A screening method for biocontrol trichoderma strains based on the dual criteria of oomycete parasitism and lethality

Abstract

This invention proposes a screening method for biocontrol Trichoderma strains based on the dual criteria of oomycete parasitism and lethality. Belonging to the field of agricultural biotechnology, this invention addresses the technical problem that existing screening techniques often rely solely on the growth inhibition rate of confrontation cultures, making it difficult to distinguish between the antibacterial and lethal effects of biocontrol bacteria. This invention establishes a progressive screening system. The method uses highly pathogenic oomycetes, namely *Phytophthora capsici* (… Phytophthora capsici ) and ultimate pyrophyllium ( Globisporangium ultimum Using oomycetes as the target, and based on the determination of growth inhibition rate through confrontation culture, this invention creatively introduces a step of "pathogen re-isolation and detection of activity based on oomycete selective culture medium" combined with microscopic observation of hyperparasitism. This method effectively eliminates false-positive strains, significantly improves the accuracy of biocontrol strain screening and field efficacy, and is suitable for large-scale exploration of biocontrol resources.

Description

Technical Field

[0001] This invention relates to the field of agricultural biotechnology, specifically to a method for screening biocontrol Trichoderma strains based on the dual criteria of oomycete parasitism and lethality. Background Technology

[0002] Soil-borne diseases caused by oomycetes, such as Phytophthora capsici (… Phytophthora capsici ) and ultimate pyrophyllium ( Globisporangium ultimum Root rot, caused by *Trichoderma*, is a persistent problem in agricultural production. Trichoderma Fungi (spp.) are widely used for the biological control of this type of disease due to their rapid growth and diverse antagonistic mechanisms.

[0003] In the research and development of biocontrol strains, the scientific nature of the screening method directly determines the effectiveness of the final product. Currently, the commonly used screening method both domestically and internationally is the "plate confrontation method," which involves inoculating pathogenic bacteria and biocontrol bacteria separately in petri dishes, and evaluating the quality of biocontrol bacteria by measuring the size of the inhibition zone or calculating the growth inhibition rate.

[0004] The main drawbacks of existing technology are: Traditional screening indicators (inhibition rate) often only reflect the ability of Trichoderma strains to delay the growth of pathogens (i.e., antifungal effect), but cannot distinguish whether they have killing ability (i.e., lethal effect). Under laboratory plate conditions, due to space and nutrient limitations, many Trichoderma strains can show high inhibition rates; however, in the complex field soil environment, once the growth vigor of Trichoderma weakens or environmental conditions change, pathogenic oomycetes (such as chlamydospores) that are only "inhibited" but not "killed" are very likely to reactivate and re-infect crops. In addition, simple plate covering does not represent effective parasitism; sometimes it is simply because the rapid growth rate of Trichoderma masks the pathogen.

[0005] Therefore, establishing a more rigorous screening system that can accurately eliminate "false positive" Trichoderma strains and select highly efficient Trichoderma strains that can both compete for growth and substantially kill oomycete pathogens through hyperparasitism is a key technical challenge in the field of biocontrol bacteria research and development. Summary of the Invention

[0006] The purpose of this invention is to propose a screening method for biocontrol Trichoderma strains based on the dual criteria of oomycete parasitism and lethality. The method introduces a key verification step of "re-isolation and lethality detection of pathogens" to ensure that the screened strains have real bactericidal activity.

[0007] The technical solution of this invention is implemented as follows: This invention provides a method for screening Trichoderma strains based on the dual criteria of parasitism and lethality against oomycete pathogens. The method includes the following steps: (1) Initial screening (growth competition test): The Trichoderma strain to be screened and the target oomycete pathogen were cultured in a confrontation culture on a solid culture medium. When the control group pathogen was fully grown or nearly fully grown, the growth inhibition rate of the Trichoderma strain against the pathogen was measured and calculated. Strains with an inhibition rate lower than the set threshold were removed. (2) Secondary screening (microscopic parasitism verification): Select the areas where the colonies in step (1) come into contact or overlap, and conduct microscopic observation to screen out Trichoderma strains that exhibit entanglement, adsorption or penetration parasitism behavior on the hyphae of pathogens. (3) Final selection (lethality verification): Select the pathogen hyphae or mycelium from the area covered by Trichoderma colonies in step (2) and inoculate them on oomycete selective medium for re-isolation culture; (4) Result determination: If the pathogen cannot grow on the oomycete selective medium, the Trichoderma strain is determined to be lethal and is taken as the target superior strain; if the pathogen can grow, the Trichoderma strain is determined to be only inhibitory to oomycetes and not lethal.

[0008] As a further improvement of the present invention, the target oomycete pathogen mentioned in step (1) is Phytophthora capsici ( Phytophthora capsici ) and ultimate pyrophyllium ( Globisporangium ultimum = Pythium ultimum ).

[0009] As a further improvement of the present invention, the confrontation culture described in step (1) is carried out on a PDA plate at 28 ± 1℃ for 5-7 days.

[0010] As a further improvement of the present invention, the selective culture medium for oomycetes described in step (3) is based on oat culture medium (30 g oat flakes boiled and filtered to retain juice, 15 g sucrose, 15 g agar powder, and water added to make up to 1 L), with the addition of a selective antibacterial agent that inhibits the growth of Trichoderma but allows the growth of oomycetes.

[0011] As a further improvement of the present invention, the antibacterial agent against Trichoderma mentioned in step (3) includes nystatin 25 mg / L and thiophanate-methyl (suspension, with a total effective ingredient content of 41%, of which thiophanate-methyl contains 34.2% and tebuconazole contains 6.8%) 50 mg / L.

[0012] As a further improvement of the present invention, the pathogenic fungal cakes picked in step (3) should be taken from the area 1-2 cm inward from the edge of the pathogenic fungal colony covered by the Trichoderma colony, so as to ensure that the Trichoderma and the pathogenic fungus have sufficient contact.

[0013] As a further improvement of the present invention, the result determination time in step (4) is 3-7 days after inoculation into the oomycete selective culture medium.

[0014] Preferably, this invention proposes a highly efficient screening method for biocontrol Trichoderma strains based on the dual criteria of oomycete parasitism and lethality. Its core lies in establishing a three-level progressive screening system of "competition-parasitism-lethality." The specific steps are as follows: 1. Initial screening (assessment of growth competition ability): using highly pathogenic *Phytophthora capsici* (… Phytophthora capsici ) and ultimate pyrophyllium ( Globisporangium ultimum The target bacteria were selected using a confrontation culture method. The Trichoderma strains to be screened and the pathogens were cultured separately on PDA plates to the appropriate stage. Mycelial discs (7 mm in diameter) were then collected and placed on opposite sides of a 9 cm diameter PDA plate, and incubated at 28°C for 5-7 days. The colony diameter was measured, and the inhibition rate was calculated using the formula: Inhibition rate (%) = [(Control colony diameter − Treated colony diameter) / Control colony diameter] × 100.

[0015] 2. Secondary Screening (Microscopic Assessment of Parasitic Ability): In the later stages of confrontation culture, when Trichoderma colonies come into contact with and cover pathogen colonies, the hyphal morphology of the contact zone and the covered area is observed under a microscope. The focus is on screening for Trichoderma strains that exhibit entanglement, adsorption, parallel growth, or penetration of pathogen hyphae. This step excludes strains that only suppress pathogens by producing antibiotics or volatile substances but lack contact parasitic ability.

[0016] 3. Final Selection (Substantial Assessment of Lethality): This is the core step of this invention. Select pathogenic fungal discs or agar blocks containing pathogenic fungi from the area completely covered by *Trichoderma* hyphae in step 2, and transfer them to an oomycete selective medium for re-isolation culture. Simultaneously, use unparasitized oomycetes as a control. Observe the growth after incubation at 28°C for 3-5 days. If the target pathogenic hyphae are observed to regrow on the selective medium, it indicates that although the pathogen is covered, it is not dead, and the *Trichoderma* strain only exhibits inhibitory activity. If no colony growth is observed, it indicates that the pathogen has been parasitized and killed by *Trichoderma*, and the *Trichoderma* strain is lethal.

[0017] This invention further protects the application of Trichoderma strains obtained by the above method in the preparation of biological pesticides for controlling oomycete diseases in plants.

[0018] The present invention has the following beneficial effects: 1. High screening accuracy: The screening strategy proposed in this invention creatively introduces lethality verification (reisolation experiment), which utilizes the difference in drug sensitivity between oomycetes and fungi to effectively eliminate "false positive" strains that perform well on plates but cannot completely kill pathogens in actual microecology, greatly improving screening efficiency and the success rate of biocontrol bacteria in field application.

[0019] 2. The selected strains exhibit superior control efficacy: The Trichoderma strains screened using this method possess a triple mechanism of spatial competition, hyperparasitism, and lethality. Experiments show that these strains demonstrate significantly better control efficacy against root rot in pot and field trials compared to strains screened solely based on inhibition rates.

[0020] 3. High versatility, no reliance on specific strains for preservation: This invention provides a universal methodological tool. Researchers can use this method to independently screen highly efficient strains adapted to local environments from soil or plant samples from various locations, making it widely applicable and valuable for promotion. Attached Figure Description

[0021] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art 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.

[0022] Figure 1 To compare the effects of different Trichoderma strains on Phytophthora capsici ( Phytophthora capsici ) and ultimate Pythium ( Globisporangium ultimum The inhibition rate comparison chart.

[0023] Figure 2 Trichoderma acicularis ( Trichoderma asperellum NY-1 against Phytophthora capsici ( Phytophthora capsici ) and ultimate pyrophyllium ( Globisporangium ultimum The plate inhibition effect of the co-culture group (inoculated with Trichoderma nystatin NY-1 and the pathogen) is shown from left to right. The front and back of the plates are from the control group (inoculated with pathogen only) and the co-culture group (inoculated with Trichoderma nystatin NY-1 and the pathogen at the same time). The top row is the target pathogen Phytophthora capsici, and the bottom row is the target pathogen Pythium cerevisiae.

[0024] Figure 3 Trichoderma acicularis ( Trichoderma asperellum NY-1 is effective against Phytophthora capsici ( Phytophthora capsici ) and ultimate Pythium ( Globisporangium ultimum Microscopic observation of parasitism (black arrows indicate the tight entanglement and adsorption sites of Trichoderma hyphae on pathogen hyphae).

[0025] Figure 4The results show the survival rate of pathogens after being reparasitized by Trichoderma strains. The left half shows the reisolation results of *Phytophthora capsici* after parasitization by *Trichoderma nystatin* NY-1, with no pathogenic hyphae growth after culture; the right half shows the reisolation results of *Phytophthora capsici* after parasitization by *Trichoderma harzianum* TH13, with *Phytophthora capsici* colonies (white aerial hyphae) visible after culture. These results distinguish between the "lethal" and "non-lethal (inhibitory)" biocontrol modes of *Phytophthora capsici* by Trichoderma strains.

[0026] Figure 5 A comparative diagram showing the control efficacy of lethal and non-lethal Trichoderma strains against Phytophthora root rot in peppers in potted plants.

[0027] A: Phenotypic characteristics of the aboveground parts and roots of pepper plants under different treatments (from left to right: water control (H2O), chemical control (oxamyl, 360 mg / L), lethal strain Trichoderma ny-1, non-lethal strain Trichoderma harzianum TH13); B: Incidence rate of Phytophthora root rot in peppers in each treatment group; C: Disease index of Phytophthora root rot in peppers in each treatment group; D: Relative control efficacy of each treatment group. Detailed Implementation

[0028] The technical solutions in the embodiments of the present invention will be clearly and completely described below. 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.

[0029] Example 1: Establishment and Implementation of the Screening System 1. Test materials Target pathogen: Phytophthora capsici, a highly pathogenic oomycete pathogen ( Phytophthora capsici ) and ultimate pyrophyllium ( Globisporangium ultimum ).

[0030] Trichoderma tested: Five Trichoderma strains isolated and purified from rhizosphere soils of different crops were identified as Trichoderma echinosporum (… Trichoderma asperellum NY-1, Trichoderma viride ( Trichoderma atroviride TH5, Trichoderma longifolia ( Trichoderma longibrachiatum TH8, Corning Trichoderma ( Trichoderma koningii TH10, Trichoderma harzianum ( Trichoderma harzianum TH13 Culture media: PDA medium (basic formula); Oomycetes selective medium: based on oat medium, with the addition of nystatin 25 mg / L and thiophanate-methyl·tebuconazole (suspension, total effective ingredient content of 41%, including 34.2% thiophanate-methyl and 6.8% tebuconazole) 50 mg / L, pH 7.0.

[0031] 2. Screening Steps (1) Inhibition rate determination (initial screening) Trichoderma and the pathogen were inoculated onto opposite sides of a PDA plate and incubated at 28°C for 5 days. The diameter of the pathogen colony was measured and the inhibition rate was calculated as follows: Inhibition rate (%) = [(Control colony diameter − Treatment colony diameter) / Control colony diameter] × 100.

[0032] The inhibition rate results show that ( Figure 1 The tested Trichoderma strains included *Trichoderma echinococcus* NY-1, *Trichoderma viride* TH5, *Trichoderma longifolia* TH8, *Trichoderma corniglans* TH10, and *Trichoderma harzianum* TH13, all of which showed inhibitory activity against the two target pathogens. The inhibition rates against *Phytophthora capsici* ranged from 40.97% to 53.71%, with *Trichoderma echinococcus* NY-1 showing the highest inhibition rate (53.71%). The inhibition rates against *Pythium cerevisiae* ranged from 47.14% to 72.47%, generally higher than the inhibition against *Phytophthora capsici*, with *Trichoderma echinococcus* NY-1 showing the highest inhibition rate (approximately 72.47%). From the plate analysis... Figure 2 In the control group, both pathogens could freely spread and grow; however, in the co-culture group, *Trichoderma echinococcus* NY-1 showed significantly better inhibitory activity against *Phytophthora capsici* and *Pythium tertii* than other tested strains. Its hyphae expanded rapidly, completely covering *Phytophthora capsici* and leaving only a very small colony for *Pythium tertii*, effectively limiting the growth and spread of the pathogens. This strain's mechanism of action includes nutrient competition, space occupation, and secretion of antimicrobial substances, possessing broad-spectrum and highly effective biocontrol potential, and can be used as a biocontrol agent to control plant diseases caused by these two pathogens.

[0033] (2) Microscopic observation of parasitism (rescreening) After the Trichoderma colonies covered the pathogen, the junction of the hyphae was observed under a microscope.

[0034] The results show that ( Figure 3 When co-cultured with two pathogens, *Trichoderma echinococcus* NY-1 exhibited clear microparasitic characteristics: on the hyphal surfaces of *Phytophthora capsici* and *Pythium cerevisiae*, typical structures (marked with black arrows) were observed where *Trichoderma echinococcus* hyphae tightly entwined the pathogen hyphae, accompanied by the adsorption and attachment of *Trichoderma* hyphae to the pathogen hyphae. This directly confirmed that this strain achieves parasitic inhibition of pathogens through hyphal entanglement and adsorption. In contrast, compared with other tested strains, only *Trichoderma echinococcus* NY-1 showed a clear and stable microparasitic structure; the other strains either did not cover the pathogen colonies or, although they did cover them, no clear microparasitic characteristics were observed. Based on these results, *Trichoderma echinococcus* NY-1 was selected as a superior biocontrol strain with active parasitic capabilities, and its microparasitic mechanism provides crucial cytological evidence for the control of target diseases.

[0035] (3) Lethal reseparation verification (final selection) From the coverage area (pathogen side) of the above 5 preferred strains, cut 5 mm mycelial cakes and transfer them to oomycete selective medium, and incubate at 28°C for 5 days.

[0036] The reseparation results show that ( Figure 4 Phytophthora capsici was re-isolated from the colony area parasitized by Trichoderma nystatin NY-1. After transferring the mycelial blocks to a selective medium (left half) and culturing for 5 days, no Phytophthora capsici mycelium grew, indicating that strain NY-1 completely killed the pathogen through reparasitism, rendering it unable to survive. Re-isolation from the parasitized area of ​​Trichoderma harzianum TH13 resulted in the growth of sparse white aerial mycelium, which was confirmed as Phytophthora capsici by microscopic examination. This indicates that strain TH13 only inhibited the growth of the pathogen, but could not completely kill it; the pathogen could resume growth after being released from Trichoderma inhibition. These results confirm that strain NY-1 is a biocontrol strain with the ability to completely kill pathogens, and its biocontrol effect is significantly better than that of strain TH13, which only inhibits the pathogen. It has more stable and thorough application potential in plant disease control.

[0037] Example 2: Verification of the effectiveness of the method (comparison of prevention and efficacy) To demonstrate the superiority of the screening method of the present invention, strain TH13 (representing the superior strain screened by the traditional method), which was identified as "antibacterial", and strain NY-1 (representing the superior strain screened by the method of the present invention), which was identified as "lethal", were selected in Example 1 for comparison of the control efficacy against root rot caused by Phytophthora capsici in potted plants.

[0038] 1. Materials and Methods 1.1 Test Materials Plant material: The chili variety "Changfeng 101" seeds were disinfected with 75% ethanol for 30 seconds and 0.1% HgCl2 for 5 minutes, then rinsed 5 times with sterile water, and sown in sterilized seedling substrate (peat moss: vermiculite = 3:1) after germination.

[0039] Test strain: Trichoderma hygroscopica ( Trichoderma asperellum NY-1, Trichoderma harzianum ( Trichoderma harzianum TH13 was cultured in PDA medium at 28°C for 7 days to prepare 1×10⁻⁶ ppm. 7 A suspension of conidia per mL was prepared for use; *Phytophthora capsici* ( Phytophthora capsici After being cultured in V8 medium (100 mL V8 vegetable juice, 3 g calcium carbonate (CaCO3), 15 g agar powder, and 900 mL distilled water) at 28°C for 7 days, 1×10⁻⁶ ppm was prepared. 6 Prepare a zoospore suspension of 1 spore / mL for later use.

[0040] Chemical reagent: Oxyphenidyl (98% active ingredient, soluble powder), diluted to 360 mg / L as a chemical control reagent.

[0041] 1.2 Pot Experiment After germination, chili seeds were sown in pots with a diameter of 12cm and a height of 10cm, with 3 seeds per pot. The pots were placed in a greenhouse (28±2℃, 16h / 8h light, 70%-80% relative humidity) to cultivate seedlings until they reached the 4-5 leaf stage. Seedlings with uniform growth were selected for pot experiments.

[0042] The experiment was set up with 4 treatment groups, each group having 3 biological replicates, and each replicate containing 15 seedlings. All pepper seedlings were first treated with 30 mL of Phytophthora capsici zoospore suspension (concentration 1×10⁻⁶). 6 Root irrigation with 30 mL of sterile water was performed at 24-hour intervals, followed by different treatments: 30 mL of sterile water was used as a control group (H2O); 30 mL of oxamyl solution (360 mg / L) was used as a chemical control (oxamyl); and 30 mL of Trichoderma nystatin NY-1 and Trichoderma harzianum TH13 spore suspensions (1×10⁻⁶) were used as a control group. 7 CFU / mL) were applied as a root drench (Trichoderma ny-1 treatment group and Trichoderma harzianum TH13 treatment group). All seedlings of the above four treatments were placed in a greenhouse (28±2℃, 16h / 8h light, 80%-90% relative humidity) for cultivation, and the substrate was kept moist during the period.

[0043] Fourteen days after inoculation, the aboveground phenotypes (leaf color, degree of wilting) and root phenotypes (degree of rot) of each group of pepper plants were observed and recorded. Disease severity was assessed according to the following grading criteria: Pepper root rot grading standards: Grade 0, no disease spots at the base of the stem; Grade 1: Lesions surround less than 25% of the stem base; Grade 3, lesions surround 25-50% of the stem base; Level 5, lesions surround 51-75% of the stem base, and the plant wilts; Level 7: Lesions surround more than 76% of the stem base, causing the plant to wilt and approach or die.

[0044] Incidence rate (%) = (Number of infected plants / Total number of plants) × 100.

[0045] Disease index = Σ (Disease level × Corresponding number of plants) / (Highest disease level × Total number of plants) × 100.

[0046] Relative efficacy: Relative efficacy (%) = (Disease index of control group - Disease index of treatment group) / Disease index of control group × 100.

[0047] 2. Results This experiment verified the field application potential of Trichoderma strains with different biocontrol mechanisms through pot inoculation. Four treatment groups were set up: a water control, a chemical agent (oxamyl) control, a lethal Trichoderma nystatin NY-1, and a non-lethal Trichoderma harzianum TH13. The results are as follows: Plant phenotype ( Figure 5 A) showed that in the water control group (H2O), the aboveground parts of the pepper plants were wilted and yellowed, and the roots showed severe rot; most plants in the hymexazol and Trichoderma NY-1 treatment groups grew vigorously, with dark green leaves and well-developed roots without rot; most plants in the Trichoderma TH13 treatment group showed severe wilting, but the roots showed slight rot symptoms, and their growth was weaker than the first two groups. Morbidity and disease index ( Figure 5 (B) and (C) show that the disease incidence rate in the water control group was close to 100%, with a disease index of 85.08; the incidence rates in the hymexazol and Trichoderma nystatus NY-1 treatment groups were both below 20%, and the disease indexes were both below 30, indicating comparable control effects; the incidence rate in the Trichoderma harzianum TH13 treatment group was approximately 26.67%, and the disease index was approximately 38.41, significantly higher than the previous two groups. Relative control efficacy ( Figure 5 D) shows that the relative efficacy of both hymexazol and Trichoderma nystatin NY-1 is over 70%, with the efficacy of Trichoderma nystatin NY-1 showing no significant difference from that of chemical agents; the relative efficacy of Trichoderma harzianum TH13 is 54.85%, significantly lower than the former two.

[0048] 3. Conclusion The lethal Trichoderma NY-1 strain showed significantly better potted plant control efficacy against Phytophthora capsici in peppers than the non-lethal Trichoderma TH13 strain, and was comparable to the chemical agent oxamyl. This indicates that strains with the ability to completely kill pathogens have more stable and superior application effects in actual disease control and can be used as potential biocontrol agents to replace chemical agents.

[0049] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for screening biocontrol Trichoderma strains based on the dual criteria of oomycete parasitism and lethality, characterized in that, Includes the following steps: (1) Initial screening: The Trichoderma strain to be screened and the target oomycete pathogen were cultured in a confrontation culture on a solid culture medium. When the pathogen in the control group was fully grown or nearly fully grown, the growth inhibition rate of the Trichoderma strain against the pathogen was measured and calculated. Strains with an inhibition rate lower than the set threshold were removed. (2) Secondary screening: Select the areas where the colonies in step (1) come into contact or overlap, and conduct microscopic observation to screen out Trichoderma strains that exhibit entanglement, adsorption or penetration parasitism behavior on the hyphae of pathogens. (3) Final selection: Select the pathogenic hyphae or mycelium from the area covered by Trichoderma colonies in step (2) and inoculate them on oomycete selective medium for re-isolation culture; (4) Result determination: If the pathogen cannot grow on the oomycete selective medium, the Trichoderma strain is determined to be lethal and is taken as the target superior strain; if the pathogen can grow, the Trichoderma strain is determined to be only inhibitory to oomycetes and not lethal.

2. The method for screening biocontrol Trichoderma strains based on the dual criteria of oomycete parasitism and lethality as described in claim 1, characterized in that, The target oomycete pathogens mentioned in step (1) are Phytophthora capsici and Pythium cerevisiae.

3. The method for screening biocontrol Trichoderma strains based on the dual criteria of oomycete parasitism and lethality as described in claim 1, characterized in that, The confrontation culture described in step (1) is carried out on a PDA plate at 28 ± 1℃ for 5-7 days.

4. The method for screening biocontrol Trichoderma strains based on the dual criteria of oomycete parasitism and lethality as described in claim 1, characterized in that, The selective medium for oomycetes mentioned in step (3) is based on oat medium with the addition of a selective antibacterial agent that inhibits the growth of Trichoderma but allows the growth of oomycetes.

5. The method for screening biocontrol Trichoderma strains based on the dual criteria of oomycete parasitism and lethality according to claim 1, characterized in that, The antifungal agent for Trichoderma mentioned in step (3) includes nystatin 25 mg / L and thiophanate-methyl 50 mg / L.

6. The method for screening biocontrol Trichoderma strains based on the dual criteria of oomycete parasitism and lethality as described in claim 1, characterized in that, In step (3), the pathogenic fungal cakes should be taken from the area 1-2 cm inward from the edge of the Trichoderma colony covering the pathogenic fungal colony.

7. The method for screening biocontrol Trichoderma strains based on the dual criteria of oomycete parasitism and lethality as described in claim 1, characterized in that, The result determination time mentioned in step (4) is 3-7 days after inoculation into the oomycete selective medium.

8. The application of the Trichoderma strains screened using the method described in any one of claims 1-8 in the preparation of biological pesticides for controlling oomycete diseases of plants.

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