Trichoderma salt-tolerant growth-promoting consortium and application thereof

Abstract

The application discloses a salt-tolerant and growth-promoting Trichoderma combined probiotic and application thereof, and improves the salt-tolerant and growth-promoting effect of Trichoderma by combining the Trichoderma strain with resource substances. The application finds that the salt-tolerant and growth-promoting effect of Trichoderma is improved by combining the Trichoderma strain with at least one of resource substances vitamin B1, p-coumaric acid and homoarginine, and the high yield of Medicago polymorpha in saline-alkali soil is improved by combining the three resource substances with the Trichoderma. The salt-tolerant and growth-promoting effect of Trichoderma on Medicago polymorpha is improved, and the high yield of Medicago polymorpha in saline-alkali soil is promoted by combining at least one of resource substances vitamin B1, p-coumaric acid and homoarginine with the Trichoderma strain to obtain the Trichoderma combined probiotic.

Description

Technical Field

[0001] This invention belongs to the field of microbial technology, specifically relating to a salt-tolerant and growth-promoting Trichoderma synbiotic and its application in promoting high yield of alfalfa in saline-alkali land; specifically relating to the application of at least one of vitamin B1, p-coumaric acid, and high arginine in combination with Trichoderma NAU-DY2 to promote high yield of alfalfa in saline-alkali land. Background Technology

[0002] Soil salinization severely jeopardizes crop yield and quality. Globally, there are approximately 950 million hectares of saline soil, exceeding 6% of the world's arable land. Statistics show that annual agricultural losses due to soil salinization reach as high as US$27.3 billion. In my country, the area of ​​saline soil exceeds 99 million hectares, accounting for 10.4% of the total soil area, and this area is increasing annually. Saline-alkali land is widely distributed in my country's semi-arid and arid regions. Compared with the distribution of saline-alkali soils in other countries, my country's saline-alkali soils exhibit more complex characteristics, specifically reflected in their wide distribution, large area, high degree of salinization, and complex and diverse soil environments. Soil salinization not only fails to meet the requirements of sustainable agricultural development but also adversely affects my country's food security and economic development. Effectively reducing soil desertification and further improving existing saline-alkali soils is of significant strategic importance for maintaining arable land and establishing a bottom line for food security.

[0003] Trichoderma possesses multiple functions, including promoting plant growth, enhancing salt tolerance, and controlling soil-borne diseases. However, as a microbial product, growth-promoting agents must reach a sufficiently high quantity to effectively exert their biological functions. Trichoderma has limited resources available during its growth process and regenerates very slowly, making it difficult for growth-promoting agents to function effectively and limiting its large-scale application. Therefore, it is necessary to find resources that can promote Trichoderma growth, provide a suitable growth environment for Trichoderma in the soil, and enhance its salt tolerance and growth-promoting ability. The inventors intend to screen resource substances that can efficiently enhance the salt tolerance of Trichoderma, and apply Trichoderma in combination with these resource substances (synbiotics) into the soil to improve the salt tolerance of alfalfa. Summary of the Invention

[0004] In view of the technical problems in the prior art, due to the limited nutrients in the soil and the slow growth of Trichoderma under salt stress, Trichoderma often fails to proliferate in large quantities in the soil and exert its due preventive effect when applied to promote plant growth. The purpose of this invention is to provide a method to improve the salt tolerance and growth promotion effect of Trichoderma.

[0005] Another objective of this invention is to provide a salt-tolerant, growth-promoting Trichoderma synbiotic and its application.

[0006] Another object of the present invention is to provide a method for promoting high yield of alfalfa in saline-alkali land.

[0007] Experiments revealed that adding vitamin B1, coumaric acid, or arginine to the culture medium significantly improved the salt tolerance of *Trichoderma* fungus NAU-DY2. In greenhouse pot experiments, the combined use of at least one of vitamin B1, coumaric acid, and arginine with *Trichoderma* fungus NAU-DY2, applied to the soil via root irrigation, significantly increased alfalfa biomass in saline-alkali soil. This indicates that any one of vitamin B1, coumaric acid, or arginine, when used in combination with *Trichoderma* fungus NAU-DY2, can significantly enhance the salt tolerance and growth-promoting ability of *Trichoderma* to alfalfa.

[0008] The objective of this invention is achieved through the following technical solution:

[0009] In a first aspect, the present invention claims protection for a method for improving the salt tolerance and growth-promoting effect of Trichoderma, which involves using Trichoderma strains in combination with resource substances to improve the salt tolerance and growth-promoting effect of Trichoderma.

[0010] Secondly, the present invention claims protection for a salt-tolerant, growth-promoting Trichoderma synbiotic, the synbiotic comprising Trichoderma strains and resource substances.

[0011] Furthermore, the Trichoderma strain mentioned in the above method and in the Synbiotic is the Trichoderma strain NAU-DY2 with the preservation number: CCTCC NO: M20241303;

[0012] The Trichoderma strain NAU-DY2, classified as Trichoderma camerunense, was deposited on June 19, 2024, at the China Center for Type Culture Collection (CCTCC) with accession number M20241303, located at Wuhan University, Wuhan, China.

[0013] The resource substance is selected from at least one of vanillic acid, p-coumaric acid, gallic acid, syringic acid, salicylic acid, cinnamic acid, ferulic acid, coumaric acid, p-hydroxybenzoic acid, S-lactylglutathione, methionine, androstenedione, homoarginine, 4-guanidinobutyric acid, 5-methoxyindole-3-carboxaldehyde, vitamin B1, vitamin B3, vitamin B4, vitamin B5, vitamin B9, and vitamin B12. More preferably, the resource substance is selected from at least one of vitamin B1, p-coumaric acid, and homoarginine.

[0014] Thirdly, this invention seeks protection for the application of the aforementioned salt-tolerant and growth-promoting Trichoderma synbiotic in promoting high yields of alfalfa in saline-alkali land. Furthermore, the aforementioned application involves applying the aforementioned Trichoderma synbiotic to the soil using a root irrigation method.

[0015] Fourthly, this invention claims protection for a method to promote high yields of alfalfa in saline-alkali land, which involves applying the aforementioned Trichoderma synbiotic to alfalfa via root irrigation, or applying Trichoderma and resource substances to alfalfa in combination via root irrigation. Further, the method specifically involves applying Trichoderma and resource substances into the soil via root irrigation.

[0016] In a specific embodiment of the present invention, in the above application and method, 0.1L of a resource substance with a concentration of 0.5-1.5mM and 0.05L of a 1×10⁻⁶ mM resource substance are applied per kilogram of soil. 7 ~1×10 9 CFU / mL Trichoderma spore suspension.

[0017] Furthermore, in the above applications and methods, the resource substance is applied in solution form. Generally, the concentration of the stock solution of the resource substance is 0.1 mol / L. When using it, it can be diluted proportionally as needed, but is not limited to this. The method for preparing the resource substance solution is as follows: dissolve the resource substance in deionized water or warm water, draw it up with a sterile syringe, and filter it through a 0.22 μm filter membrane to obtain the resource substance solution.

[0018] The Trichoderma spore suspension is a Trichoderma strain NAU-DY2 spore suspension. Further, the concentration of the Trichoderma strain NAU-DY2 spore suspension is 1×10⁻⁶. 7 ~10 9 CFU / mL, preferably 1×10⁻⁶ 8 CFU / mL.

[0019] In a specific embodiment of the present invention, the Trichoderma strain NAU-DY2 spore suspension is obtained by the following method: strain NAU-DY2 is activated using PDA solid medium; cultured at 28°C for 7 to 10 days, and the bacterial cells are scraped into sterile physiological saline; the concentration of Trichoderma spores is adjusted with sterile physiological saline to obtain the Trichoderma NAU-DY2 spore suspension.

[0020] A further preferred method is to apply a resource substance (e.g., at least one of vitamin B1, p-coumaric acid, and high-arginine) and a suspension of Trichoderma strain NAU-DY2 spores to the soil via root irrigation 5-7 days after transplanting alfalfa seedlings with 2-3 true leaves. More specifically, 0.1 L of a 1 mM resource substance and 0.05 L of a 1 × 10⁻⁶ spore suspension are applied per kilogram of soil. 8 CFU / mL Trichoderma spore suspension.

[0021] The beneficial effects of this invention are:

[0022] This invention combines at least one of vitamin B1, p-coumaric acid, and high arginine with Trichoderma strain NAU-DY2 to obtain a Trichoderma synbiotic that enhances the salt tolerance and growth promotion ability of Trichoderma. This synbiotic can significantly improve the salt tolerance and growth promotion effect of Trichoderma on alfalfa, while increasing the yield of alfalfa in saline-alkali land. Attached Figure Description

[0023] Figure 1 Photographs showing the effects of different resource substances on the growth promotion of Trichoderma NAU-DY2 under salt stress.

[0024] Among them, CK is the treatment without resource substances; the rest are the treatments with the corresponding resource substances added; the colony photos are taken of fresh Trichoderma mycelia added to 1 / 4 PDA medium containing 400mM NaCl and incubated at 28℃ for 48h.

[0025] Figure 2 Colony diameter representing the growth-promoting effect of different resource substances on Trichoderma NAU-DY2 under salt stress.

[0026] Among them, CK is the treatment without resource substances; the rest are the treatments with the corresponding resource substances added; the colony diameter is measured by adding fresh Trichoderma mycelia to 1 / 4 PDA medium containing 400mM NaCl and culturing at 28℃ for 48h.

[0027] Figure 3 The effects of different resources on the growth promotion of alfalfa plants in saline-alkali soil were studied.

[0028] Figure 4 The results show the effects of different resource substances on the growth promotion of alfalfa plants in saline-alkali soil.

[0029] Figure 5 To investigate the growth-promoting effects of different Trichoderma prebiotics and Trichoderma on alfalfa plants in saline-alkali soil.

[0030] Figure 6 Results of the determination of the growth-promoting effects of different Trichoderma prebiotics and Trichoderma fungi on alfalfa plants in saline-alkali soil. (Biomaterial preservation information:)

[0031] Trichoderma strain NAU-DY2, classified as Trichoderma camerunense, was deposited at the China Center for Type Culture Collection (CCTCC) on June 19, 2024, with accession number CCTCC NO: M20241303, at Wuhan University, Wuhan, China. Detailed Implementation

[0032] The technical solution of the present invention will be further described below with reference to specific embodiments.

[0033] Experimental Example 1: Isolation, Screening and Identification of Trichoderma

[0034] Strain screening: Soil samples were collected from Dongying City, Shandong Province, China, and approximately 5g was weighed using a balance. A pre-sterilized conical flask containing 45ml of sterile water and several sterile glass beads was taken out. The soil sample was poured into the flask, sealed with a sealing film, and placed in a single-layer rotary shaker at 200 rpm for 1 hour. This is 10... -1 The dilution solution. On the laminar flow hood, select a test tube containing 4.5 ml of sterile water, and use a sterile pipette tip to transfer 0.5 ml of the suspension from the conical flask into it, and aspirate and blow several times. This is 10. -2 Dilute the solution, then transfer 0.5 ml of the suspension to the next test tube; this makes 10. -3 For diluents, prepare 10 solutions respectively, following this method. -3 10 -4 10 -5 Diluent. Three dilution levels were selected, and Trichoderma selective medium was used for spread culture. 0.1 ml of diluent was placed on each plate, and the spread should be as uniform as possible. Three replicates were set up for each dilution level. The uniformly spread Trichoderma selective medium plates were placed in an incubator at 28℃. Trichoderma colonies will grow in about two to three days. Colonies resembling Trichoderma were observed visually. Mycelia were picked and placed on PDA medium for further culture. Multiple subculturing was required for strain purification. The final strain was inoculated onto PDA slant for preservation. Finally, molecular biology ITS sequence identification identified it as *Trichoderma camerunense*, and it was named NAU-DY2.

[0035] The selected strain NAU-DY2 was classified and named Trichoderma camerunense. It was deposited at the China Center for Type Culture Collection on June 19, 2024, with accession number CCTCC NO: M20241303, and the deposit address is Wuhan University, Wuhan, China.

[0036] Example 2 investigated the effect of resource substances on salt tolerance and growth promotion of Trichoderma.

[0037] Resource substances: vanillic acid, p-coumaric acid, gallic acid, syringic acid, salicylic acid, cinnamic acid, ferulic acid, coumaric acid, p-hydroxybenzoic acid, S-lactylglutathione, methionine, androstenedione, high arginine, 4-guanidinobutyric acid, 5-methoxyindole-3-carboxaldehyde, vitamin B1, vitamin B3, vitamin B4, vitamin B5, vitamin B9, vitamin B12.

[0038] Preparation of the stock solution of resource substances: Accurately weigh each resource substance using a 0.01 g balance, dissolve it in deionized water or hot water, and aspirate it with a sterile syringe. Filter the solution through a 0.22 μm filter membrane into a 5 mL sterile centrifuge tube to obtain a sterile stock solution of resource substances with a concentration of 100 mM. Store the solution at -4 °C for later use.

[0039] Preparation of culture medium: When the pre-sterilized 1 / 4 PDA medium containing 400mM NaCl is cooled to about 40°C, add the pre-prepared stock solution of the resource substance to the medium to make the concentration of the resource substance in the medium 100uM, and then quickly pour it into a 9cm petri dish for later use.

[0040] Experimental setup: Experimental group: Added resource material and Trichoderma NAU-DY2; Control group: Added only Trichoderma NAU-DY2, and replaced the resource material with an equal volume of water.

[0041] A 5mm Trichoderma NAU-DY2 mycelial cake was inoculated in the middle of a 9cm culture medium containing different resources. The culture was carried out at 28℃ for 48h, and the colony diameter was measured with vernier calipers.

[0042] The promoting effects of different resource substances on the growth of Trichoderma NAU-DY2 are shown in [the table below]. Figure 1 and Figure 2 The results showed that, compared with the control group, vitamin B1, high arginine and p-coumaric acid could significantly promote the increase of Trichoderma NAU-DY2 biomass (P<0.05).

[0043] Example 3: Effects of combined application of resource substances and Trichoderma activator on alfalfa growth under salt stress

[0044] Soil: Saline-alkali soil from Yancheng, Dongtai City, Jiangsu Province (pH 8.36, EC 2.69 ms / cm)

[0045] Preparation of Trichoderma NAU-DY2 spore suspension: Strain NAU-DY2 was activated using PDA solid medium; after incubation at 28℃ for 7–10 days, the mycelial cells were scraped and placed in sterile physiological saline; the concentration of Trichoderma spores was adjusted to 1 × 10⁻⁶ using sterile physiological saline. 8 CFU / mL was used to obtain a suspension of Trichoderma NAU-DY2 spores.

[0046] Based on the effect of resource substances on promoting the salt tolerance growth of Trichoderma, superior resource substances (Trichoderma NAU-DY2, resource substances: vitamin B1, high arginine and p-coumaric acid) were screened out, and the effects of the above resource substances on the ability of Trichoderma to promote the salt tolerance growth of alfalfa were further investigated.

[0047] Alfalfa variety: Aurora.

[0048] After surface sterilization, alfalfa seeds were placed on sterile plates lined with filter paper moistened with sterile deionized water and germinated at 4℃ for 2 days. Seedlings with uniform growth (2-3 true leaves) were transplanted into 4-well seedling trays containing 200g of Dongtai saline-alkali soil. Seven days after transplanting, Trichoderma and resource substances were evenly applied to the roots of the alfalfa using a root drenching method. Each pot was also inoculated with 20mL of a 1mM resource substance solution (to achieve a resource substance concentration of 100µl / kg soil) and simultaneously with 10mL of a 10% concentration solution. 8 CFU / mL Trichoderma spore suspension; Experimental settings: 1) Trichoderma NAU-DY2+ resource group: added resource substance and Trichoderma spore suspension; 2) Trichoderma only group: added only Trichoderma spores, with an equal volume of water replacing the resource substance; 3) Resource only group: added only resource substance, with an equal volume of water replacing the Trichoderma spores; 4) Control group: water replacing both resource substance and Trichoderma spores. After approximately 60 days of growth, the growth-promoting effect of Trichoderma prebiotics on alfalfa under salt-alkali stress was observed.

[0049] In this embodiment, 3) only resource groups are added: the effects of different resource substances, such as purple flowers, on the growth of alfalfa seedlings in saline-alkali soil are as follows: Figure 3 As shown; the effect of inoculating only with the resource substance purple flowers on the biomass of alfalfa seedlings is as follows. Figure 4 As shown.

[0050] The results showed that the plant height and fresh weight of alfalfa plants did not increase significantly compared with the control group CK after the addition of resource substances alone, indicating that a single resource substance cannot promote the growth of alfalfa under salt stress.

[0051] In this embodiment, the effects of 1) the Trichoderma NAU-DY2+ resource group and 2) the Trichoderma-only group on the growth of alfalfa seedlings are as follows: Figure 5 As shown; the effect on alfalfa seedling biomass is as follows: Figure 6 As shown.

[0052] The results showed that, after inoculation with only Trichoderma NAU-DY2, the plant height and fresh weight of alfalfa plants did not increase significantly compared with the control group (CK). However, it was found that the combined use of any one of vitamin B1, coumaric acid, or arginine with Trichoderma NAU-DY2 significantly improved the growth-promoting effect of alfalfa under salt-alkali stress compared with the control group (CK). This indicates that the combined use of any one of vitamin B1, coumaric acid, or arginine with Trichoderma NAU-DY2 can better improve the salt tolerance and growth-promoting ability of alfalfa under salt stress, alleviate salt damage, increase biomass, and thus promote alfalfa growth.

Claims

1. A salt-tolerant, growth-promoting Trichoderma synbiotic, characterized in that, This synbiotic contains Trichoderma strains and resource substances; The Trichoderma strain mentioned is Trichoderma strain NAU-DY2 with preservation number: CCTCC NO: M20241303; The resource substance is at least one of vitamin B1, p-coumaric acid, and high arginine.

2. The application of the salt-tolerant Trichoderma synbiotic of claim 1 in promoting high yield of alfalfa in saline-alkali land.

3. The application according to claim 2, characterized in that, The Trichoderma synbiotic described in claim 1 was applied to the soil using the root irrigation method.

4. The application according to claim 3, characterized in that, Apply 0.1L of a 0.5-1.5mM resource substance and 0.05L of a 1×10⁻⁶ solution per kilogram of soil. 7 ~1×10 9 Trichoderma spore suspension at CFU / mL.

5. A method for promoting high yield of alfalfa in saline-alkali land, characterized in that, The Trichoderma synbiotic of claim 1 is applied to alfalfa by root irrigation, or Trichoderma and resource substances are applied to alfalfa in combination by root irrigation.

6. The method according to claim 5, characterized in that, Trichoderma and other resources were applied to the soil using a root irrigation method.

7. The method according to claim 6, characterized in that, Apply 0.1L of a 0.5-1.5mM resource substance and 0.05L of a 1×10⁻⁶ solution per kilogram of soil. 7 ~1×10 9 Trichoderma spore suspension at CFU / mL.

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