Trichoderma sp. suitable for supplementing alfalfa in degraded grassland to promote growth and improve competitiveness and its microbial fertilizer product

Abstract

The present application discloses a strain of Trichoderma suitable for promoting growth and improving competitiveness of alfalfa in degraded grassland, and a bacterial fertilizer product thereof, the strain NAU-HL7 is classified and named as Trichoderma pini (Trichoderma pini) Trichoderma pinicola ), and was preserved in China Center for Type Culture Collection on October 30, 2024, with a strain preservation number of CCTCC NO: M 20242377. The strain NAU-HL7 is used to prepare a bacterial fertilizer product suitable for promoting growth and improving competitiveness of alfalfa in degraded grassland, and the mass ratio of the Trichoderma solid fermentation, calcium superphosphate and controlled-release nitrogen fertilizer in the product is 1:(15-25):(8-12). The product can be used for seedling stage planting and rapid growth of alfalfa in degraded grassland in the north, so as to compete faster than the original grassland vegetation, ensure successful supplemental planting of high-quality forage, overwintering and regreening, and improve grassland productivity.

Description

Technical Field

[0001] This invention belongs to the field of agricultural fertilizer production technology, specifically relating to a Trichoderma strain suitable for promoting alfalfa growth and enhancing competitiveness in degraded grasslands, and its microbial fertilizer products. Background Technology

[0002] Grassland degradation is mainly manifested in reduced biodiversity, increased toxic weeds, and severe soil erosion. Reseeding degraded grasslands can effectively improve grassland yield and quality, and is one of the effective measures for renewing and rejuvenating grassland communities. In developed countries such as the United States and Canada, reseeding leguminous forage grasses is one of the main methods for grassland restoration and renewal. In mixed leguminous and grass forage grasslands, leguminous forage grasses not only have a high protein content themselves, but also provide fixed nitrogen to surrounding non-nitrogen-fixing plants, thereby improving the nutritional value of the entire grassland community. Alfalfa, a widely cultivated perennial leguminous forage grass in my country, has the advantages of single planting, multiple harvests, rich nutrition, and good palatability, and is known as the "King of Forage Grasses," making it an excellent target variety for degraded reseeding. In the grassland areas of northern my country, especially in the high-latitude Hulunbuir region, winters are characterized by extremely cold temperatures, posing a significant challenge to the successful establishment and regrowth of newly reseeded species. Alfalfa seedlings grow slowly and have low competitiveness, which affects their later growth and reduces grassland productivity. Therefore, the ability of reseeded species to grow rapidly after emergence, thereby enhancing their systemic resistance and improving their competitiveness among species, is crucial for their successful overwintering. Furthermore, nutrient deficiencies in degraded grasslands, especially phosphorus, also reduce the competitiveness of reseeded forage grasses, exacerbating overwintering difficulties and leading to low productivity. Therefore, developing new fertilizer products suitable for reseeding degraded grasslands is essential for restoring degraded grasslands and improving productivity.

[0003] Utilizing agricultural microorganisms is an effective way to ensure forage yield and develop a healthy and sustainable forage industry. The application of growth-promoting and stress-resistant functional microorganisms and their products has received increasing attention both domestically and internationally, becoming an important field of modern bioscience research and comprehensive development. Trichoderma spp., a type of fungus widely distributed in soil, decaying wood, and plant debris, plays a crucial role in green and efficient agricultural production. Previous studies have shown that applying Trichoderma products to grassland soil can significantly increase the number of soil microorganisms and the activities of soil invertases, phosphatases, peroxidases, and ureases, thereby improving soil quality, increasing soil fertility and fertilizer utilization, promoting forage growth and development, enhancing plant disease resistance, and ultimately increasing yield and quality. It also plays a vital role in reducing environmental pollution. Our previous research has also confirmed that Trichoderma biofertilizer products can effectively improve the soil microbial community of Leymus chinensis grassland and significantly increase its yield. Therefore, in response to the actual needs of degraded grassland improvement, the development of a new type of high-efficiency microbial fertilizer product specifically designed for the reseeding of degraded grasslands will be beneficial for the restoration of degraded grasslands in northern my country and will provide new productivity for grassland production, ecological security and the promotion of sustainable economic and social development in my country. Summary of the Invention

[0004] The purpose of this invention is to provide a Trichoderma strain suitable for promoting alfalfa growth and enhancing competitiveness in degraded grasslands, and its microbial fertilizer products.

[0005] Another objective of this invention is to provide a method for promoting alfalfa seedling establishment, growth, and competitiveness in degraded grasslands. A specialized Trichoderma fertilizer product for alfalfa reseeding in degraded grasslands, prepared using the Trichoderma strain NAU-HL7 screened in this invention, facilitates rapid seedling establishment, significantly promotes alfalfa growth, enhances the competitiveness of alfalfa reseeded in degraded grasslands, and helps it successfully overwinter and regrow.

[0006] The objective of this invention can be achieved through the following technical solutions:

[0007] Firstly, this invention seeks protection for a Trichoderma strain, NAU-HL7, classified as *Trichoderma pinicola*, which was deposited on October 30, 2024, at the China Center for Type Culture Collection (CCTCC), Wuhan University, China, with accession number CCTCC NO: M 20242377. This strain is a Trichoderma suitable for promoting growth and / or enhancing competitiveness when reseeding alfalfa in degraded grasslands.

[0008] The NAU-HL7 strain was identified as *Trichoderma mapinicola* based on morphological, physiological, and biochemical characteristics and fungal ITS. Its optimal growth conditions were: 28℃, pH 5.0–6.0, with glucose or sucrose as the carbon source, and cultured on PDA medium. The *Trichoderma* strain NAU-HL7 grew rapidly; after 60 hours of cultivation under optimal conditions, colonies completely covered a 9cm plate. Initially, the colonies were cottony and grayish-white; as the conidia matured, the colonies gradually turned green from the center to the edge; and as the hyphae matured, the surface of the culture medium was densely covered with dark green spores.

[0009] Secondly, the present invention seeks protection for the use of the above-mentioned Trichoderma strain NAU-HL7 in the following (1) or (2):

[0010] (1) Application in promoting growth and / or enhancing competitiveness of alfalfa in reseeding degraded grasslands;

[0011] (2) Application in the preparation of microbial fertilizer products suitable for promoting growth and / or enhancing competitiveness of alfalfa when reseeding degraded grasslands.

[0012] Thirdly, the present invention claims protection for a Trichoderma fertilizer product specifically for reseeding, the fertilizer product containing solid fermentation material (granules) of the aforementioned Trichoderma strain NAU-HL7.

[0013] Furthermore, the content of Trichoderma strain NAU-HL7 in the aforementioned microbial fertilizer product is not less than 4.0 × 10⁻⁶. 9 CFU / g.

[0014] Furthermore, the aforementioned microbial fertilizer products also include superphosphate and controlled-release nitrogen fertilizer.

[0015] Furthermore, the above-mentioned microbial fertilizer product is prepared using a method including the following steps:

[0016] (1) Prepare the fermentation broth of the aforementioned Trichoderma strain NAU-HL7, wherein the viable cell count of the fermentation broth is not less than 1.0 × 10⁻⁶. 8 CFU / mL;

[0017] (2) Add 7.5% corn flour and 10.0% rice bran to the crushed, sterilized pure alfalfa straw and inoculate with 10% of the fermentation broth prepared in step (1). Adjust the moisture content and pH value until the sporulation of Trichoderma strain NAU-HL7 in the fermentation culture system is ≥4.0×10⁻⁶. 9 Trichoderma solid fermentation product was obtained by collecting 1 / g of the product;

[0018] (3) Evaporate the moisture content of the Trichoderma solid fermentation product prepared in step (2) to below 30% under normal temperature conditions, and mechanically extrude and granulate it; after mixing with superphosphate granules and controlled-release nitrogen fertilizer granules, a microbial fertilizer product suitable for reseeding alfalfa in degraded grassland to promote growth and improve competitiveness is obtained.

[0019] Furthermore, the process of preparing the fermentation broth of the Trichoderma strain NAU-HL7 in step (1) is as follows: the Trichoderma strain NAU-HL7 is inoculated into 1 / 8 PDB medium and fermented at 28°C and 200 r / min for 3 to 5 days.

[0020] Furthermore, in step (2), the water content is adjusted to 60-65%, the pH value is adjusted to 5-8, and the culture is carried out in shallow trays at 28°C for 4-7 days.

[0021] Further, in step (3), the mass ratio of the Trichoderma solid fermentation product (granules), superphosphate (P2O5≥12%) and controlled-release nitrogen fertilizer (N≥44%) is 1:(15~25):(8~12); preferably 1:(18~22):(9~11); further preferably 1:(19~21):(10~11); most preferably 1:20:(10~11).

[0022] Fourthly, this invention claims protection for a method to promote the growth and / or enhance the competitiveness of alfalfa reseeded in degraded grasslands. This method involves applying a microbial fertilizer product containing the aforementioned Trichoderma strain NAU-HL7 to the degraded grassland to promote seedling establishment, growth, and enhance competitiveness of alfalfa reseeded in degraded grasslands. Experimental results show that the prepared microbial fertilizer product achieves a germination rate of over 90% for alfalfa.

[0023] This invention isolated a Trichoderma strain (NAU-HL7) with significant growth-promoting effects on alfalfa in situ from degraded grassland, and classified it as *Trichoderma pinicola*. Based on a solid-state shallow-tray fermentation system, using alfalfa straw powder as the main substrate, with 7.5% corn flour added as the carbon source and 10.0% rice bran as the nitrogen source, a Trichoderma solid-state fermentation product (≥4×10⁻⁶) was obtained. 9The product (CFU / g) is extruded and granulated for ease of production and application. Considering the difficulty of overwintering alfalfa replanting in degraded grasslands in northern regions, and to promote rapid growth and enhanced resistance of replanted alfalfa after establishment, as well as improve its competitiveness in degraded grasslands and facilitate successful overwintering and regrowth, a special Trichoderma fertilizer product for degraded grassland replanting has been developed based on Trichoderma solid fermentation product (granules). This product is a mixed granular form, containing Trichoderma solid fermentation product (granules), superphosphate (P2O5≥12%), and controlled-release nitrogen fertilizer (N≥44%), with a mixing mass ratio of 1:(15~25):(8~12). This product can be used for seedling establishment and growth of alfalfa replanted in degraded grasslands in northern regions, enabling it to compete faster than native grassland vegetation, ensuring successful replanting of high-quality forage, smooth overwintering and regrowth, and improving grassland productivity.

[0024] The beneficial effects of this invention are:

[0025] (1) The Trichoderma strain NAU-HL7 (Trichoderma pinicola) was a highly efficient strain screened in the in-situ environment of grassland. It has not been reported in other research results. It is the first time that the growth-promoting effect and competitiveness enhancement of reseeded species have been studied in the field of degraded grassland restoration.

[0026] (2) This Trichoderma fertilizer contains NAU-HL7, a specific and highly efficient growth-promoting bacterial strain suitable for reseeding alfalfa in degraded grasslands. Its growth-promoting effect is significant, serving the seedling establishment and rapid growth of alfalfa in degraded grassland reseeding scenarios, and enhancing competitiveness. This product is convenient for field use; the growth-promoting bacteria it contains can function through multiple growth-promoting mechanisms, thereby achieving the goals of restoring degraded grasslands, improving grassland productivity, and increasing the proportion of high-quality forage.

[0027] (3) This microbial fertilizer product has a reasonable nutrient ratio, which can assist alfalfa seedling emergence in degraded grasslands and significantly improve its competitiveness, ensuring that it can successfully overwinter and turn green in severe cold environments. It is a microbial fertilizer product specifically developed for the reseeding of degraded grasslands. At the same time, this product is also beneficial to improving grassland resistance and forage quality, promoting soil nutrient transformation and cycling, improving soil structure, and increasing soil fertility, thereby improving the grassland soil environment, reducing pollution, effectively preventing soil-borne diseases, and realizing pollution-free production of forage grass, which is a sustainable agricultural production method. Attached Figure Description

[0028] Figure 1 Colony morphology of NAU-HL7, a growth-promoting strain used to promote alfalfa growth.

[0029] Figure 2 Image showing the ITS identification results for NAU-HL7.

[0030] Figure 3Pot experiments were conducted to determine the optimal growth-promoting effect of the growth-promoting strain NAU-HL7 on alfalfa.

[0031] Figure 4 The effect of NAU-HL7 on alfalfa biomass.

[0032] Figure 5 Product display image of Trichoderma fertilizer.

[0033] Figure 6 The optimal formula for Trichoderma fertilizer products is selected.

[0034] Figure 7 The effect of Trichoderma fertilizer products on reseeding alfalfa in degraded grasslands.

[0035] Figure 8 The impact of Trichoderma fertilizer products on reseeding alfalfa in degraded grasslands. Detailed Implementation

[0036] The present invention will be described in detail below with reference to specific embodiments. These embodiments are provided to better understand the present invention, but are not intended to limit the invention. The experimental methods used in the following embodiments are all conventional methods, and the experimental reagents involved are all conventional biochemical reagents.

[0037] Example 1

[0038] (I) Isolation, identification and screening of growth-promoting bacterial strains in degraded grassland soil

[0039] Isolation of growth-promoting strains: The soil used was collected from degraded grassland in Hailar, Hulunbuir City, Inner Mongolia Autonomous Region (49°26'26"N", 120°10'10"E). Trichoderma strains were screened by dilution and spread method, and more than 200 Trichoderma strains were obtained. All strains were stored in glycerol at -80℃.

[0040] The growth-promoting strain NAU-HL7 was identified as *Trichoderma pinicola*.

[0041] The main biological characteristics of NAU-HL7 are (e.g.) Figure 1 The NAU-HL7 strain was identified as *Trichoderma pinicola* based on morphological, physiological, and biochemical characteristics and fungal ITS. Its optimal growth conditions were: 28℃, pH 5.0–6.0, with glucose or sucrose as the carbon source, and cultured on PDA medium. The *Trichoderma* strain NAU-HL7 grew rapidly; after 60 hours of cultivation under optimal conditions, colonies completely covered a 9cm plate. Initially, the colonies were cottony and grayish-white; as the conidia matured, the colonies gradually turned green from the center to the edge; and as the hyphae matured, the surface of the culture medium was densely covered with dark green spores.

[0042] The ITS identification results of NAU-HL7 are as follows: Figure 2 .

[0043] This strain was deposited at the China Center for Type Culture Collection (CCTCC) on October 30, 2024, at Wuhan University, Wuhan, China, with accession number CCTCC NO: M 20242377.

[0044] Primary and secondary screening of growth-promoting strains: Since the most important growth-promoting mechanism of *Trichoderma* is competition, which is closely related to its growth rate, the growth rate of *Trichoderma* was used as the primary screening indicator, resulting in six representative strains: HL5-1, HL2-2, HL12-2, HL7-2, HL8-1, and HL3-1. Based on this, the effect on alfalfa seedling growth was used as the secondary screening indicator: *Trichoderma* strains with faster growth rates were applied to the roots of alfalfa seedlings. By observing the growth of the alfalfa seedlings, the strains that best promoted alfalfa growth were selected. HL7-2 showed the best growth-promoting effect on alfalfa seedlings (e.g., HL5-1, HL2-2, HL12-2, HL7-2, HL8-1, and HL3-1). Figure 3 , 4 Based on the results of the initial screening and secondary screening, HL7-2 was selected as the optimal strain for the production of Trichoderma-specific fertilizer products for subsequent reseeding, and was renamed NAU-HL7.

[0045] (II) Production of Trichoderma-specific fertilizer products for reseeding

[0046] (1) Inoculate the growth-promoting strain NAU-HL7 into 1 / 8 PDB culture medium for liquid fermentation. Control the fermentation temperature range to 28℃, the stirring speed to 200 r / min, and the fermentation time to 3-5 days, ensuring that the number of NAU-HL7 spores in the fermentation broth is ≥10. 8 CFU / mL.

[0047] 1 / 8PDB medium: Measured in 1L volume, the preparation method is as follows: Peel 50g of potatoes, cut them into small pieces, put them in water and boil them. After boiling, boil for 30 minutes, then filter through four layers of medical gauze. Add 5g of glucose to the filtrate and make up to 1L. Set the pH to natural and sterilize at 115℃ for 30 minutes.

[0048] (2) Add 7.5% (m / m, g / 100g) corn flour and 10.0% (m / m, g / 100g) rice bran to crushed, sterilized pure alfalfa straw (2-5mm), and inoculate with 10% (v / m, ml / 100g) Trichoderma fermentation broth (3.0×10⁻⁶). 8 The mixture was prepared by adjusting the water content to 60% and the pH to 5-8 using 0.05 mol / L nitric acid. It was then incubated in shallow dishes at 28°C for 4-7 days to achieve a Trichoderma sporulation rate of 10-1. 10Trichoderma solid fermentation product was obtained by 1 / g.

[0049] (3) Under normal temperature conditions, the moisture content of the Trichoderma solid fermentation product is evaporated to below 30%, and mechanically extruded and granulated to obtain Trichoderma solid fermentation product (granules); after mixing with superphosphate granules and controlled-release nitrogen fertilizer granules, a microbial fertilizer product suitable for reseeding alfalfa seedlings in degraded grasslands and improving competitiveness is obtained (e.g., Figure 5 ).

[0050] (III) Pot experiment on controlled-release nitrogen fertilizer application

[0051] The soil used in the experiment was derived from in-situ degraded grassland, collected from degraded grassland in Hailar, Hulunbuir City, Inner Mongolia Autonomous Region (49°26'26"N", 120°10'10"E). The experiment adopted a single-factor experimental design with a total of 7 treatments, each with 3 replicates: (1) CK; (2) P application (225kg superphosphate / hm). 2 (3) Apply P+Trichoderma solid fermentation product (granules) (11.25 kg / hm) 2 (4) Apply P + Trichoderma solid fermentation product (granules) + N1 (60 kg N / hm) 2 (5) Apply P + Trichoderma solid fermentation product (granules) + N2 (90 kg N / hm) 2 (6) Apply P + Trichoderma solid fermentation product (granules) + N3 (120 kg N / hm) 2 (7) Apply P + Trichoderma solid fermentation product (granules) + N4 (150 kg N / hm) 2 In the test results (such as...) Figure 6 The dry and fresh weights of treatment (6) increased by 3.31 and 3.09 times respectively compared with the control, and were significantly higher (P<0.05) than other treatments. Therefore, we selected this formula to form a new type of fertilizer product for reseeding and verified its growth on actual degraded grasslands. The mixture of Trichoderma solid fermentation product (granules), superphosphate (P2O5≥12%) and controlled-release nitrogen fertilizer (N≥44%) was in a mass ratio of 1:20:10.67.

[0052] (IV) The impact of novel growth-promoting microbial fertilizers on the growth of alfalfa reseeded in degraded grasslands

[0053] The experiment was conducted on degraded grassland in Hailar, Hulunbuir City, Inner Mongolia Autonomous Region (49°26'29"N", 120°10'4"E). The experiment consisted of five treatments, with five plots per treatment and each plot measuring 24 m². 2(1) Re-sow alfalfa; (2) Re-sow alfalfa + [phosphate fertilizer (15kg / mu)]; (3) Re-sow alfalfa + [slow-release nitrogen fertilizer (8kg / mu)]; (4) Re-sow alfalfa + [phosphate fertilizer + nitrogen fertilizer]; (5) Re-sow alfalfa + [phosphate fertilizer + nitrogen fertilizer + Trichoderma solid fermentation material (granules) (0.75kg / mu)].

[0054] In the test results (such as) Figure 7 , 8 After applying Trichoderma fertilizer, the plant height and dry weight of reseeded alfalfa increased by 4.36 and 10.10 times respectively compared with the control, significantly assisting and promoting the growth of reseeded alfalfa and improving grassland productivity. The growth competitiveness of reseeded alfalfa was characterized by the ratio of the dry weight of reseeded alfalfa in degraded grassland to the dry weight of other grassland plants. After applying Trichoderma fertilizer, the competitiveness of reseeded alfalfa increased by 11.04 times compared with the control, significantly improving the competitiveness of reseeded alfalfa in degraded grassland. This indicates that the fertilizer product provides a reliable guarantee for the safe overwintering and regrowth of reseeded alfalfa.

Claims

1. A Trichoderma strain, NAU-HL7, used to promote alfalfa growth during reseeding in degraded grasslands, is classified and named Trichoderma pinnifolia (NAU-HL7). Trichoderma pinicola The strain was deposited on October 30, 2024, at the China Center for Type Culture Collection (CCTCC), Wuhan University, Wuhan, China, with accession number CCTCC NO: M 20242377.

2. The use of Trichoderma strain NAU-HL7 in claim 1 in promoting growth or / and improving competitiveness of alfalfa in degraded grassland.

3. The use of Trichoderma strain NAU-HL7 in claim 1 in preparing a microbial fertilizer product suitable for promoting growth or / and improving competitiveness of alfalfa in degraded grassland.

4. A Trichoderma-based inoculant product for use in cover cropping, characterized in that, The microbial fertilizer product comprises the solid fermentation of Trichoderma strain NAU-HL7 in claim 1.

5. The mycofertilizer product of claim 4, wherein, The content of the Trichoderma strain NAU-HL7 in the bacterial fertilizer product is not less than 4.0 x 10 9 CFU / g.

6. The mycofertilizer product of claim 4, wherein, The microbial fertilizer product further comprises superphosphate and controlled-release nitrogen fertilizer.

7. The mycofertilizer product of claim 6, wherein, The microbial fertilizer product is prepared by the following method: (1) the fermentation liquor of the Trichoderma strain NAU-HL7 according to claim 1, wherein the viable cell count of the fermentation liquor is not less than 1.0×10 8 CFU / mL; (2) adding 7.5% of corn powder and 10.0% of rice bran into the crushed, sterilized pure alfalfa straw, inoculating 10% of the fermentation broth prepared in step (1), adjusting the moisture content and pH value until the spore yield of the Trichoderma strain NAU-HL7 in the fermentation culture system is ≥4.0×10 9 spores / g to obtain a Trichoderma solid fermentation product; (3) adding 7.5% of corn powder and 10.0% of rice bran into the crushed, sterilized pure alfalfa straw, inoculating 10% of the fermentation broth prepared in step (1), adjusting the moisture content and pH value until the spore yield of the Trichoderma strain NAU-HL7 in the fermentation culture system is ≥4.0×10 9 spores / g to obtain a Trichoderma solid fermentation product; (3) Evaporate the moisture content of the Trichoderma solid fermentation prepared in step (2) to below 30% at room temperature, and mechanically extrude and granulate; after mixing with superphosphate particles and controlled-release nitrogen fertilizer particles, a microbial fertilizer product suitable for promoting growth or / and improving competitiveness of alfalfa in degraded grassland is obtained.

8. The mycofertilizer product of claim 7, wherein, The process for preparing the fermentation broth of Trichoderma strain NAU-HL7 in step (1) is as follows: inoculate Trichoderma strain NAU-HL7 into 1 / 8 PDB medium, and ferment and cultivate at 28°C and 200 r / min for 3-5 days.

9. The mycofertilizer product of claim 7, wherein, In step (2), the moisture content is adjusted to 60-65%, the pH value is adjusted to 5-8, and shallow plate cultivation is carried out at 28°C for 4-7 days.

10. The mycofertilizer product of claim 7, wherein, In step (3), the mass ratio of the Trichoderma solid fermentation, superphosphate, and controlled-release nitrogen fertilizer is 1:(15-25):(8-12).

11. A method of promoting the growth or / and competitiveness of a sown alfalfa in a degraded grassland, characterized in that, Apply the microbial fertilizer product in any one of claims 4-10 to degraded grassland to promote the growth or / and improve the competitiveness of alfalfa in the degraded grassland.

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