A strain of Trichoderma yunnanensis and its application

Abstract

The present invention relates to the field of microbial technology, and discloses a strain of Trichoderma yunnanensis and its application. Trichoderma yunnanensis )CCTCC NO: M 2025645 is a high-yield siderophore strain with a siderophore production activity of 69.79%, capable of synthesizing and secreting carboxylic acid siderophores; and the strain has sulfur oxidation function; In addition, the present invention also provides the use of the Trichoderma in regulating plant diseases, and the Trichoderma of the present invention can antagonize Fusarium solani ( Fusarium solani ), Fusarium graminearum ( Fusarium gramineae )、Fusarium rotundifolia( Fusarium concentricum ) and brown crustacean ( Rosellinia necatrix ), Alternaria brassicae ( Alternaria brassicae (Fr.) Keissler); The Yunnan Trichoderma fungus provided by the present invention is a strain isolated from tobacco-growing soil. Compared with strains screened in extreme environments and laboratory mutagenesis, it has better adaptability to the planting environment and can stably colonize in the rhizosphere and soil of plants, thereby exerting a long-term and stable growth-promoting effect. It is safe and environmentally friendly and has broad application prospects.

Description

Technical Field

[0001] The present invention relates to the technical field of microorganisms, and in particular to a strain of Trichoderma yunnanensis and applications thereof. Background Art

[0002] Soil health is crucial for sustainable agricultural development. However, due to excessive fertilizer and pesticide use and improper cropping practices, my country faces a range of soil degradation issues, including soil compaction, continuous cropping problems, salinization, and heavy metal contamination, which severely impact crop yields and food security. Therefore, finding green, environmentally friendly, and effective measures to improve soil health is urgent.

[0003] The rhizosphere is the interface between crops and the environment. Rhizosphere microorganisms participate in various soil biochemical processes. Plant growth-promoting rhizobacteria (PGPR) are rhizosphere microorganisms that are beneficial to plant growth. They promote crop nutrient absorption, inhibit pathogenic microorganisms, improve abiotic stress environments, and secrete plant hormones. They are important for promoting plant growth and maintaining soil ecological health. The diverse microbial communities in the soil assist in completing a series of important soil ecological functions by regulating soil nutrient cycling, decomposing soil organic matter, inhibiting soil-borne diseases, improving soil structure, and supporting plant production. They are a vital link in the material cycle and energy flow of ecosystems, maintaining the sustainable development of ecosystems. Using soil microorganisms to assist in soil improvement and the removal and remediation of toxic pollutants is a cost-effective and environmentally friendly method.

[0004] Currently, PGPR's role in remediating soil barriers and promoting crop growth is gaining increasing attention. Microbial fertilizers, biopesticides, and seedling substrates developed with rhizosphere microorganisms as core components are already widely used. Therefore, further exploring and screening more PGPR strains with efficient colonization and broad functionality will not only facilitate the development of new biofertilizer products and improve agricultural production efficiency, but also further promote the development of agriculture towards a greener, more environmentally friendly, and sustainable direction. Summary of the Invention

[0005] The present invention aims to solve the problem that the existing technology lacks effective plant biocontrol bacteria and their products in practical applications, and provides a Yunnan Trichoderma strain and its application. The Yunnan Trichoderma strain provided by the present invention is isolated from tobacco-growing farmland soil in Zhaotong City, Yunnan Province. The strain has strong siderophore production activity and sulfur oxidation function, and can also antagonize Fusarium solani ( Fusarium nightshade ), Fusarium graminearum ( Fusarium gramineae )、Fusarium rotundifolia( Fusarium concentricum ) and brown crustacean ( Rosellinia necatrix ), Alternaria brassicae ( Alternaria brassicae(Fr.) Keissler).

[0006] To achieve the above object, the present invention provides a first aspect of a Trichoderma yunnanensis strain ( Trichoderma from Yunnan ), the strain number is YNK-FG0001, and the preservation number is CCTCC NO: M 2025645.

[0007] The second aspect of the present invention provides a bacterial agent, the active ingredient of which is the Trichoderma yunnanensis of the first aspect.

[0008] The third aspect of the present invention provides the use of the strain of the first aspect or the bacterial agent of the second aspect in producing siderophores, sulfur oxidation and antagonizing pathogens; the pathogens include Fusarium solani ( Fusarium solani ), Fusarium graminearum ( Fusarium gramineae )、Fusarium rotundifolia( Fusarium concentricum ) and brown crustacean ( Rosellinia necatrix ), Alternaria brassicae ( Alternaria brassicae (Fr.) Keissler).

[0009] Through the above technical solution, the present invention can achieve at least the following beneficial effects:

[0010] (1) The Yunnan Trichoderma provided by the present invention has a strong siderophore production function and sulfur oxidation function, and can also antagonize Fusarium solani ( Fusarium solani ), Fusarium graminearum ( Fusarium gramineae )、Fusarium rotundifolia( Fusarium concentricum ) and brown crustacean ( Rosellinia necatrix ), Alternaria brassicae ( Alternaria brassicae (Fr.) Keissler), which can effectively prevent and control diseases and improve soil quality.

[0011] (2) The Yunnan Trichoderma provided by the present invention is a strain isolated from flue-cured tobacco soil. Compared with strains screened in extreme environments and laboratory mutagenesis, it has better adaptability to the planting environment and can stably colonize in the rhizosphere and soil of plants, thereby exerting a long-term and stable growth-promoting effect. Moreover, since it is a strain isolated from farmland soil, the risk of the strain causing adverse effects on the local environment and the microbial ecology in the soil during its application in farmland is reduced. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Figure 1 1 is a colony morphology diagram of the YNK-FG0001 strain cultured on a plate in Example 1;

[0013] Figure 2 is the phylogenetic tree constructed based on the ITS gene sequence of the YNK-FG0001 strain in Example 1;

[0014] Figure 3 2 is a diagram showing the effect of siderophore production by the YNK-FG0001 strain in Example 2, wherein A is the front view of the culture plate and B is the back view of the culture plate;

[0015] Figure 4 is the absorbance value of the siderophore produced by the YNK-FG0001 strain in Example 3 under the full wavelength range;

[0016] Figure 5 This is a diagram of the sulfur oxidation effect of the YNK-FG0001 strain in Example 4, in which: (A) is a physical picture of the strain cultured in MST medium with a color developer, and (B) is a comparison of CK (color developer) and a mixture of fermentation broth and MTS liquid culture medium.

[0017] Figure 6 The antagonistic effect of strain YNK-FG0001 against pathogens in Example 5 is shown in FIG: (A) Antagonistic effect against Fusarium solani ( Fusarium solani ) effect diagram; (B) Antagonism against Fusarium graminearum ( Fusarium gramineae ) effect diagram; (C) Antagonistic to Fusarium rotundifolia ( Fusarium concentricum ) effect diagram; (D) Antagonism against Alternaria brassicae ( Alternaria cabbages (Fr.) Keissler) effect diagram; (E) Antagonism against brown soil crust fungus ( Rosellinia necatrix ) effect diagram.

[0018] Biological Deposits

[0019] The Yunnan Trichoderma provided by the present invention is classified and named as: Trichoderma yunnanensis YNK-FG0001 was deposited in the China Center for Type Culture Collection on March 31, 2025, located at 299 Bayi Road, Wuchang District, Wuhan City, Hubei Province, Wuhan University, with the deposit number: CCTCC NO: M 2025645. DETAILED DESCRIPTION

[0020] The endpoints of the ranges and any values ​​disclosed herein are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoints of each range, the endpoints of each range and individual point values, and the individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered to be specifically disclosed herein.

[0021] In the present invention, unless otherwise specified, the term "siderophore" refers to a small molecule compound that chelates iron; "strain with high siderophore production activity" refers to a microorganism that can efficiently synthesize siderophores; "fermentation metabolites" refers to a strain that secretes many metabolites through fermentation; and "antibacterial activity" refers to the activity of inhibiting or hindering the growth of plant pathogens.

[0022] During the research, the inventors of the present invention isolated a strain of Trichoderma yunnanensis ( Trichoderma yunnanensis ). It was found that the strain has the ability to produce siderophores and antagonize Fusarium solani ( Fusarium solani ), Fusarium graminearum ( Fusarium gramineae )、Fusarium rotundifolia( Fusarium concentricum ) and brown crustacean ( Rosellinia necatrix ), Alternaria brassicae ( Alternaria brassicae (Fr.) Keissler) pathogens, the strain not only promotes nutrient absorption but also antagonizes pathogens. Further research has also revealed that it can effectively promote plant growth, increase agricultural productivity, and promote the sustainable development of ecological agriculture.

[0023] Based on the above findings, the first aspect of the present invention provides a strain of Trichoderma yunnanensis ( Trichoderma from Yunnan ), the deposit number of this strain is CCTCC NO: M 2025645.

[0024] The second aspect of the present invention provides a microbial agent, which comprises the Trichoderma yunnanensis as described in the first aspect and an optional carrier.

[0025] The third aspect of the present invention provides use of the Trichoderma yunnanensis described in the first aspect or the bacterial agent described in the second aspect in producing siderophores.

[0026] The present invention further provides a quantitative analysis method for verifying the siderophore production activity of the strain, which comprises:

[0027] YNK-FG0001 strain was inoculated into culture medium and cultured.

[0028] According to the present invention, the culture medium can be a PDA solid culture medium, which can be a conventional culture medium suitable for the growth of Trichoderma. In a preferred embodiment, the solid culture medium contains: 200-400g of potatoes, 20-40g of glucose, 18-36g of agar, 1-2L of deionized water, pH 7.0. The color developer contains: CAS detection solution (0.0605g of chrome azurol S, 1mmol·L -1 FeCl3·6H2O+10mmol·L-1 HCl, deionized water, etc.). Liquid culture medium contains: glucose 30.0-60.0 g·L -1 , sodium nitrate 2.0-40.0g·L -1 , potassium phosphate trihydrate 1.0-2.0g·L -1 , potassium chloride 0.5-1.0g·L -1 , magnesium sulfate heptahydrate 0.5g·L -1 , 8-hydroxyquinoline 0.75-1.5g·L -1 , deionized water 1.0-2.0L.

[0029] According to the present invention, the preferred conditions for culturing the strain include: a preferred temperature of 25-30° C., and a preferred culturing time of 3-4 days.

[0030] Any method and conditions that can verify Trichoderma yunnanensis and enable it to produce siderophores can be applied to the present invention.

[0031] The fourth aspect of the present invention provides the use of the Trichoderma yunnanensis as described above in the preparation of carboxylic acid type iron carrier.

[0032] A fifth aspect of the present invention provides a method for sulfur oxidation, comprising culturing the Trichoderma yunnanensis described in the first aspect and collecting the culture product.

[0033] Any method and conditions capable of fermenting and culturing Trichoderma yunnanensis and causing it to undergo sulfur oxidation can be applied to the present invention.

[0034] The sixth aspect of the present invention provides the Yunnan Trichoderma described in the first aspect in antagonizing Fusarium solani ( Fusarium nightshade ), Fusarium graminearum ( Fusarium gramineae )、Fusarium rotundifolia( Fusarium concentricum ) and brown crustacean ( Rosellinia necatrix ), Alternaria brassicae ( Alternaria brassicae (Fr.) Keissler)'s application.

[0035] A seventh aspect of the present invention provides a bacterial agent having the function of promoting plant growth, wherein the active ingredient in the bacterial agent includes the Trichoderma yunnanensis described in the first aspect.

[0036] According to a preferred embodiment of the present invention, the bacterial agent is a liquid bacterial agent.

[0037] Preferably, the content of Trichoderma yunnanensis in the liquid bacterial agent is less than 1×10 6 —1×10 8 pieces / mL.

[0038] The present invention will be described in detail below by way of examples. It should be understood that the following examples are only used to further explain and illustrate the present invention, and are not intended to limit the present invention.

[0039] In the following examples, unless otherwise specified, all reagents and materials used were purchased from regular chemical / biological reagent or material suppliers, and all reagents were of analytical grade.

[0040] In the following examples, unless otherwise specified, the operating temperature is room temperature (25±5° C.).

[0041] Example 1

[0042] This example is used to illustrate the acquisition, identification and preservation of Trichoderma yunnanensis CCTCC NO: M 2025645.

[0043] (1) Strain isolation and purification

[0044] A fungus was isolated from the soil of a flue-cured tobacco farmland in Yiliang County, Zhaotong City, Yunnan Province, China (104°21'E, 27°37'N) in August 2024 and deposited in the General Microbiology Center of the China Culture Collection of Microorganisms with the deposit number CCTCC NO: M 2025645.

[0045] PDA medium was used in the isolation and purification of the strain. The preparation method was as follows: 200 g of potato, 20 g of glucose, 15-20 g of agar, 1000 mL of deionized water, adjusted to pH 7.0 ± 0.1, and sterilized by high pressure at 121°C for 25 min.

[0046] A fungus was isolated and purified from soil samples collected from flue-cured tobacco fields in Yiliang County, Zhaotong City, Yunnan Province by using the dilution spread plate method and tip hyphae picking method. It was named Trichoderma yunnanensis.

[0047] (2) Strain identification

[0048] 1. Identification of fungal morphological characteristics and molecular genetic classification

[0049] Colony: Yunnan Trichoderma strain YNK-FG0001 was inoculated on PDA medium and cultured at 28℃. The colony on the PDA medium showed the following changes: at the beginning, the strain was white, dense, and round, and expanded to the surrounding areas; after growing for a period of time, light green spores were produced from the center of the colony, and the center turned green; finally, the entire colony turned green. Figure 1 As shown. Trichoderma yunnanensis) strain was inoculated into PDA solid medium and cultured at 30°C for 48 hours. The strain was then sent to the Chongqing branch of Beijing Qingke Biotechnology Co., Ltd. for ITS sequencing. The sequence result was: (SEQ ID NO.1). The sequencing results were imported into NCBI for comparison and compared with standard strain sequences with a homology of more than 80%. Trichoderma yunnanense , NR134419) with a homology of up to 98.98% (see Figure 2 ), so it was identified as a microorganism of the subphylum Deuteromycotina, class Hypomycetes, order Hypomycetales, family Hypomycetaceae, and genus Trichoderma. A phylogenetic tree of the strain was constructed using MEGA7. Figure 2 The drawn phylogenetic tree of the strains is shown.

[0050] 3. Identification results

[0051] Combined with the molecular detection results of strain YNK-FG0001 and the fungal morphological characteristics detection results, the strain was identified as Trichoderma yunnanensis ( Trichoderma yunnanensis )

[0052] (3) Strain preservation

[0053] The classifications obtained above are named as: Trichoderma yunnanensis The Trichoderma yunnanensis strain YNK-FG0001 was deposited on March 31, 2025, at the China Center for Type Culture Collection, Wuhan University, No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province, China, with the deposit number CCTCCNO: M 2025645.

[0054] Example 2

[0055] This example is used to illustrate the siderophore production performance of the strains involved in the present invention.

[0056] The culture method is as follows:

[0057] Preparation of iron-free Czapek solid medium: glucose 30.0 g·L -1 , sodium nitrate 2g·L -1 , potassium phosphate trihydrate 1g·L -1 , potassium chloride 0.5g·L -1 , magnesium sulfate heptahydrate 0.5g·L -1 , 8-hydroxyquinoline 0.75 g·L -1 , 1L of deionized water, 18g of solid agar. Double-layer color development medium: lower layer: 100ml of deionized water, 1.8g of agar, 15ml of CAS solution; upper layer: iron-free Czapek solid medium. Then inoculate the YNK-FG0001 strain on the double-layer plate medium. The appearance of red indicates that the strain secretes iron carriers. Figure 3 As shown in A and B, a red halo appeared around the colony, indicating that the strain secreted siderophore.

[0058] The siderophore production activity of the strain was quantitatively determined as follows:

[0059] Strains whose bottom layer of the culture medium turned red were selected and cultured in iron-free Czapek liquid medium at 28°C and 150 rpm on a constant temperature shaker (model: HS-200B) for 48 hours. After incubation, 2–5 mL of the culture medium was filtered through a 0.22 μm sterile filter membrane and an equal volume of CAS detection solution was added. After 1 hour of filtration, the OD630 value was measured using a full-wavelength microplate reader (model: MultiskaGO), denoted as As. The absorbance of the uninoculated liquid medium was measured using the same method as a reference value, denoted as Ar. The siderophore concentration was expressed in siderophore activity units (SU): SU = [(Ar - As) / Ar] × 100%. The determination was repeated three times, and the average value was calculated for comparison and analysis. The results are shown in Table 1. After 5 days of culture, the siderophore production activity of the strain was 69.79%.

[0060] Table 1: Quantitative determination of siderophore production by strains

[0061]

[0062] Note: The data in the table are the average values ​​(standard deviation) of the soil sample analysis results; Ar; the blank group at OD 630 Absorbance value (reference value); As; treatment group at OD 630 Absorbance value.

[0063] Example 3

[0064] This example is used to illustrate the chemical structure type identification of the siderophore compound produced by the strain of this research invention. The identification method is as follows:

[0065] Carboxylate-type siderophores: Identification using the copper sulfate test (CuSO4 test): 2 ml of 0.25 μmol / L CuSO4 solution and 4 ml of pH 4.0 acetate buffer were added to a volumetric flask containing 2 ml of strain test solution (each strain was tested separately). The sample was then scanned using a high-performance liquid chromatograph (HPLC) (Agilent 1260) with full-band UV light. If a maximum absorption peak appeared between 190 nm and 280 nm, the strain secreted carboxylates. The results showed that the strain culture filtrate exhibited a maximum absorption peak between 190 nm and 280 nm, indicating that the strain YNK-FG0001 produced carboxylates. Figure 4 .

[0066] Example 4

[0067] This example is used to illustrate the sulfur oxidation effect of Trichoderma yunnanensis CCTCC NO: M 2025645. The effect testing method is as follows:

[0068] MST (Modified Thiosulfate) Medium (g / L): Weigh 5.0 g of sodium thiosulfate, 0.1 g of potassium dihydrogen phosphate, 0.2 g of sodium bicarbonate, 0.1 g of ammonium chloride, 5.0 g of glucose, and 5.0 g of yeast extract powder and dissolve in 1000 mL of water. Add 0.008 g of bromocresol purple as a color developer, adjust the pH to 8 ± 0.1, and add 20 g of agar. Autoclave at 121°C for 20 min.

[0069] Method 1: The strain YNK-FG0001 obtained in Example 1 was cultured on potato agar solid medium and placed in a 25°C constant temperature incubator for inverted culture. A 5mm diameter cake was taken from the cultured Yunnan Trichoderma using a hole punch and inoculated into MST medium with a color developer. Each treatment was repeated 3 times. The plate was placed in a 30°C incubator and continuously observed for yellow circles on the colony plate. Method 2: The Yunnan Trichoderma strain was inoculated into sterilized PDB liquid culture medium and cultured on a shaker at 25°C and 160r / min for 8 days. A large number of bacteria and spores were produced in the culture medium. The culture medium was filtered with 4 layers of sterile gauze to obtain the fermentation stock solution of Trichoderma. Take 2ml of the fermentation stock solution and mix it with 2ml of MST (modified thiosulfate) liquid to see if the MST liquid turns yellow.

[0070] Figure 5 (AB) show the sulfur oxidation effect of strain YNK-FG0001. As can be seen from Figure (A), the culture medium at the bottom of the colony produces a yellow circle, and Figure (B) the MST liquid turns yellow, indicating that the strain is capable of sulfur oxidation.

[0071] Example 5

[0072] This example is used to illustrate the interaction between Trichoderma yunnanensis YNK-FG0001 and Fusarium solani ( Fusarium solani ), Fusarium graminearum ( Fusarium gramineae )、Fusarium rotundifolia( Fusarium concentricum ) and brown crustacean ( Rosellinia necatrix ), Alternaria brassicae ( Alternaria brassicae (Fr.) Keissler)'s antagonistic effect.

[0073] Test pathogen: Fusarium solani ( Fusarium solani ), Fusarium graminearum ( Fusarium grasses )、Fusarium rotundifolia( Fusarium concentricum ) and brown crustacean ( Rosellinia murderer ), Alternaria brassicae ( Alternaria brassicae (Fr.) Keissler) was provided by the laboratory of the Institute of Agricultural Resources and Environment, Yunnan Academy of Agricultural Sciences.

[0074] Plate confrontation test: Fusarium solani ( Fusarium solani ), Fusarium graminearum ( Fusarium grasses )、Fusarium rotundifolia( Fusarium concentricum ) and brown crustacean ( Rosellinia murderer ), Alternaria brassicae ( Alternaria brassicaeKeissler) was used as the indicator pathogen. A 5 mm pathogen cake was inoculated in the center of the PDA culture medium, and the strain YNK-FG0001 obtained in Example 1 was inoculated in a cross pattern at a distance of 25 mm. The uninoculated plate served as a control. Three replicates were used for each strain. The plates were cultured in a constant temperature incubator at 25-30°C in the dark for 5-7 days, and the inhibition rate was calculated.

[0075] After the culture was completed, the average value and inhibition rate were calculated. Inhibition rate (%) = (colony diameter of the control group - colony diameter of the treatment group) / (colony diameter of the control group - 5) × 100.

[0076] Figure 6 (A)-(E) show the effects of strain YNK-FG0001 on Fusarium solani ( Fusarium solani ), Fusarium graminearum ( Fusarium gramineae )、Fusarium rotundifolia( Fusarium concentricum ) and brown crustacean ( Rosellinia necatrix ), Alternaria brassicae ( Alternaria brassicae The results of the plate confrontation assay showed that strain YNK-FG0001 could antagonize the above five pathogens, with inhibition rates of 65.40%, 33.64%, 54.60%, 45.15% and 50.26% respectively.

[0077] The preferred embodiments of the present invention have been described in detail above, but the present invention is not limited thereto. Within the technical concept of the present invention, various simple variations of the technical solution of the present invention may be made, including combining the various technical features in any other appropriate manner. These simple variations and combinations should also be regarded as disclosed in the present invention and fall within the scope of protection of the present invention.

Claims

1. A strain of Trichoderma yunnanensis ( Trichoderma yunnanense ), characterized in that The deposit number of this strain is: CCTCC NO: M2025645, and its classification name is: Trichoderma yunnanense .

2. A bacterial agent, characterized in that The active ingredient of the bacterial agent includes the Trichoderma yunnanensis according to claim 1.

3. The microbial agent according to claim 2, wherein The bacterial agent is a liquid bacterial agent; the content of Trichoderma yunnanensis in the bacterial agent is 1×10 6 -1×10 8 pieces / mL.

4. The use of the Yunnan Trichoderma according to claim 1 or the bacterial agent according to claim 2 in producing siderophores, sulfur oxidation and antagonizing pathogens; the pathogens include Fusarium solani ( Fusarium solani ), Fusarium graminearum ( Fusarium graminearum )、Fusarium rotundifolia( Fusarium concentricum ) and brown crustacean ( Rosellinia necatrix ), Alternaria brassicae ( Alternaria brassicae (Fr.) Keissler); among them, The siderophore is a carboxylic acid siderophore.

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