganisms from the soil on their body surface. Their cell walls and body surface mucus will firmly adhere to microbial cells. Traditional washing methods (such as rinsing with water or soaking in a single solvent) are difficult to completely remove them, resulting in a large number of surface bacteria mixed in with the extracted microbial samples, which cannot truly reflect the original microbial community structure in the digestive tract.

[0005] The extraction efficiency and integrity of DNA from nematode digestive tract microorganisms are low: the digestive tract tissue of nematodes is delicate and extremely difficult to dissect. Direct dissection to obtain digestive tract tissue is not only time-consuming and laborious, but also prone to loss of digestive tract contents. When grinding the whole worm body for extraction, impurities such as the worm exoskeleton and tissue proteins will inhibit the DNA extraction efficiency. At the same time, traditional extraction kits are designed for soil or metagenomic samples and are not adapted to the trace characteristics of nematode digestive tract microorganisms, which can easily lead to problems such as low DNA yield, fragment breakage, and inhibitor residues, affecting the subsequent PCR amplification effect.

[0006] The specificity and accuracy of amplification and sequencing analysis are insufficient: the abundance and diversity of the nematode digestive tract microbial community are low, and the primers used in some existing studies have limited coverage, making it difficult to comprehensively capture the bacterial and fungal groups in the digestive tract; in addition, non-specific amplification and chimeric sequence generation are prone to occur during PCR amplification, and the sequencing data processing process lacks standardization, resulting in large species annotation bias and making it impossible to accurately quantify the community composition and relative abundance of digestive tract microorganisms.

[0007] The experimental process lacks systematic optimization: existing technologies mostly focus on improving single steps (such as isolation methods or extraction kits), without forming a complete standardized process from nematode isolation, body surface purification, DNA extraction, amplification and sequencing to data analysis. The operation differences between different laboratories are large, resulting in poor reproducibility and comparability of experimental results, making it difficult to promote the standardized advancement of soil nematode digestive tract microbiology research.

[0008] Therefore, developing a systematic method for efficiently isolating nematodes, thoroughly removing surface contamination, accurately extracting digestive tract microbial DNA, and combining specific amplification with standardized data analysis has become an urgent need to solve current technical bottlenecks and promote in-depth research on the digestive tract microorganisms of soil nematodes. Summary of the Invention

[0009] The purpose of this invention is to provide a method for extracting and determining microorganisms in the digestive tract of soil nematodes, so as to solve the problems existing in the prior art.

[0010] To achieve the above objectives, the present invention provides the following solution: One of the technical solutions of this invention is a method for extracting and determining the digestive tract microorganisms of soil nematodes, comprising the following steps: (1) Separation and extraction of nematodes: The shallow tray method is used to separate nematodes from the soil, which are then washed and sieved, placed in a shallow tray for settling, starved, concentrated and preserved. (2) Extraction of digestive tract microorganisms: After identifying the species of nematodes, individuals of nematodes of specific nutritional groups were selected, and after elution treatment of the surface microorganisms, the DNA of digestive tract microorganisms was extracted. (3) DNA amplification: PCR amplification was performed using primers on the V3-V4 region of the 16S rRNA gene of digestive tract bacteria and the ITS region of the ITS gene of fungi. (4) Sequencing and data analysis: After purification and quantification of PCR amplification products, paired-end sequencing was performed using the Illumina MiSeq platform. The raw data was processed by splitting, quality filtering, sequence splicing, chimeric sequence removal, ASV clustering and species annotation.

[0011] Based on the above technical solution, the present invention has the following technical effects: The method for extracting and determining the digestive tract microorganisms of soil nematodes provided by this invention has significant advantages: It employs a combination of washing with 60-mesh, 100-mesh, and 400-mesh sieves and shallow-plate starvation treatment to efficiently separate nematodes while ensuring their integrity and removing digestive tract residue. Multiple rounds of low-speed centrifugation (5-6 seconds) followed by elution with anhydrous ethanol thoroughly remove surface bacteria and prevent sample loss. Optimized extraction using the Fast DNA® Spin Kit for Soil yields high-purity digestive tract microbial DNA with low inhibitor residues. The synchronous and identical process for soil samples ensures data comparability. PCR amplification of the 16S rRNA gene V3V4 region using primers 515F / 806R, and PCR amplification of the ITS1 region of the ITS gene using primers ITS5 / ITS2, combined with Illumina MiSeq sequencing and standardized data processing, and flattening of the ASV table, improves the accuracy of species annotation. The entire process is standardized, with high repeatability and reliability, providing reliable support for analyzing nematode-microbe symbiotic relationships and assessing soil quality, and is suitable for research in multiple fields. Attached Figure Description

[0012] Figure 1 is an appendix to the abstract. Figure 2 shows the apparatus for separating nematodes using the shallow dish method.

[0013] Figure 3 shows the washing of nematode surface microorganisms with anhydrous ethanol followed by low-speed centrifugation.

[0014] Figure 4 shows the process of transferring the DNA solution to the Spin filter in stages.

[0015] Figure 5 shows the filter being washed with SEWS-M solution.

[0016] Figure 6 shows the horizontal community composition of fungi in the digestive tract of nematodes.

[0017] Figure 7 shows the horizontal community composition of fungi in the digestive tract of nematodes.

[0018] Figure 8 shows the horizontal community composition of bacteria in the digestive tract of nematodes.

[0019] Figure 9 shows the horizontal community composition of bacteria in the digestive tract of nematodes.

[0020] Figure 10 shows the α-diversity of the fungal community in the digestive tract of nematodes. Among them, (A) is the Chao1 index of fungi in the digestive tract of nematodes, and (B) is the Shannon index of fungi in the digestive tract of nematodes.

[0021] Figure 11 shows the α-diversity of the bacterial community in the digestive tract of nematodes. Among them, (A) is the Chao1 index of bacteria in the digestive tract of nematodes, and (B) is the Shannon index of bacteria in the digestive tract of nematodes.

[0022] Figure 12 shows the species diversity analysis of the fungal community in the digestive tract of nematodes. Among them, (A) is the differential species annotation branch diagram of fungi in the digestive tract of nematodes, and (B) is the LDA-score diagram of fungi in the digestive tract of nematodes.

[0023] Figure 13 shows the species diversity analysis of the bacterial community in the digestive tract of nematodes. Among them, (A) is the differential species annotation branch diagram of bacteria in the digestive tract of nematodes, and (B) is the LDA-score diagram of bacteria in the digestive tract of nematodes.

[0024] Figure 14 shows the analysis of fungal community construction in the digestive tract of nematodes, where (A), (B), (C) and (D) represent groups A10, A20, A30 and A40, respectively.

[0025] Figure 15 The bacterial community of the digestive tract of nematodes was constructed and analyzed, where (A), (B), (C) and (D) represent groups A10, A20, A30 and A40, respectively. Detailed Implementation

[0026] Unless otherwise specified, the technical solutions described in this invention are all conventional solutions in the field, and the reagents or raw materials used are all purchased from commercial channels or are publicly available unless otherwise specified.

[0027] This invention provides a method for extracting and determining microorganisms from the digestive tract of soil nematodes, comprising the following steps: (1) Separation and extraction of nematodes: The shallow tray method is used to separate nematodes from the soil, which are then washed and sieved, placed in a shallow tray for settling, starved, concentrated and preserved. (2) Extraction of digestive tract microorganisms: After identifying the species of nematodes, individuals of nematodes of specific nutritional groups were selected, and after elution treatment of the surface microorganisms, the DNA of digestive tract microorganisms was extracted. (3) DNA amplification: PCR amplification was performed using primers on the V3-V4 region of the 16S rRNA gene of digestive tract bacteria and the ITS region of the ITS gene of fungi. (4) Sequencing and data analysis: After purification and quantification of PCR amplification products, paired-end sequencing was performed using the Illumina MiSeq platform. The raw data was processed by splitting, quality filtering, sequence splicing, chimeric sequence removal, ASV clustering and species annotation.

[0028] In some specific implementation schemes, the washing and sieving method in step (1) is as follows: weigh fresh soil and pour it into a beaker, add water and stir well, then let it stand. Pour it into a 60-mesh sieve, a 100-mesh sieve in the middle and a 400-mesh sieve in the lower, and shake it while pouring. Repeat the water addition, mixing, standing and sieving operation 3 times. Then use a spray nozzle to rinse the nematode suspension in the 100-mesh and 400-mesh sieves and collect them into a container. The 100-mesh sieve contains larger nematodes such as omnivorous nematodes, while the 400-mesh sieve contains smaller nematodes such as bacteriophages, fungiophages and plant parasitic nematodes.

[0029] In some specific implementation schemes, the method for allowing the shallow dish to settle in step (1) is as follows: ①Preparation of shallow dish device: Take an 18-mesh stainless steel mesh sieve, lay two layers of paper towels on it and fix them with bamboo skewers, and place it in a shallow dish; ② Pour the mixture of water, nematodes and mud obtained from washing and sieving into the sieve through a watch glass. Rinse the remaining mud with clean water and pour it in. Let it stand. ③ Remove the sieve, shake the shallow dish, transfer the water to another container, let it stand for more than 2 hours, remove the top layer of water, shake it well, pour it into a large test tube, and let it stand for another 2 hours.

[0030] In some specific implementation schemes, the method of concentration and preservation in step (1) is as follows: remove the water from the top of the large test tube, retain 2-3 ml of the solution containing nematodes, add anhydrous ethanol to ensure that the ethanol content is more than 75%, shake well and store in a -20℃ refrigerator.

[0031] In some specific implementation schemes, the method for eluting surface microorganisms in step (2) is as follows: (1) Centrifuge the sterile centrifuge tube containing nematodes at low speed for 5-6 seconds; (2) Remove the supernatant, retaining some anhydrous ethanol to prevent the loss of nematodes; (3) Add anhydrous ethanol to one-third of the centrifuge tube, centrifuge again and remove the supernatant; (4) Repeat the above operation 5 times and check whether there are nematodes in the supernatant. If so, pick them back into the precipitate.

[0032] In some specific implementation schemes, the method for extracting digestive tract microbial DNA in step (2) is as follows: (1) Add 978 μL of sodium phosphate buffer and 122 μL of MT buffer to the nematode sample in the lysis matrix tube; (2) Homogenize at 6.0 m / s for 40 seconds, then centrifuge at 14000 x g for 5-10 minutes; (3) Take the supernatant, add 250 μL of PPS solution and mix well. Centrifuge at 14000 x g for 5 minutes and then take the supernatant. (4) Add 1 mL of binding matrix solution, invert and mix for 2 minutes, let stand for 3 minutes, and discard 500 μL of supernatant; (5) Transfer the DNA solution to a Spin filter in portions, centrifuge at 14000xg for 1 minute, and discard the liquid in the collection tube; (6) Add 500 μL of SEWS-M solution to wash the filter, centrifuge at 14000 x g for 1 minute, and discard the liquid in the collection tube; (7) Centrifuge at 14000xg for 2 minutes to dry the filter, replace with a sterile centrifuge tube, and air dry at room temperature for 5 minutes; (8) Add 50 μL of DES elution buffer, centrifuge at 14000 x g for 1 minute, and collect the elution buffer as the digestive tract microbial DNA.

[0033] In some specific implementation schemes, the PCR amplification in step (3) is performed on bacteria and fungi, respectively; Primers for amplifying bacteria are shown in SEQ ID NO. 1~2, and primers for amplifying fungi are shown in SEQ ID NO. 3~4; The PCR amplification reaction system consisted of 25 μL of quantified DNA template, 1.25 μL of upstream and downstream primers (10 μM), 5 μL of Q5 reaction buffer (5×), 0.5 μL of dNTPs (10 mM), 0.25 μL of Q5 high-fidelity DNA polymerase, and 5 μL of Q5 high-fidelity GC buffer (5×). The remainder was made up with Nuclease-Free Water. The bacterial PCR amplification reaction conditions were: 98℃ pre-denaturation for 3 min, 98℃ denaturation for 30 s, 52℃ annealing for 30 s, 72℃ extension for 45 s, for a total of 26 cycles, and a final extension at 72℃ for 5 min. The reaction conditions for fungal PCR amplification were: 98℃ pre-denaturation for 3 min, 98℃ denaturation for 30 s, 55℃ annealing for 30 s, 72℃ extension for 45 s, for a total of 30 cycles, and a final extension at 72℃ for 5 min.

[0034] In some specific implementation schemes, the specific operations of sequencing and data analysis in step (4) include: PCR amplification products are purified and quantified after detection by 2% agarose gel electrophoresis; purified products are mixed at equimolar concentrations and then subjected to paired-end sequencing; raw data are split and quality filtered by Fastp software, sequence is assembled by Flash software, chimeric sequence identification and amplicon sequence variant (ASV) clustering are completed through the DADA2 process; bacterial ASV taxonomic allocation uses the SILVA database (version 138), and fungal ASV taxonomic allocation uses the UNITE database (version 9.0), with confidence thresholds set to 70% for both.

[0035] In some specific implementations, the ASV table is flattened in step (4).

[0036] Example 1 1. Materials and Methods 1.1 The shallow dish method was used to isolate and extract nematodes. The specific steps are as follows: (1) Washing and sieving nematodes: Weigh 100g of fresh soil and pour it into a beaker. Add water to the 1000mL mark and stir well. Let it stand for 1 minute. Pour it into a set of mesh sieves. The upper layer is 60 mesh, the middle layer is 100 mesh, and the lower layer is 400 mesh. While pouring, shake the sieve to prevent water from filling the middle 100 mesh sieve and the lower 400 mesh sieve and overflowing from the sieve. Then, add water to the beaker and mix well. Let it stand for 1 minute and pour it into the mesh sieve. Repeat this process three times. Remove the 100-mesh and 400-mesh sieves and use a spray nozzle to rinse the mud off the nematode suspensions in the 100-mesh and 400-mesh sieves. Pour each nematode into a beaker. The 100-mesh sieve contains larger nematodes, such as omnivorous nematodes, while the 400-mesh sieve contains smaller nematodes, such as bacteriophages, fungiophages, and plant parasitic nematodes.

[0037] (2) Let it stand in a shallow dish: Preparation of the shallow dish apparatus: Take an 18-mesh stainless steel sieve, number it, and line the sieve with two layers of paper towels, securing them with bamboo skewers. Place the sieve in a shallow dish. Gently pour the water and mud from the beaker onto the sieve surface through a watch glass (the watch glass prevents the paper towels from tearing when pouring the water and mud). Pour in all the mixture of water, nematodes, and mud. Any remaining mud can be rinsed with water and poured back onto the sieve surface. Let the shallow dish apparatus stand for 24 hours to allow the nematodes to settle and starve, thus obtaining nematode specimens with clearly visible organs.

[0038] After 24 hours, gently remove the sieve and slowly shake the shallow dish (without spilling the water). Transfer all the water from the shallow dish to a 250ml beaker and let it stand for at least 2 hours. Carefully remove the water from the top of the beaker using a vacuum pump, leaving about 1cm (about 50ml) of water. Shake the remaining water well and pour it all into a large test tube, then let it stand for at least 2 hours.

[0039] (3) Nematode concentration and fixation. Carefully remove the supernatant water from the test tube that has been standing for more than 2 hours, leaving only about 2-3 ml. The nematodes will concentrate in the water at the bottom of the test tube. Avoid shaking the test tube during the operation to prevent the nematodes from being stirred up again. Add anhydrous ethanol solution to the test tube after removing the water and shake well. Pour the fixed nematode suspension into a specimen bottle, tighten the cap, write a label and serial number, place it in a specimen box for identification, and store it frozen at -20℃.

[0040] 1.2 Elution of surface microorganisms from nematode samples After identifying the nematode samples isolated from the soil, surface soil nematodes were collected under a 40x inverted optical microscope. Three replicates of 10, 20, 30, and 40 nematode individuals were collected using cell clamps and preserved in sterile centrifuge tubes containing anhydrous ethanol for cryopreservation to stabilize the microbial DNA. Due to the small size of the nematodes and the difficulty in dissecting their digestive tracts, a standard washing procedure was first used to remove microbial DNA contamination from the surface of the soil nematodes.

[0041] The specific elution procedure is as follows: (1) Centrifuge the centrifuge tube that originally contained nematodes for 5-6 seconds using a low-speed centrifuge. The time should not be too long (to prevent the nematodes from breaking off); (2) Gently place the centrifuge tube on the centrifuge tube rack and use a dropper to remove an appropriate amount of supernatant into a new sterile centrifuge tube (leave a small amount of anhydrous ethanol in the centrifuge tube to avoid the loss of nematodes); (3) Add anhydrous ethanol again (one-third of the way), centrifuge again, and remove the supernatant; (4) Repeat the above steps 3 times and check whether there are nematodes in the removed supernatant. If there are, pick them out and put them into the sediment. If not, pour the supernatant into the waste liquid bucket.

[0042] 1.3 Extraction of soil nematode digestive tract microorganisms using soil Fast DNA ®The Spin Kit for Soil (MPBiomedicals, USA) was used to extract DNA from the digestive tract microorganisms. The extraction was performed according to the manufacturer's instructions. The specific steps were as follows: (1) Sample preparation: Add nematode sample, 978 μL sodium phosphate buffer, and 122 μL MT buffer to the lysis matrix tube; (2) Homogenization: Place the tube into a FastPrep (or similar) instrument and centrifuge at 6.0 m / s for 40 seconds; then centrifuge at 14000 x g for 5-10 minutes to precipitate impurities; (3) Protein precipitation: Transfer the supernatant to a clean 2 mL centrifuge tube, add 250 μL of PPS solution and mix 10 times; centrifuge at 14000 × g for 5 minutes and collect the supernatant; (4) Adjusting binding conditions: Transfer the supernatant to a 15 mL tube, add 1 mL of binding matrix solution, invert and mix for 2 minutes, then let stand for 3 minutes; discard 500 μL of supernatant; (5) DNA (6) Wash the filter: Add 500 μL of the prepared SEWS-M solution to the filter, centrifuge at 14000 xg for 1 minute, and discard the liquid in the collection tube; if the volume of the mixture exceeds 600 μL, repeat this step; (7) Dry the filter: Centrifuge again at 14000 xg for 2 minutes, replace with a sterile centrifuge tube, and then air dry the Spin filter at room temperature for 5 minutes; (8) Elute DNA: Add 50 μL of DES elution buffer to the filter, centrifuge at 14000 xg for 1 minute, and the liquid in the collection tube is the DNA that can be used directly.

[0043] 1.4 PCR amplification and data acquisition PCR amplification of gastrointestinal microorganisms was performed using primer combinations 338F / 806R (SEQ ID NO.1: 5'-ACTCCTACGGGAGGCAGCA-3') / (SEQ ID NO.2: 5'-GGACTACHVGGGTWTCTAAT-3') and ITS5F / ITS1R (SEQ ID NO.3: 5'-GGAAGTAAAAGTCGTAACAAGG-3') / (SEQ ID NO.4: 5'-GCTGCGTTCTTCATCGATGC-3'), targeting the V3-V4 region of bacterial 16S rRNA genes and the ITS region of fungal ITS genes, respectively. The PCR reaction system was 25 μL, including a quantitative DNA template, 1.25 μL of forward and reverse primers (10 μM), 5 μL of Q5 reaction buffer (5×), 0.5 μL of dNTPs (10 mM), 0.25 μL of Q5 high-fidelity DNA polymerase, and 5 μL of Q5 high-fidelity GC-MS. Buffer solution (5×), the remainder made up with nuclease-free water; bacterial reaction conditions: 98℃ pre-denaturation for 3 min, 98℃ denaturation for 30 s, 52℃ annealing for 30 s, 72℃ extension for 45 s, for a total of 26 cycles, with a final extension at 72℃ for 5 min. Fungal reaction conditions: 98℃ pre-denaturation for 3 min, 98℃ denaturation for 30 s, 55℃ annealing for 30 s, 72℃ extension for 45 s, for a total of 30 cycles, with a final extension at 72℃ for 5 min.

[0044] PCR amplification products were detected by 2% agarose gel electrophoresis. Target products were purified using AgencourtAMPureBeads (Beckman Coulter, Indianapolis, USA) and quantified using the PicoGreends DNA assay kit (Invitrogen, Carlsbad, California, USA). The purified amplification products were mixed at equimolar concentrations and subjected to paired-end sequencing (2×300 bp) using the Illumina MiSeq platform (Illumina, San Diego, USA) by Shanghai Paisenno Biotechnology Co., Ltd.

[0045] Raw data were processed using the QIIME2 2024.5 analysis workflow: demultiplexed reads were quality filtered using Fastp and merged using Flash. Chimeric sequence identification and amplicon sequence variant (ASV) clustering were performed using the DADA2 workflow. Bacterial ASV taxonomic assignments were performed using the SILVA database (version 138), and fungal ASV taxonomic assignments were performed using the UNITE database (version 9.0), with a confidence threshold of 70% for both.

[0046] This study obtained 898,976 and 1,046,541 high-quality fungal and bacterial sequences from nematode samples (n=12). After removing mitochondrial and chloroplast sequences, the ASV tables for fungi and bacteria were diluted to 61,165 and 58,710 sequences per sample, respectively. These ASV tables were used for subsequent community analysis.

[0047] 1.5 Data Analysis Taxonomic composition analysis: Based on the taxonomic identification results, the specific species composition of each sample at the phylum and genus levels was obtained. Different taxonomic levels, composed of phylum and genus, represent different scales for viewing the community composition structure. First, the number of microbial taxa in different samples at the two taxonomic levels was compared. Then, R software was used to plot the data into bar charts to visually compare the number of taxa at the same level in different samples. Alpha diversity index analysis: The following alpha diversity indices, including the Chao1 index and the Shannon index, were calculated for each sample using QIIME2 software, and box plots were plotted to compare the richness and evenness of ASVs among different samples. Analysis of differences between groups at each taxonomic level: Using QIIME2 software, the composition and abundance tables of each sample at the six taxonomic levels (phylum, class, order, family, genus, and species) were obtained, and the analysis results were presented through bar charts. PERMANOVA (Adonis / PERMANOVA analysis) was used to evaluate the significance of differences in microbial community structure between groups. The linear discriminant analysis effect size (LEfSe) method was used to detect taxa with significant differences between groups. Nonlinear least squares (NLS) was used to fit the species abundance distribution model. The nlsLM function attempts to adjust the parameters in the model (here, m) to minimize the sum of squared residuals between observed values ​​(i.e., species frequency) and model predictions. It uses an iterative algorithm to continuously adjust the parameter values ​​until the optimal parameter values ​​that minimize the residuals are found. Visualization is performed in R (version 4.3.3).

[0048] 2 Results 2.1 Composition of the digestive tract microbial community of nematodes At the phylum level (Figure 6), Ascomycota was the dominant fungal group in all treatment groups, and its relative abundance increased with increasing treatment gradient. Conversely, the relative abundance of Basidiomycota and other groups decreased. At the genus level (Figure 7), the community composition response was more refined, with the A10 group showing a richer variety of fungal genera, including… Aspergillus , Malassezia , Hamigera , Alternaria , Corcicreas , Pseudopeyronellaea , Cladosporium , Amphinema , Penicillium , Athelia , Talaremyces , Leptcdiscella As the treatment gradient increases, the genus *Aspergillus* (etc.) Aspergillus The relative abundance of ) increased slightly, while the proportion of Malassezia spp., which was higher in group A10, was higher. Malassezia Genera such as ) show some attenuation in high-gradient groups. Pseudopeyronellaea Genera such as *Proteobacteria* remained stable in some treatment groups. At the phylum level, *Proteobacteria* was the dominant phylum in all samples, exhibiting the highest relative abundance in A10, which gradually decreased with increasing sample gradient (Figure 8). At the genus level, the bacterial genus composition of A10 included... Massila , Enhydrobacte , Cutibacterium , Acinetobacter , Corynebacterium , Pseudomonas ,by Massilia , Enhydrobacter This is the dominant genus (Figure 9). Overall, different experimental gradients only showed slight trends in the phylum and genus-level composition of bacterial and fungal communities, but the overall differences were not significant. Both types of microorganisms had a high proportion of "other" rare groups at the genus level, suggesting that all four experimental gradients could maintain the presence of rare groups, indicating that 10 nematodes are sufficient to determine the community composition of the nematode digestive tract microorganisms.

[0049] 2.2 Diversity of nematode digestive tract microorganisms In the alpha diversity analysis of fungi, the Chao1 index showed no significant difference among different groups, exhibiting a trend of first increasing and then decreasing. p >0.05). The Shannon index also showed no significant difference between groups ( p>0.05). Overall, different experimental gradients did not significantly affect the richness and diversity of the fungal community (Figure 10). In the bacterial alpha diversity analysis, the Chao1 index showed a trend of first increasing and then decreasing in different experimental gradient groups, but the differences between groups were not statistically significant ( p >0.05). The Shannon index also showed no significant difference between groups ( p = 0.47), different experimental gradients did not significantly affect the richness and diversity of the bacterial community (Figure 11). These results indicate that the experimental gradient had no significant impact on the overall richness and diversity of the community, and selecting 10 nematodes to study the α-diversity of digestive tract microorganisms was appropriate. The trends in the changes of the two types of microbial diversity were consistent, reflecting that the effects of the experimental treatments on the microbial community have a certain degree of universality.

[0050] 2.3 LEfSe analysis of nematode digestive tract microorganisms LEfSe analysis (LDA threshold = 2) showed no significant indicative taxa differences in fungal and bacterial communities across different experimental gradients: group A20 was significantly enriched. Aureobasidium It belongs to the genus *Fungi*; the characteristic genus of fungi in group A30 is... Pseudocoleophoma (Fig. 12), group A10 was significantly enriched. Aeromicrobium genus, Rubellimicrobium genus, Tardiphaga genus, Flavitalea genus and Faecalibaeterium Belongs to the bacteria, group A20. Chthonomonas genus and Bradyrhizobium The genus is the core characteristic of bacterial groups, while group A40 is only characterized by... Neisseria The genera are key indicator bacterial groups (Figure 13). These results indicate that the experimental treatment gradients only produced a slight selective enrichment effect on fungal and bacterial communities. The data from group A10 are sufficiently representative, and it is appropriate to select 10 nematodes to study the digestive tract microbiota.

[0051] 2.4 Construction of the digestive tract microbial community of nematodes Neutral model prediction analysis revealed highly similar NCM curve morphologies for fungal communities A10, A20, A30, and A40. When the horizontal axis value was less than -3, the species frequency remained extremely low (close to 0); when the horizontal axis value was greater than -2, the frequency rapidly increased and approached 1 (Figure 14). All bacterial community fitting curves exhibited the same pattern: when the horizontal axis value was less than -5, the species frequency remained extremely low (close to 0); when the horizontal axis value was greater than -4, the frequency rapidly increased and approached 1 (Figure 15). This indicates that under different gradient treatments, the species frequency of both fungal and bacterial communities followed a basically consistent pattern with the environmental gradient. There were no significant differences in the distribution range and density of black, red, and light blue scatter dots for all fungal communities (Figure 14); similarly, there were no significant differences in the distribution range and density of black, gray, and cyan scatter dots for all bacterial communities (Figure 15), indicating that there was no significant shift in the species frequency of communities across different groups. The median (med) of all fungal communities was around 95% (A10: 95.96%, A20: 95.51%, A30: 95.93%, A40: 89.29%), with only A40 slightly lower but still within the same level. There was no order-of-magnitude difference between the low and high value ranges (Figure 14). The median (med) of all bacterial communities was in the range of 81%–90% (A10: 81.53%, A20: 90.06%, A30: 81.88%, A40: 88.86%). There was also no order-of-magnitude difference between the low and high value ranges (Figure 15). This further indicates that the community stability characteristics of both fungal and bacterial communities were similar across different gradient treatments. In summary, the gradient change from A10 to A40 did not significantly affect the community building patterns of fungal and bacterial communities, meaning that selecting 10 nematodes to study the construction of the digestive tract microbial community is already representative.

[0052] Obviously, the above embodiments of the present invention are merely examples for clearly illustrating the present invention, and are not intended to limit the implementation of the present invention. For those skilled in the art, other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively describe all embodiments here. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the scope of protection of the claims of the present invention.

Claims

1. A method for extracting and determining microorganisms from the digestive tract of soil nematodes, characterized in that, Includes the following steps: (1) Separation and extraction of nematodes: The shallow tray method is used to separate nematodes from the soil, which are then washed and sieved, placed in a shallow tray for settling, starved, concentrated and preserved. (2) Extraction of digestive tract microorganisms: After identifying the species of nematodes, individuals of nematodes of specific nutritional groups were selected, and after elution treatment of the surface microorganisms, the DNA of digestive tract microorganisms was extracted. (3) DNA amplification: PCR amplification was performed using primers on the V3-V4 region of the 16S rRNA gene of digestive tract bacteria and the ITS region of the ITS gene of fungi. (4) Sequencing and data analysis: After purification and quantification of PCR amplification products, paired-end sequencing was performed using the Illumina MiSeq platform. The raw data was processed by splitting, quality filtering, sequence splicing, chimeric sequence removal, ASV clustering and species annotation.

2. The extraction and determination method according to claim 1, characterized in that, The washing and sieving method in step (1) is as follows: Weigh fresh soil and pour it into a beaker, add water and stir well, then let it stand. Pour it into a 60-mesh sieve, a 100-mesh sieve in the middle, and a 400-mesh sieve in the bottom, while stirring. Repeat the process of adding water, stirring, standing, and sieving three times. Then, use a spray nozzle to rinse the nematode suspension in the 100-mesh and 400-mesh sieves and collect them into a container. The 100-mesh sieve contains larger nematodes such as omnivorous nematodes, while the 400-mesh sieve contains smaller nematodes such as bacteriophages, fungiophages, and plant parasitic nematodes.

3. The extraction and determination method according to claim 2, characterized in that, The method for allowing the shallow dish to settle in step (1) is as follows: ①Preparation of shallow dish device: Take an 18-mesh stainless steel mesh sieve, lay two layers of paper towels on it and fix them with bamboo skewers, and place it in a shallow dish; ② Pour the mixture of water, nematodes and mud obtained from washing and sieving into the sieve through a watch glass. Rinse the remaining mud with clean water and pour it in. Let it stand. ③ Remove the sieve, shake the shallow dish, transfer the water to another container, let it stand for more than 2 hours, remove the top layer of water, shake it well, pour it into a large test tube, and let it stand for another 2 hours.

4. The extraction and determination method according to claim 3, characterized in that, The concentration and preservation method in step (1) is as follows: remove the water from the top of the large test tube, retain 2-3 ml of the solution containing nematodes, add anhydrous ethanol to ensure that the ethanol content is above 75%, shake well and store in a -20℃ refrigerator.

5. The extraction and determination method according to claim 4, characterized in that, The method for eluting surface microorganisms in step (2) is as follows: (1) Centrifuge the sterile centrifuge tube containing nematodes at low speed for 5-6 seconds; (2) Remove the supernatant, retaining some anhydrous ethanol to prevent the loss of nematodes; (3) Add anhydrous ethanol to one-third of the centrifuge tube, centrifuge again and remove the supernatant; (4) Repeat the above operation 5 times and check whether there are nematodes in the supernatant. If so, pick them back into the precipitate.

6. The extraction and determination method according to claim 5, characterized in that, The method for extracting microbial DNA from the digestive tract in step (2) is as follows: (1) Add 978 μL of sodium phosphate buffer and 122 μL of MT buffer to the nematode sample in the lysis matrix tube; (2) Homogenize at 6.0 m / s for 40 seconds, then centrifuge at 14000 x g for 5-10 minutes; (3) Take the supernatant, add 250 μL of PPS solution and mix well. Centrifuge at 14000 x g for 5 minutes and then take the supernatant. (4) Add 1 mL of binding matrix solution, invert and mix for 2 minutes, let stand for 3 minutes, and discard 500 μL of supernatant; (5) Transfer the DNA solution to a Spin filter in portions, centrifuge at 14000xg for 1 minute, and discard the liquid in the collection tube; (6) Add 500 μL of SEWS-M solution to wash the filter, centrifuge at 14000 x g for 1 minute, and discard the liquid in the collection tube; (7) Centrifuge at 14000xg for 2 minutes to dry the filter, replace with a sterile centrifuge tube, and air dry at room temperature for 5 minutes; (8) Add 50 μL of DES elution buffer, centrifuge at 14000 x g for 1 minute, and collect the elution buffer as the digestive tract microbial DNA.

7. The extraction and determination method according to claim 6, characterized in that, In step (3), PCR amplification is performed on bacteria and fungi, respectively; Primers for amplifying bacteria are shown in SEQ ID NO. 1~2, and primers for amplifying fungi are shown in SEQ ID NO. 3~4; The PCR amplification reaction system consisted of 25 μL of quantified DNA template, 1.25 μL of upstream and downstream primers (10 μM), 5 μL of Q5 reaction buffer (5×), 0.5 μL of dNTPs (10 mM), 0.25 μL of Q5 high-fidelity DNA polymerase, and 5 μL of Q5 high-fidelity GC buffer (5×). The remainder was made up with Nuclease-Free Water. The bacterial PCR amplification reaction conditions were: 98℃ pre-denaturation for 3 min, 98℃ denaturation for 30 s, 52℃ annealing for 30 s, 72℃ extension for 45 s, for a total of 26 cycles, and a final extension at 72℃ for 5 min. The reaction conditions for fungal PCR amplification were: 98℃ pre-denaturation for 3 min, 98℃ denaturation for 30 s, 55℃ annealing for 30 s, 72℃ extension for 45 s, for a total of 30 cycles, and a final extension at 72℃ for 5 min.

8. The extraction and determination method according to claim 7, characterized in that, The specific operations of sequencing and data analysis in step (4) include: PCR amplification products are purified and quantified after detection by 2% agarose gel electrophoresis; purified products are mixed at equimolar concentrations and then subjected to paired-end sequencing; raw data are split and quality filtered by Fastp software, sequence is assembled by Flash software, chimeric sequence identification and amplicon sequence variant (ASV) clustering are completed through the DADA2 workflow; ASV taxonomic allocation of bacteria is based on the SILVA database (version 138), and ASV taxonomic allocation of fungi is based on the UNITE database (version 9.0), with confidence thresholds set at 70% for both.

9. The extraction and determination method according to claim 8, characterized in that, In step (4), the ASV table is flattened.

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