Salt-tolerant growth-promoting trichoderma synbiotics and application thereof

Abstract

The invention discloses a salt-tolerant and growth-promoting trichoderma synbiotics and application thereof. Trichoderma strains and resource substances are combined for use, so that the salt-tolerant and growth-promoting effects of trichoderma are improved. Researches find that the salt-tolerant and growth-promoting effects of trichoderma are improved by combined use of trichoderma strains and at least one of resource substances including vitamin B1, p-coumaric acid and homoarginine, and the high yield of alfalfa in saline-alkali soil can be improved by combined use of the three resource substances and trichoderma. At least one of resource substances including vitamin B1, p-coumaric acid and homoarginine is combined with a trichoderma strain to obtain the trichoderma synbiotics, and the trichoderma synbiotics can improve the salt-tolerant and growth-promoting effects of trichoderma on alfalfa and promote the high yield of alfalfa in saline-alkali soil.

Description

Technical Field

[0001] The present invention belongs to the technical field of microbiology, and specifically relates to a salt-tolerant and growth-promoting Trichoderma synbiotic and the application of the Trichoderma synbiotic in promoting high yield of Medicago sativa in saline-alkali soil; specifically relates to the application of at least one of vitamin B1, p-coumaric acid, and homoarginine in combination with Trichoderma NAU-DY2 in promoting high yield of Medicago sativa in saline-alkali soil. Background Art

[0002] The problem of soil salinization seriously endangers the yield and quality of crops. The global saline soil area is about 950 million hectares, exceeding 6% of the global cultivated land area. It is estimated that the annual loss caused by soil salinity to agriculture is as high as $27.3 billion. The salinized soil area in China exceeds 99 million hectares, accounting for 10.4% of the total soil area, and the area is increasing year by year. Saline-alkali soils are widely distributed in semi-arid and arid regions of China. Compared with the distribution of saline-alkali soils in other countries, China has more complex characteristics, specifically reflected in the wide distribution range, large area, high degree of salinization, and complex diversity of the soil environment. Soil salinization not only does not meet the requirements of agricultural sustainable development, but also has an adverse impact on China's food security and economic development. Whether it can effectively reduce soil desertification and improve the existing saline-alkali soil to a greater extent is of great strategic significance for maintaining the area of agricultural cultivated land and building the bottom line of food security.

[0003] Trichoderma has multiple functions such as promoting plant growth, improving the salt tolerance of plants, and preventing soil-borne diseases. However, as a microbial product, the growth-promoting bacteria preparation must reach a sufficiently high order of magnitude to effectively exert its biological function in application. The resources that Trichoderma can utilize during growth are limited, and the regeneration is very slow, resulting in the growth-promoting bacteria being difficult to play a role, which limits its large-scale application and promotion. Therefore, it is necessary to find resources that can promote the growth of Trichoderma, provide a suitable growth resource environment for Trichoderma in the soil, and enhance its salt-tolerant and growth-promoting ability. The inventor intends to screen resource substances that can efficiently enhance the salt tolerance of Trichoderma, and apply Trichoderma and the resource substances (synbiotic) into the soil to improve the salt tolerance of Medicago sativa. Summary of the Invention

[0004] Aiming at the technical problem in the prior art that due to limited nutrients in the soil and slow growth of Trichoderma under salt stress, when applying Trichoderma to promote plant growth, Trichoderma is often difficult to proliferate in large numbers in the soil and exert its due control effect, the purpose of the present invention is to provide a method for improving the salt-tolerant and growth-promoting effect of Trichoderma.

[0005] Another object of the present invention is to provide a salt-tolerant and 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] It is found through experiments that adding vitamin B1, p-coumaric acid or homoarginine to the culture medium can significantly improve the salt tolerance of Trichoderma strain NAU-DY2; in greenhouse pot experiments, at least one of vitamin B1, p-coumaric acid and homoarginine is used in combination with Trichoderma strain NAU-DY2 and applied to the soil by the root irrigation method, which can significantly increase the biomass of alfalfa in saline-alkali soil. This shows that any one of vitamin B1, p-coumaric acid and homoarginine used in combination with Trichoderma strain NAU-DY2 can significantly improve the salt tolerance and growth-promoting ability of Trichoderma on alfalfa.

[0008] The object of the present invention is achieved by the following technical solutions:

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

[0010] In the second aspect, the present invention claims a salt-tolerant and growth-promoting Trichoderma symbiotic agent, which comprises Trichoderma strains and resource substances.

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

[0012] This Trichoderma strain NAU-DY2 is taxonomically named Trichoderma camerunense, deposited in the China Center for Type Culture Collection on June 19, 2024, with the preservation number: CCTCC NO: M20241303, and the deposit address is: Wuhan University, Wuhan, China.

[0013] The resource substances are 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-lactoylglutathione, methionine, androsterone, homoarginine, 4-guanidinobutyric acid, 5-methoxyindole-3-carboxaldehyde, vitamin B1, vitamin B3, vitamin B4, vitamin B5, vitamin B9, and vitamin B12. Further preferably, the resource substances are selected from at least one of vitamin B1, p-coumaric acid, and homoarginine.

[0014] In the third aspect, the present invention claims the application of the above salt-tolerant and growth-promoting Trichoderma symbiotic agent in promoting high yield of alfalfa in saline-alkali land. Furthermore, the above application is to apply the above Trichoderma symbiotic agent to the soil by the root irrigation method.

[0015] Fourthly, the present invention claims a method for promoting high yield of alfalfa in saline-alkali land, which applies the above-mentioned Trichoderma symbiotic bacteria to alfalfa by the root irrigation method, or jointly applies Trichoderma and resource substances to alfalfa by the root irrigation method. Further, the method specifically is to apply Trichoderma and resource substances into the soil by the root irrigation method.

[0016] In the specific embodiments of the present invention, in the above applications and methods, 0.1 L of a resource substance with a concentration of 0.5 - 1.5 mM and 0.05 L of a Trichoderma spore suspension with a concentration of 1×10 7 ~1×10 9 CFU / mL are applied to each kilogram of soil.

[0017] Further, in the above applications and methods, the resource substance is applied in the form of a solution. Generally, the concentration of the resource substance solution stock solution is 0.1 mol / L. When in use, it can be diluted according to needs in proportion, but it is not limited thereto. The preparation method of the resource substance solution is: the resource substance is dissolved in deionized water or warm water, and is sucked with a sterile syringe and filtered through a 0.22 μm filter membrane to obtain the resource substance solution.

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

[0019] In the specific embodiments of the present invention, the Trichoderma strain NAU-DY2 spore suspension is obtained by the following method: the strain NAU-DY2 is activated with a PDA solid medium; cultured at 28 °C for 7 d - 10 d, and the mycelium is 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] Further preferably, 5 - 7 days after transplanting alfalfa seedlings with 2 - 3 true leaves, a resource substance (such as at least one of vitamin B1, p-coumaric acid, and homoarginine) and the Trichoderma strain NAU-DY2 spore suspension are applied into the soil by the root irrigation method. More specifically, 0.1 L of a resource substance with a concentration of 1 mM and 0.05 L of a Trichoderma spore suspension with a concentration of 1×10 8 CFU / mL are applied to each kilogram of soil.

[0021] Advantages of the present invention:

[0022] The present invention uses at least one of vitamin B1, p-coumaric acid and homoarginine in combination with the Trichoderma strain NAU-DY2 to obtain a Trichoderma synbiotic that improves the salt tolerance and growth-promoting ability of Trichoderma. The synbiotic can significantly enhance the salt tolerance and growth-promoting effect of Trichoderma on alfalfa, while increasing the yield of alfalfa in saline-alkali land. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 Photos showing how different resource materials promote the growth of Trichoderma NAU-DY2 under salt stress.

[0024] Among them, CK is the treatment without resource substances; the others are the treatments with the addition of corresponding resource substances; photos of colonies obtained by adding fresh Trichoderma mycelium to 1 / 4 PDA medium containing 400 mM NaCl and culturing at 28°C for 48 hours.

[0025] Figure 2 The colony diameter is the growth-promoting effect of different resource materials on Trichoderma NAU-DY2 under salt stress.

[0026] Among them, CK is the treatment without resource substances; the others are the treatments with the addition of corresponding resource substances; the colony diameter of fresh Trichoderma mycelium was added to 1 / 4 PDA medium containing 400 mM NaCl and cultured at 28°C for 48 hours.

[0027] Figure 3 The growth-promoting effect of different resource materials on alfalfa plants in saline-alkali soil.

[0028] Figure 4 These are the results of the growth-promoting effects of different resource materials on alfalfa plants in saline-alkali soil.

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

[0030] Figure 6 The results show the effect of different Trichoderma prebiotics and Trichoderma on the growth promotion of alfalfa plants in saline-alkali soil.

[0031] The Trichoderma strain NAU-DY2 is classified as Trichoderma camerunense and was deposited in the China Center for Type Culture Collection on June 19, 2024 with the deposit number: CCTCC NO: M20241303. The deposit address is: Wuhan University, Wuhan, China. DETAILED DESCRIPTION

[0032] The technical solution of the present invention will be further described below in conjunction with specific implementation methods.

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

[0034] Strain Screening: Soil samples were collected from Dongying City, Shandong Province, China, and about 5 g was weighed with a balance. A pre-sterilized conical flask containing 45 ml of sterile water and several sterile glass beads was taken out, and the weighed soil sample was poured into the conical flask. The flask was covered with a sealing film and placed in a single-layer rotary shaker at 200 r / min for 1 h. This was the 10 -1 dilution. In the ultra-clean bench, a test tube containing 4.5 ml of sterilized water was selected, and 0.5 ml of the conical flask suspension was transferred into it with a sterilized pipette tip and pipetted several times. This was the 10 -2 dilution. Then, 0.5 ml of the suspension was transferred to the next test tube. This was the 10 -3 dilution. Referring to this method, 10 -3 , 10 -4 , and 10 -5 dilutions were prepared respectively. Three dilution levels were selected and spread on Trichoderma selective medium for cultivation. 0.1 ml of the dilution was added to each plate, and the spreading should be as even as possible. Three replicates were set for each dilution level. The Trichoderma selective medium plates after even spreading were placed in an incubator at 28 °C. After about two to three days, Trichoderma colonies would grow. The colonies similar to Trichoderma were observed by naked eyes, and the hyphae were picked and cultured on PDA medium. Multiple subcultures were needed for purification of the strain. The final strain was inoculated and preserved on a PDA slant. Finally, it was identified as Trichoderma camerunense by molecular biology ITS sequence and named NAU-DY2.

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

[0036] Example 2: Investigation of the Salt Tolerance and Growth Promotion Effects of Resource Substances on 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 -Lactoylglutathione, Methionine, Androsterone, Homoarginine, 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: Weigh each resource substance accurately with a ten-thousandth balance, dissolve it with deionized water or hot water, and then suck it up with a sterile syringe. Filter it under 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 it in a refrigerator at -4°C for later use.

[0039] Preparation of the culture medium: When the 1 / 4 PDA medium containing 400 mM NaCl, which has been sterilized in advance, is cooled to about 40°C, add the previously prepared stock solution of resource substances to the medium so that the concentration of the resource substances in the medium is 100 μM. Then quickly pour it into a 9-cm Petri dish for later use.

[0040] Experimental setup: Experimental group: Add resource substances and Trichoderma asperellum NAU-DY2; Control group: Only add Trichoderma asperellum NAU-DY2, and replace the resource substances with an equal volume of water.

[0041] Inoculate a 5-mm mycelial disc of Trichoderma asperellum NAU-DY2 in the middle of a 9-cm culture medium containing different resource substances. Incubate it at 28°C for 48 h, and measure the colony diameter with a vernier caliper.

[0042] The promoting effect of different resource substances on the growth of Trichoderma asperellum NAU-DY2 can be seen in Figure 1 and Figure 2 , and the results show that compared with the control group, vitamin B1, homoarginine, and p-coumaric acid can significantly promote the increase in the biomass of Trichoderma asperellum NAU-DY2 (P < 0.05).

[0043] Example 3 Effect of the combined application of resource substances and Trichoderma on the growth of Medicago sativa under salt stress

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

[0045] Preparation of the spore suspension of Trichoderma asperellum NAU-DY2: The strain NAU-DY2 is activated with PDA solid medium; incubate it at 28°C for 7 - 10 d, scrape the mycelium into sterile physiological saline; adjust the spore concentration of Trichoderma asperellum to 1×10 8 CFU / mL with sterile physiological saline to obtain the spore suspension of Trichoderma asperellum NAU-DY2.

[0046] According to the effect of resource substances in promoting the salt-tolerant growth of Trichoderma, select the relatively excellent resource substances (Trichoderma asperellum NAU-DY2, resource substances: vitamin B1, homoarginine, and p-coumaric acid), and further investigate the effect of the above resource substances on promoting the salt-tolerant growth ability of Medicago sativa by Trichoderma.

[0047] Medicago sativa variety: Aurora.

[0048] After surface sterilization, the purple flower seeds were placed on a sterile plate lined with filter paper moistened with sterile deionized water and germinated at 4°C for 2 days. Seedlings with consistent growth (having 2-3 true leaves) were transplanted into a 4-hole seedling tray containing 200 g of Dongtai saline-alkali soil. Seven days after seedling transplantation, Trichoderma and resource substances were evenly poured into the roots of alfalfa by the method of root irrigation. A 20 mL of 1 mM resource substance solution was added to each pot (so that the concentration of the resource substance applied to each pot was 100 μmol / kg of soil), and at the same time, 10 mL of a Trichoderma spore suspension with a concentration of 10 8 CFU / mL was added; the following treatments were set up in the experiment: 1) Trichoderma NAU-DY2 + resource substance group: resource substance and Trichoderma spore suspension were added; 2) Trichoderma-only group: only Trichoderma spores were added, and an equal volume of water was used to replace the resource substance; 3) Resource-only group: only the resource substance was added, and an equal volume of water was used to replace the Trichoderma spores; 4) Control group: an equal volume of water was used to replace the resource substance and Trichoderma spores. After growing for about 60 days, the growth-promoting effect of Trichoderma prebiotics on alfalfa under saline-alkali stress was observed.

[0049] In this example, 3) Resource-only group: The growth status of alfalfa seedlings with different resource substances in saline-alkali soil was as Figure 3 shown; the effect of only inoculating the resource substance on the biomass of alfalfa seedlings was as Figure 4 shown.

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

[0051] In this example, 1) Trichoderma NAU-DY2 + resource substance group and 2) Trichoderma-only group: The growth status of alfalfa seedlings was as Figure 5 shown; the effect on the biomass of alfalfa seedlings was as Figure 6 shown.

[0052] The results showed that the plant height and fresh weight of alfalfa plants after only inoculating a single Trichoderma NAU-DY2 did not increase significantly compared with the control group CK. However, it was found that when any one of vitamin B1, p-coumaric acid, or homoarginine was used in combination with Trichoderma NAU-DY2, the growth-promoting effect on alfalfa under saline-alkali stress was significantly better than that of the control group CK; it was indicated that the combination of one of vitamin B1, p-coumaric acid, or homoarginine with Trichoderma NAU-DY2 could better significantly improve the salt tolerance and growth-promoting ability of alfalfa under salt stress, alleviate the salt damage phenomenon, increase the biomass, and thus promote the growth effect of alfalfa.

Claims

1. A method for improving the salt tolerance and growth promotion effect of Trichoderma, characterized in that, Combined use of Trichoderma strains and resource substances to improve the salt tolerance and growth promotion effect of Trichoderma The resource substances are 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 - lactoylglutathione, methionine, androsterone, homoarginine, 4 - guanidinobutyric acid, 5 - methoxyindole - 3 - carbaldehyde, vitamin B1, vitamin B3, vitamin B4, vitamin B5, vitamin B9, and vitamin B12; preferably, at least one selected from vitamin B1, p - coumaric acid, and homoarginine.

2. The method according to claim 1, wherein The Trichoderma strain is Trichoderma strain NAU - DY2 with the preservation number: CCTCC NO: M20241303.

3. A salt-tolerant growth-promoting Trichoderma symbiotic agent, characterized in that, This symbiotic bacterium contains Trichoderma strain and resource substances; The resource substances are 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 - lactoylglutathione, methionine, androsterone, homoarginine, 4 - guanidinobutyric acid, 5 - methoxyindole - 3 - carbaldehyde, vitamin B1, vitamin B3, vitamin B4, vitamin B5, vitamin B9, and vitamin B12; preferably, at least one selected from vitamin B1, p - coumaric acid, and homoarginine.

4. The salt-tolerant growth-promoting Trichoderma symbiotic bacteria according to claim 3, wherein The Trichoderma strain is Trichoderma strain NAU - DY2 with the preservation number: CCTCC NO: M20241303.

5. Application of the salt - tolerant and growth - promoting Trichoderma symbiotic bacterium according to claim 3 or 4 in promoting high - yield of Medicago sativa in saline - alkali land.

6. The application according to claim 5, wherein The Trichoderma symbiotic bacterium according to claim 3 or 4 is applied into the soil by the root - irrigation method.

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

8. A method for promoting high yield of alfalfa in saline-alkali land, characterized in that, The Trichoderma symbiotic bacterium according to claim 3 or 4 is applied to Medicago sativa by the root - irrigation method, or Trichoderma and resource substances are jointly applied to Medicago sativa by the root - irrigation method.

9. The method according to claim 8, characterized in that Trichoderma and resource substances are applied into the soil by the root - irrigation method.

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

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