Arbuscular mycorrhizal fungal genetically engineered strain and application thereof

Abstract

The present application relates to a kind of oligosporous arthrobotrys genetic engineering strain and its application, belong to the technical field of microbial pesticide.The oligosporous arthrobotrys genetic engineering strain (Arthrobotrys oligospora FLP18shRNA) is preserved in China General Microbiological Culture Collection Center on October 18, 2023;Preservation unit address: Beijing Chaoyang District, North Star West Road No. 1, No. 3;Preservation number is CGMCC No.40899.The engineering strain provided in the present application has strong root-knot nematode control ability while having the same growth rate as the original strain, by applying the conidium of the engineering strain in soil, the southern root-knot nematode infection rate can be effectively reduced, and crops are protected from root-knot nematode infection.Whether in plate or soil environment, the constructed oligosporous arthrobotrys genetic engineering strain can effectively control southern root-knot nematode, and the control efficiency is 41.6%.

Description

Technical Field

[0001] This invention belongs to the field of microbial pesticide technology, specifically relating to a genetically engineered strain of Oligosporium nobile and its application. Background Technology

[0002] Plant parasitic nematodes are widespread infectious diseases affecting plants globally, with a broad host range and severe damage, causing economic losses of up to one hundred billion US dollars annually worldwide. Root-knot nematodes are an important type of plant parasitic nematode, hosting over 3,000 plant species, including many vegetable crops, particularly tomatoes and cucumbers. Furthermore, root-knot nematode diseases exacerbate soil-borne fungal diseases such as wilt and root rot, as well as some bacterial diseases, becoming a major obstacle to vegetable production. In conclusion, root-knot nematodes pose a significant threat to agriculture, and the development of safe, efficient, and low-cost nematode control methods has become an urgent need in the agricultural sector.

[0003] Currently, chemical pesticides remain the primary means of controlling plant parasitic nematodes due to their simplicity, high efficacy, and rapid effect. However, chemical pesticides pose numerous safety risks, such as toxicity to non-target organisms while killing nematodes, and the tendency to leave residues in soil and crops, seriously affecting food and environmental safety. In recent years, to ensure agricultural production safety, agricultural product quality safety, and ecological environment safety, and to effectively prevent, control, and reduce the risks of pesticide use, 70 types of chemical pesticides have been banned or restricted. Furthermore, with rapid economic and social development, there are higher requirements for ecological environmental protection, people's living needs, and agricultural product quality. "Green agriculture" is increasingly becoming a focus of public attention. Exploring and applying efficient and non-toxic green governance methods such as biological control and RNA pesticides to prevent excessive chemical pesticide residues and ensure the safety of agricultural products is an essential path for the modernization of agriculture.

[0004] Oligosporium argentea is a typical nematode natural enemy fungus and a very important biocontrol fungal resource. Compared with traditional chemical pesticides, microbial agents have the advantages of being safe, pollution-free, and residue-free, but they also suffer from low efficacy. Furthermore, existing reports indicate that Oligosporium argentea is effective against *C. elegans* but less effective against root-knot nematodes. Therefore, molecular editing using genetic engineering techniques may be one way to improve its efficacy against root-knot nematodes. Nematode neuropeptides play many key roles in nematode physiology, including host recognition, movement, navigation, digestion, infection, and reproduction. They have been identified as potential drug targets. Therefore, given the known cross-species exchange of substances and information between nematode-preying fungi and nematodes, engineering the original Oligosporium argentea strain to express shRNA that targets and interferes with root-knot nematode neuropeptides could be an effective way to obtain strains that effectively control root-knot nematodes.

[0005] FMRF amide-like peptides (FLPs) are the most diverse and major neuropeptide group in nematodes, with a C-terminal Arg-Phe-NH2 characteristic structure. They play a key role in the neurosensory-motor function of root-knot nematodes and are crucial to the parasitic function of nematodes.

[0006] shRNA (short hairpin RNA) is an RNA sequence with a tight hairpin loop, consisting of two short inverted repeat sequences and a circular region connecting them. As a common gene silencing tool in RNA interference technology, shRNA can be introduced into cells via vectors, where it is digested with enzymes to form siRNA, which then forms a silencing complex that can specifically target and degrade mRNA to achieve gene silencing. Summary of the Invention

[0007] The purpose of this invention is to provide a genetically engineered strain of *Armillaria oligospora* and its application. This strain has a strong ability to control root-knot nematodes. By applying the conidia of this engineered strain to the soil, the infection rate of root-knot nematodes in southern China can be effectively reduced, protecting crops from root-knot nematode infection.

[0008] This study found that after expressing shRNA targeting FMRF amide-like peptide 18 in *Arctium oliguriae*, its extracellular vesicles can self-assemble the shRNA. The extracted extracellular vesicles, when applied to *Symplocos spp.*, effectively silenced the expression of FMRF amide-like peptide 18 (flp-18), interfering with the nematode's host recognition. In practical applications in large-scale soil environments, the spore suspension of this engineered strain can be used as a microbial pesticide, directly sprayed into the soil for long-term and effective control of *Symplocos spp.*

[0009] To achieve the above objectives, the present invention provides the following solution: This invention provides a genetically engineered strain of Oligosporium argentea (… Arthrobotrys oligospora flp- 18shRNA The Oligosporium argentis genetically engineered strain was deposited on October 18, 2023, at the China General Microbiological Culture Collection Center; address of the depository: No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing; accession number: CGMCC No. 40899.

[0010] The present invention also provides an extracellular polymeric compound, wherein the extracellular polymeric compound is a metabolite of the Oligosporium argentea genetically engineered bacterium.

[0011] Furthermore, the extracellular polymeric material is an extracellular vesicle.

[0012] The present invention also provides the application of the following substances (1) or (2) in the control of southern root-knot nematodes: (1) The oligosporous Aranea spp. genetically engineered bacteria described above; (2) The extracellular polymers mentioned above.

[0013] Furthermore, the control of southern root-knot nematodes aims to reduce the infection rate of southern root-knot nematodes.

[0014] The present invention also provides a biological agent for controlling southern root-knot nematodes, the biological agent comprising the aforementioned Oligosporium argentea genetically engineered bacteria or the aforementioned extracellular polymer.

[0015] Furthermore, the specific preparation method of the biological agent includes the steps of inoculating the Oligosporium argentea genetically engineered bacteria onto PDA slant medium to obtain test tube culture; and then inoculating the test tube culture onto PDA solid medium for culture.

[0016] Furthermore, the PDA slant culture medium formula includes: 2% glucose, 200g potato juice, and 1.8-2% agar.

[0017] Furthermore, the preparation method of the PDA solid culture medium is as follows: 20g of corn flour is boiled in 1400mL of water for 30 minutes, then filtered through 4 layers of gauze, and 20g of agar powder is added to the filtrate, and water is added to 1000mL.

[0018] The present invention discloses the following technical effects: The engineered strain provided by this invention, while possessing the same growth rate as the original strain, exhibits stronger root-knot nematode control capabilities. By applying the conidia of this engineered strain to the soil, the infection rate of southern root-knot nematodes can be effectively reduced, protecting crops from nematode infestation. Whether on agar plates or in soil environments, the constructed oligosporous Araneae genetically engineered strain (…) Arthrobotrys oligospora flp-18shRNA Both of these methods can effectively control southern root-knot nematodes, with a control efficacy of 41.6%. The product of this invention has the outstanding advantages of low cost, environmental friendliness, and good control effect against southern root-knot nematodes, and has good potential application prospects. Attached Figure Description

[0019] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0020] Figure 1 A schematic diagram of the flp-18 shRNA expression vector; Figure 2 This is a PCR verification image of E. coli transformants. Figure 3PCR verification image of protoplast transformants; Figure 4 The image is a semi-quantitative validation diagram of flp18-shRNA expression. In the diagram, 1, 2, and 3 are the engineered strains of Oligosporium to be validated, 4 is a positive control of artificially synthesized siRNA of the same size as expected, and 5 is the wild-type strain of Oligosporium. Figure 5 This is a schematic diagram of the Planck gel plate experiment; Figure 6 This is a schematic diagram of nematode aggregation on tomato roots under a microscope. Figure 7 A statistical chart showing the number of nematodes in the roots of tomatoes; Figure 8 A graph showing the transcriptional level of the flp-18 gene in the southern root-knot nematode. Figure 9 A schematic diagram showing the disease status of each group of plants; Figure 10 The graph shows the measurement of relevant indicators for each group of plants. Detailed Implementation

[0021] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0022] The technical solution of the present invention will be clearly and completely described below with reference to specific embodiments. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0023] This invention utilizes genetic engineering techniques to construct an engineered strain of Oligosporium argentis expressing flp-18 shRNA (targeting and interfering with the root-knot nematode neuropeptide FMRF amide-like peptide 18), which can be used as a biological agent for the control of southern root-knot nematodes.

[0024] This invention involves genetically modifying the standard strain of *Arthrobotrys oligospora* (purchased from the American Center for Standard Biological Collections (ATCC), strain number: ATCC24927), constructing an shRNA expression vector using plasmid molecular construction technology, and obtaining a genetically engineered strain (*Arthrobotrys oligospora flp-18shRNA*) that targets and interferes with the expression of FMRF amide-like peptide 18 in root-knot nematodes via protoplast transformation. This engineered strain was deposited on October 18, 2023, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC No. 40899.

[0025] The expression backbone of shRNA in the shRNA expression vector is shown below (the red part is the loop structure): 5'-AATTGTTCATTAAATCGGCTTCAAGAGAGCCGATTTAATGAACAATT-3' 3'-TTAACAAGTAATTTAGCCGAAGTTCTCTCGGCTAAATTACTTGTTAA-5' The expressed shRNA sequence is as follows:

[0026] The constructed shRNA expression vector is as follows Figure 1 As shown.

[0027] The gene sequence of FMRF amide-like peptide 18 is as follows (GenBank: AY729022.1): ATGTTTATTTTATTAACATCGTTATTCCTTTGTTTTGGAGAGATAATTGCAAATGGAGAGGCGGGTCATAATAATGAAGAAATTGGAATGGAAAAAAGAATGCTCTCATATGCCGATTTAATGAACAATTTTGGAGGACCTAGTGCCCTCGATTTGGCGGGTCAAGGTCTCCTTATAGACGATGAAAGACCAAAACGTGAACAAAATTATAATAATGACGATGTACATATAATGACTCTTTGGAAACGTTCACCTTCC TATGGACCTTTCTTTCTTTAACACAGCTGGGGATTTGACTAAAAAGGATGACTTTATTGCTCCAGGAGTTTTGAGATTCGGGAAGCGAATGCCTGGGGTACTTCGATTTGGTAAAAGGGACCGAGTTGTT ATACAAGAAAAGAAGGCCGTCCCAGGAGTACTCCGTTTTGGCAAAAGACAATCTCAAGAGAGTGGAGCAGTCCCTGGCGTGCTCAGATTCGGTAAAAGAATGCCTCAAGTCCTCCGATTTGGAAAATAA The amino acid sequence of FMRF amide-like peptide 18 is as follows (GenBank: AAW56943.1): MFILLTSLFLCFGEIIANGEAGHNNEEIGMEKRMLSYADLMNNFGGPSALDLAGQGLLIDDERPKREQNYNNDDVHIMTLWKRSPSYGPSFFNTAGDLTKKDDFIAPGVLRFGKRMPGVLRFGKRDRVVIQEKKAVPGVLRFGKRQSQESGAVPGVLRFGKRMPQVLRFGK The genetically engineered strain of Arthrobotrys oligosporaflp-18shRNA, which reduces the infectivity of root-knot nematodes in this invention, is mainly used to prepare biological agents for the control of southern root-knot nematodes.

[0028] Example 1: Construction of the genetically engineered strain of *Arthrobotrys oligospora* flp-18 shRNA The specific construction method is as follows: Original strain for molecular construction: wild-type strain of Arthrobotrysoligospora (ATCC24927).

[0029] The strain used for plasmid transformation was Escherichia coli DH5α (Qingke Biotechnology).

[0030] Experimental vector: pCAMBIA-Hyg-1303 plasmid vector.

[0031] The enzymes and reagents used are shown in Table 1: Table 1 Enzymes and reagents used

[0032] 1. Constructing the flp-18 shRNA expression vector 1.1 Construction of flp-18 shRNA expression backbone Two oligonucleotide segments (Qingke Biotechnology) were synthesized to form the two strands of the expression backbone. The oligonucleotide sequences are as follows: flp-18shRNA-F: 5'-CATGAATTGTTCATTAAATCGGCTTCAAGAGAGCCGATTTAATGAACAATT-3' flp-18shRNA-R: 5'-CTAGAATTGTTCATTAAATCGGCTCTCTTGAAGCCGATTTAATGAACAATT-3' Note: The blue area on the left is the NcoⅠ viscous end, the green area is the SpeⅠ viscous end, and the red part in the middle is the loop structure.

[0033] The two oligonucleotide chains were annealed, and the reaction system is shown in Table 2: Table 2 Reaction System

[0034] The reaction procedure is as follows: (1) 95℃ for 2 minutes; (2)95℃ 90s, -1℃per cycler; (3) Go to step 2, 70×; (4) Keep warm at 4℃.

[0035] 1.2 Linearization of PCA-Hyg-1303 vector The reaction system was established on ice, and the reaction system is shown in Table 3: Table 3 Reaction System

[0036] Slowly pipette the reaction mixture to mix it thoroughly, centrifuge briefly, incubate at 37°C for 3 hours, and then incubate at 80°C for 20 minutes to heat-inactivate the restriction endonuclease.

[0037] 1.3 Ligation of PCA-Hyg-1303 linearized vector with double-stranded oligonucleotides The reaction system was established on ice, and the reaction system is shown in Table 4: Table 4 Reaction System

[0038] The above reaction system was incubated at 22℃ for 1 hour, then the DNA ligase was heat-inactivated at 65℃ for 10 minutes, and stored at -20℃.

[0039] 1.4 Transformation of ligation products into E. coli DH5α competent cells (1) Melt Escherichia coli DH5α competent cells in an ice bath; (2) Add 5 µL of the ligation product; (3) Ice bath for 40 minutes; (4) Heat shock at 42℃ for 60 seconds; (5) Ice bath for 5 minutes; (6) Add 900µL of LB medium preheated at 37℃, and incubate at 37℃ and 180rpm for 1h with shaking. (7) Add 100µL of bacterial solution to an LB plate containing kanamycin, spread it evenly, and incubate it in a 37℃ incubator for 16h.

[0040] 1.5 Colony PCR verification of E. coli positive transformants The PCR verification primers used are as follows: PCA-F:5'-CCGTCGGCGAAATAGCA-3' PCA-R: 5'-GTTGTGGCGGGTCTTGAA-3' (1) Prepare the PCR premix solution according to the system shown in Table 5: Table 5 Reaction System

[0041] (2) Pick a single colony from a plate with a sterile toothpick, spot a single colony on a new resistant plate and preserve it. Then place the toothpick in the prepared PCR premix and stir to ensure that the colony on the toothpick is fully detached from the PCR system.

[0042] (3) Perform the PCR reaction according to the following procedure. ① 94℃ for 10 minutes; ②94℃ 30s, 58℃ 30s, 72℃ 1min; ③Go to step ② 30×; ④ Keep warm at 4℃; (4) Take 2.5µL of the above PCR reaction solution and perform agarose electrophoresis verification (120V, 30min); (5) Sequencing the PCR stock solution of transformants whose electrophoretic bands are consistent with the expected ones; those with correct sequencing are positive transformants.

[0043] The final experimental results are as follows Figure 2 As shown, the obtained electrophoresis results were consistent with the expected band size (1175bp). Those that were sent for testing and sequenced without error were considered positive transformants.

[0044] 2. Protoplast transformation 2.1 Amplification of the full-length fragment containing the flp-18 shRNA expression framework The amplification primers used are as follows: PCA-FLP18-F: 5'-CAGAAGGCAATGTCATACCACT-3' PCA-FLP18-R: 5'- CACATACAAATGGACGAACGGAT-3' Using the flp-18 shRNA expression vector as a template, PCR was performed using the primers described above.

[0045] The obtained PCR reaction solution was subjected to PCR product recovery to obtain a full-length fragment of 5919 bp with a concentration of 400 ng / µL.

[0046] 2.2 Preparation of protoplasts (1) The starting strain, *Synthetium solani*, was inoculated onto a 6 cm PDA plate and cultured at 28°C for 5 days. Then, the inoculated mycelium was transferred to a 500 ml Erlenmeyer flask containing 250 ml of TG liquid medium and cultured statically at 28°C for 12 h, followed by shaking at 28°C and 180 rpm for 24 h. (2) Collect 1.0 g of tender wet mycelium by filtering with a sterile funnel containing 6 layers of lens paper, wash the mycelium 3 times with sterile water, and then wash the mycelium 3 times with MN solution. (3) The collected mycelium was suspended in 10 ml of cell wall degrading enzyme solution (MN solution contains 12.5 mg / ml snailase and 12.5 mg / ml cellulase, and the two enzymes were sterilized by filtration through a 0.22 µm filter) and enzymatically digested at 30℃ and 180 rpm for 3-5 h. Microscopic examination revealed a large number of spherical protoplasts lysed in the mycelium. (4) Filter the undigested mycelium using a sterile funnel containing three layers of lens paper, and put the collected solution containing protoplasts into a 1.5 ml centrifuge tube. Centrifuge at 6000 rpm for 5 minutes and discard the supernatant. (5) Add 1.5 ml of KTC solution to wash the precipitate, centrifuge again under the same conditions, discard the supernatant, repeat the washing of the precipitate three times, suspend the protoplasts in KTC solution, and examine the number of protoplasts under a microscope to make the concentration reach 10⁸ / ml.

[0047] 2.3 Protoplast Transformation (1) Add 25µL of the full-length fragment (about 10µg) to a 1.5 ml centrifuge tube containing 100 μL of protoplasts, mix gently, and incubate on ice for 40 min; (2) Add 1 mL of PTC solution, mix gently, and let stand at 28°C for 20 min; (3) Add 200 μL of the mixture to a Tp3 plate, spread it evenly, and incubate at 28°C for 16 hours; (4) After 16 hours of incubation, pour a thin layer of Tp3 medium containing 200 μg / ml hygromycin onto the top of the plate and incubate at 28°C. Transformant colonies will appear in 6-9 days. (5) After the transformants appear, pick a single colony and transfer the transformants 4 times on a TYGA plate with 200 µg / ml hygromycin B. Store the transformed cells for later use.

[0048] 2.4. Screening Transformants (1) Scrape the transformant hyphae into a 1.5 mL centrifuge tube containing urea extract and break the hyphae using a tissue disruptor; (2) After centrifugation, transfer the supernatant, add an equal volume of DNA extraction solution to the supernatant, and vortex to mix; (3) Centrifuge, take the upper layer of liquid into a new 1.5mL centrifuge tube, add an equal volume of isopropanol and 1 / 10 volume of 3M sodium acetate; (4) After placing at -20℃ for 20 min, centrifuge, remove the supernatant, wash the precipitate twice with 70% ethanol, and then remove the ethanol; (5) Open the centrifuge tube to allow the ethanol to evaporate naturally; (6) When the precipitate has become translucent, add 100 μL of sterile water and slowly dissolve the precipitate at 4°C. (7) Using the dissolved genome as a template, PCA-F and PCA-R as verification primers, positive transformants were preliminarily screened by PCR and agarose gel electrophoresis to detect the presence of flp18shRNA expression framework. (8) RNA was extracted from transformants containing the target band, and reverse transcription of the RNA was performed according to the reaction system and procedure shown in Table 6. Table 6 Reaction System

[0049] Reaction procedure: 42℃ for 60 minutes; 95℃ for 3 minutes; (9) Using cDNA obtained by reverse transcription as a template, transformants expressing flp18shRNA were semi-quantitatively screened using validation primers; The primer sequences for verification are as follows: flp-18yz-F:5'-GCCGCCGAATTGTTCATTAAATCGGCAT-3' The Reverse Prime:TIANGEN miRcute Enhanced miRNA Quantitative Detection Kit contains poly-T.

[0050] Experimental results are as follows Figure 3 and Figure 4 As shown, electrophoresis confirmed that three transformants produced bands consistent with expectations, and sequencing was also error-free. Therefore, these three transformants were selected for further semi-quantitative verification. The results showed that all three transformants expressed flp-18shRNA, making them the desired positive transformants.

[0051] Example 2: Specific culture method of Arthrobotrys oligospora flp-18shRNA genetically engineered strain 1. In vitro culture Culture medium formulation: PDA medium: 2% glucose; 1000ml potato juice (200g potato boiled in 1400ml water and filtered), 2% agar. Inoculate the mycelium of the Oligosporium argentea genetically engineered strain onto the culture medium and incubate at 20-28 ℃ for 3-5 days to obtain in vitro culture.

[0052] 2. Scale-up culture in petri dishes Using the aforementioned PDA medium and a 6 cm diameter culture dish, the mycelium of the Oligosporium argentis genetically engineered strain was inoculated, sealed with sealing film, and cultured at 28°C for 5 days to obtain a strain for liquid expansion culture.

[0053] 3. Culturing on CMA solid medium The culture medium formula is: 0.5% yeast extract, 1000mL corn juice (20g corn flour added to 1400ml water, boiled for 30 minutes and filtered), and 2% agar.

[0054] When culturing (sporulation) in Erlenmeyer flasks, first dispense 50ml of culture medium into each 250ml Erlenmeyer flask, sterilize at 121℃ for 20 minutes, and after solidification, directly inoculate the strain into the Erlenmeyer flask.

[0055] 4. TG liquid culture The liquid TG medium consists of the following components: 1% glucose and 1% Tryptone.

[0056] Dispense 250ml of culture medium into each 500ml Erlenmeyer flask, sterilize at 121℃ for 20 minutes, inoculate with bacterial blocks, and incubate at 28℃ on a shaker (180rpm) for 5 days.

[0057] Example 3: Extraction of extracellular vesicles from the genetically engineered strain *Arthrobotrys oligospora* flp-18 shRNA The specific extraction method is as follows: (1) Inoculate fresh bacterial blocks into liquid TG medium and incubate at 28 ℃ in a shaker (180 rpm) for 5 days; (2) The mycelium was removed by filtration through a sterile funnel containing 12 layers of lens paper; (3) Centrifuge the fermentation broth at 4℃, 10000rpm, for 30min to remove residual mycelium and cell debris; (4) The fermentation broth was filtered with a 0.22µm filter to remove larger vesicles; (5) The fermentation broth was concentrated using a 100 kDa ultrafiltration tube; (6) Centrifuge 130,000 g of concentrate at 4°C for 70 min at ultra-high speed; (7) Discard the supernatant. The precipitate is the extracellular vesicle. Resuspend the precipitate in 200µL PBS solution to obtain the extracellular vesicle solution. (8) The protein concentration of vesicle solution was determined by BCA method.

[0058] Example 4: Taxis experiment of Southern Root-knot Nematode 1. Collection of root-knot nematodes Southern root-knot nematodes were preserved and propagated on greenhouse tomatoes. Infected plants were carefully removed from the soil, and the roots were cleaned with water to remove adhering soil. Egg masses were picked up with sterile forceps and placed in sterile glass petri dishes. The egg masses were cleaned with a 1% sodium hypochlorite solution for 1 minute, and then washed with sterile water to remove any residual sodium hypochlorite solution. The egg masses were then transferred to a 300-mesh sieve to separate second-instar larvae. The sieve was placed on a plate containing sterile water, ensuring the water completely submerged the egg masses. The plates were incubated in the dark at 28°C for 3 days. The hatched nematodes were used in the experiments.

[0059] 2. Preparation of Planck gel Place a volumetric flask containing 500mL of sterile water on ice, slowly add 115g of Pluronic F-127, stir well with a magnetic stirrer, and store in a 4℃ refrigerator for later use.

[0060] 3. Seed preparation Disinfect tomato seeds with a 1% sodium hypochlorite solution for 20 minutes, then wash the seeds with sterile water to remove residual sodium hypochlorite. Soak the seeds at 50°C for 30 minutes, then sow them in a petri dish containing moistened filter paper. Place the petri dishes at 28°C for 3-5 days, until the seeds have sprouted roots of about 1.5 cm, at which point the next step of the experiment can be carried out.

[0061] 4. Nematode treatment The soaking system is configured as shown in Table 7: Table 7 Immersion System

[0062] Approximately 1000 second-instar larvae of the southern root-knot nematode were added to the soaking system and incubated overnight at room temperature with gentle shaking in the dark. The nematodes were washed three times with M9 buffer, centrifuged at 5000 rpm for 1 min, and resuspended in approximately 50 µL of M9 solution for the next step of the experiment.

[0063] 5. Planck gel experiment (1) Place a seed with a root length of 1.5cm on one side of a 6cm flat plate; (2) Add 200 Southern Root-Knot Nematode larvae 1.5cm away from the root tip; (3) Incubate in the dark at 28℃ for 6 hours, and observe the number of nematodes near the roots under a microscope.

[0064] Experimental diagram as follows Figure 5 As shown; experimental results are as follows Figure 6 and Figure 7As shown in the figure. The results showed that after treating southern root-knot nematodes with extracellular vesicles of the engineered strain Arthrobotrys oligospora flp-18shRNA, the number of tomato root nematodes decreased by 40% compared with the CK group. This indicates that the vesicles secreted by the engineered strain can effectively reduce the host recognition ability of southern root-knot nematodes, thereby blocking their infection process.

[0065] Example 5: Detection of gene silencing efficiency of engineered bacteria Miflp18. The reagents used are as follows: Magen Rapure Total RNA Microkit, Tiangen one-step premixed reagent for first-strand genomic cDNA synthesis, LightCycler 480 SYBR Green Master.

[0066] 1. Extraction of RNA from Southern Root-knot Nematode After treating Southern root-knot nematodes with engineered bacterial vesicles for 16 hours, approximately 2000 nematodes were collected in 1.5 mL centrifuge tubes, and RNA was extracted according to the following steps.

[0067] (1) Add 200µL LRTL lysis buffer to the centrifuge tube, use a hand-held mechanical grinder to homogenize the sample at high speed intermittently, and add 150µL LRTL lysis buffer after the sample is completely homogenized. (2) Centrifuge at 12000 rpm at room temperature for 5 min, and transfer the supernatant to a new 1.5 mL centrifuge tube; (3) Add an equal volume of RNA Binding Buffer and mix gently; (4) Load HiPure RNA Mini Column1 into a collection tube, transfer the mixture into the column, and centrifuge at 8000g for 30s; (5) Discard the filtrate, put the column back into the collection tube, add 500µL Buffer Rw1, let stand for 3 min, and centrifuge at 8000g for 30 s; (6) Discard the filtrate, put the column back into the collection tube, add 500µL Buffer Rw2, let stand for 3 min, and centrifuge at 8000g for 30 s; (7) Repeat step (6); (8) Discard the filtrate and centrifuge the empty column at 10000g for 3 min; (9) Transfer the column to a 1.5 mL centrifuge tube, add 30 µL of RNAse Free Water to the center of the column membrane, let stand for 1 min, centrifuge at 10000 g for 1 min, and use the obtained RNA for subsequent reverse transcription experiments.

[0068] 2. Reverse transcription of total RNA from *Strombus heterophyllum* The RNA of *Strombus simonii* was reverse transcribed into cDNA using the following system and conditions. The reaction system is shown in Table 8: Table 8 Reaction System

[0069] The reaction conditions are as follows: 42℃ for 15 minutes 95℃ for 3 minutes 3. Target gene silencing efficiency determination Using the cDNA generated in the previous step as a template, the transcription level of the Flp18 gene of *Strombus stenoptera* was detected by qRT-PCR using primers MiF18-RT-F / MiF18-RT-R. *Strombus stenoptera* Mi-Actin was used as an internal reference gene, and the amplification primers were Mi-βactin-F / Mi-βactin-R.

[0070] The primer sequences are as follows: MiF18-RT-F: 5'-CGATTTGGCGGGTCAAGGTCT-3' MiF18-RT-R: 5'-AGAAGGTCCATAGGAAGGTGAACG-3' Mi-βactin-F: 5'-GCTGCTTCGTCTTCTTCTTGGAA-3' Mi-βactin-R:5'-CGGATGTCAATGTCGCACTTCA-3' The experimental results are as follows Figure 8 As shown, applying an extracellular vesicle solution of an engineered strain to the southern root-knot nematode can reduce the expression of the flp18 gene by approximately 2.8 times.

[0071] Example 6: Pot experiment on the control of southern root-knot nematodes by the genetically engineered strain Arthrobotrys oligospora FLP18shRNA. To achieve stable and long-term control of southern root-knot nematodes, this embodiment selected a genetically engineered strain of *Arthropoda oligospora* to colonize the soil and then tested the strain's control efficacy.

[0072] Tomato seeds were germinated with sterile water for 72 hours and then sown in seedling trays containing sterile soil. In a 26℃ constant temperature and light incubator, when the tomatoes had 3-4 true leaves, they were transplanted into 10cm diameter plastic pots containing 700g of soil. For the experimental group, approximately 4 million spores per pot were added to the soil one week in advance and stirred thoroughly. The control group received an equal amount of sterile water. One week after transplanting, four holes were made around the tomato rhizome for inoculation with 1000 nematodes per plant, with three plants per treatment.

[0073] After 30 days of treatment, relevant plant indicators were measured, and the disease severity was graded according to the national standard GB / T 17980.38-2000 to calculate the relative control efficacy.

[0074] The experimental results are as follows: The condition of the roots of each group of plants is as follows: Figure 9 As shown (the red arrow points to the root node): Relevant indicators of each group of plants are as follows Figure 10 As shown:

[0075]

[0076] Grading standards for root-knot nematode disease: Grade 0: No root knots; Grade 1: Root knot ratio <25%; Grade 2: Root knot ratio 25%~50%; Grade 3: Root knot ratio 50%~75%; Grade 4: Root knot ratio 75% or more.

[0077]

[0078]

[0079] The results showed that the Arthrobotrys oligospora flp-18shRNA strain we constructed could effectively control southern root-knot nematodes, with a control efficacy of 41.6%, whether on agar plates or in soil.

[0080] Example 7

[0081] The engineered strain of *Arthrum oligosporum* was inoculated onto PDA slant. The culture medium formula was: 2% glucose; 200g potato juice; 1.8-2% agar; pH: natural. The mycelium of the engineered strain of *Arthrum oligosporum* was then inoculated onto the culture medium and cultured at 25-28℃ for 3-5 days to obtain in vitro culture.

[0082] Inoculate the test tubes with PDA solid medium, the formulation of which is the same as that for PDA slant. Incubate at room temperature for 5 days. Then, pick mycelial blocks and inoculate them with sterilized CMA medium, the formulation of which is: 20g corn flour, 1400mL water, boiled for 30 minutes. Filter through four layers of gauze, add 20g agar powder to the filtrate, and bring the water to 1000mL. Dispense 50ml into 250mL Erlenmeyer flasks and sterilize at 121℃ for 20 minutes. Incubate statically at 28℃ for 15 days to obtain a large number of conidia. Add 50ml of sterile water and shake. The resulting spore suspension can be used as a pesticide to control southern root-knot nematodes.

[0083] Of course, the above description is not limited to the examples above. Technical features not described in this invention can be implemented by or using existing technology, and will not be repeated here. The above embodiments and drawings are only used to illustrate the technical solutions of this invention and are not intended to limit this invention. This invention has been described in detail with reference to preferred embodiments. Those skilled in the art should understand that any changes, modifications, additions or substitutions made by those skilled in the art within the scope of this invention do not depart from the spirit of this invention and should also fall within the scope of protection of the claims of this invention.

Claims

1. The application of extracellular polymers in the control of southern root-knot nematodes, characterized in that, The extracellular polymer is Oligosporium argentis (… Arthrobotrys oligospora Extracellular vesicles of the genetically engineered bacteria; the Oligosporium argentis genetically engineered bacteria was deposited on October 18, 2023, at the China General Microbiological Culture Collection Center; address of the depository: No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing; accession number: CGMCC No. 40899; The control of southern root-knot nematodes aims to reduce the infection rate of southern root-knot nematodes.

2. An extracellular polymer, characterized in that, The extracellular polymeric material is an extracellular vesicle of the genetically engineered strain Oligosporium nobile; the genetically engineered strain Oligosporium nobile was deposited on October 18, 2023, at the China General Microbiological Culture Collection Center; the address of the depository is No. 3, No. 1, Beichen West Road, Chaoyang District, Beijing; the accession number is CGMCC No. 40899.

3. A biological agent for controlling southern root-knot nematodes, characterized in that, The biological agent includes the extracellular polymer as described in claim 2.

4. The biological agent according to claim 3, characterized in that, The specific preparation method of the biological agent includes the steps of inoculating the oligosporous Araneae sp. genetically engineered bacteria as described in claim 2 onto PDA slant medium to obtain test tube culture; and then inoculating the test tube culture onto PDA solid medium for culture.

5. The biological agent according to claim 4, characterized in that, The PDA slant culture medium formula includes: 2% glucose, 1000ml potato juice obtained by boiling and filtering 200g potatoes with 1400ml of water, and 1.8-2% agar.

6. The biological agent according to claim 4, characterized in that, The method for preparing the PDA solid culture medium is as follows: 20g of corn flour is boiled in 1400mL of water for 30 minutes, then filtered through 4 layers of gauze, and 20g of agar powder is added to the filtrate, and water is added to 1000mL.

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