Trichoderma harzianum, trichoderma graminearum, trichoderma graminearum and application thereof in agriculture

Abstract

The invention relates to the field of microorganisms, and particularly provides three types of trichoderma strains, namely, a trichoderma harzianum strain T31818-A (the preservation number is GDMCC (Gramineae Microorganism Center for March Center) NO.64772), a trichoderma gramineae strain YNE00410-A (the preservation number is GDMCC NO.64822), a trichoderma gramineae strain YNE00489-A (GDMCC NO.64775), a trichoderma viride strain YNE00489-A (the preservation number is GDMCC NO.64775) and a trichoderma viride strain YNE00489-A (the preservation number is GDMCC NO.64775). The invention also provides application of the three trichoderma strains in agriculture: promoting plant growth, preventing and treating plant diseases and improving plant disease resistance.

Description

Technical Field

[0001] The invention relates to the field of microorganisms, and in particular to three Trichoderma strains and their application in agriculture. Background Art

[0002] Trichoderma is an important type of biocontrol fungus. It has become a highly respected biocontrol agent due to its low cost, high efficiency and environmental protection. In the field of biological control of various plant fungal diseases, the application of Trichoderma is particularly common. In the biological control of plant diseases, Trichoderma relies on competition, parasitism, antibiotics and induced resistance to achieve control of plant diseases.

[0003] Trichoderma belongs to the kingdom Fungi, phylum Ascomycota, order Hypocreales, family Hypocreaceae. The genus is diverse and numerous, with more than 500 species. Trichoderma is rich in resources and widely distributed, and has important biocontrol functions. Trichoderma preparations used to prevent and control plant diseases account for an increasingly large proportion of the market. Trichoderma harzianum, Trichoderma virens, Trichoderma viride, Trichoderma longibrachiatum, Trichoderma reesei, Trichoderma koningii, Trichoderma atroviride, Trichoderma asperellum, Trichoderma ghanense, Trichoderma guizhouense, etc. have been developed into biocontrol agents for plant diseases. However, the resources of Trichoderma species used as biocontrol are still very limited. Therefore, fully tapping Trichoderma resources, screening and evaluating the biocontrol activity of Trichoderma, and developing new Trichoderma resources can lay a solid foundation for in-depth biological control of plant diseases.

[0004] The invention of CN109112072A, "Application of Trichoderma in Promoting Plant Growth", states that the Trichoderma is Trichoderma asperellum, strain number: HTTA-Z0002, and deposit number: CGMCC No. 15677. The HTTA-Z0002 strain can degrade phosphorus, potassium and cellulose, produce plant hormones and iron carriers, and promote plant growth in many aspects, and can induce plant resistance to stress and salt. It is a strain that has a multifunctional effect on promoting plant growth and can reduce the use of chemical fertilizers.

[0005] The invention of CN114891645B "Trichoderma asperellum HB40619 and its application": the Trichoderma is Trichoderma asperellum, strain number: HB40619, and preservation number CGMCC No.40143. HB40619 is used in preventing and controlling tomato leaf wilt, in preparing biocontrol agents for preventing and controlling diseases caused by Fusarium oxysporum, and in preparing agents for preventing and controlling tomato leaf wilt; Trichoderma aspergillus HB40619 can effectively inhibit Fusarium oxysporum; tomato seedlings treated with a mixed spore solution of Trichoderma aspergillus HB40619 strain and Fusarium oxysporum showed a significant disease resistance to Fusarium oxysporum; at the same time, after measuring chlorophyll fluorescence-related parameters and hormone content in leaves, it was found that Trichoderma aspergillus HB40619 strain can protect photosynthetic system II in tomato seedlings and can induce changes in plant hormone content to improve disease resistance; and can regulate changes in resistance hormones in plants, induce defense responses in tomato seedlings, and improve disease resistance.

[0006] The invention of CN 114836329 B, "Trichoderma harzianum HB40609 and its application": the Trichoderma is Trichoderma harzianum, the strain number is HB40609, and the deposit number is CGMCC No. 40142. HB40609 is used in the prevention and treatment of tomato leaf wilt, in the preparation of biological control preparations for the prevention and treatment of diseases caused by Fusarium oxysporum, and in the preparation of preparations for the prevention and treatment of tomato leaf wilt; HB40609 can effectively inhibit Fusarium oxysporum; the treated tomato seedlings show a more significant disease resistance to Fusarium oxysporum; at the same time, after measuring the chlorophyll fluorescence related parameters and hormone content in the leaves, it is found that the HB40609 strain can protect the photosynthetic system II in the tomato seedlings and can induce changes in the hormone content of the plant to improve disease resistance.

[0007] The invention of CN 113862160 B, "A strain of Trichoderma pseudokoningii Tk905 with biological control and induced disease resistance and its application": the Trichoderma is Trichoderma pseudokoningii, strain number Tk905, and the deposit number is GDMCC No: 61934. The Tk905 strain and its fermentation liquid have a significant inhibitory effect on the growth of various plant pathogenic fungi, and can cover a variety of pathogenic fungi in the later stage, and produce a large number of conidia around them, showing extremely strong competitiveness and broad-spectrum anti-plant pathogenic fungi activity. At the same time, the Trichoderma pseudokoningii Tk905 strain can also induce plants to produce disease resistance, has huge biological control potential, and has a great development and application prospect.

[0008] Alternaria sp. is a widely distributed, broad-spectrum, important pathogenic fungus that causes leaf spot diseases in many plants. Botrytis aclada causes gray mold in onions. Botrytis cinerea is a widely distributed, broad-spectrum, plant pathogenic fungus that causes gray mold in many plants. Fusarium graminearum, also known as Fusarium graminearum, is a widely distributed, important pathogenic fungus that causes head blight in wheat and barley. This species also causes root rot, wilt, and corn stalk rot in many plants. Fusarium oxysporum, also known as Fusarium oxysporum, is a widely distributed, important plant pathogenic fungus that causes wilt in many plants. Plectosphaerella cucumerina is an important plant pathogenic fungus that causes root rot, blackleg, and leaf spot diseases in many plants. Rhizoctonia solani is an important plant pathogenic fungus that causes rice sheath blight, corn sheath blight, and damping-off diseases of many plants. Sclerotinia sclerotiorum is an important broad-spectrum plant pathogenic fungus that causes sclerotinia disease in many plants including Cruciferae, Leguminosae, Cucurbitaceae, etc. Pythium sp. is an important plant pathogenic oomycete that causes damping-off disease in the seedling stage of many plants and rot in the adult stage. Ustilaginoidea virens is an important and widely distributed plant pathogenic fungus that causes false smut disease of rice.

[0009] Cai and Druzhinina (Fungal Diversity, 2021, 107(1): 1-69) developed a standard scheme for molecular identification of Trichoderma, which was recognized and promoted by the International Committee on Taxonomy of Trichoderma (ICTT). That is, if a Trichoderma fungus meets the sequence similarity values ​​of rpb2 ≥ 99% and tef1 ≥ 97% of a unique species, it can be identified as that Trichoderma species. If it does not meet the requirements, further phylogenetic analysis is performed to determine whether it is a new species. Summary of the invention

[0010] The technical problem to be solved by the present invention is to provide the application of three Trichoderma strains in agriculture. The three Trichoderma strains are Trichoderma densissimum strain T31818-A, Trichoderma graminis strain YNE00410-A and Trichoderma graminicola strain YNE00489-A. The application in agriculture includes promoting plant growth and preventing and controlling plant diseases (improving plant disease resistance).

[0011] In order to solve the above technical problems, the present invention provides the following three solutions:

[0012] Solution 1:

[0013] The invention provides Trichoderma densissimum T31818-A, whose deposit number is GDMCCNO:64772.

[0014] The strain Trichoderma densissimum T31818-A of the present invention has a deposited name of Trichodermadensissimum, a deposited unit of Guangdong Microbiological Culture Collection Center, a deposited address of 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, a deposited number of GDMCC NO: 64772, and a deposited date of June 20, 2024.

[0015] The invention also provides the use of Trichoderma densissimum T31818-A: it is used for promoting plant growth and preventing and controlling plant diseases (improving plant disease resistance) in plant cultivation.

[0016] As an improvement on the use of Trichoderma densissimum T31818-A of the present invention: strain T31818-A can produce iron carriers, degrade cellulose, inhibit the growth of plant pathogenic fungi and plant pathogenic oomycetes, has induced resistance to gray mold on aboveground parts, and can multiply in large quantities in a matrix.

[0017] Plant pathogenic fungi include: Botrytis aclada, Fusarium graminearum, Plectosphaerella cucumerina, Pythium aristosporum, Rhizoctonia solani and Sclerotiniasclerotiorum; plant pathogenic oomycetes include Pythium aristosporum.

[0018] Compared with the Trichoderma described in the background technology, the strain T31818-A of the present invention has the following technical advantages:

[0019] 1. Provide a new Trichoderma species;

[0020] 2. Ability to reproduce in large numbers in the substrate;

[0021] 3. Used to prevent and control soil-borne Fusarium wilt;

[0022] 4. Induced resistance to gray mold on aboveground parts

[0023] 5. It has broad-spectrum antibacterial activity: It has antibacterial activity against plant pathogenic fungi and plant pathogenic oomycetes such as Botrytis aclada, Fusarium graminearum, Plectosphaerella cucumerina, Pythium aristosporum, Rhizoctonia solani and Sclerotiniasclerotiorum;

[0024] 6. Degrade cellulose and produce iron carriers;

[0025] 7. Promote plant growth.

[0026] Option 2:

[0027] The invention provides Trichoderma graminis YNE00410-A, whose deposit number is GDMCCNO:64822.

[0028] The strain Trichoderma graminis YNE00410-A of the present invention has a deposited name of Trichoderma graminis, a deposited unit of Guangdong Microbiological Culture Collection Center, a deposited address of 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, a deposited number of GDMCCNO:64822, and a deposited date of June 20, 2024.

[0029] The present invention also provides the use of the Trichoderma graminis YNE00410-A: for promoting plant growth and preventing and controlling plant diseases (improving plant disease resistance) in plant cultivation.

[0030] As an improvement of the use of Trichoderma graminis YNE00410-A of the present invention: YNE00410-A can produce iron carriers, degrade cellulose, inhibit the growth of plant pathogenic fungi, has induced resistance to gray mold on the aboveground parts, and can multiply in large quantities in a substrate.

[0031] Plant pathogenic fungi include: Rhizoctonia solani, Sclerotinia sclerotiorum, Fusarium graminearum and Ustilaginoideavirens.

[0032] Compared with the Trichoderma described in the background technology, the strain YNE00410-A of the present invention has the following technical advantages:

[0033] 1. Provide a new Trichoderma species;

[0034] 2. Ability to reproduce in large numbers in the substrate;

[0035] 3. Used to prevent and control soil-borne Fusarium wilt;

[0036] 4. Induced resistance to gray mold on aboveground parts;

[0037] 5. It has broad-spectrum antibacterial activity: It has antibacterial activity against plant pathogenic fungi such as Rhizoctonia solani, Sclerotinia sclerotiorum, Fusarium graminearum and Ustilaginoidea virens;

[0038] 6. Degrade cellulose and produce iron carriers.

[0039] 7. Promote plant growth.

[0040] Option 3:

[0041] The invention provides Trichoderma graminicola YNE00489-A, whose deposit number is GDMCC NO:64775.

[0042] The Trichoderma graminicola YNE00489-A of the present invention has a deposit name of Trichoderma graminicola, a deposit unit of Guangdong Microbiological Culture Collection Center, a deposit address of 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, a deposit number of GDMCC NO: 64775, and a deposit date of June 20, 2024.

[0043] The present invention also provides the use of the Trichoderma graminicola YNE00489-A: for promoting plant growth and preventing and controlling plant diseases (improving plant disease resistance) in plant cultivation.

[0044] As an improvement of the use of Trichoderma graminicola YNE00489-A of the present invention: Trichoderma graminicola YNE00489-A can produce iron carriers, degrade cellulose, inhibit the growth of plant pathogenic fungi, has induced resistance to gray mold on the aboveground parts, and can reproduce in large quantities in a substrate.

[0045] Plant pathogenic fungi include Rhizoctonia solani, Sclerotinia sclerotiorum, Fusarium graminearum and Ustilaginoideavirens.

[0046] Compared with the Trichoderma described in the background technology, the strain YNE00489-A of the present invention has the following technical advantages:

[0047] 1. Provide a new Trichoderma species;

[0048] 2. Ability to reproduce in large numbers in the substrate;

[0049] 3. Used to prevent and control soil-borne Fusarium wilt;

[0050] 4. Induced resistance to gray mold on aboveground parts;

[0051] 5. It has broad-spectrum antibacterial activity: It has antibacterial activity against plant pathogenic fungi such as Rhizoctonia solani, Sclerotinia sclerotiorum, Fusarium graminearum and Ustilaginoidea virens;

[0052] 6. Degrade cellulose and produce iron carriers.

[0053] 7. Promote plant growth. BRIEF DESCRIPTION OF THE DRAWINGS

[0054] The specific implementation modes of the present invention are further described in detail below with reference to the accompanying drawings.

[0055] Figure 1-1 This is the colony picture of T31818-A cultured on CMD medium for 7 days;

[0056] Figure 1-2 This is the microscopic morphology of T31818-A cultured in CMD medium;

[0057] Figure 1-3 is a phylogenetic tree based on rpb2 and tef1 gene sequences;

[0058] Figure 1-4 The figure shows the formation of hydroxamic acid siderophore by T31818-A in CAS medium;

[0059] Figure 1-5 This is a diagram showing the hydrolysis transparent circle formed by T31818-A on sodium carboxymethylcellulose medium;

[0060] Figure 1-6 This is a graph showing the antibacterial activity of T31818-A measured by plate confrontation culture;

[0061] Figure 1-7 This is the antibacterial activity diagram of T31818-A determined by the cellophane method;

[0062] Figure 1-8 Figure 2. The effect of T31818-A on the growth of cucumber seedlings in sterile potted culture.

[0063] Figure 1-9 The graph shows the induced resistance of T31818-A to cucumber gray mold in sterile matrix potted plants.

[0064] Figure 2-1 This is the colony picture of YNE00410-A cultured on CMD medium for 7 days;

[0065] Figure 2-2The microscopic morphology of YNE00410-A cultured in CMD medium;

[0066] Figure 2-3 is a phylogenetic tree based on rpb2 and tef1 gene sequences;

[0067] Figure 2-4 The figure shows the formation of hydroxamic acid siderophore by YNE00410-A in CAS medium;

[0068] Figure 2-5 The diagram shows the hydrolysis transparent circle formed by YNE00410-A on sodium carboxymethylcellulose medium;

[0069] Figure 2-6 This is the antibacterial activity diagram of YNE00410-A determined by the cellophane method;

[0070] Figure 2-7 The growth promotion of cucumber seedlings by YNE00410-A was measured in sterile substrate potted plants;

[0071] Figure 2-8 The diagram shows the induced resistance of YNE00410-A to cucumber gray mold in sterile potted plants.

[0072] Figure 3-1 This is the colony picture of YNE00489-A cultured on CMD medium for 7 days;

[0073] Figure 3-2 The microscopic morphology of YNE00489-A cultured in CMD medium;

[0074] Figure 3-3 is a phylogenetic tree based on rpb2 and tef1 gene sequences;

[0075] Figure 3-4 The figure shows the formation of hydroxamic acid siderophore by YNE00489-A in CAS medium;

[0076] Figure 3-5 The diagram shows the hydrolysis transparent circle formed by YNE00489-A on sodium carboxymethylcellulose medium;

[0077] Figure 3-6 This is the antibacterial activity diagram of YNE00489-A determined by the cellophane method;

[0078] Figure 3-7 Figure 1 shows the effect of YNE00489-A on the growth of cucumber seedlings in sterile potted culture.

[0079] Figure 3-8 The graph shows the induced resistance of YNE00489-A to cucumber gray mold in sterile potted plants. DETAILED DESCRIPTION

[0080] The present invention is further described below in conjunction with specific embodiments, but the protection scope of the present invention is not limited thereto:

[0081] The following plant pathogenic fungi belong to the ascomycetes among fungi: Botrytis cinerea, Fusarium graminearum, Sclerotinia sclerotiorum, Ustilaginoidea virens, for example, can be selected from ACCC36027, ACCC 39294, ACCC 39269, and ACCC 36443 of the China Agricultural Microbiological Culture Collection Center, respectively; Botrytis aclada can be selected from CCTCC HF 2008152 of the China Type Culture Collection Center; Plectosphaerella cucumerina can be selected from CGMCC 3.17050 of the China General Microbiological Culture Collection Center. Rhizoctonia solani, which belongs to the basidiomycetes among fungi, can be selected from ACCC 36246 of the China Agricultural Microbiological Culture Collection Center. Pythium aristosporum is a plant pathogenic oomycete, and ACCC 39248 of the China Agricultural Microbiological Culture Collection Center can be used.

[0082] Solution 1:

[0083] Example 1.1 Isolation and identification of Trichoderma densissimum strain T31818-A

[0084] 1. Separation

[0085] Potato Dextrose Agar (PDA): potato extract powder 10g, glucose 20g, agar 15g, chloramphenicol 0.1g, distilled water 1000ml, sterilize at 121℃ for 15min.

[0086] Rose Bengal Agar (RBA): potato extract powder 10g, glucose 20g, agar 15g, chloramphenicol 0.1g, rose Bengal 0.02g, distilled water 1000ml, sterilize at 121℃ for 15min.

[0087] Soil samples from wheat fields were collected in Jinan, Shandong Province, China, and separated using the dilution plate method. The process is as follows: First, remove 1-2 cm of the topsoil from the sampling plot, use a 25 cm deep soil sampler to collect soil samples at a depth of about 20 cm, take 200 g of soil samples, put them into sterile bags, seal them, keep records, and store them at 4°C. Weigh 10 g of soil samples, put them into a 250 mL Erlenmeyer flask containing small steel balls and 90 mL of sterile water, and shake them on a shaker at 120 r·min. -1 Shake for 10 minutes to fully disperse the soil sample. -1 times dilution; then pipette 10 -1 Transfer 1 mL of the dilution into a centrifuge tube containing 9 mL of sterile water to form a 10-fold dilution. -2 10 times dilution; -3 Pour the melted Bengal red culture medium into a 9 cm diameter culture dish, 15 mL per dish, and wait for the culture medium to cool to make a Bengal red culture medium plate; then take 10 -2 , 10 -3 0.1 mL of the soil sample dilution was applied on a red Bengal medium plate, and then the dilution was evenly spread with a sterile applicator. Each concentration was repeated 3 times. After culturing in a light incubator at 25°C for 3 days, the colonies were observed, and the hyphae at the edge of the colonies were picked and placed on a new PDA medium plate to obtain strain T31818-A.

[0088] 2. Taxonomic characteristics

[0089] Corn Dextrose Agar (CMD): 40 g corn flour, 20 g glucose, 15 g agar, 1000 mL distilled water, sterilized at 121°C for 15 min.

[0090] Culture characteristics: T31818-A was cultured on CMD medium at 25℃ for 3 days, and the colony radius was 38-45mm; the colony outline was clear, white, thin, and the aerial hyphae were sparse; conidia were observed about 2 days after inoculation, which were white at the beginning, turned yellow-green after 3-4 days, and turned dark green after 5-6 days; conidia were produced in the conidial clusters, which formed 4 obvious bands; there was no diffuse pigment, and the odor was not obvious ( Figure 1-1 ).

[0091] Microscopic features: Conidiophores are pyramidal ( Figure 1-2), with opposite branches, main axis with lateral branches sometimes at right angles or inclined upward; main axis and each branch usually terminate in a whorl of 3-4 phialides, sometimes in crossed whorls, sometimes solitary; phialides usually ampullate, sometimes globose, 5.7-8.0 × 2.5-2.9 μm; conidia green, subglobose to globose, 2.8-3.1 × 2.4-2.9 μm; chlamydospores solitary, terminal or intercalary, globose or subglobose, 13.3-16.4 × 12.8-14.6 μm.

[0092] 3. T31818-A Identification

[0093] The strain T31818-A was cultured with PDA, and genomic DNA was extracted. The second largest subunit of RNA polymerase II (rpb2) and translation elongation factor 1-α gene (tef1) were sequenced. The sequence similarity values ​​of rpb2 and tef1 of strain T31818-A were 100% and 100% with those of Trichoderma densissimum model strain T32434, 97.54% and 99.4% with those of Trichoderma paradensissimum model strain T31823, and 97.30% and 99.36% with those of Trichoderma pholiotae model strain JZBQH12. A phylogenetic tree was constructed based on the rpb2 and tef1 sequence datasets ( Figure 1-3 ), the maximum likelihood bootstrap value (MLBS) higher than 70% and the Bayesian posterior probability (BIPP) greater than 0.90 were given above the phylogenetic tree nodes, strains T31818-A and T32434 formed an independent branch (MLBP / BIPP=100% / 1.00), Trichoderma paradensissimum strains T31823 and T31824 formed an independent branch (MLBP / BIPP=85% / 0.974); Trichoderma pholiotae strains JZBQH12 and JZBQH11 formed an independent branch (MLBP / BIPP=99% / 0.99). T31818-A was identified as Trichoderma densissimum based on the sequence similarity values ​​of rpb2 and tef1 and the results of phylogenetic analysis.

[0094] The deposit information of the strain Trichoderma densissimum T31818-A of the present invention is as follows:

[0095] Deposit name: Trichoderma densissimum, deposit unit: Guangdong Microbiological Culture Collection Center, deposit address: 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, deposit number: GDMCC NO:64772, deposit date June 20, 2024.

[0096] Example 1.2, determination of siderophore activity

[0097] CAS dye solution: add 100mL ferric chloride solution to 500mL CAS solution and shake well to mix, slowly add the mixed solution to 400mL HDTMA solution along the wall of the beaker, gently shake to mix the solution, and you can get a blue CAS dye solution. CAS solution: Chrome azurol S (CAS) 0.7g, deionized water 500mL. Ferric chloride solution: ferric chloride 0.270g, concentrated hydrochloric acid (12M) 0.83mL, deionized water 1000mL. Hexadecyltrimethyl ammonium Bromide (HDTMA) solution: HDTMA 1.5g, deionized water 1000mL.

[0098] CAS medium: Take 100 mL of 20% sucrose solution, 30 mL of 10% (mass%) acid hydrolyzed casein, 1 mL of 1 mM calcium chloride, 200 mL of 1 mM magnesium sulfate heptahydrate solution, and 18 g of agar, add deionized water to 900 mL, and sterilize at 121°C for 15 min. When the CAS medium is cooled to about 60°C, slowly add 50 mL each of the separately sterilized 0.1 M phosphate buffer and CAS dye solution, avoid generating bubbles, mix well, and pour the melted medium into a 90 mm diameter plate to obtain a blue CAS medium plate.

[0099] Take a 5mm Trichoderma T31818-A cake cultured in PDA medium at 25℃ for 2 days, inoculate it in a CAS medium plate, and culture it at 25℃ for 5 days to observe whether it produces a color change circle to determine whether it produces iron carriers. The color of the CAS medium plate changes from blue to orange or yellow, indicating the production of hydroxamic acid iron carriers, and from blue to purple, indicating the production of catechol iron carriers. The colony diameter and the color change circle diameter are measured respectively, and the ratio of the color change circle diameter to the colony diameter is calculated. The size of the strain's iron carrier production activity is qualitatively evaluated based on the ratio of the color change circle diameter to the colony diameter. Each treatment is repeated 3 times. Trichoderma T31818-A changes the CAS medium from blue to orange or yellow, indicating the production of hydroxamic acid iron carriers; the colony diameter of Trichoderma T31818-A on the CAS medium plate is 81.5mm, the color change circle diameter is 79.0mm, and the ratio of the color change circle diameter to the colony diameter is about 0.97( Figure 1-4 ).

[0100] Example 1.3. Determination of cellulose degradation activity

[0101] Carboxymethyl cellulose sodium (CMC-Na) culture medium: carboxymethyl cellulose sodium (CMC-Na) 10g, glucose 5g, ammonium sulfate 2.0g, potassium hydrogen phosphate 1.0g, magnesium sulfate heptahydrate 0.5g, potassium chloride 0.5g, ferrous sulfate heptahydrate 0.01g, agar 15g, distilled water 1000ml, sterilize at 121℃ for 15min.

[0102] Take a 5mm Trichoderma T31818-A cake cultured in PDA medium at 25℃ for 2 days, inoculate it in CMC-Na medium, culture it at 28℃ for 48h, record the colony diameter, stain it with 1 mg / mL Congo red solution for 2h, discard the stain, wash it with 1 M NaOH solution, observe and record the diameter of the hydrolysis transparent zone, repeat each treatment 3 times, and qualitatively evaluate the activity of the strain in degrading cellulose based on the ratio of the hydrolysis transparent zone diameter to the colony diameter. The hydrolysis transparent zone diameter of Trichoderma T31818-A on CMC-Na medium is 53.3mm, the colony diameter is 65.7mm, and the ratio of the hydrolysis transparent zone diameter to the colony diameter is about 0.8, indicating that Trichoderma T31818-A has cellulose degradation activity ( Figure 1-5 ).

[0103] Example 1.4: Determination of parasitic ability by plate confrontation culture

[0104] The antifungal activity of Trichoderma sp. T31818-A against Botrytis aclada, Fusarium graminearum, Plectosphaerella cucumerina, Pythium aristosporum, Rhizoctonia solani and Sclerotinia sclerotiorum was determined by plate confrontation culture. 5 mm Trichoderma T31818-A cake and target pathogenic fungus cake cultured in PDA medium at 25°C for 5 days were inoculated on the edges of both sides of the PDA medium plate, respectively, and cultured at 25°C in light and dark (light:dark=12h:12h, light intensity 15000Lux). The pathogenic fungus cake inoculated alone was used as the control. Each treatment was repeated 3 times. After 10 days, the contact between the pathogen and Trichoderma on the front of the culture plate was observed. The grading standard is as follows: 45 Trichoderma hyphae completely covered the pathogen; 30 Trichoderma hyphae moderately covered the pathogen; 15 Trichoderma hyphae weakly covered the pathogen; 0 Trichoderma and pathogen hyphae did not cover each other. The confrontation culture experiment found that the hyphae coverage of Trichoderma T31818-A on Botrytisaclada, Pythium aristosporum, and Rhizoctonia solani was 45, the hyphae coverage on Fusarium graminearum and Plectosphaerella cucumerina was 30, and the hyphae coverage on Sclerotinia sclerotiorum was 15, indicating that Trichoderma T31818-A has good parasitic ability against pathogenic fungi ( Figure 1-6 ).

[0105] Example 1.5. Determination of antibacterial activity by cellophane method

[0106] The antimicrobial activity of the metabolites produced by Trichoderma T31818-A against target pathogenic fungi were determined by the cellophane method.

[0107] Cut the cellophane into 9 cm pieces and soak them in sterile water, sterilize them at 121°C for 15 min, and spread a double layer of cellophane on a 9 cm diameter PDA culture medium plate.

[0108] A 5 mm cake of Trichoderma T31818-A cultured in PDA medium at 25℃ for 2 days was pre-inoculated on a double layer of glass paper on a PDA medium plate, and cultured at 25℃ with light and dark (light: dark = 12h: 12h, light intensity 15000Lux) for 2 days. The glass paper and the Trichoderma growing on it were removed with sterile tweezers (the metabolites produced by Trichoderma can penetrate into the culture medium through the glass paper, but Trichoderma itself cannot penetrate into the culture medium). Then a 5 mm cake of the target pathogenic fungus was inoculated into the center of the PDA medium plate; the target pathogenic fungus not pre-inoculated with Trichoderma was used as the control, and cultured at 25℃ with light and dark (light: dark = 12h: 12h, light intensity 15000Lux) for 5 days, and each treatment was repeated 3 times. The colony diameter of the pathogen was determined by the cross method, and the inhibition rate of Trichoderma against the pathogenic fungus was calculated.

[0109] Inhibition rate (%) = (control colony diameter - treated colony diameter) / (control colony diameter - bacterial cake diameter) × 100%.

[0110] The cellophane method showed that the inhibition rates of Trichoderma T31818-A against Fusarium graminearum were 98%, against Rhizoctonia solani was 95%, and against Botrytisaclada, Plectosphaerella cucumerina, Pythiumaristosporum and Sclerotinia sclerotiorum were all 100%, indicating that Trichoderma T31818-A had good antibacterial activity against pathogenic fungi ( Figure 1-7 ).

[0111] Example 1.6: Potted plant test to determine the effect on cucumber growth

[0112] 1. Determination of the effect of Trichoderma T31818-A on cucumber growth using sterile matrix potted plants

[0113] The matrix is ​​peat: vermiculite = 3:1 (mass ratio), 400 mg of a large amount of water-soluble compound fertilizer (N: P2O5: K2O + TE, 20:20:20) / 100 g of matrix. The water-soluble compound fertilizer containing a large amount of elements is added to the matrix according to the above dosage ratio, and then the water content is adjusted to 60% to obtain the test matrix.

[0114] 100 g of the test matrix was placed in a tissue culture bottle and sterilized at 121°C for 30 min. Trichoderma T31818-A was cultured in a PDA medium plate at 28°C for 5-7 days. The Trichoderma spores were washed with sterile water to prepare a Trichoderma spore suspension mother solution. The spore concentration of the mother solution was counted with a hemocytometer. The concentration of the Trichoderma spore suspension mother solution was diluted with sterile water to 1×10 7 Each tissue culture bottle was inoculated with 1×10 7 1 mL of Trichoderma spore suspension was stirred evenly with a triangular rod to make the matrix contain 1×10 5 / g substrate. The cucumber seeds were disinfected with 75% alcohol and 1% sodium hypochlorite for 10 minutes, then rinsed with sterile water 3-4 times, placed in a clean bench to air dry, and placed in a 1 / 5MS medium (a medium in which the content of all components in the MS medium is reduced to 1 / 5 of the original content) plate, and germinated in the dark at 28°C until 70% of the cucumber seeds germinated. The cucumber seeds with consistent germination were sown in the above-mentioned tissue culture bottle matrix, with 1 cucumber seed per bottle, and the control without Trichoderma inoculation was used. The seeds were cultured in a light incubator at 28°C light: 22°C dark = 16h: 8h, and the light intensity was 22000Lux. The plant height, aboveground and root freshness of the cucumber seedlings were measured on the 20th day after sowing, and each treatment was repeated 3 times. The results showed that the plant height, aboveground fresh weight and root fresh weight of cucumber seedlings treated with CK were about 3.9 cm, 0.45 g and 0.16 g, respectively, while the plant height, aboveground fresh weight and root fresh weight of cucumber seedlings treated with Trichoderma T31818-A were 7.73 cm, 2.864 g and 0.389 g, respectively, which were about 100.0%, 540.3% and 149.9% higher than those of the control, respectively, significantly promoting the growth of cucumber plants ( Figure 1-8 ).

[0115] 2. Determination of the effect of Trichoderma T31818-A on cucumber growth using garden soil potted plants

[0116] Each tissue culture bottle contained: 100 g of garden soil, 50 g of substrate (peat: vermiculite = 3:1, mass ratio), 400 mg of water-soluble compound fertilizer of macroelements (N:P2O5:K2O+TE, 20:20:20), and 60 mL of water. Each tissue culture bottle was inoculated with 1×10 71mL of Trichoderma spore suspension with spores / mL was added and stirred evenly with a triangular stick. The surface disinfection, germination and sowing of cucumber seeds were the same as above. The plant height, stem diameter and whole plant dry weight of cucumber seedlings were measured on the 14th day after sowing, and each treatment was repeated 3 times. The growth potential was calculated, growth potential = (stem diameter / plant height) × dry weight. The results showed that the growth potential of cucumber seedlings treated with CK was about 0.12 (stem diameter was 2.4mm, plant height was 8.4cm, and dry weight was 0.42g), and the growth potential of cucumber seedlings treated with Trichoderma T31818-A was 0.18 (stem diameter was 3.44mm, plant height was 8.13cm, and dry weight was 0.425g), which was about 50.7% higher than the control, significantly promoting the growth of cucumber plants.

[0117] Example 1.7 Effect on the Induced Disease Resistance of Cucumber Gray Mold

[0118] Each tissue culture bottle contained: a cultivation medium consisting of 100 g of garden soil and 50 g of substrate, 400 mg of a large amount of water-soluble compound fertilizer (N: P2O5: K2O + TE, 20:20:20), and 60 mL of water. Each tissue culture bottle was inoculated with 1×10 7 spores / mL of Trichoderma spore suspension 1mL, stir evenly with a triangular stick. Use sterile tweezers to take cucumber seeds with consistent germination and sow them in potted plants, 1 cucumber seed per pot, set a potted plant without Trichoderma as a control, and culture in a 28℃ incubator. 20 days after sowing, inoculate 5mm Botrytis cinerea cake on cucumber leaves, and observe the disease condition of leaves after culturing at 20℃ for 3 days ( Figure 1-9 ), measure the diameter of leaf lesions, and calculate the control effect.

[0119] Control effect (%) = (control lesion diameter - treatment lesion diameter) / (control lesion diameter - fungus cake diameter) × 100%.

[0120] The results showed that the diameter of the lesions in the control group was about 3.2 cm, while the diameter of the lesions treated with Trichoderma T31818-A was 1.40 cm. The size of the gray mold lesions on cucumber leaves treated with Trichoderma T31818-A was significantly reduced, indicating that the inoculation of Trichoderma increased the resistance of cucumber to gray mold. The control effect of Trichoderma T31818-A on cucumber gray mold was 66.5%, and the spore content of Trichoderma T31818-A in the potting matrix was 7.7×10 8 / g cultivation substrate, indicating that Trichoderma T31818-A can effectively reproduce in the substrate and thus occupy the ecological niche.

[0121] Option 2:

[0122] Example 2.1 Isolation and identification of Trichoderma graminis strain YNE00410-A

[0123] 1. Separation

[0124] Malt Extract Agar (MEA): 20 g malt extract, 15 g agar, 1000 mL distilled water, sterilized at 121°C for 15 min.

[0125] Oplismenus undulatifolius samples were collected from the Naban River Basin Nature Reserve in Xishuangbanna, Yunnan Province, China, and isolated by tissue separation. The process is as follows: select healthy leaf sheaths without disease symptoms, rinse the leaf sheaths with tap water, and cut the leaf sheaths into segments of about 10 cm in length; then disinfect the leaf sheath segments with 75% alcohol and 1% (available chlorine) sodium hypochlorite for 3 minutes and 10 minutes respectively, and rinse with sterile water 3 times; cut them into segments of about 0.5 cm in length with a sterile scalpel, place them on MEA culture medium plates containing 50 mg / L ampicillin and streptomycin sulfate, 10 small segments per plate, 5 plates per sample, culture in the dark at 25°C, transfer the hyphae grown on the edge of the leaf sheath segments to PDA culture medium plates for culture, and purify the strain YNE00410-A.

[0126] 2. Taxonomic characteristics

[0127] Culture characteristics: YNE00410-A was cultured on CMD medium at 25℃ for 3 days. The colony radius was 36-43mm and it covered the plate in 5 days. The white hyphae were thin and close to the plate. After 3 days, green and white conidia were formed, and finally 4-5 indistinct bands were formed. There was no special smell and diffusible pigment was produced ( Figure 2-1 ).

[0128] Microscopic features: Conidiophores ( Figure 2-2 ) Pyramidal, lateral branches solitary or opposite, with an angle of nearly 90° to the main axis, with secondary branching; phialides solitary or in whorls of 2-5, or cross-shaped at the top, flask-shaped, 5.2-8.1×2.7-3.9μm in size; conidia green, spherical to subspherical, 3.1-4.2×2.9-3.9μm in size; thick-walled spores mostly terminal, globose to elliptical, 5.6-7.6×5.3-7.1μm in size.

[0129] 3. Identification of YNE00410-A

[0130] Strain YNE00410-A was cultured with PDA, genomic DNA was extracted, and the second largest subunit of RNA polymerase II (rpb2) and translation elongation factor 1-α gene (tef1) were sequenced. The sequence similarity values ​​of rpb2 and tef1 of strain YNE00410-A with those of Trichoderma graminis model strain YNE00410 were 100% and 100%, respectively, and the sequence similarity values ​​of rpb2 and tef1 with those of Trichodermabannaens model strain HMAS248840 were 97.42% and 96.06%, respectively. A phylogenetic tree was constructed based on the rpb2 and tef1 sequence datasets ( Figure 2-3 ), a maximum likelihood bootstrap value (MLBS) higher than 70% and a Bayesian posterior probability (BIPP) greater than 0.90 were given above the phylogenetic tree nodes, strains YNE00410-A and YNE00410 formed an independent branch (MLBP / BIPP=100% / 1.00), T. bannaense strains HMAS248840 and HMAS248865 formed an independent branch (MLBP / BIPP=100% / 1.00), T. graminis and T. bannaense clustered into a branch with a higher support rate (MLBP / BIPP=76 / 0.947). YNE00410-A was identified as Trichoderma graminis based on the sequence similarity values ​​of rpb2 and tef1 and the results of phylogenetic analysis.

[0131] The deposit information of the strain Trichoderma graminis YNE00410-A of the present invention is as follows:

[0132] Deposit name: Trichoderma graminis, deposit unit: Guangdong Microbiological Culture Collection Center, deposit address: 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, deposit number: GDMCC NO:64822, deposit date June 20, 2024.

[0133] Example 2.2, determination of siderophore activity

[0134] Take a 5mm Trichoderma YNE00410-A cake cultured in PDA medium at 25℃ for 2 days, inoculate it in a CAS medium plate, and culture it at 25℃ for 5 days to observe whether it produces a color change circle to determine whether it produces siderophores. The color of the CAS medium plate changes from blue to orange or yellow, indicating the production of hydroxamic acid siderophores, and from blue to purple, indicating the production of catechol siderophores. The colony diameter and the color change circle diameter are measured respectively, and the ratio of the color change circle diameter to the colony diameter is calculated. The size of the siderophore production activity of the strain is qualitatively evaluated according to the ratio of the color change circle diameter to the colony diameter. Each treatment is repeated 3 times. Trichoderma YNE00410-A changes the CAS medium from blue to orange or yellow, indicating that it has the ability to produce hydroxamic acid siderophores. The colony diameter of Trichoderma YNE00410-A on the CAS medium plate is 85.0mm, the color change circle diameter is 85.0mm, and the ratio of the color change circle diameter to the colony diameter is 1.0( Figure 2-4 ).

[0135] Example 2.3. Cellulose degradation activity assay

[0136] Take a 5mm cake of Trichoderma YNE00410-A cultured in PDA medium at 25℃ for 2 days, inoculate it in CMC-Na medium, culture it at 28℃ for 48h, record the colony diameter, stain it with 1 mg / mL Congo red solution for 2h, discard the solution, wash it with 1 M NaOH solution, observe and record the diameter of the hydrolysis transparent zone, repeat each treatment 3 times, and qualitatively evaluate the activity of the strain in degrading cellulose based on the ratio of the hydrolysis transparent zone diameter to the colony diameter. The hydrolysis transparent zone diameter of Trichoderma YNE00410-A on CMC-Na medium is 51.3mm, the colony diameter is 66.3mm, and the ratio of the hydrolysis transparent zone diameter to the colony diameter is about 0.8, indicating that Trichoderma YNE00410-A has cellulose degradation activity ( Figure 2-5 ).

[0137] Example 2.4: Determination of antibacterial activity by cellophane method

[0138] Rhizoctonia solani, Sclerotinia sclerotiorum, Fusarium graminearum and Ustilaginoidea virens were used as target pathogenic fungi, and the antibacterial activity of the metabolites produced by Trichoderma YNE00410-A against pathogenic fungi was determined by the cellophane method.

[0139] Cut the cellophane into 9 cm pieces and soak them in sterile water, sterilize them at 121°C for 15 min, and spread a double layer of cellophane on a 9 cm diameter PDA culture medium plate.

[0140] A 5 mm cake of Trichoderma YNE00410-A cultured in PDA medium at 25℃ for 2 days was pre-inoculated on a double layer of glass paper on a PDA medium plate, and cultured at 25℃ with light and dark (light: dark = 12h: 12h, light intensity 15000Lux) for 2 days. The glass paper and the Trichoderma growing on it were removed with sterile tweezers (the metabolites produced by Trichoderma can penetrate into the culture medium through the glass paper, but Trichoderma itself cannot penetrate into the culture medium). Then a 5 mm cake of the target pathogenic fungus was inoculated into the center of the PDA medium plate; the target pathogenic fungus not pre-inoculated with Trichoderma was used as the control, and cultured at 25℃ with light and dark (light: dark = 12h: 12h, light intensity 15000Lux) for 5 days, and each treatment was repeated 3 times. The colony diameter of the pathogen was determined by the cross method, and the inhibition rate of Trichoderma against the pathogenic fungus was calculated.

[0141] Inhibition rate (%) = (control colony diameter - treated colony diameter) / (control colony diameter - bacterial cake diameter) × 100%.

[0142] The cellophane method showed that the inhibition rates of Trichoderma YNE00410-A against Rhizoctonia solani, Sclerotinia sclerotiorum, Fusarium graminearum and Ustilaginoidea virens were 78.6%, 96.8%, 87.1% and 58.0%, respectively, indicating that Trichoderma YNE00410-A had good antibacterial activity against pathogenic fungi ( Figure 2-6 ).

[0143] Example 2.5: Potted plant test to determine the effect on cucumber growth

[0144] 1. Determination of the effect of Trichoderma YNE00410-A on cucumber growth using sterile matrix potted plants

[0145] The matrix is ​​peat: vermiculite = 3:1 (mass ratio), 400 mg of a large amount of water-soluble compound fertilizer (N: P2O5: K2O + TE, 20:20:20) / 100 g of matrix. The water-soluble compound fertilizer containing a large amount of elements is added to the matrix according to the above dosage ratio, and then the water content is adjusted to 60% to obtain the test matrix.

[0146] 100 g of the test matrix was placed in a tissue culture bottle and sterilized at 121°C for 30 min. Trichoderma YNE00410-A was cultured in a PDA medium plate at 28°C for 5-7 days. The Trichoderma spores were washed with sterile water to prepare a Trichoderma spore suspension mother solution. The spore concentration of the mother solution was counted with a hemocytometer. The concentration of the Trichoderma spore suspension mother solution was diluted with sterile water to 1×10 7Each tissue culture bottle was inoculated with 1×10 7 1 mL of Trichoderma spore suspension was stirred evenly with a triangular rod to make the matrix contain 1×10 5 / g substrate. The cucumber seeds were disinfected with 75% alcohol and 1% sodium hypochlorite for 10 minutes, then rinsed with sterile water 3-4 times, placed in a clean bench to air dry, and placed in a 1 / 5MS culture medium plate. The cucumber seeds were germinated in the dark at 28℃ until 70% of the cucumber seeds germinated. The cucumber seeds with consistent germination were sown in the above-mentioned tissue culture bottle matrix, with 1 cucumber seed per bottle. The control was no Trichoderma inoculation. The seeds were cultured in a light incubator at 28℃ light: 22℃ dark = 16h: 8h, and the light intensity was 22000Lux. The plant height, aboveground and root freshness of the cucumber seedlings were measured on the 20th day after sowing, and each treatment was repeated 3 times. The results showed that the plant height, aboveground fresh weight and root fresh weight of cucumber seedlings treated with CK were equivalent to those obtained in scheme 1. The plant height, aboveground fresh weight and root fresh weight of cucumber seedlings treated with Trichoderma YNE00410-A were 5.53 cm, 1.801 g and 0.371 g, respectively, which were about 43.1%, 302.7% and 138.3% higher than those of the control, respectively, significantly promoting the growth of cucumber plants ( Figure 2-7 ).

[0147] 2. Determination of the effect of Trichoderma YNE00410-A on cucumber growth using garden soil potted plants

[0148] Each tissue culture bottle contained: 100 g of garden soil, 50 g of substrate (peat: vermiculite = 3:1, mass ratio), 400 mg of water-soluble compound fertilizer of macroelements (N:P2O5:K2O+TE, 20:20:20), and 60 mL of water. Each tissue culture bottle was inoculated with 1×10 7 1mL of Trichoderma spore suspension with spores / mL was added and stirred evenly with a triangular stick. The surface disinfection, germination and sowing of cucumber seeds were the same as above. The plant height, stem diameter and whole plant dry weight of the cucumber seedlings were measured on the 14th day after sowing, and each treatment was repeated 3 times. The growth potential was calculated, growth potential = (stem diameter / plant height) × dry weight. The results showed that the growth potential of the cucumber seedlings treated with CK was equivalent to the results obtained in scheme 1, and the growth potential of the cucumber seedlings treated with Trichoderma YNE00410-A was 0.15 (stem diameter was 3.27mm, plant height was 7.93cm, and dry weight was 0.356g), which was about 23.2% higher than the control, significantly promoting the growth of cucumber plants.

[0149] Example 2.6 Effect on the Induced Disease Resistance of Cucumber Gray Mold

[0150] Each tissue culture bottle contained: a cultivation medium consisting of 100 g of garden soil and 50 g of substrate, 400 mg of a large amount of water-soluble compound fertilizer (N: P2O5: K2O + TE, 20:20:20), and 60 mL of water. Each tissue culture bottle was inoculated with 1×10 7 spores / mL of Trichoderma spore suspension 1mL, stir evenly with a triangular stick. Use sterile tweezers to take cucumber seeds with consistent germination and sow them in potted plants, 1 cucumber seed per pot, set a potted plant without Trichoderma as a control, and culture in a 28℃ incubator. 20 days after sowing, inoculate 5mm Botrytis cinerea cake on cucumber leaves, and observe the disease condition of leaves after culturing at 20℃ for 3 days ( Figure 2-8 ), measure the diameter of leaf lesions, and calculate the control effect.

[0151] Control effect (%) = (control lesion diameter - treatment lesion diameter) / (control lesion diameter - fungus cake diameter) × 100%.

[0152] The results showed that the diameter of the lesions in the control group was equivalent to that in the first scheme. The diameter of the lesions treated with Trichoderma YNE00410-A was 1.55 cm. The size of the gray mold lesions on cucumber leaves treated with Trichoderma YNE00410-A was significantly reduced, indicating that the inoculation of Trichoderma increased the resistance of cucumber to gray mold. The control effect of Trichoderma YNE00410-A on cucumber gray mold was 60.9%. The spore content of Trichoderma YNE00410-A in the potting medium was 3.1×10 8 / g cultivation substrate, indicating that Trichoderma YNE00410-A can effectively reproduce in the substrate and thus occupy the ecological niche.

[0153] Option 3:

[0154] Example 3.1 Isolation and identification of Trichoderma graminicola strain YNE00489-A

[0155] 1. Separation

[0156] Malt Extract Agar (MEA): 20 g malt extract, 15 g agar, 1000 mL distilled water, sterilized at 121°C for 15 min.

[0157] The stem samples of Oplismenus undulatifolius, a grass plant, were collected from the Naban River Basin Nature Reserve in Xishuangbanna, Yunnan Province, China, and separated by tissue separation. The process is as follows: select healthy stems without disease symptoms, rinse the roots with tap water, and cut the stems into segments of about 10 cm in length; then disinfect the stem segments with 75% alcohol and 1% (available chlorine) sodium hypochlorite for 3 minutes and 10 minutes respectively, and rinse with sterile water 3 times; cut them into segments of about 0.5 cm in length with a sterile scalpel, place them on MEA culture medium plates containing 50 mg / L ampicillin and streptomycin sulfate, 10 small segments per plate, 5 plates per sample, culture in the dark at 25°C, transfer the hyphae grown on the edge of the stem segments to PDA culture medium plates for culture, and purify the strain YNE00489-A.

[0158] 2. Taxonomic characteristics

[0159] Corn Dextrose Agar (CMD): 40 g corn flour, 20 g glucose, 15 g agar, 1000 mL distilled water, sterilized at 121°C for 15 min.

[0160] Culture characteristics: strain YNE00489-A was cultured on CMD medium at 25°C for 3 days, the colony radius was 42-43 mm, and the plate was full in 5 days; the colony was transparent, and the aerial hyphae were pale white, loose and sparse; conidia aggregated into larger conidia clusters, distributed in 3-4 inconspicuous zones; no obvious odor or diffuse pigment was observed ( Figure 3-1 ).

[0161] Microscopic features: Conidiophores ( Figure 3-2 ) The main axis is obvious, gradually tapering towards the top, the top is sterile, the branches often grow at right angles, the secondary branches are short, the branch distance is irregular, the branches at the base are generally paired, or arranged in 3 whorls, and generally solitary at the top; the phialide is ampoule-shaped or subglobose, generally arranged in 3-5 whorls, occasionally solitary, measuring 4.3-6.9×3.3-4.4μm; conidia are green, cylindrical or elliptical, measuring 3.3-3.9×2.1-2.6μm; thick-walled spores are intercalary or terminal, round or elliptical, measuring 6.2-9.0×5.6-8.7μm.

[0162] 3. Identification of YNE00489-A

[0163] Strain YNE00489-A was cultured with PDA, genomic DNA was extracted, and the second largest subunit of RNA polymerase II (rpb2) and translation elongation factor 1-α gene (tef1) were sequenced. The sequence similarity values ​​of rpb2 and tef1 of strain YNE00489-A were 100% and 100% with those of Trichoderma graminicola model strain YNE00490, 97.66% and 97.42% with those of Trichoderma velutinum model strain DAOM 230013, and 98.89% and 96.58% with those of Trichoderma polypori model strain HMAS248855. A phylogenetic tree was constructed based on the rpb2 and tef1 sequence datasets ( Figure 3-3 ), a maximum likelihood bootstrap value (MLBS) higher than 70% and a Bayesian posterior probability (BIPP) greater than 0.90 were given above the phylogenetic tree nodes, strains YNE00489-A and YNE00490 formed an independent branch (rate MLBP / BIPP=100% / 1.00), Trichoderma velutinum strains DAOM 230013 and LESF132 formed an independent branch (MLBP / BIPP=100% / 1.00), these two branches formed a large branch with Trichoderma polypori, with a high support rate (MLBP / BIPP=74% / 0.997). YNE00489-A was identified as Trichoderma graminicola based on the sequence similarity values ​​of rpb2 and tef1 and the results of phylogenetic analysis.

[0164] The deposit information of Trichoderma graminicola YNE00489-A of the present invention is as follows:

[0165] Deposit name: Trichoderma graminicola, deposit unit: Guangdong Microbiological Culture Collection Center, deposit address: 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, deposit number: GDMCC NO:64775, deposit date June 20, 2024.

[0166] Example 3.2, determination of siderophore activity

[0167] Take a 5mm Trichoderma YNE00489-A cake cultured in PDA medium at 25℃ for 2 days, inoculate it in a CAS medium plate, and culture it at 25℃ for 5 days to observe whether it produces a color change circle to determine whether it produces siderophores. The color of the CAS medium plate changes from blue to orange or yellow, indicating the production of hydroxamic acid siderophores, and from blue to purple, indicating the production of catechol siderophores. The colony diameter and the color change circle diameter are measured respectively, and the ratio of the color change circle diameter to the colony diameter is calculated. The size of the siderophore production activity of the strain is qualitatively evaluated according to the ratio of the color change circle diameter to the colony diameter. Each treatment is repeated 3 times. Trichoderma YNE00489-A changes the CAS medium from blue to orange or yellow, indicating that it has the ability to produce hydroxamic acid siderophores. The colony diameter of Trichoderma YNE00489-A on the CAS medium plate is 85.0mm, the color change circle diameter is 85.0mm, and the ratio of the color change circle diameter to the colony diameter is 1.0( Figure 3-4 ).

[0168] Example 3.3. Cellulose degradation activity assay

[0169] Take a 5mm Trichoderma YNE00489-A cake cultured in PDA medium at 25℃ for 2 days, inoculate it in CMC-Na medium, culture it at 28℃ for 48h, record the colony diameter, stain it with 1 mg / mL Congo red solution for 2h, discard the stain, wash it with 1 M NaOH solution, observe and record the diameter of the hydrolysis transparent zone, repeat each treatment 3 times, and qualitatively evaluate the activity of the strain in degrading cellulose based on the ratio of the hydrolysis transparent zone diameter to the colony diameter. The hydrolysis transparent zone diameter of Trichoderma YNE00489-A on CMC-Na medium is 39.3mm, the colony diameter is 67.0mm, and the ratio of the hydrolysis transparent zone diameter to the colony diameter is about 0.6, indicating that Trichoderma YNE00489-A has cellulose degradation activity ( Figure 3-5 ).

[0170] Example 3.4. Determination of antibacterial activity by cellophane method

[0171] Rhizoctonia solani, Sclerotinia sclerotiorum, Fusarium graminearum and Ustilaginoidea virens were used as target pathogenic fungi, and the antibacterial activity of the metabolites produced by Trichoderma YNE00489-A against pathogenic fungi was determined by the cellophane method.

[0172] Cut the cellophane into 9 cm pieces and soak them in sterile water, sterilize them at 121°C for 15 min, and spread a double layer of cellophane on a 9 cm diameter PDA culture medium plate.

[0173] A 5 mm cake of Trichoderma YNE00489-A cultured in PDA medium at 25℃ for 2 days was pre-inoculated on a double layer of glass paper on a PDA medium plate, and cultured at 25℃ with light and dark (light: dark = 12h: 12h, light intensity 15000Lux) for 2 days. The glass paper and the Trichoderma growing on it were removed with sterile tweezers (the metabolites produced by Trichoderma can penetrate into the culture medium through the glass paper, but Trichoderma itself cannot penetrate into the culture medium). Then a 5 mm cake of the target pathogenic fungus was inoculated into the center of the PDA medium plate; the target pathogenic fungus not pre-inoculated with Trichoderma was used as the control, and cultured at 25℃ with light and dark (light: dark = 12h: 12h, light intensity 15000Lux) for 5 days, and each treatment was repeated 3 times. The colony diameter of the pathogen was determined by the cross method, and the inhibition rate of Trichoderma against the pathogenic fungus was calculated. Inhibition rate (%) = (control colony diameter - treated colony diameter) / (control colony diameter - bacterial cake diameter) × 100%. The cellophane method showed that the inhibition rates of Trichoderma YNE00489-A against Rhizoctonia solani, Sclerotinia sclerotiorum, Fusarium graminearum and Ustilaginoidea virens were 84.4%, 32.2%, 87.0% and 33.3%, respectively, indicating that Trichoderma YNE00489-A had good antibacterial activity against pathogenic fungi ( Figure 3-6 ).

[0174] Example 3.5: Potted plant test to determine the effect on cucumber growth

[0175] 1. Determination of the effect of Trichoderma YNE00489-A on cucumber growth using sterile matrix potted plants

[0176] The matrix is ​​peat: vermiculite = 3:1 (mass ratio), 400 mg of a large amount of water-soluble compound fertilizer (N: P2O5: K2O + TE, 20:20:20) / 100 g of matrix. The water-soluble compound fertilizer containing a large amount of elements is added to the matrix according to the above dosage ratio, and then the water content is adjusted to 60% to obtain the test matrix.

[0177] 100 g of the test matrix was placed in a tissue culture bottle and sterilized at 121°C for 30 min. Trichoderma YNE00489-A was cultured in a PDA medium plate at 28°C for 5-7 days. The Trichoderma spores were washed with sterile water to prepare a Trichoderma spore suspension mother solution. The spore concentration of the mother solution was counted with a hemocytometer. The concentration of the Trichoderma spore suspension mother solution was diluted with sterile water to 1×10 7 Each tissue culture bottle was inoculated with 1×10 71 mL of Trichoderma spore suspension was stirred evenly with a triangular rod to make the matrix contain 1×10 5 / g substrate. The cucumber seeds were disinfected with 75% alcohol and 1% sodium hypochlorite for 10 minutes, then rinsed with sterile water 3-4 times, placed in a clean bench to air dry, and placed in a 1 / 5MS culture medium plate. The cucumber seeds were germinated in the dark at 28℃ until 70% of the cucumber seeds germinated. The cucumber seeds with consistent germination were sown in the above-mentioned tissue culture bottle matrix, with 1 cucumber seed per bottle. The control was no Trichoderma inoculation. The seeds were cultured in a light incubator at 28℃ light: 22℃ dark = 16h: 8h, and the light intensity was 22000Lux. The plant height, aboveground and root freshness of the cucumber seedlings were measured on the 20th day after sowing, and each treatment was repeated 3 times. The results showed that the plant height, aboveground fresh weight and root fresh weight of cucumber seedlings treated with CK were equivalent to those obtained in scheme 1. The plant height, aboveground fresh weight and root fresh weight of cucumber seedlings treated with Trichoderma YNE00489-A were 7.07 cm, 2.209 g and 0.377 g, respectively, which were about 82.8%, 393.9% and 142.4% higher than those of the control, respectively, significantly promoting the growth of cucumber plants ( Figure 3-7 ).

[0178] 2. Determination of the effect of Trichoderma YNE00489-A on cucumber growth using garden soil potted plants

[0179] Each tissue culture bottle contained: 100 g of garden soil, 50 g of substrate (peat: vermiculite = 3:1, mass ratio), 400 mg of water-soluble compound fertilizer of macroelements (N:P2O5:K2O+TE, 20:20:20), and 60 mL of water. Each tissue culture bottle was inoculated with 1×10 7 1 mL of Trichoderma spore suspension with spores / mL was added and stirred evenly with a triangular stick. The surface disinfection, germination and sowing of cucumber seeds were the same as above. The plant height, stem diameter and whole plant dry weight of the cucumber seedlings were measured on the 14th day after sowing, and each treatment was repeated 3 times. The growth potential was calculated, growth potential = (stem diameter / plant height) × dry weight. The results showed that the growth potential of the cucumber seedlings treated with CK was equivalent to the results obtained in scheme 1, and the growth potential of the cucumber seedlings treated with Trichoderma YNE00489-A was 0.14 (stem diameter was 2.77 mm, plant height was 6.73 cm, and dry weight was 0.340 g), which was about 16.7% higher than the control, significantly promoting the growth of cucumber plants.

[0180] Example 3.6 Effect on the Induced Disease Resistance of Cucumber Gray Mold

[0181] Each tissue culture bottle contained: a cultivation medium consisting of 100 g of garden soil and 50 g of substrate, 400 mg of a large amount of water-soluble compound fertilizer (N: P2O5: K2O + TE = 20:20:20), and 60 mL of water. Each tissue culture bottle was inoculated with 1×10 7spores / mL of Trichoderma spore suspension 1mL, stir evenly with a triangular stick. Use sterile tweezers to take cucumber seeds with consistent germination and sow them in potted plants, 1 cucumber seed per pot, set a potted plant without Trichoderma as a control, and culture in a 28℃ incubator. 20 days after sowing, inoculate 5mm Botrytis cinerea cake on cucumber leaves, and observe the disease condition of leaves after culturing at 20℃ for 3 days ( Figure 3-8 ), measure the diameter of leaf lesions, and calculate the control effect.

[0182] Control effect (%) = (control lesion diameter - treatment lesion diameter) / (control lesion diameter - fungus cake diameter) × 100%.

[0183] The results showed that the diameter of the lesions in the control group was equivalent to that in the first scheme. The diameter of the lesions treated with Trichoderma YNE00489-A was 1.52 cm. The size of the gray mold lesions on cucumber leaves treated with Trichoderma YNE00489-A was significantly reduced, indicating that the inoculation of Trichoderma increased the resistance of cucumber to gray mold. The control effect of Trichoderma YNE00489-A on cucumber gray mold was 62.1%. The spore content of Trichoderma YNE00489-A in the potting medium was 6.0×10 8 / g cultivation substrate, indicating that Trichoderma YNE00489-A can effectively reproduce in the substrate and thus occupy the ecological niche.

[0184] contrast:

[0185] HTTA-Z0002, HB40619, HB40609, and Tk905 were experimented according to the method described in Example 1.7 (Example 2.6, Example 3.6) above, and the diameter of the lesions was about 2.5 to 3 cm, which was much larger than the diameter of the lesions treated with Trichoderma T31818-A of the present invention (1.40 cm), much larger than the diameter of the lesions treated with Trichoderma YNE00410-A of the present invention (1.55 cm), and much larger than the diameter of the lesions treated with Trichoderma YNE00489-A of the present invention (1.52 cm).

[0186] The corresponding spore contents of HTTA-Z0002, HB40619, HB40609, and Tk905 in the culture medium are approximately (2-8)×10 5 The breeding effect is far inferior to that of the present invention.

[0187] Finally, it should be noted that the above examples are only some specific embodiments of the present invention. Obviously, the present invention is not limited to the above embodiments, and there are many variations. All variations that can be directly derived or associated with the content disclosed by a person skilled in the art should be considered as the protection scope of the present invention.

Claims

1. Trichoderma densissimum T31818-A, characterized by: The deposit number is GDMCC NO:64772.

2. The use of Trichoderma densissimum T31818-A as claimed in claim 1, characterized in that: Promote plant growth and prevent and control plant diseases.

3. The use of Trichoderma densissimum T31818-A according to claim 2, characterized in that: The strain T31818-A can produce iron carriers, degrade cellulose, inhibit the growth of plant pathogenic fungi and plant pathogenic oomycetes, has induced resistance to gray mold on aboveground parts, and can reproduce in large quantities in the matrix.

4. Trichoderma graminis YNE00410-A, characterized in that: the deposit number is GDMCC NO:64822.

5. The use of Trichoderma graminis YNE00410-A according to claim 4, characterized in that: Promote plant growth and prevent and control plant diseases.

6. The use of Trichoderma graminis YNE00410-A according to claim 5, characterized in that: YNE00410-A can produce iron carriers, degrade cellulose, inhibit the growth of plant pathogenic fungi, induce resistance to gray mold on aboveground parts, and can reproduce in large quantities in the matrix.

7. Trichoderma graminicola YNE00489-A, characterized in that: the deposit number is GDMCC NO:64775.

8. The use of Trichoderma graminicola YNE00489-A according to claim 7, characterized in that: Promote plant growth and prevent and control plant diseases.

9. The use of Trichoderma graminicola YNE00489-A according to claim 8, characterized in that: Trichoderma graminicola YNE00489-A can produce iron carriers, degrade cellulose, inhibit the growth of plant pathogenic fungi, induce resistance to gray mold on the aboveground parts, and can reproduce in large quantities in the matrix.

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