Coniothecium conicum ac-chi e01 strain and applications thereof

Abstract

The present application relates to the technical field of microorganism, and particularly relates to a conical arthrobotus AC-ChiE01 strain and application thereof. The present application provides a conical arthrobotus AC-ChiE01 strain, the chitinase activity of the strain is significantly higher than that of a wild type strain, and the predation efficiency on the second instar larvae of horse roundworm is also significantly improved compared with the wild type strain. In addition, the conical arthrobotus AC-ChiE01 strain provided by the present application has good growth characteristics, and the sporulation capacity of the strain does not have obvious disadvantages compared with the wild type strain, and the conical arthrobotus AC-ChiE01 strain has a broad application prospect in the biological control of livestock digestive tract nematodiasis.

Description

Technical Field

[0001] This invention relates to the field of microbial technology, specifically to a *Conophytum comosum* strain AC-ChiE01 and its applications. Background Technology

[0002] Chemical control of gastrointestinal nematode diseases in livestock has many limitations and easily causes environmental pollution. Therefore, finding safe and effective alternative control methods has become an important research topic. Predatory fungi show good application prospects because they can reduce the number of parasitic nematodes without harming the host animal, and are widely distributed in nature and environmentally friendly.

[0003] Enhancing the key virulence factors of biocontrol fungi through genetic engineering is an effective strategy to improve their biocontrol efficacy. However, the introduction of exogenous genes is often accompanied by changes in the growth traits of the host fungus. Obtaining an engineered strain that can efficiently express exogenous enzymes while maintaining good growth and reproduction capabilities is quite difficult and involves a lot of randomness.

[0004] Conical plexiformis ( Arthrobotrys conoides *Arthrum conocytosum* is a typical predatory fungus belonging to the genus *Arthrum*. During predation and invasion, *Arthrum conocytosum* relies on the mechanical force of its predatory structures and the combined action of secreted extracellular hydrolytic enzymes to fixate and decompose the nematode's body wall, thus invading and growing within the nematode. Currently, there is limited research on engineered strains of *Arthrum conocytosum*; therefore, this invention is proposed. Summary of the Invention

[0005] To address the aforementioned technical problems, this invention provides a *Platycodon conocytosporum* strain AC-ChiE01 and its applications.

[0006] Specifically, the technical solution of the present invention is as follows:

[0007] In a first aspect, the present invention provides a *Plasmodium conocytosum* strain AC-ChiE01, which is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 42276.

[0008] The AC-ChiE01 strain of *Conophytum comosum* provided by this invention has higher chitinase expression activity than the wild type. In the induction medium with chitin as the sole carbon source, the chitinase activity produced reaches more than 45 U / mL.

[0009] The AC-ChiE01 strain of *Conophytum coniculatum* provided by this invention has a higher nematode predation rate than the wild type. Under the test conditions of 7 days of culture, 25°C, and 100 nematodes / plate (plate diameter 90 mm), its terminal predation efficiency against second-stage larvae of *Nematodeum equinoides* is 89.47%~93.49%.

[0010] Secondly, the present invention provides a method for culturing the *Plasmodiophora conocytoides* AC-ChiE01 strain: using CMA or PDA solid medium and culturing at 25±2℃.

[0011] Thirdly, the present invention provides a screening method for the AC-ChiE01 strain of *Conophytum conocytosum*: using CMA or PDA solid medium, culturing at 25±2℃, the medium containing not less than 400 μg / L of hygromycin.

[0012] Fourthly, the present invention provides a microbial preparation comprising the aforementioned *Conophytum comosum* AC-ChiE01 strain.

[0013] Fifthly, the present invention provides the use of the *Conophytum comosum* AC-ChiE01 strain or the microbial preparation in the preparation of a drug for the prevention and treatment of digestive tract nematode diseases in livestock (including digestive tract nematode diseases in cattle, horses, sheep, etc.).

[0014] Preferably, the livestock digestive tract nematode disease is caused by Equine roundworm.

[0015] Preferably, the *Equisetum equinoides* includes second-stage larvae of *Equisetum equinoides*.

[0016] Preferably, in the drug, the AC-ChiE01 strain of *Trichoderma conocytosum* is the sole active pharmaceutical ingredient that kills *Strombus equinofluvicalis*; or, the AC-ChiE01 strain of *Trichoderma conocytosum* works in conjunction with other active pharmaceutical ingredients to kill *Strombus equinofluvicalis*.

[0017] Beneficial effects:

[0018] This invention provides a strain of *Trichoderma conocytoides* AC-ChiE01, which exhibits significantly higher chitinase activity than the wild-type strain and shows a significantly improved predation efficiency against second-stage larvae of *Strombus equineus*. Furthermore, the *Trichoderma conocytoides* AC-ChiE01 strain provided by this invention exhibits good growth characteristics and its sporulation capacity is not significantly inferior to that of the wild-type strain, demonstrating broad application prospects in the biological control of nematode diseases in the digestive tract of livestock. Attached Figure Description

[0019] To more clearly illustrate the technical solutions in this invention or the prior art, the accompanying drawings used in the description of the embodiments or the prior art will be described below.

[0020] Figure 1 The results of hygromycin resistance testing for *Armillaria conchae* AC_XJ01 in Example 1 of this invention are shown. A through E correspond to hygromycin concentrations of 0, 200, 400, 600, and 800 μg / L, respectively.

[0021] Figure 2 This is an electrophoresis image of the pCAMBIA1303-Chi6195 plasmid double enzyme digestion identification in Example 1 of the present invention. Wherein, M: 1kb Plus DNA Marker; 1: pCAMBIA1303-Chi6195 plasmid double enzyme digestion.

[0022] Figure 3 This is an electrophoresis image of the colony PCR verification of Agrobacterium tumefaciens GV3101 transformants in Example 1 of this invention. Wherein, M: 2kDNA Marker; 1-6: single colonies of Agrobacterium tumefaciens GV3101; 7: negative control.

[0023] Figure 4 This is the result of hygromycin resistance screening in the genetically transformed transformants of Example 1 of the present invention. Wherein, A: genetically transformed transformant colonies; B: *Conophytum comosum* AC_XJ01 control group.

[0024] Figure 5 This is an electrophoresis diagram for PCR verification of the hygromycin resistance gene in the recombinant *Conophytum comosum* positive transformants in Example 1 of this invention. Wherein, M: 2kDNA Maker; 1: Positive control; 2: Negative control; 3-5: Recombinant *Conophytum comosum* positive transformants.

[0025] Figure 6 This is an electrophoresis diagram for PCR verification of the chitinase gene Chi6195 in the recombinant *Conophytum comosum* positive transformant from Example 1 of this invention. Wherein, M: 2kDNA Maker; 1: Positive control; 2: Negative control; 3-5: Recombinant *Conophytum comosum* positive transformants.

[0026] Figure 7 This is a phenotypic comparison of the recombinant *Conophytum comosum* positive transformant colonies in Example 1 of the present invention. Wherein, A: wild group; B: experimental group 1 (AC-ChiE01); C: experimental group 2 (AC-ChiE02); D: experimental group 3 (AC-ChiE03).

[0027] Figure 8 This is a comparative growth diagram of the recombinant *Conophytum comosum* positive transformant hyphae in Example 1 of the present invention. From left to right, they are: wild group (200×), experimental group 1 (AC-ChiE01, 200×), experimental group 2 (AC-ChiE02, 200×), and experimental group 3 (AC-ChiE03, 200×).

[0028] Figure 9This is a photomicrograph of nematode larvae captured by the recombinant *Conophytum constrictum* positive transformant in Example 1 of this invention. From left to right, the groups are: wild group (200×), experimental group 1 (AC-ChiE01, 200×), experimental group 2 (AC-ChiE02, 200×), and experimental group 3 (AC-ChiE03, 200×). Detailed Implementation

[0029] Enhancing the key virulence factors of biocontrol fungi through genetic engineering is an effective strategy to improve their biocontrol efficacy. However, the introduction of exogenous genes is often accompanied by changes in the growth traits of the host fungus, and obtaining an engineered strain that can efficiently express exogenous enzymes while maintaining good growth and reproduction capabilities is quite difficult and random.

[0030] This invention utilizes Agrobacterium tumefaciens GV3101-mediated genetic transformation technology, employing the genetic transformation vector pCAMBIA1303-Chi6195 containing the chitinase gene Chi6195, to introduce the chitinase gene Chi6195, an infective extracellular protease, into *Armillaria truncatula* (a species of cauliflower). Arthrobotrys conoides AC_XJ01 (deposited at the China General Microbiological Culture Collection Center, accession number: CGMCC No. 41025); positive transformants were screened using PDA medium containing 400 μg / L hygromycin, and after PCR verification and genetic stability analysis, a recombinant strain AC-ChiE01 that stably inherited and overexpressed chitinase Chi6195 was finally obtained. This strain was deposited on October 13, 2025, at the China General Microbiological Culture Collection Center (CGMCC, address: No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, 100101, China), and classified as *Trichophyton conocytosum*. Arthrobotrys conoides The accession number is CGMCC No.42276.

[0031] The AC-ChiE01 engineered strain provided by this invention exhibits significantly higher chitinase activity than the wild-type strain, achieving a predation efficiency of up to 93.49% against second-stage larvae of *Stromboma equina*, a significant improvement over the wild-type strain's highest predation rate (76.84%). Furthermore, this engineered strain demonstrates excellent growth characteristics, with its sporulation capacity not significantly affected compared to the wild-type, indicating broad application prospects in the biological control of nematode diseases in the digestive tract of livestock.

[0032] To make the above-mentioned objects, features and advantages of the present invention more apparent and understandable, the specific embodiments of the present invention will be described in detail below with reference to the examples in the specification.

[0033] Many specific details are set forth in the following description in order to provide a full understanding of the invention. However, the invention may also be practiced in other ways different from those described herein, and those skilled in the art can make similar extensions without departing from the spirit of the invention. Therefore, the invention is not limited to the specific embodiments disclosed below.

[0034] In the description of this specification, the references to terms such as "one embodiment," "some embodiments," "specific implementation," or "some specific implementations," etc., refer to specific features, structures, materials, or characteristics described in connection with that embodiment or example, which are included in at least one embodiment or example of the present invention. In this specification, the illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples. Moreover, without contradiction, those skilled in the art can combine and integrate the different embodiments or examples described in this specification, as well as the features of different embodiments or examples.

[0035] The endpoints and any values ​​of the ranges disclosed in this specification are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of the various ranges, the endpoint values ​​of the various ranges and individual point values, and individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed herein.

[0036] In the embodiments provided in this specification, unless specific techniques or conditions are specified, the techniques or conditions described in the literature in this field, or the product instructions, shall be followed. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased from legitimate channels.

[0037] The materials involved in the following embodiments include:

[0038] Strains, nematodes, and plasmids: *Platysporium conocyticum* (AC_XJ01) (deposited at the China General Microbiological Culture Collection Center, accession number: CGMCC No. 41025); *Nematodeus equinoides* larvae, provided and preserved by the Parasitology Laboratory of Xinjiang Agricultural University; *Agrobacterium tumefaciens* GV3101, purchased from Sangon Biotech Co., Ltd. (Shanghai); pAN7-1 plasmid and pCAMBIA1303 plasmid, purchased from Fenghui Biotechnology Co., Ltd.

[0039] Reagents and kits: Restriction endonucleases, Thermo Fisher Scientific; Acetone and chitin powder, Beijing Huamaike Biotechnology Co., Ltd.; pMD19-T (simple) and DH5α competent cells, Shanghai Sangon Biotech Co., Ltd. (for plasmid transformation and cloning); 2×TaqPCR Mix, Guangzhou Dongsheng Biotechnology Co., Ltd.; DNA standard DL 2000, Beijing TransGen Biotechnology Co., Ltd. (for...) Figure 3 , Figure 5 , Figure 6 DNA standard DL 10000, Tiangen Biotech (Beijing) Co., Ltd. (for use) Figure 2 Chitinase activity assay kit, BoxBio; gel recovery kit (for recovery of enzyme digestion products), plasmid extraction kit, OMEGA (for extraction of pCAMBIA-Chi6195 plasmid); DNA extraction kit, Beijing Solarbio Technology Co., Ltd.

[0040] Instruments and equipment: Biochemical incubator, Shanghai Qixin; Water bath, Delite Technology Co., Ltd.; Ultra-clean workbench, Wuxi Yichun Chemical Equipment Co., Ltd.; HNY-200B shaker, Nanbei Instrument Co., Ltd.; HICO-21 benchtop high-speed centrifuge, Sangon Biotech Co., Ltd.

[0041] Example 1

[0042] Construction, screening and identification of recombinant paniculatum.

[0043] (1) Determination of the selective pressure sensitivity of Conophytum comosum AC_XJ01 to hygromycin.

[0044] The fungal species AC_XJ01 was inoculated onto PDA solid medium and cultured at 25℃ for 7 days. After that, the mycelium was collected and small pieces of the same size were inoculated onto PDA solid medium containing 0, 200, 400, 600, 800, and 1000 μg / L hygromycin. After culturing at 25℃ for 7 days, the fungal growth was observed.

[0045] The experimental results are shown in Figure 1 .

[0046] according to Figure 1 By observing the growth of *Platystomium conocyticum* AC_XJ01 on hygromycin-resistant PDA solid medium at different concentrations for 7 days, it was found that growth completely stopped at a hygromycin concentration of 400 μg / L. Therefore, 400 μg / L was selected as the screening concentration.

[0047] (2) Construction of fungal expression vectors.

[0048] The promoter PgpdA and terminator TtrpC fragments were obtained by PCR. Using pAN7-1 plasmid as a template, a 2129 bp promoter PgpdA fragment was cloned (primers: PgpdA-F:5'-GCC). CTAGG GAATTCCCTCTACA-3';PgpdA-R:5'-GC AAGCTT The primer CTGCTCAAGCGGGT-3' (with an Avr II restriction site introduced upstream and a Hind III restriction site introduced downstream, the underlined sites being the restriction sites) was cloned into the pMD19-T vector. Using pAN7-1 plasmid as a template, a 763 bp terminator TtrpC fragment (primer: TtrpC-F:5'-) was cloned. GGATCC ACTTAACGTTAC -3';TtrpC-R:5'- CGCGCGCG The primer TCGAGTGGAGAT-3' (with an upstream BamHI restriction site and a downstream MauBII restriction site, indicated by underlined areas) was cloned into the pMD19-T vector. The correctly sequenced pMD19-T-PgpdA plasmid was double-digested with restriction endonucleases Avr II and Hind III to obtain the PgpdA fragment. The correctly sequenced pMD19-T-TtrpC plasmid was double-digested with restriction endonucleases BamHI and MauBII to obtain the TtrpC fragment. These fragments were ligated to obtain the PgpdA-TtrpC fragment, which was then ligated to the larger fragment obtained from the pCAMBIA1303 plasmid, which was also double-digested with restriction endonucleases Avr II and MauBII, to construct the fungal expression vector pCAMBIA1303-PgpdA-TtrpC.

[0049] Using plasmid 28a-AC-Chi6195 as a template, the 1176 bp target sequence of Chi6195 was cloned (primers: Chi6195-F:5'- AAGCTT TCTCTGGCTGTACTGG-3'; Chi6195-R: 5'- GGATCC GCGTTAAGAAGACAGA-3'; the upstream primer introduces the Hind III restriction site, and the downstream primer introduces the BamHI restriction site (the underlined part is the restriction site), and it is cloned into the pMD19-T vector.

[0050]

[0051] The correctly sequenced plasmid was double-digested with restriction endonucleases Hind III and BamHI to obtain the Chi6195 target fragment. This fragment was then ligated with the larger fragment obtained from the plasmid pCAMBIA1303-PgpdA-TtrpC, which was also double-digested with Hind III and BamHI, to construct the fungal expression vector pCAMBIA1303-Chi6195. The vector was then identified by double digestion with restriction endonucleases Avr II and MauBI I.

[0052] The identification results are as follows Figure 2 .

[0053] according to Figure 2 The pCAMBIA1303-Chi6195 plasmid was identified by double digestion with Avr II and MauB I. The size of the digested fragment matched that of the target fragment, indicating that the vector pCAMBIA1303-Chi6195 was successfully constructed.

[0054] The expression vector pCAMBIA1303-Chi6195 was transformed into Agrobacterium tumefaciens GV3101 via freeze-thaw method. The Chi6195 gene was verified by PCR amplification of the monoclonal strain grown on LB solid medium containing 100 mg / L kanamycin.

[0055] The verification results are shown below. Figure 3 .

[0056] according to Figure 3 After transforming Agrobacterium tumefaciens GV3101 with the constructed expression vector pCAMBIA1303-Chi6195 using the freeze-thaw method, single colonies were picked and the TtrpC gene was amplified by PCR, confirming that the plasmid was successfully transformed into Agrobacterium tumefaciens GV3101.

[0057] (3) Genetic transformation of *Agrobacterium tumefaciens* GV3101 into *Platysporum conocytosum* AC_XJ01.

[0058] Genetic transformation system of *Agrobacterium tumefaciens* GV3101-mediated *Armillaria tumefaciens* AC_XJ01: Agrobacterium tumefaciens GV3101 was used as the infecting strain, and the recipient was the conidia of Arbuscular mycorrhizal AC_XJ01, with a conidial suspension concentration of 1×10⁻⁶. 6 The concentration of acetylsalicylic acid was 200 μmol / L, the co-culture temperature was 25℃, and the co-culture time was 48 h.

[0059] Follow these steps to convert: Agrobacterium tumefaciens GV3101 containing the expression vector pCAMBIA1303-Chi6195 was placed in 10 mL of LB liquid medium containing 100 mg / L kanamycin and cultured overnight at 28 °C and 200 r / min. After centrifugation to collect the bacterial cells, they were resuspended in 3 mL of IM medium to a concentration of OD600 of 0.15–0.2, and cultured further until the OD600 reached 0.5–1.0. 100 μL of the pre-cultured Agrobacterium tumefaciens GV3101 bacterial suspension was mixed with an equal volume of Panthera chinensis AC_XJ01 spore suspension and co-cultured at 28 °C in the dark for 24 h. 100 μL of the mixture was then spread on PDA solid medium containing 400 μg / L hygromycin for selection culture.

[0060] The screening and culture results are shown in Figure 4 .

[0061] according to Figure 4 100 μL of co-cultured *Pseudomonas conocyticus* AC_XJ01 spore suspension was screened on CMA medium containing hygromycin resistance at a final concentration of 400 μg / L for 96 h. Single colony images were obtained, and *Pseudomonas conocyticus* colonies were clearly visible compared to the control group. Preliminary verification proved that the genetic transformation of *Pseudomonas conocyticus* AC_XJ01 was successful.

[0062] (4) Screening of recombinant paniculatum.

[0063] Positive transformants of *Conophytum comosum* were inoculated onto PDA solid medium without hygromycin and cultured at 25°C for 7–9 days. Well-grown recombinant *Conophytum comosum* positive transformants were then transferred to new PDA solid medium containing 400 μg / L hygromycin for rescreening.

[0064] (5) Molecular identification and genetic stability testing of recombinant paniculatum.

[0065] Genetic stability analysis was performed by extracting genomic DNA from recombinant Conophytum positive transformants that grew well on PDA solid medium using the Solarbio fungal DNA extraction kit, and then amplifying the Chi6195 and TtrpC genes for verification.

[0066] The specific procedures are as follows: First, the three recombinant *Platycodon grandiflorus* positive transformants detected by PCR were inoculated onto PDA solid medium and cultured for 7 days. Mycelia were picked from the edge and inoculated onto PDA solid medium for three passages. Then, the recombinant *Platycodon grandiflorus* positive transformants were transferred again onto PDA solid medium containing 400 μg / L hygromycin for rescreening. Genomic DNA was extracted from the normally growing recombinant *Platycodon grandiflorus* transformants, and the Chi6195 and TtrpC genes were amplified by PCR for verification.

[0067] The verification results are shown below. Figure 5 and Figure 6 .

[0068] according to Figure 5 and Figure 6 Three recombinant *Armillaria confusa* positive transformants (AC-ChiE01, AC-ChiE02, AC-ChiE03) were selected and inoculated into fresh PDA medium without hygromycin B for subculturing. After three consecutive subculturings, each transformant was again transferred to PDA selection medium containing 400 μg / L hygromycin resistance, and the hygromycin resistance marker gene was verified by PCR. The pCAMBIA1303-Chi6195 plasmid containing the hygromycin resistance gene was used as the positive control, and *Armillaria confusa* strain AC_XJ01 was used as the negative control. Primers Hyg-F (5'-GAGTTCAGGCTTTTTCAT-3') and Hyg-R (5'-CCGAGGGCAAAGAAATAG-3') were used to detect the hygromycin resistance gene (…). Figure 5 PCR amplification and verification of the chitinase gene using Chi6195-F and Chi6195-R were performed. Figure 6 The results showed that all the test transformants amplified a single and bright target band, and the size was consistent with the positive control. The hygromycin resistance gene and chitinase gene were both about 1100 bp. The results indicated that the plasmid carrying the hygromycin resistance gene and chitinase gene fragments had been integrated into the genome of *Conophytum comosum* AC_XJ01.

[0069] Example 2

[0070] Phenotypic analysis of recombinant Panicellar simulans positive transformants.

[0071] After obtaining a stable genetically derived transformant based on Example 1, the Chi6195 gene transformant and wild-type Conophytum comosum AC_XJ01 were inoculated onto PDA and CMA plates and cultured at 25°C for 7 days.

[0072] Observation revealed that some Chi6195 gene transformants exhibited colony phenotypes similar to wild-type *Conophytum comosum* AC_XJ01. Figure 7 , Figure 8 The growth rate differs from the growth rate (Table 1).

[0073] To observe the changes in sporulation capacity between Chi6195 gene transformants (AC-ChiE01, AC-ChiE02, AC-ChiE03) and wild-type *Conophytum comosum* AC_XJ01, in this example, spore suspensions containing the same number of spores were spread on CMA plates of the same area. After incubation at 25°C for 10 days, all spores were collected separately, and spore suspensions of the same volume were prepared. The spores were counted using a hemocytometer (Table 1), and their germination efficiency was determined (Table 2).

[0074] Note: In Tables 1 and 2, the same letter after the data in the same column indicates no significant difference (P>0.05), while the values ​​marked with letters a, b, c and d show significant differences between columns (P<0.05).

[0075] Table 1. Results of growth rate and sporulation of *Platycodon grandiflorus*

[0076] Table 2. Results of germination efficiency determination of *Platysporium conocytum* transformants

[0077] Example 3

[0078] Chitinase induction and activity assay of recombinant Panicellarioides positive transformants.

[0079] (1) Preparation of colloidal chitin (10 mg / mL): Place 1.0 g of chitin powder in a mortar, add 4 mL of acetone pre-cooled at 4 °C, and grind thoroughly under ice bath and ventilation conditions. During grinding, acetone can be added as needed. While grinding, slowly add 30 mL of concentrated hydrochloric acid, and it will dissolve completely after a few minutes. Centrifuge at 8000 g for 15 min at 4 °C. Collect the supernatant, and slowly add 50% ethanol (more than 5 times the volume of the supernatant) under vigorous stirring. After stirring thoroughly, let it stand to allow the colloidal chitin to precipitate. Filter with filter paper and discard the supernatant. Wash the chitin precipitate repeatedly with deionized water until the pH reaches 7.0, and make up to 100 mL. Store in a refrigerator away from light.

[0080] (2) Preparation of crude chitinase solution: 30 mL of basal culture medium was added to a 300 mL Erlenmeyer flask, and 3 mL of *Armillaria coniculatus* spore suspension (1×10⁻⁶) was added. 7 Inoculate with (number of cells / mL), and culture on a rotating shaker at 28℃ and 160 r / min for 96 h. Inoculate with chitinase induction medium under the same conditions and culture for 24 h. The supernatant from centrifuged fermentation broth (3000 r / min, 20 min) is the crude enzyme solution, and enzyme activity is measured.

[0081] (3) Chitinase activity assay: The chitinase activity assay kit from BoxBio was used for the assay.

[0082] Using *Conophytum comosum* strain AC_XJ01 as the wild-type control, the chitinase activity of recombinant chitinase strains AC-ChiE01, AC-ChiE02, and AC-ChiE03 of Chi6195 was measured in chitin induction medium for 90 h. The enzyme activity results are shown in Table 3.

[0083] Table 3 shows that, under induction culture conditions, the wild-type *Conophytum constrictum* strain AC_XJ01 had an average protease activity of 40.15 U / mL. The three recombinant *Conophytum constrictum* positive transformants (AC-ChiE01, AC-ChiE02, and AC-ChiE03) had protease activities of 45.75 U / mL, 42.75 U / mL, and 42.95 U / mL, respectively. The protease activity of AC-ChiE01 group was approximately 13.95% higher than that of the wild-type group. This indicates that the recombinant strains successfully achieved high expression of the Chi6195 chitinase.

[0084] Note: In Table 3, the same letter after the data in the same column indicates no significant difference (P>0.05), while the values ​​marked with letters a, b, and c show significant differences between columns (P<0.05).

[0085] Table 3. Chitinase activity assay of positive transformants of *Platysporum contortus*

[0086] Example 4

[0087] Activity assay of recombinant Panicellarioides positive transformants preying on nematodes.

[0088] Three recombinant positive transformants of *Trichoderma conocytoides* (AC-ChiE01, AC-ChiE02, and AC-ChiE03) cultured in CMA medium at 25 °C for 7 days, and the wild-type control group *Trichoderma conocytoides* AC_XJ01, were selected. Second-stage larvae of *Nematoda* were added, 100 larvae per petri dish (90 mm diameter). The dishes were then incubated at 23 °C–25 °C, and the number of nematode predators was observed and recorded at 12-hour intervals. The terminal predation rate was calculated (terminal predation rate = number of live nematodes in each group / number of live nematodes in the control group × 100%). Differences in predation rates among the groups were analyzed using SPSS.

[0089] See results Figure 9 Table 4.

[0090] according to Figure 9 The paniculatum captured second-stage larvae of the horse nematode at 48 hours.

[0091] According to Table 4, the predation rate of recombinant *Trichoderma paniculatum* AC-ChiE01 in experimental group 1 was the highest, at 93.49%. The predation rate of *Trichoderma paniculatum* AC_XJ01 in the wild control group was 76.84%. The predation rate of recombinant *Trichoderma paniculatum* AC-ChiE01 was more than 20% higher than that of *Trichoderma paniculatum* AC_XJ01.

[0092] Note: In Table 4, the same letter after the data in the same column indicates no significant difference (P>0.05), while the values ​​marked with letters a, b, and c show significant differences between columns (P<0.05).

[0093] Table 4 Results of nematode predation activity of *Platysporum contortus* transformants

[0094] In summary, this invention successfully constructed and screened the *Trichoderma conocytoides* AC-ChiE01 strain. This strain exhibits significantly higher chitinase activity than the wild-type strain, and its predation efficiency against second-stage larvae of *Strombus equine* reaches a maximum of 93.49%, a significant improvement compared to the wild-type *Trichoderma conocytoides* AC_XJ01 (approximately 76.84%). Furthermore, the *Trichoderma conocytoides* AC-ChiE01 strain provided by this invention demonstrates good growth characteristics, and its sporulation capacity is not significantly affected, showing broad application prospects in the biological control of nematode diseases in the digestive tract of livestock.

[0095] It should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, and all such modifications or substitutions should be covered within the scope of the present invention.

Claims

1. Paniculate plexiformis ( Arthrobotrys conoides AC-ChiE01 strain, characterized in that, It is deposited at the China General Microbiological Culture Collection Center, with accession number CGMCC No. 42276.

2. The *Platymonas conocytosporum* AC-ChiE01 strain according to claim 1, characterized in that, In an induction medium with chitin as the sole carbon source, the chitinase activity produced reached over 45 U / mL.

3. The *Platymonas conocytosporum* AC-ChiE01 strain according to claim 1, characterized in that, Under the test conditions of 7 days of culture, 25℃, and 100 nematodes / plate, the terminal predation efficiency against the second-stage larvae of *Streptococcus equi* was 89.47%~93.49%.

4. The method for culturing the *Platymonas conocytosporum* AC-ChiE01 strain according to claim 1, characterized in that, Use CMA or PDA solid medium and culture at 25±2℃.

5. The screening method for the *Plasmodiophora conocytoides* AC-ChiE01 strain according to claim 1, characterized in that, Use CMA or PDA solid medium and culture at 25±2℃; the medium contains not less than 400 μg / L of hygromycin.

6. A microbial preparation, characterized in that, The microbial preparation comprises the AC-ChiE01 strain of *Conophytum comosum* as described in claim 1.

7. The use of the AC-ChiE01 strain of *Conophytum comosum* according to claim 1 or the microbial preparation according to claim 4 in the preparation of a drug for the prevention and treatment of digestive tract nematode diseases in livestock.

8. The application according to claim 7, characterized in that, The aforementioned nematode disease of the livestock digestive tract is caused by Equine roundworm.

9. The application according to claim 7, wherein the *Equisetum equinoides* comprises a second-stage larva of *Equisetum equinoides*.

10. The application according to claim 7, characterized in that, In the drug, the AC-ChiE01 strain of *Conophytum constrictum* is the sole active pharmaceutical ingredient that kills *Equisetum equinofluipes*; or, the AC-ChiE01 strain of *Conophytum constrictum* works in combination with other active pharmaceutical ingredients to kill *Equisetum equinofluipes*.

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