Parasitic wasp-friendly multifunctional trichoderma asperellum ZJ12 and application thereof

Abstract

The invention discloses a parasitic wasp-friendly multifunctional trichoderma asperellum ZJ12 and application thereof, the preservation number of the strain is CCTCC (China Center For Type Culture Collection) NO: M2025805, and the strain has broad-spectrum and efficient antagonistic activity; various phytopathogens such as fusarium oxysporum, fusarium solani, fusarium pseudograminearum, verticillium dahliae, tea pestalotiopsis, phytophthora nicotianae, northern leaf spot helminthosporium bombycis and globulosporium melanocarpum are efficiently inhibited, and a good prevention and treatment effect is shown in a potting prevention effect experiment; the biocontrol microbial inoculum shows transboundary prevention and control potential to lepidoptera pests (plutella xylostella and cabbage caterpillars) and has good biological safety to natural enemy insect parasitic wasps (chrysalis pupae and lateral cotesia manilensis), and the problem that an existing biocontrol microbial inoculum is single in function is effectively solved.

Description

Technical Field

[0001] This invention belongs to the field of microbial technology, specifically relating to a strain of Trichoderma echinosporum ZJ12 and its applications. Background Technology

[0002] Soil-borne plant diseases caused by fungi and oomycetes are a key factor restricting global agricultural production. These pathogens can survive in the soil for a long time and spread covertly, often causing devastating diseases such as crop wilt, root rot, stem rot, and verticillium wilt, which are extremely difficult to control. Among them, Verticillium dahliae (… Verticillium dahliae Cotton Verticillium wilt caused by *Fusarium oxysporum*, and by *Fusarium oxysporum* (… Fusarium oxysporum Phytophthora ( ) Phytophthora Diseases caused by lepidopteran pests (such as diamondback moths) are widespread and have diverse hosts, posing a persistent threat to major economic crops such as cotton, chili peppers, wheat, and tobacco. At the same time, lepidopteran pests (such as diamondback moths) also pose a threat. Plutella xylostella Green caterpillars Pieris turnips (This is a type of agricultural pest) is another important agricultural pest. Its larvae directly feed on crop leaves, stems and fruits, causing serious yield losses and quality decline, and the pressure of prevention and control is enormous.

[0003] Currently, agricultural production still heavily relies on chemical pesticides for the control of such diseases. However, the long-term and excessive use of chemical pesticides not only easily leads to pesticide resistance in pathogens but also may cause problems such as pesticide residues, soil microecological imbalance, and environmental pollution. With increasing global emphasis on agricultural product quality and safety and ecological environmental protection, developing efficient, green, and sustainable disease control strategies has become an urgent need in the agricultural sector.

[0004] Utilizing beneficial microorganisms to control plant diseases is an important development direction for green agriculture. Trichoderma fungi, as important biocontrol microorganisms, can achieve integrated control of plant fungal diseases through multiple mechanisms. In disease control, Trichoderma effectively inhibits pathogen infection and spread by competing for ecological niches, hyperparasitizing pathogens (secreting hydrolytic enzymes such as chitinase and glucanase to degrade the pathogen cell wall), producing antimicrobial secondary metabolites, and inducing systemic resistance in plants. Building on this, this invention further focuses on the potential pest control value of Trichoderma fungi beyond its traditional disease control functions. Unlike the existing single-target development approach of microbial pesticides, this invention discovers that specific Trichoderma strains may directly affect the feeding behavior and survival of lepidopteran pest larvae through their metabolite activity or parasitic characteristics, thereby achieving integrated biological control of diseases and pests. Furthermore, current registration and safety evaluation of most microbial pesticides primarily focus on their efficacy against target organisms (pathogens or pests), generally lacking systematic safety evaluations of non-target organisms, especially natural enemy insects (such as parasitic wasps, which play a crucial role in ecological regulation). Existing Trichoderma agents still face challenges in practical applications, including limited functionality, unstable efficacy, and unclear potential impacts on non-target organisms. In particular, multifunctional strains possessing high antibacterial, significant insecticidal, ecologically safe, and compatible with natural enemies remain scarce, hindering their further promotion and application.

[0005] Therefore, isolating and screening Trichoderma strains with multiple functions, including high efficiency in antibacterial activity, significant insecticidal effects, and safety for natural enemies, has become a key task in developing biopesticides and promoting the green transformation of agriculture. By breeding such high-quality strains with integrated functions and eco-friendly characteristics, we can not only effectively overcome the bottleneck of existing single-function microbial agents, but also improve crop health and reduce dependence on chemical pesticides through a comprehensive control model of "one strain, multiple effects" and synergy with natural enemies. This provides crucial resources and technical support for building a stable and sustainable plant protection system. Summary of the Invention

[0006] To address the aforementioned problems, this invention provides a strain of *Trichoderma echinococcus* ZJ12, a microbial inoculant, and its applications. This strain possesses broad-spectrum and highly efficient antagonistic activity, effectively inhibiting various plant pathogens. It demonstrated good control efficacy in potted plant experiments and exhibited cross-species control potential against lepidopteran pests, while also showing good safety against parasitic wasps, effectively overcoming the limitation of existing biocontrol agents with limited functionality.

[0007] To address the above problems, the present invention provides the following technical solution: Firstly, this application provides a strain of Trichoderma acicularis ( Trichoderma asperellumZJ12, with accession number CCTCC NO: M2025805, was deposited on April 16, 2025, at the China Center for Type Culture Collection (address: Wuhan University, Wuhan, China). This strain was isolated from a rice leaf roller that died naturally in a paddy field in Zhijiang City, Hubei Province. Cnaphalocrocis medinalis The strain was isolated from the cadavers of adult insects. Its morphological characteristics are as follows: on PDA plates, the hyphae are initially white and dense, producing abundant, flocculent aerial hyphae; with prolonged culture time, the colonies gradually turn dark green, indicating the production of numerous conidia. The conidiophore-producing cells are phialidated, and the conidia are nearly spherical or elliptical. Morphological and molecular biological (based on ITS sequence) identification results indicate that this strain is *Trichoderma echinosporum* (…). Trichoderma asperellum It was named ZJ12. Further indoor biosafety tests showed that this strain was resistant to the parasitic wasp *Gymnocypris pupa* (Gymnocypris pupa). Pteromalus pupae ) and Manila lateral groove braconid wasps ( Microplitis manila It is safe from natural enemy insects such as Ashmead, demonstrating its enemy-friendly nature. Secondly, this invention provides the application of the aforementioned *Trichoderma echinococcus* ZJ12 in inhibiting various plant pathogenic fungi. Plate confrontation experiments have demonstrated that *Trichoderma echinococcus* ZJ12 exhibits strong antifungal activity against various plant pathogens, especially *Fusarium oxysporum* (…). Fusarium oxysporum Fusarium solani () Fusarium solani Fusarium pseudograss () Fusarium pseudogramineum Verticillium dahliae Verticillium dahliae ), Tea-like spores ( Pestalotiopsis theae ), Tobacco Phytophthora ( Phytophthora nicotianae ), large spot disease bulging umbelliferous worms ( Turkish sedge ) and *Ulva globosum* ( Nigrospora spherica The ZJ12 strain exhibited significant antagonistic effects against pathogens such as [list of pathogens]. After 7 days of plate confrontation culture, the inhibition rate of the ZJ12 strain against the above pathogens reached over 75%, with the inhibition rate against cotton Verticillium dahliae, corn leaf blight pathogen Helicobacter pylori, tobacco black shank pathogen Phytophthora, and tea black spot pathogen Helicobacter globosum reaching over 95%, demonstrating highly efficient biological control potential.

[0008] Thirdly, the present invention also provides a microbial inoculant, comprising *Trichoderma echinococcus* ZJ12. The active ingredient in the microbial inoculant is *Trichoderma echinococcus* ZJ12 cells, fermentation broth, or conidia. The fermentation broth of *Trichoderma echinococcus* ZJ12 is used to soak plant seeds, or to irrigate plant roots, or the *Trichoderma echinococcus* cells are mixed with an organic carrier to prepare a biological inoculant for application. Based on the broad-spectrum and highly effective antibacterial properties of *Trichoderma echinococcus* ZJ12, it can be used as an active ingredient to prepare microbial inoculants for the prevention and control of various plant diseases.

[0009] Fourthly, the present invention also provides the above-mentioned *Trichoderma echinococcus* ZJ12 or microbial agents containing *Trichoderma echinococcus* ZJ12 for the control of lepidopteran pests such as the diamondback moth. Plutella xylostella ) and cabbage caterpillar ( Pieris turnips Applications of *Trichoderma echinococcus* ZJ12 in [the study / investigation]. *Trichoderma echinococcus* ZJ12 demonstrated cross-species control capabilities against lepidopteran pests. Indoor bioassays showed that the fermentation broth of ZJ12 had significant lethal effects on the first and second instar larvae of the diamondback moth and cabbage caterpillar, with corrected mortality rates reaching 78.35% and 78.95%, respectively.

[0010] Fifthly, this invention also provides the application of the *Trichoderma echinococcus* ZJ12 microbial inoculant in the control of cotton Verticillium wilt. Efficacy experiments using plug seedling cultivation have confirmed that this inoculant has a significant and stable control effect against cotton Verticillium wilt. During the cotton seedling stage, the conidial fermentation broth of *Trichoderma echinococcus* ZJ12 (concentration ≥ 1×10⁻⁶) is used. 7 The cotton seedlings were treated with a root drenching solution containing CFU / mL of *Verticillium dahliae* spores. Three days later, the seedlings were inoculated with a spore suspension of *Verticillium dahliae*, the pathogen causing cotton Verticillium wilt, to simulate pathogen stress. The results showed that, under this treatment, the *Trichoderma echinococcus* ZJ12 microbial inoculant achieved an 83.05% control effect against cotton Verticillium wilt. Compared with the untreated positive control group, the disease index of the cotton seedlings in the ZJ12 treatment group was significantly reduced, and the plant height, stem diameter, and biomass were all significantly better than the diseased control, exhibiting growth close to that of healthy plants. This demonstrates that the inoculant not only effectively controls the disease but also promotes plant growth.

[0011] Compared with the prior art, the multifunctional Trichoderma echinocandiformis ZJ12 strain with both insecticidal and antibacterial activities provided by the present invention has at least the following beneficial effects: I. Broad-spectrum and Highly Effective Biocontrol Function: The *Trichoderma echinococcus* strain ZJ12 provided by this invention has been demonstrated through plate confrontation experiments to possess broad-spectrum and highly effective antagonistic effects against various important plant pathogenic fungi, including *Fusarium oxysporum*, *Verticillium dahliae*, and *Phytophthora tobaccoii*. Its inhibition rate against multiple pathogens exceeds 75%, and its inhibition rate against pathogens such as cotton verticillium wilt and tobacco black shank exceeds 95%, demonstrating its great potential as a core biocontrol strain.

[0012] II. Cross-species pest control: This strain not only controls plant diseases, but its fermentation broth also exhibits significant lethality against diamondback moth larvae and cabbage caterpillar larvae, with corrected mortality rates reaching 78.35% and 78.95%, respectively, demonstrating its potential for cross-species control of lepidopteran pests. This characteristic overcomes the limitation of traditional biocontrol agents having only one function, providing a new resource for developing "one-strain-multiple-effect" biological pesticide products.

[0013] III. Stable Control Efficacy with Growth-Promoting Effect: Through efficacy experiments using tray seedling cultivation, this strain was formulated into a microbial inoculant and applied to cotton production. The control effect against cotton Verticillium wilt remained consistently above 83.05%. Furthermore, the treated cotton seedlings maintained near-healthy growth even under pathogen stress, indicating a synergistic effect of effectively controlling the disease while promoting plant growth, thus effectively ensuring crop health and yield.

[0014] IV. Environmentally Friendly and Compatible with Natural Enemies: Indoor safety tests on representative parasitic wasps such as the Manila parasitic wasp and the Manila lateral groove wasp demonstrated that this strain is safe for non-target natural enemy insects. It provides a core resource for constructing a green integrated pest management system that combines fungal control with fungal control with insect control and wasp control with insect control, overcoming the negative impacts of traditional chemical pesticides and some biological agents on the ecosystem. Attached Figure Description

[0015] Figure 1 This is a colony morphology diagram of Trichoderma echinosporum ZJ12.

[0016] Figure 2 Phylogenetic tree of Trichoderma echinocandes ZJ12 constructed based on ITS sequence.

[0017] Figure 3 This is a plate confrontation experiment of Trichoderma echinosporum ZJ12 against 8 plant pathogenic fungi.

[0018] Figure 4 This is a diagram showing the indoor bioassay of diamondback moth by fermentation broth of Trichoderma echinococcus ZJ12.

[0019] Figure 5 This is a diagram illustrating the efficacy of Trichoderma echinosporum ZJ12 in controlling Verticillium wilt in cotton seedling trays.

[0020] Figure 6 This is a colony morphology diagram of Trichoderma hygroscopicum ZJ12 that was re-isolated from the surface of the parasitic wasp after coexistence. Detailed Implementation

[0021] To make the objectives, technical solutions, and advantages of this invention clearer, the implementation methods of this invention will be described in detail below with reference to specific embodiments. It should be understood that the following embodiments are only for explaining this invention and are not intended to limit the scope of protection of this invention. All other embodiments obtained by those skilled in the art based on the embodiments of this invention without inventive effort are within the scope of protection of this invention.

[0022] In the following embodiments of the present invention, unless otherwise specified, the plant pathogenic fungi (such as Verticillium dahliae, Fusarium oxysporum, etc.), test insects (diamond moth, cabbage caterpillar), and test plants (cotton, etc.) involved were all provided by the Hubei Provincial Engineering Technology Research Center for Biological Pesticides. Other reagents, culture medium components, cultivation substrates, and conventional instruments and equipment involved are all commercially available products or can be obtained through conventional methods in the art. Unless otherwise specified, the methods used in the following embodiments are conventional experimental methods in the fields of microbiology, plant pathology, and entomology.

[0023] Example 1: Isolation and Identification of Trichoderma echinococcus strain ZJ12 1. Strain Isolation In July 2024, naturally infected and dead adult leafrollers were collected from rice paddies in Zhijiang City, Yichang City, Hubei Province. After rinsing the insect samples with sterile water to remove surface dust, they were immersed in 75% ethanol for 30 seconds for surface disinfection, followed by rinsing three times with sterile water to remove residual ethanol. A small amount of tissue was excised from the insect body using a sterile scalpel and inoculated into the center of potato dextrose agar (PDA) plates. The plates were incubated in the dark at 28°C for 3-5 days. Once new hyphae appeared at the edge of the plate, the tips of the hyphae at the edge of the colony were picked using a sterile inoculation needle and transferred to a new PDA plate for streaking purification. The above purification steps were repeated three times until morphologically uniform pure culture colonies were obtained. This isolate was named ZJ12.

[0024] 2. Morphological identification After being cultured on PDA medium at 28°C for 3 days, strain ZJ12 exhibited white, dense mycelia that expanded outwards in a cotton-like pattern. After 5-7 days of culture, spores began to appear in the center of the colony, and the colony color gradually changed from white to dark green. By day 10, the entire colony was actively producing spores and was dark green. Figure 1 Under a microscope, its conidiophores are lateral and branched in a tree-like manner; the conidiophores are flask-shaped; the conidia are spherical or nearly spherical with a smooth surface. These morphological characteristics are similar to those of the genus *Trichoderma*. Trichoderma (This is consistent with the typical characteristics of Trichoderma hydatids, in particular.)

[0025] 3. Molecular biological identification The purified strain ZJ12 plates were sent to the Wuhan branch of Beijing Qingke Biotechnology Co., Ltd. for ITS sequence sequencing. The sequence is shown in SEQ ID NO.1: The above sequence was subjected to homology alignment (BLAST analysis) in the GenBank database of the National Center for Biotechnology Information (NCBI). The results showed that the sequence shared over 99% ITS sequence similarity with multiple standard strains of *Trichoderma echinococcosis*. Multigene phylogenetic analysis revealed that this strain... Trichoderma asperellum Clustered on the same branch ( Figure 2 Based on its morphological characteristics, the isolate was ultimately identified as *Trichoderma hygroscopica*. Trichoderma asperellum It was named Trichoderma hygroscopicum ZJ12.

[0026] 4. Strain preservation This strain was deposited at the China Center for Type Culture Collection (CCTCC) on April 16, 2025, at Wuhan University, Wuhan, China, with accession number CCTCC NO: M2025805.

[0027] Example 2: Plate confrontation experiment of Trichoderma echinococcus ZJ12 against plant pathogenic fungi 1. Experimental Materials Test strain: The biocontrol strain was *Trichoderma echinococcus* ZJ12 as described in Example 1. Target pathogens included: *Fusarium oxysporum* (…). Fusarium oxysporum Fusarium solani () Fusarium solani Fusarium pseudograss () Fusarium pseudograsses Verticillium dahliae Verticillium dahliae ), Tea-like spores ( Pestalotiopsis theae ), Tobacco Phytophthora ( Phytophthora nicotianae ), large spot disease bulging umbelliferous worms ( Turkish sedge ) and *Ulva globosum* ( Nigrospora spherica All strains were provided by the Hubei Provincial Engineering Technology Research Center for Biological Pesticides.

[0028] Culture medium: Potato glucose agar (PDA) medium.

[0029] 2. Experimental Methods (1) Preparation of mycelial cakes: Trichoderma echinosporum ZJ12 and each of the tested pathogens were inoculated into the center of a fresh PDA plate and incubated at 28℃ for 5-7 days. After the colonies grew to a suitable size, mycelial cakes were cut from the vigorous and uniformly growing area at the edge of the colony using a sterile punch (5 mm inner diameter) for later use.

[0030] (2) Confrontation culture setup: The two-end plate confrontation method was used. Under aseptic conditions, ZJ12 mycelial discs and a pathogenic mycelial disc were inoculated at equal intervals (approximately 6-7 cm apart) at both ends of the same PDA plate, with the mycelial surface in close contact with the culture medium. Each pair of combinations was set up in triplicate. At the same time, each pathogen was individually inoculated in the center of the plate as a control to determine its normal growth diameter.

[0031] (3) Cultivation and observation: Place the inoculated plates in a constant temperature incubator at 28℃. Regularly observe and record the growth of colonies on the antagonistic line and the antagonistic phenomenon.

[0032] (4) Data Measurement and Calculation: After 7 days of incubation, the diameter of pathogen colonies facing the opposing direction in each plate was measured using the cross-crossing method. The specific method was as follows: the length of the two perpendicular intersecting lines passing through the pathogen inoculation point was measured with vernier calipers, and the average value was taken as the opposing growth diameter (D1) of the pathogen. The individual growth diameter (D0) of the pathogen in the control plate was measured in the same way.

[0033] The antibacterial rate is calculated using the following formula: Antibacterial rate = [(D0 - D1) / D0] × 100% 3. Experimental Results After 7 days of plate confrontation culture, Trichoderma echinococcus ZJ12 showed significant antagonistic effects against all 8 tested plant pathogenic fungi (e.g., Figure 3 The results are summarized in the table below: Table 1. Inhibition rate of Trichoderma echinocandes ZJ12 against various pathogenic fungi. Experimental results show that Trichoderma echinocandes ZJ12 has broad-spectrum and highly efficient inhibitory activity against the listed plant pathogenic fungi, with inhibition rates all exceeding 75%. In particular, its inhibitory effect on the pathogens of cotton Verticillium wilt, corn leaf blight, tobacco black shank, and tea black spot is particularly outstanding (inhibition rate > 95%), demonstrating its application potential in the field of biological control.

[0034] Example 3: Indoor bioassay of diamondback moth and cabbage caterpillar by fermentation broth of Trichoderma echinococcus ZJ12 1. Experimental Materials Test strain and fermentation broth preparation: The biocontrol strain was *Trichoderma echinocandes* ZJ12 as described in Example 1. Its mycelial cake was inoculated into potato dextrose liquid (PDB) medium and fermented on a shaker at 28°C and 180 rpm for 14 days. After fermentation, the mycelium was removed by filtration through double-layer sterile gauze to obtain the original fermentation broth. The conidial concentration in the fermentation broth was adjusted to 1 × 10⁻⁶ using a hemocytometer and sterile water. 7 Approximately CFU / mL, store at 4℃ for later use.

[0035] Test insects: healthy 1st-2nd instar larvae of diamondback moth and cabbage caterpillar, provided by Hubei Provincial Engineering Technology Research Center for Biological Pesticides, and reared to the required instar using artificial feed or cabbage leaves in the center's standard artificial climate chamber (temperature 25±1℃, relative humidity 70%±5%, photoperiod 14L:10D).

[0036] Feed and equipment: Special artificial feed for lepidopteran insects (provided by Hubei Provincial Engineering Technology Research Center for Biological Pesticides); fresh cabbage leaves free of disease spots, washed with clean water and air-dried for later use; disposable plastic petri dishes (9 cm in diameter); qualitative filter paper; sterile forceps, scalpel, etc.

[0037] 2. Experimental Methods (1) Preparation of raw test feed or leaves: For the raw test of diamondback moth, the artificial feed was cut into small, uniform pieces using a sterile scalpel. The treatment group immersed the feed pieces in Trichoderma echinocandes ZJ12 fermentation broth (concentration approximately 1×10⁻⁶). 7 Soak in sterile water (CFU / mL) for 5 minutes, then remove with sterile tweezers and place on sterile filter paper to air dry for later use; the control group was treated in the same way with an equal amount of sterile water.

[0038] For the bioassay of cabbage caterpillars, uniformly sized cabbage leaves were used as host materials. The treatment group immersed the leaves in a Trichoderma echinococcus fermentation broth (concentration approximately 1×10⁻⁶). 7 Immerse in sterile water (CFU / mL) for 10 seconds, then remove and air dry on sterile filter paper; the control group is treated with sterile water in the same way.

[0039] (2) Inoculation and treatment: Place a circular filter paper of the same size as the bottom of each disposable petri dish at the bottom to absorb excess moisture. Place an equal amount of the above-treated feed or leaf material in each petri dish, and then use a soft brush to inoculate 20 1-2 instar diamondback moth or cabbage caterpillar larvae of the same size into each petri dish. Each treatment is repeated 3 times.

[0040] (3) Cultivation and observation: The culture dishes were placed in a standard artificial climate chamber for cultivation. The number of dead larvae, the number of surviving larvae, and their growth and development status were observed and recorded daily. Freshly treated feed or leaves were replaced in a timely manner, and residues and excrement were cleaned up. The experiment continued until most of the larvae in the control group had emerged from their larvae.

[0041] (4) Data statistics and analysis: On the 10th day of treatment, the mortality of larvae was counted. Those that successfully pupated and eventually emerged as adults were considered to have survived normally. The Abbott formula was used to calculate the corrected mortality rate.

[0042] 3. Experimental Results Table 2. Results of indoor bioassays of Trichoderma echinococcus ZJ12 against diamondback moth and cabbage caterpillar. Conclusion: Indoor bioassay results (Table 2) showed that the fermentation broth of *Trichoderma echinococcus* ZJ12 had significant stomach poison lethal activity against 1st and 2nd instar larvae of *Plutella xylostella*. On day 7 of treatment, the corrected mortality rate reached 78.35%, which was significantly different from the water control group (P < 0.01). The number of larvae that successfully pupated and emerged from the ZJ12 fermentation broth was significantly lower than that in the control group. Furthermore, the surviving larvae in the treatment group exhibited significant growth and development retardation; their pupation and emergence times were significantly later than those in the control group, and the emerging adults were less vigorous (e.g., ...). Figure 4 Meanwhile, indoor bioassays using the leaf-dipping method on cabbage caterpillars showed that the terminal survival rate of larvae in the ZJ12 fermentation broth treatment group was significantly lower than that in the control group, with a corrected mortality rate of 78.95%, indicating that the fermentation broth also has a significant lethal effect on cabbage caterpillars. Considering its strong lethal activity and developmental retardation effect, it is speculated that *Trichoderma echinococcus* ZJ12 may exert its control effect through insecticidal secondary metabolites produced in its fermentation broth or by disrupting the balance of the larval gut microbiota.

[0043] This embodiment confirms that Trichoderma echinocandes ZJ12 not only has a good inhibitory effect on plant pathogenic fungi, but its fermentation broth also has significant biocontrol activity against lepidopteran pests such as diamondback moth and cabbage caterpillar, providing experimental evidence for its role as a core strain of a multifunctional biological pesticide with both antibacterial and insecticidal functions.

[0044] Example 4: Efficacy experiment of Trichoderma echinocandes ZJ12 against cotton Verticillium wilt in plug seedling cultivation 1. Experimental Materials The biocontrol strain was *Trichoderma echinococcus* ZJ12 as described in Example 1; the target pathogen was *Verticillium dahliae*, the pathogen of Verticillium dahliae in cotton; the test plant was the susceptible cotton variety "Zhongmian Institute 63"; the spore suspension of ZJ12 and *Verticillium dahliae* used in the experiment was prepared according to the method described in Example 3, and the concentration of *Trichoderma echinococcus* ZJ12 spores was adjusted to 1×10⁻⁶. 7 CFU / mL, the concentration of Verticillium dahliae spores was adjusted to 1×10⁻⁶. 6 CFU / mL; cultivation was carried out using 72-well trays and commercially sterilized seedling substrate.

[0045] 2. Experimental Methods (1) Seedling raising and grouping: Cotton seeds were sown in 72-cell trays and cultivated in a greenhouse at 25±2℃ until the four-true-leaf stage for experimental use. The experiment consisted of four treatment groups, with three replicates per group: CK (blank control): No microorganisms were inoculated (water treatment); T1 (Treatment 1): Only ZJ12 fermentation broth was inoculated, without pathogen inoculation; T2 (Treatment 2): Inoculation with pathogens only, without biocontrol treatment; T3 (Biocontrol Treatment Group): First, inoculate with ZJ12 fermentation broth, and then inoculate with pathogens 3 days later.

[0046] (2) Inoculation treatment: Biocontrol treatment: For cotton seedlings in groups T1 and T3, root irrigation was performed using ZJ12 spore suspension, with approximately 20 mL applied to each seedling.

[0047] Pathogen inoculation: Three days after biocontrol treatment (or water treatment), cotton seedlings in groups T2 and T3 were inoculated with pathogens. Approximately 20 mL of the prepared Verticillium dahliae spore suspension was poured into the root zone of each seedling.

[0048] (3) Cultivation and Investigation: All treated cotton seedlings were placed in the same greenhouse (temperature 25±1℃, relative humidity 85%±5%, photoperiod 16L:18D) for routine management. Disease investigation was conducted 30 days after pathogen inoculation. The disease severity grading standards are as follows: Table 3 Disease Rating Standards The prevention and control effect is calculated based on the disease index, using the following formula: Disease index = [∑(Number of diseased plants at each level × Corresponding disease level) / (Total number of plants surveyed × Highest disease level)] × 100 Prevention and control effect = [(Control disease index - Treatment disease index) / Control disease index] × 100% 3. Experimental Results Table 4. Control efficacy of Trichoderma echinocandes ZJ12 against cotton Verticillium wilt. Thirty days after inoculation with the pathogen, cotton seedlings in group T2 (pathogen-treated) showed severe disease, with an average disease index of 85.30. In contrast, group T3, pretreated with ZJ12 fermentation broth, showed a significantly reduced disease index to 14.46, achieving a control effect of 83.05% against cotton Verticillium wilt. Group T1 (treated only with ZJ12) showed no disease symptoms, indicating that ZJ12 itself is safe for cotton seedlings. Compared to the infected T2 group, the growth vigor of cotton seedlings in the T3 treatment group significantly recovered. Figure 5 ).

[0049] Conclusion: This example demonstrates that using Trichoderma echinocandes fermentation broth (≥1×10⁻⁶) during the seedling stage is effective. 7 Pretreatment with root irrigation (using CFU / mL) can effectively achieve a stable control efficacy of over 80%. Simultaneously, this treatment significantly alleviates the pathogen's inhibitory effect on plant growth, demonstrating a dual synergistic effect of "disease prevention + growth promotion," providing crucial practical evidence for developing this strain into a cotton-specific biological agent.

[0050] Example 5: Indoor safety test of Trichoderma echinosporum ZJ12 on parasitic wasps 1. Experimental Materials The biocontrol strain was *Trichoderma echinocandes* ZJ12 as described in Example 1; the test insects were healthy butterfly pupae (*Trichoderma hymenopterus*) within 24 hours of emergence and adult *Brachysmus manila*, both provided by the Hubei Provincial Engineering Technology Research Center for Biological Pesticides. The ZJ12 conidial suspension used in the experiment was prepared according to the method described in Example 3, and the concentration was adjusted to 1×10⁻⁶. 7 CFU / mL.

[0051] 2. Experimental Methods (1) Set up treatment groups: Spray ZJ12 spore suspension evenly onto the inner wall of a transparent rearing tube (specification: Φ3 cm × 8 cm). The amount of spraying should be such that the tube wall is evenly moistened but does not pool into droplets or run. After spraying, let it stand in a ventilated place to dry for 5-10 minutes. Control group: Spray an equal amount of sterile water and treat as above. Each parasitic wasp was set up independently, with 3 replicates per group and 10 wasps per replicate.

[0052] (2) Feeding and observation: All treatments were carried out in a standard artificial climate chamber (temperature 25±1℃, relative humidity 70%±5%, photoperiod 14L:10D), and the parasitic wasps were continuously fed with 20% honey water through a capillary feeder. The number of surviving parasitic wasps was observed and recorded at regular intervals every day for 5 consecutive days.

[0053] (3) Data statistics and analysis: After the experiment, the cumulative survival rate of each group was calculated. The difference in survival rate between the treatment group and the control group was analyzed by independent samples t test, and the significance level was set at α=0.05.

[0054] 3. Experimental Results Table 5. Results of the determination of the effect of Trichoderma echinosporum ZJ12 on the survival rate of parasitic wasps. After the experiment, the survival rates of the two parasitic wasps are shown in Table 5. Data analysis indicates that, under the experimental conditions, the conidia of *Trichoderma echinosporum* ZJ12 (1×10⁻⁶) were the most viable. 7 CFU / mL showed no acute toxicity to adult parasitic wasps and *Trichoderma hymenopsula* (P > 0.05), and *Trichoderma hymenopsula* ZJ12 was re-isolated from the surface of the parasitic wasps after the experiment. Figure 6 This embodiment demonstrates that *Trichoderma echinococcus* ZJ12 exhibits good biosafety against two important natural enemy insects, the pupa *Gnaphalium affine* and *Brachystomum manilaense*. This strain and its microbial inoculum, as "parasite-friendly" biocontrol agents, meet the core requirements of environmental friendliness and non-target biosafety in the integrated pest management (IPM) system.

Claims

1. A parasitic wasp-friendly, multifunctional Trichoderma hygroscopicum strain ( Trichoderma asperellum ZJ12, characterized in that, The accession number is CCTCC NO: M2025805.

2. A microbial inoculant, characterized in that, Includes Trichoderma acicularis ZJ12 as described in claim 1.

3. The microbial agent according to claim 2, characterized in that, The active ingredient of the microbial agent is the mycelium, fermentation broth, or conidia of Trichoderma echinocandes ZJ12.

4. The application of Trichoderma echinocandes ZJ12 as described in claim 1 or the microbial agent as described in claim 2 in the prevention and control of plant diseases.

5. The application according to claim 4, characterized in that, The plant diseases mentioned include: pepper wilt, pepper root rot, wheat stem base rot, cotton verticillium wilt, tea leaf spot, tobacco black shank, corn leaf blight, and tea black spot.

6. The application of *Trichoderma echinocandes* ZJ12 as described in claim 1 or the microbial inoculant as described in claim 2 in inhibiting plant pathogens, characterized in that... The pathogens include: Fusarium oxysporum (… Fusarium oxysporum Fusarium solani () Fusarium solani Fusarium pseudograss ( ), Fusarium pseudograminearum Verticillium dahliae Verticillium dahliae ), Tea-like spores ( Pestalotiopsis theae ), Tobacco Phytophthora ( Phytophthora nicotianae ), large spot disease bulging umbelliferous worms ( Exserohilum turcicum ) and *Ulva globosum* ( Nigrospora sphaerica ).

7. The application according to claim 4 or 5, characterized in that, The application method is as follows: soak plant seeds in the fermented spore liquid of Trichoderma echinocandes ZJ12, or irrigate the plant roots, or mix the ZJ12 strain with an organic carrier to prepare a biological agent for application.

8. The application of Trichoderma echinocandes ZJ12 as described in claim 1 or the microbial agent as described in claim 2 in the control of insect pests.

9. The application according to claim 8, characterized in that, The insect pests include the diamondback moth ( Plutella xylostella ) and cabbage caterpillar ( Pieris rapae ).

10. The application according to claim 8 or 9, characterized in that, The aforementioned Trichoderma ZJ12 or microbial inoculant can be used in conjunction with parasitic wasps for pest and disease control.

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