Hongyred trichoderma near, Hongyred trichoderma new and application of Hongyred trichoderma near and Hongyred trichoderma new to plant growth promotion and plant disease control

Abstract

The invention belongs to the field of microorganisms, and particularly relates to two trichoderma strains for promoting plant growth and preventing and treating plant diseases and application of the two trichoderma strains. The invention provides a red trichoderma paragongkii strain T31355-A, and the preservation number of the red trichoderma paragongkii strain T31355-A is GDMCC (Graphical Databine Microorganism Center) NO.64773. The red trichoderma paragongkii strain T31355-A is a strain of a The invention further provides a red trichoderma new hoongkui strain YNE00787-A, and the preservation number of the red trichoderma new hoongkui strain YNE00787-A is GDMCC (China General Microbiological Culture Collection Center) NO. 64774. The invention also provides application of the two trichoderma strains at the same time, wherein the two trichoderma strains are used for promoting plant growth and preventing and treating plant diseases in plant cultivation.

Description

Technical Field

[0001] The invention belongs to the field of microorganisms, and specifically relates to two Trichoderma strains for promoting plant growth and preventing and controlling plant diseases and applications thereof. Background Art

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

[0003] Competition refers to the phenomenon of competition for space, nutrition, oxygen, etc. between two or more microorganisms existing in the same microhabitat. Among them, spatial competition is the competition between biocontrol bacteria for the surface space or internal space of plants, especially the sites where pathogens invade plants. The pre-colonization of biocontrol bacteria on the plant surface prevents the colonization of pathogens; nutritional competition is the competition between biocontrol bacteria and pathogens for limited available materials such as plant secretions, plant residues, and root shedding, including carbohydrates, amino acids, inorganic salts, vitamins, and secondary metabolites necessary for the growth and development of microorganisms. Competition is one of the important biocontrol mechanisms of Trichoderma. Trichoderma grows and reproduces rapidly, can decompose cellulose, lignin, chitin, etc., occupy the surface space or internal space of plants, prevent the living space and infection and colonization sites of harmful pathogens, thereby effectively reducing the infection of plant pathogens. In addition, iron, as a cofactor of many enzymes, is indispensable for the normal growth and development of plants and microorganisms. However, under aerobic conditions, iron is easy to form insoluble iron oxide, which makes it unable to be effectively utilized. As a low molecular weight chelating agent, siderophore has a high affinity for iron. 3+ ) and converts it into soluble Fe 2+ , which makes it easier for plants and microorganisms to absorb and utilize it. In the competition between Trichoderma and pathogens, siderophores play a vital role. Trichoderma that can secrete siderophores can not only improve the availability of iron to plants, but also the siderophores of Trichoderma can effectively consume the available iron sources in the soil, thereby effectively inhibiting the growth of pathogens.

[0004] Antibiotic effect refers to the phenomenon that the metabolites produced by one microorganism have a direct inhibitory effect on another microorganism, which inhibits the growth of the other microorganism, while the microorganism itself is not affected. Antibiotic effect is one of the important biocontrol mechanisms of Trichoderma. Trichoderma inhibits the growth and reproduction of pathogenic fungi by producing metabolites with antimicrobial activity, thereby reducing the occurrence of diseases.

[0005] Induced resistance refers to the phenomenon that various biological or abiotic induction factors activate the natural defense mechanism of plants, triggering the resistance of plants to pathogenicity of pathogens, thus protecting plants from pathogenic damage or reducing damage. Induced resistance is one of the important biological control mechanisms of Trichoderma. Trichoderma activates the induced resistance of plants, thereby improving the defense ability of plants against a variety of pathogens.

[0006] Trichoderma asperellum HTTA-Z0002 (CGMCC No.15677, CN109112072A), Trichoderma asperellum HB40619 (CGMCC No.40143, CN114891645A), Trichoderma harzianum HB40609 (CGMCC No.40142, CN114836329 A), and Trichoderma pseudokoningii Tk905 (GDMCC No: 61934, CN 113862160A) have the following properties: growth-promoting effect, inhibitory effect on the growth of some plant pathogenic fungi, and disease resistance.

[0007] The genus Trichoderma belongs to the kingdom Fungi, phylum Ascomycota, order Hypocreales, family Hypocreaceae. The genus is rich in species and number, and the number of species in the genus Trichoderma has exceeded 500. 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 Trichoderma Taxonomy Committee (ICTT). That is, if a Trichoderma species 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. Trichoderma is abundant in resources, 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, Trichodermaatroviride, 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. Summary of the invention

[0008] The technical problem to be solved by the present invention is to provide two kinds of Trichoderma and their application in promoting plant growth and preventing and controlling plant diseases; the two kinds of Trichoderma are respectively Trichoderma parahongkuii strain T31355-A and Trichoderma neohongkuii strain YNE00787-A.

[0009] In order to solve the above technical problems, the present invention provides the following two solutions:

[0010] Solution 1:

[0011] The invention provides Trichoderma parahongkuii T31355-A, whose deposit number is GDMCC NO.64773.

[0012] The strain Trichoderma parahongkuii T31355-A of the present invention has a preservation name of Trichoderma parahongkuii, a preservation unit: Guangdong Microbiological Culture Collection Center, a preservation address: 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, a preservation number: GDMCC NO.64773, and a preservation time of June 20, 2024.

[0013] The present invention also provides the use of the Trichoderma spp. strain T31355-A: for promoting plant growth and preventing and controlling plant diseases in plant cultivation.

[0014] The present invention also provides the use of Trichoderma parahongkuii T31355-A:

[0015] The strain T31355-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.

[0016] As an improvement of the use of Trichoderma parahongkuii T31355-A of the present invention:

[0017] Plant pathogenic fungi include Fusarium graminearum, Sclerotiniasclerotiorum, Ustilaginoidea virens, and Rhizoctonia solani.

[0018] Compared with the Trichoderma described in the background technology, the strain T31355-A of the present invention has the following technical advantages:

[0019] 1. Provide a new Trichoderma species;

[0020] 2. It can reproduce in large numbers in the matrix, thereby occupying the ecological niche and preventing the invasion of other pathogens;

[0021] 3. Induced resistance to gray mold on aboveground parts;

[0022] 4. Broad-spectrum antibacterial activity: It has antibacterial activity against plant pathogenic fungi such as Fusarium graminearum, Sclerotinia sclerotiorum, Ustilaginoidea virens, which belong to Ascomycetes, and Rhizoctonia solani, which belongs to Basidiomycetes; that is, it can inhibit the growth of the above-mentioned plant pathogenic fungi, thereby improving the disease resistance of plants;

[0023] 5. Degrade cellulose;

[0024] 6. Produce iron carriers;

[0025] 7. Promote plant growth.

[0026] Option 2:

[0027] Provided is a Trichoderma (Trichoderma neohongkuii) strain YNE00787-A, whose preservation number is GDMCC NO.64774.

[0028] The strain Trichoderma neohongkuii YNE00787-A of the present invention has a preservation name of Trichoderma neohongkuii, a preservation unit: Guangdong Microbiological Culture Collection Center, a preservation address: 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, a preservation number: GDMCC NO.64774, and a preservation time of June 20, 2024.

[0029] The present invention also provides the use of the Trichoderma neohongkuii YNE00787-A: used for promoting plant growth and preventing and controlling plant diseases in plant cultivation.

[0030] Improvements of the use of Trichoderma neohongkuii YNE00787-A of the present invention:

[0031] 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.

[0032] As a further improvement of the use of Trichoderma neohongkuii YNE00787-A of the present invention:

[0033] Plant pathogenic fungi include Fusarium graminearum, Fusarium oxysporum, Sclerotinia sclerotiorum, Ustilaginoidea virens, and Rhizoctonia solani.

[0034] Compared with the Trichoderma described in the background technology, the strain YNE00787-A of the present invention has the following technical advantages:

[0035] 1. Provide a new Trichoderma species;

[0036] 2. Ability to reproduce in large numbers in the substrate;

[0037] 3. Induced resistance to gray mold on aboveground parts;

[0038] 4. It has broad-spectrum antibacterial activity: It has antibacterial activity against pathogenic fungi such as Fusarium graminearum, Fusarium oxysporum, Sclerotinia sclerotiorum, Ustilaginoidea virens, which belong to Ascomycetes, and Rhizoctonia solani, which belongs to Basidiomycetes; that is, it can inhibit the growth of the above-mentioned plant pathogenic fungi, thereby improving the disease resistance of plants;

[0039] 5. Degrade cellulose;

[0040] 6. Produce iron carriers;

[0041] 7. Promote plant growth. BRIEF DESCRIPTION OF THE DRAWINGS

[0042] The specific implementation modes of the present invention are further described in detail below with reference to the accompanying drawings.

[0043] Figure 1-1 This is the colony picture of T31355-A cultured on CMD medium for 7 days;

[0044] Figure 1-2 This is the microscopic morphology of T31355-A cultured in CMD medium;

[0045] Figure 1-3 is a phylogenetic tree based on rpb2 and tef1 gene sequences;

[0046] Figure 1-4 The figure shows the formation of hydroxamic acid siderophore by T31355-A in CAS medium;

[0047] Figure 1-5 This is a diagram showing the hydrolysis transparent circle formed by T31355-A on sodium carboxymethylcellulose medium;

[0048] Figure 1-6 Figure 3. Antibacterial activity of T31355-A determined by the cellophane method;

[0049] Figure 1-7 Figure 2. The growth promotion of cucumber seedlings by T31355-A in sterile substrate potted culture.

[0050] Figure 1-8 The graph shows the induced resistance of T31355-A to cucumber gray mold in sterile matrix potted plants.

[0051] Figure 2-1 This is the colony picture of YNE00787-A cultured on CMD medium for 7 days;

[0052] Figure 2-2 The microscopic morphology of YNE00787-A cultured in CMD medium;

[0053] Figure 2-3 is a phylogenetic tree based on rpb2 and tef1 gene sequences;

[0054] Figure 2-4 The figure shows the formation of hydroxamic acid siderophore by YNE00787-A in CAS medium;

[0055] Figure 2-5 The diagram shows the hydrolysis transparent circle formed by YNE00787-A on sodium carboxymethylcellulose medium;

[0056] Figure 2-6 This is the antibacterial activity diagram of YNE00787-A determined by the cellophane method;

[0057] Figure 2-7 Figure 1 shows the effect of YNE00787-A on the growth of cucumber seedlings in sterile potted culture.

[0058] Figure 2-8 The diagram shows the induced disease resistance of YNE00787-A to cucumber gray mold in sterile potted plants. DETAILED DESCRIPTION

[0059] The present invention is further described below in conjunction with specific embodiments, but the protection scope of the present invention is not limited thereto:

[0060] Fusarium graminearum, Fusarium oxysporum, Sclerotinia sclerotiorum and Ustilaginoidea virens, the above plant pathogenic fungi belong to ascomycetes among fungi; ACCC 39294, ACCC 39326, ACCC 39269 and ACCC 36443 of China Agricultural Microbiological Culture Collection Center can be selected respectively.

[0061] Rhizoctonia solani, a plant pathogenic fungus belonging to the basidiomycetes among fungi; ACCC 36246 of the China Agricultural Microbiological Culture Collection Center can be used.

[0062] Solution 1:

[0063] Example 1.1 Isolation and identification of Trichoderma parahongkuii strain T31355-A I. Isolation

[0064] Potato Dextrose Agar (PDA): potato extract powder 10g, glucose 20g, agar 15g, chloramphenicol 0.1g, distilled water 1000ml, sterilize at 121℃ for 15min.

[0065] 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.

[0066] Soil samples from paddy fields were collected from Lishui City, Zhejiang 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 triangular 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; then make 10 -3Pour 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 T31355-A.

[0067] 2. Taxonomic characteristics

[0068] 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.

[0069] Culture characteristics: T31355-A was cultured on CMD medium at 25℃ for 3 days. The colony radius was 60-65mm and it covered the plate in 4 days. The mycelium was white and closely attached to the culture medium. Aerial mycelium was sparse. White cottony spore clusters were visible on the 3rd day. They turned yellow-green on the 4th day. The banded structure was not obvious. It had a fruity odor and no diffusible pigment was produced ( Figure 1-1 ).

[0070] Microscopic features: Conidiophores have a distinct main axis ( Figure 1-2 ), branches are paired or solitary, with an angle of less than or close to 90° relative to the main axis, and the branches terminate in a single phialide or a whorl of 2-3 phialides; the phialides are ampoule-shaped, slightly swollen in the middle, and measure 5.4-11.0×2.7-4.1μm; conidia are green, ellipsoidal to short cylindrical, and measure 3.6-5.1×2.6-3.5μm; chlamydospores are solitary, terminal or intercalary, spherical or subspherical, and measure 4.5-8.3×4.3-7.8μm.

[0071] 3. T31355-A Identification

[0072] The strain T31355-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 T31355-A and Trichoderma parahongkuii model strain T31356 were 100% and 100%, respectively. A phylogenetic tree was constructed based on the rpb2 and tef1 sequence data sets ( Figure 1-3), 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 T31355-A and T31356 formed an independent branch (MLBS / BIPP=100% / 1.0), strains T32026 and T32027 formed an independent branch (MLBS / BIPP=100% / 1.0), endophytic strains YNE00720 and YNE00787 formed an independent branch (MLBS / BIPP=100% / 1.0), and these three branches clustered into a large branch with a high support rate (MLBS / BIPP=98% / 1.0). T31355-A was identified as Trichoderma parahongkuii based on the sequence similarity values ​​of rpb2 and tef1 and the results of phylogenetic analysis.

[0073] Example 1.2, determination of siderophore activity

[0074] 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.

[0075] 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.

[0076] Take a 5mm Trichoderma T31355-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-red, indicating the production of catechol siderophores; measure the colony diameter and the color change circle diameter, calculate the ratio of the color change circle diameter to the colony diameter, and qualitatively evaluate the siderophore production activity of the strain according to the ratio of the color change circle diameter to the colony diameter. Repeat each treatment 3 times. Trichoderma T31355-A changes the CAS medium from blue to purple-red, indicating the production of catechol siderophores; the colony diameter of Trichoderma T31355-A on the CAS medium plate is 61.0mm, the color change circle diameter is 85.0mm, and the ratio of the color change circle diameter to the colony diameter is 1.4( Figure 1-4 ).

[0077] LNM culture medium: 2 mL of 1 mM magnesium sulfate heptahydrate, 0.1 mL of 1 mM calcium chloride, 1 mL of 20% sucrose solution, 3 mL of 10% acid hydrolyzed casein, and 0.1 M phosphate buffer to make up to 100 mL.

[0078] Take a 5mm Trichoderma T31355-A cake cultured in PDA medium at 25℃ for 2 days, inoculate it in LNM medium, and culture it at 25℃ and 120r / min for 6 days. Take samples on the 4th, 5th and 6th days, centrifuge at 10000r / min for 10min, aspirate the supernatant, add an equal volume of CAS dye, mix evenly, keep it away from light for more than 1h, and use a spectrophotometer to measure the absorbance (As) at 680nm. Mix the liquid culture medium with the uninoculated PDA medium cake and the CAS dye in equal volumes, and use its absorbance as the reference value (Ar). Each treatment was repeated 3 times. Siderophore production is expressed as the relative content of siderophore activity units (SU), SU (%) = [(Ar-As) / Ar] × 100%. The results showed that the SU (%) of Trichoderma sp. T31355-A on the 4th, 5th and 6th days were 37.88%, 41.69% and 34.53%, respectively.

[0079] Example 1.3. Determination of cellulose degradation activity

[0080] 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.

[0081] Take a 5mm Trichoderma T31355-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 dye for 2h, discard the dye, 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 T31355-A on CMC-Na medium is 45.3mm, the colony diameter is 61.3mm, and the ratio of the hydrolysis transparent zone diameter to the colony diameter is greater than 0.7, indicating that Trichoderma T31355-A has cellulose degradation activity ( Figure 1-5 ).

[0082] Example 1.4. Determination of antibacterial activity by cellophane method

[0083] The antimicrobial activity of the metabolites produced by Trichoderma sp. T31355-A against Fusarium graminearum, Rhizoctonia solani, Sclerotinia sclerotiorum and Ustilaginoidea virens were determined by the membrane filtration method.

[0084] 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.

[0085] A 5 mm cake of Trichoderma T31355-A cultured in PDA medium at 25℃ for 2 days was pre-inoculated on a double-layer glass paper of 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 grown on it were removed with sterile tweezers, and then a 5 mm cake of the target pathogenic fungus was inoculated in 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.

[0086] Inhibition rate (%) = (control colony diameter - treated colony diameter) / (control colony diameter - bacterial cake diameter) × 100%. The membrane filter method showed that the inhibition rate of Trichoderma T31355-A against Fusarium graminearum was 98%, and the inhibition rate against Rhizoctonia solani, Sclerotinia sclerotiorum and Ustilaginoidea virens was 100%, indicating that Trichoderma T31355-A has good antibacterial activity against pathogenic fungi ( Figure 1-6 ).

[0087] Example 1.5: Potted plant test to determine the effect on cucumber growth

[0088] 1. Determination of the effect of Trichoderma T31355-A on cucumber growth using sterile matrix potted plants

[0089] 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.

[0090] 100 g of the test matrix was placed in a tissue culture bottle and sterilized at 121°C for 30 min. Trichoderma T31355-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 3.87 cm, 0.447 g and 0.156 g, respectively, while the plant height, aboveground fresh weight and root fresh weight of cucumber seedlings treated with Trichoderma T31355-A were 6.73 cm, 1.691 g and 0.352 g, respectively, which were 74.1%, 277.9% and 126.1% higher than those of the control, respectively, significantly promoting the growth of cucumber plants ( Figure 1-7 ).

[0091] 2. Determination of the effect of Trichoderma T31355-A on cucumber growth using garden soil potted plants

[0092] 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 dry weight of the whole plant of the cucumber seedlings were measured on the 14th day after sowing, and each treatment was repeated 3 times. The growth potential was calculated, and the growth potential = (stem diameter / plant height) × dry weight. The results showed that the growth potential of the cucumber seedlings treated with CK was 0.12 (stem diameter was 2.38mm, plant height was 8.35cm, and dry weight was 0.418g), and the growth potential of the cucumber seedlings treated with Trichoderma T31355-A was 0.17 (stem diameter was 3.52mm, plant height was 8.10cm, and dry weight was 0.383g), which was 40.2% higher than the control, significantly promoting the growth of cucumber plants.

[0093] Example 1.6 Effect on the Induced Disease Resistance of Cucumber Gray Mold

[0094] 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 1-8 ), measure the diameter of leaf lesions, and calculate the control effect.

[0095] Control effect (%) = (control lesion diameter - treatment lesion diameter) / (control lesion diameter - fungus cake diameter) × 100%. The results showed that the lesion diameter of the control group was 3.18 cm, and the lesion diameter of the Trichoderma T31355-A treatment was 1.52 cm. The size of the gray mold lesions on cucumber leaves treated with Trichoderma T31355-A was significantly reduced, indicating that inoculation of Trichoderma increased the resistance of cucumber to gray mold. The control effect of Trichoderma T31355-A on cucumber gray mold was 62.1%, and the spore content of Trichoderma T31355-A in the potting medium was 1.4×10 9 / g cultivation substrate, indicating that Trichoderma T31355-A can effectively reproduce in the substrate and thus occupy the ecological niche.

[0096] Option 2:

[0097] Example 2.1 Isolation and identification of Trichoderma neohongkuii strain YNE00787-A

[0098] 1. Separation

[0099] Malt Extract Agar (MEA): 20 g malt extract, 15 g agar, 1000 mL distilled water, sterilized at 121°C for 15 min.

[0100] Root samples of Thysanolaena latifolia, a grass plant, were collected from the Naban River Basin Nature Reserve in Xishuangbanna, Yunnan Province, China, and isolated using the tissue separation method. The process is as follows: select healthy roots without disease symptoms, rinse the roots with tap water, and cut the roots into segments of about 10 cm in length; then disinfect the root 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 root segments to PDA culture medium plates for culture, and purify the strain YNE00787-A.

[0101] 2. Taxonomic characteristics

[0102] Culture characteristics: YNE00787-A was cultured on CMD medium at 25°C for 3 days. The colony radius was 49-53 mm and it covered the plate in 4 days. The colony was transparent and radial. The aerial hyphae were loose and pale white. Conidia formed in large white cotton-like conidia after 3 days of culture. After 4-5 days, they turned green and spread in 2-3 inconspicuous concentric rings. No odor or pigment was found ( Figure 2-1 ).

[0103] Microscopic features: Conidiophores are tree-like ( Figure 2-2 ), main axis is obvious, the angle between branches and main axis is slightly less than 90°, opposite or solitary, with terminal branches or phialides at the top, and long branches are located at the base of the main axis; phialides are arranged in 2-3 whorls, with basal swells, phialides are straight or slightly swollen in the middle, ampoule-shaped, and measure 5.3-10.7×2.4-3.5μm; conidia are elliptical, green or yellow-green, and measure 4.3-3.5×3.5-2.7μm; thick-walled spores are solitary, terminal or intercalary, spherical or nearly spherical, and measure 6.5-9.7×6.1-9.5μm.

[0104] 3. Identification of YNE00787-A

[0105] Strain YNE00787-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 YNE00787-A with those of Trichoderma neohongkuii model strain YNE00787 were 100% and 100%, respectively, and the sequence similarity values ​​of rpb2 and tef1 with those of Trichoderma hongkuii model strain T32026 were 99.63% and 96.66%, respectively, and the sequence similarity values ​​of rpb2 and tef1 with those of Trichoderma parahongkuii model strain T31356 were 99.02% and 96.30%, respectively. A phylogenetic tree was constructed based on the rpb2 and tef1 sequence datasets ( Figure 2-3), the maximum likelihood support rate (left) higher than 70% and the Bayesian posterior probability (right) greater than 0.90 are given above the phylogenetic tree nodes. Strains YNE00787-A and YNE00787 form an independent branch (maximum likelihood bootstrap probability MLBS / Bayesian posterior probability BIPP=100% / 1.00), Trichoderma hongkuii strains T32026 and T32027 form an independent branch (MLBS / BIPP=100% / 1.00), and Trichoderma parahongkuii strains T31356 and T31355 form an independent branch (MLBS / BIPP=100% / 1.00). These three branches form a large branch (MLBS / BIPP=98% / 1.0). Based on the sequence similarity values ​​of rpb2 and tef1 and the results of phylogenetic analysis, YNE00787-A was identified as Trichoderma neohongkuii.

[0106] Example 2.2, determination of siderophore activity

[0107] Take a 5mm Trichoderma YNE00787-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-red, 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 YNE00787-A changes the CAS medium from blue to purple-red, indicating that it has the ability to produce catechol siderophores. The colony diameter of Trichoderma YNE00787-A on the CAS medium plate is 58.3mm, the color change circle diameter is 85.0mm, and the ratio of the color change circle diameter to the colony diameter is 1.5( Figure 2-4 ).

[0108] Take a 5mm Trichoderma YNYNE00787-A cake cultured in PDA medium at 25℃ for 2 days, inoculate it in LNM medium, and culture it at 25℃ and 120r / min for 6 days. Take samples on the 4th, 5th and 6th days, centrifuge at 10000r / min for 10min, aspirate the supernatant, add an equal volume of CAS dye, mix evenly, keep it away from light for more than 1h, and use a spectrophotometer to measure the absorbance (As) at 680nm. Add an equal volume of liquid culture medium to which uninoculated PDA medium cakes are added and CAS dye is mixed, and its absorbance is used as the reference value (Ar). Each treatment is repeated 3 times. Siderophore production is expressed as the relative content of siderophore activity units (SU), SU (%) = [(Ar-As) / Ar] × 100%. The contents of Trichoderma YNE00787-A on the 4th, 5th and 6th days were 52.17%, 47.42% and 46.28% respectively.

[0109] Example 2.3. Cellulose degradation activity assay

[0110] Take the 5mm Trichoderma YNE00787-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 YNE00787-A on CMC-Na medium is 42.7mm, the colony diameter is 50.7mm, and the ratio of the hydrolysis transparent zone diameter to the colony diameter is greater than 0.8, indicating that Trichoderma YNE00787-A has cellulose degradation activity ( Figure 2-5 ).

[0111] Example 2.4: Determination of antibacterial activity by membrane filtration method

[0112] The antimicrobial activity of the metabolites produced by Trichoderma YNE00787-A against Fusarium graminearum, Fusarium oxysporum, Rhizoctonia solani, Sclerotinia sclerotiorum and Ustilaginoidea virens were determined by the filter membrane method.

[0113] 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.

[0114] A 5 mm cake of Trichoderma YNE00787-A cultured in PDA medium at 25℃ for 2 days was pre-inoculated on a double-layer glass paper of a PDA medium plate, and cultured at 25℃ for 2 days in light and dark (light: dark = 12h: 12h, light intensity 15000Lux). The glass paper and the Trichoderma grown on it were removed with sterile tweezers, and then a 5 mm cake of the target pathogenic fungus was inoculated in 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℃ for 5 days in light and dark (light: dark = 12h: 12h, light intensity 15000Lux), 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.

[0115] Inhibition rate (%) = (control colony diameter - treated colony diameter) / (control colony diameter - bacterial cake diameter) × 100%.

[0116] The membrane filtration method showed that the inhibition rates of Trichoderma YNE00787-A against Fusarium graminearum, Fusarium oxysporum, Rhizoctonia solani, Sclerotiniasclerotiorum and Ustilaginoidea virens were 84.3%, 62.9%, 99.4%, 90.0% and 100%, respectively, indicating that Trichoderma YNE00787-A has good antibacterial activity against pathogenic fungi ( Figure 2-6 ).

[0117] Example 2.5: Potted plant test to determine the effect on cucumber growth

[0118] 1. Determination of the effect of Trichoderma YNE00787-A on cucumber growth using sterile matrix potted plants

[0119] 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.

[0120] 100 g of the test matrix was placed in a tissue culture bottle and sterilized at 121°C for 30 min. Trichoderma YNE00787-A was cultured in a PDA culture 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 0.2MS culture medium plate. Germination was accelerated at 28℃ in the dark until 70% of the cucumber seeds germinated. The cucumber seeds with consistent germination were sown in the above 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 YNE00787-A were 7.07 cm, 1.811 g and 0.332 g, respectively, which were 82.8%, 304.8% and 113.5% higher than those of the control, respectively, significantly promoting the growth of cucumber plants ( Figure 2-7 ).

[0121] 2. Determination of the effect of Trichoderma YNE00787-A on cucumber growth using garden soil potted plants

[0122] 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. Calculate the growth potential, 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 that obtained in Scheme 1, and the growth potential of the cucumber seedlings treated with Trichoderma YNE00787-A was 0.16 (stem diameter was 3.45mm, plant height was 8.30cm, and dry weight was 0.390g), which was 36.2% higher than the control, significantly promoting the growth of cucumber plants.

[0123] Example 2.6 Effect on the Induced Disease Resistance of Cucumber Gray Mold

[0124] 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.

[0125] Control effect (%) = (control lesion diameter - treatment lesion diameter) / (control lesion diameter - fungus cake diameter) × 100%.

[0126] The results showed that the diameter of the lesions in the control group was the same as that in scheme 1, and the diameter of the lesions treated with Trichoderma YNE00787-A was 1.48 cm. The size of the gray mold lesions on cucumber leaves treated with Trichoderma YNE00787-A was significantly reduced, indicating that the inoculation of Trichoderma increased the resistance of cucumber to gray mold. The control effect of Trichoderma YNE00787-A on cucumber gray mold was 63.4%, and the spore content of Trichoderma YNE00787-A in the potting medium was 1.3×10 9 / g cultivation substrate, indicating that Trichoderma YNE00787-A can effectively reproduce in the substrate and thus occupy the ecological niche.

[0127] contrast:

[0128] HTTA-Z0002, HB40619, HB40609, and Tk905 were tested according to the method described in Example 1.6 (Example 2.6). 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 T31355-A of the present invention (1.52 cm) and the diameter of the lesions treated with Trichoderma YNE00787-A of the present invention (1.48 cm).

[0129] The corresponding spore contents in the culture medium of HTTA-Z0002, HB40619, HB40609, and Tk905 are approximately (2-8)×10 5 The propagation effect is far inferior to that of T31355-A and YNE00787-A of the present invention.

[0130] 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 parahongkuii T31355-A, Features: The deposit number is GDMCC NO.64773.

2. The use of Trichoderma parahongkuii T31355-A as claimed in claim 1, Features: Promote plant growth and prevent and control plant diseases.

3. The use of Trichoderma parahongkuii T31355-A according to claim 2, Features: The strain T31355-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.

4. The use of Trichoderma parahongkuii T31355-A according to claim 3, Features: Plant pathogenic fungi include Fusarium graminearum, Sclerotiniasclerotiorum, Ustilaginoidea virens, and Rhizoctonia solani.

5. Trichoderma neohongkuii YNE00787-A, characterized in that: the deposit number is GDMCC NO.64774.

6. The use of Trichoderma neohongkuii YNE00787-A as claimed in claim 5, Features: Promote plant growth and prevent and control plant diseases.

7. The use of Trichoderma neohongkuii YNE00787-A according to claim 6, Features: 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.

8. The use of Trichoderma neohongkuii YNE00787-A according to claim 7, Features: Plant pathogenic fungi include Fusarium graminearum, Fusarium oxysporum, Sclerotinia sclerotiorum, Ustilaginoidea virens, and Rhizoctonia solani.

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