Steviol glycoside-based trichoderma conidiation promoting fermentation bio- preparation, and preparation method and application thereof

Abstract

The present application relates to the technical field of microorganisms, in particular to a fermentation biological agent for promoting sporulation of trichoderma based on stevia rebaudiana polysaccharide, a preparation method and application thereof, the present application realizes secondary utilization of the waste after processing of stevia rebaudiana, i.e., stevia rebaudiana polysaccharide, replaces traditional carbon sources, finds that it can significantly promote the growth of trichoderma colonies, is superior to traditional glucose carbon sources, and the promoting effect of the component on the growth and sporulation of a strain TB2 is particularly obvious, the strain can be fermented in a MSM culture medium in the presence of stevia rebaudiana polysaccharide, and a fermentation biological agent can be prepared after filtration, the fermentation biological agent has good promoting function on plants, reduces the dependence of agricultural production on synthetic fertilizers, and meets the demand of green agriculture.In addition, the present application optimizes the liquid fermentation process of trichoderma, adjusts the concentration of stevia rebaudiana polysaccharide, pH and culture conditions, and improves the production efficiency, and the sporulation amount in liquid fermentation can reach 10 7 million / mL, and the output of active ingredients is stable.

Description

Technical Field

[0001] This invention relates to the field of microbial technology, and in particular to a fermentation biological agent based on stevia polysaccharide that promotes sporulation of Trichoderma, its preparation method, and its application. Background Technology

[0002] Stevia polysaccharides are a byproduct of stevia, obtained after extraction, settling, and membrane concentration, yielding a solid content of 50%. They are classified as industrial waste. Primarily composed of dietary fiber polysaccharides (crude polysaccharides), they possess low sugar and low calorie content combined with antioxidant properties, demonstrating potential for functional applications.

[0003] Trichoderma is widely distributed in nature and has important biocontrol applications. Many species can not only antagonize various soil-borne fungal pathogens but also promote plant growth. Due to its complex and diverse biocontrol mechanisms, including competition, hyperparasitism, antagonism, and induction of plant resistance, Trichoderma occupies an irreplaceable position in the sustainable management of plant diseases.

[0004] Liquid fermentation is a commonly used modern fermentation technology that primarily utilizes free liquid substrates in the fermentation broth as raw materials, supplemented by a certain rotation speed and aeration rate. Compared with solid-state fermentation, liquid submerged fermentation can ensure the uniformity of nutrients in the culture medium and easily control parameters such as temperature and pH. It can achieve efficient production of high-quality Trichoderma propagules, and liquid fermentation has a high degree of automation, making it easy to realize industrial production.

[0005] Currently, most Trichoderma products on the market are used in production as biological agents. While Trichoderma preparations have many advantages, they also have several drawbacks. For example, Trichoderma preparation is mostly done through solid-state culture, resulting in low automation, high costs, and compromised product quality. Furthermore, the widespread use of stevia in food additives, sweeteners, and the pharmaceutical industry generates a large amount of stevia polysaccharides from the waste residue after processing. If these polysaccharides are not properly utilized, they not only have adverse environmental impacts but also waste significant nutrient resources. To address these problems, this invention optimizes Trichoderma culture efficiency by using stevia polysaccharides as a high-quality carbon source for liquid fermentation of Trichoderma, thereby achieving high-value utilization of stevia polysaccharides. Summary of the Invention

[0006] In view of the above, it is necessary to utilize stevia polysaccharides, explore their potential in microbial fermentation, effectively utilize stevia polysaccharides at a high value, and reduce environmental pollution.

[0007] To achieve the above objectives, the technical solution adopted by this invention is: a fermentation biological agent based on stevia polysaccharides that promotes sporulation of Trichoderma, wherein the fermentation biological agent is derived from Trichoderma brevis. Trichoderma brevicompactum The strain TB2 was obtained by fermentation with stevia polysaccharide solution and MSM medium; the *Trichoderma shortensis* was obtained by fermentation. Trichoderma brevicompactumTB2, its classification name is: Trichoderma brevicompactum TB2, classified in Chinese as *Trichoderma septum* TB2, with accession number CCTCC NO: M20231913, is deposited at the China Center for Type Culture Collection, No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province, on October 16, 2023. This strain has been published in patent application 202311753128.0.

[0008] Furthermore, the stevia polysaccharide solution is prepared by diluting it 50-100 times.

[0009] Furthermore, the MSM culture medium obtained by fermentation consists of the following components: 2.0 g of (NH4)2SO4, 0.2 g of MgSO4·7H2O, 0.01 g of CaCl2·2H2O, 0.001 g of FeSO4·7H2O, 1.5 g of Na2HPO4·12H2O, 41.5 g of KH2PO, and 1000 mL of distilled water.

[0010] The present invention also includes a method for preparing the fermented biological agent, the method being as follows.

[0011] (1) Preparation of spore suspension: Trichoderma brevicornu Trichoderma brevicompactum After the TB2 strain was activated, it was inoculated onto a PDA plate and cultured. Sterile water was added in a sterile environment, and the spore suspension was obtained by scraping with a spreading stick and filtering through a filter cloth.

[0012] (2) Preparation of fermented biological agent: Prepare MSM medium, add stevia polysaccharide to prepare a 50-100 times dilution concentration, adjust the pH to 7, and then inoculate it into the liquid medium at a volume percentage of 1%. Ferment for 120 hours, centrifuge to remove the precipitate, and take the supernatant to obtain the fermented biological agent.

[0013] The short-dense Trichoderma Trichoderma brevicompactum The preservation number of strain TB2 is CCTCC NO: M20231913.

[0014] Furthermore, the dilution concentration in step (2) is 100 times.

[0015] Furthermore, the PDA plate culture conditions in step (1) are as follows: cultured in a 28℃ light incubator for 72 h.

[0016] Furthermore, the liquid culture medium culture conditions in step (2) are: 30°C, 170 rpm shaker culture for 48 hours.

[0017] The present invention also includes the application of the fermented biological agent or the fermented biological agent prepared by the method in promoting cucumber growth.

[0018] The present invention also includes the application of the fermented biological agent or the fermented biological agent prepared by the method in the preparation of bio-organic fertilizer.

[0019] This invention has the following beneficial effects: It utilizes stevia polysaccharides, a waste product from stevia processing, for secondary use. Experiments have shown that stevia polysaccharides can replace traditional carbon sources, significantly promoting the growth of Trichoderma colonies, superior to traditional glucose carbon sources. Simultaneously, the utilization of stevia polysaccharides turns waste into treasure, reducing environmental pollution and lowering costs. This component has a particularly significant promoting effect on the growth and sporulation of strain TB2. Combined with the growth-promoting effect of this strain, verification revealed that fermentation of this strain in MSM medium in the presence of stevia polysaccharides followed by filtration can prepare a fermented biological agent. This fermented biological agent has a significant promoting effect on cucumbers. Experiments showed that although strain TB2 has growth-promoting functions, its effect on cucumber growth is not significant. This indicates that the fermented biological agent of this application effectively solves the technical defect of the TB2 strain's insignificant growth-promoting effect on cucumbers, achieving efficient resource utilization of stevia polysaccharides. This fermented biological agent has good growth-promoting functions for plants, reducing agricultural production's dependence on synthetic fertilizers and meeting the needs of green agriculture. Pot experiments have demonstrated that fermented biological agents can significantly increase seedling biomass, improve root development, and promote plant growth. The fermented biological agent prepared in this invention optimizes the Trichoderma liquid fermentation process. By adjusting the concentration of stevia polysaccharides, pH, and culture conditions, production efficiency is improved, and the spore production during liquid fermentation can reach 10... 7 The output of active ingredients is stable at 1 / mL. Attached Figure Description

[0020] Figure 1 The results show the colony diameter of Trichoderma strain TB2 at 24 h and 48 h; the left side is for 24 h and the right side is for 48 h.

[0021] Figure 2 The results for the colony diameter of Trichoderma strain NJAU4742 at 24 h and 48 h are shown; the left side is for 24 h and the right side is for 48 h.

[0022] Figure 3 The results show the colony diameter of Trichoderma strain WJG7 at 24 h and 48 h; the left side is for 24 h and the right side is for 48 h.

[0023] Figure 4 The results show the colony diameter of Trichoderma strain JR701 at 24 h and 48 h; the left side is for 24 h and the right side is for 48 h.

[0024] Figure 5The results show the colony diameter of Trichoderma strain SY39-2 at 24 h and 48 h; the left side is for 24 h and the right side is for 48 h.

[0025] Figure 6 The results show the colony diameter of Trichoderma strain HN36-1 at 24 h and 48 h; the left side is for 24 h and the right side is for 48 h.

[0026] Figure 7 The results show the colony diameter of Trichoderma strain HN2 at 24 h and 48 h; the left side is for 24 h and the right side is for 48 h.

[0027] Figure 8 The image shows the growth of seven Trichoderma strains under different treatments.

[0028] Figure 9 The graph shows the sporulation results of seven Trichoderma strains under different treatments.

[0029] Figure 10 Microscopic images of Trichoderma NJAU4742 under different liquid fermentation treatments.

[0030] Figure 11 Microscopic images of Trichoderma TB2 under different liquid fermentation treatments.

[0031] Figure 12 The results of different treatments on cucumber growth are shown in the figure; from left to right, they are T1, T2, T3, T4, T5, and T6.

[0032] Figure 13 The figure shows the effect of different treatments on the root growth of cucumbers; the three plants on the left are the T6 treatment, and the three plants on the right are the CK treatment.

[0033] Figure 14 The graph shows the effect of different treatments on the aboveground / underground dry weight of cucumber plants; the left graph shows the aboveground dry weight, and the right graph shows the underground dry weight.

[0034] Figure 15 PCA analysis plot showing the differences between different treatments.

[0035] Figure 16 Statistical analysis of metabolites in different treatments.

[0036] Figure 17 A graph showing the enrichment analysis of differentially expressed metabolic pathways under different treatments.

[0037] Information on the preservation of biological materials

[0038] The preservation information for strain TB2 in this application is: Trichoderma brevis. Trichoderma brevicompactum TB2, its classification name is: Trichoderma brevicompactumTB2, classified in Chinese as *Trichoderma septum* TB2, with accession number CCTCC NO: M20231913, is deposited at the China Center for Type Culture Collection, No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province, on October 16, 2023. This strain has been published in patent application 202311753128.0. Detailed Implementation

[0039] All features disclosed in this specification, or all steps in all disclosed methods or processes, may be combined in any way, except for mutually exclusive features and / or steps.

[0040] Unless otherwise stated, each feature disclosed in this specification (including any appended claims and abstract) is merely one example of a series of equivalent or similar features. Example 1

[0041] This embodiment studies the effect of stevia polysaccharide on the growth of Trichoderma strains, as detailed below.

[0042] The stevia polysaccharides used in the embodiments of this application were all purchased from Dongtai Haorui Biotechnology Co., Ltd., and their main indicators and properties are shown in Table 1.

[0043]

[0044] 2. Experimental Methods: Seven Trichoderma strains (TB2, NJAU4742, WJG7, JR701, SY39-2, HN36-1, and HN2) were selected. Carbon-free basal medium (MSM) was prepared, and stevia polysaccharide was added to prepare concentrations of 50, 100, 500, and 1000 times. 20 g / L agar was added to each solution. -1 Adjust the pH to 7 and autoclave at 115°C for 30 minutes. A medium supplemented with glucose (a common carbon source) was set up as a positive control (Glu) to verify the growth ability of the strains, while a medium without stevia polysaccharide served as a negative control (CK). Using a pipette, 5 μL of spores from seven Trichoderma strains were inoculated onto the surface of a solid medium and incubated at 30°C for 48 h. The presence and growth of colonies at different stevia polysaccharide concentrations were observed and recorded at 24 h and 48 h, and the diameter was measured.

[0045] The test results are as follows Figures 1-8 As shown: Figure 1 The colony diameter of Trichoderma strain TB2 at 24 h and 48 h is... Figure 2 The colony diameter of Trichoderma strain NJAU4742 at 24 h and 48 h is... Figure 3 For Trichoderma strain WJG7, the colony diameter at 24 h and 48 h is... Figure 4The colony diameter of Trichoderma strain JR701 at 24 h and 48 h is... Figure 5 The colony diameter of Trichoderma strain SY39-2 at 24 h and 48 h is... Figure 6 The colony diameter of Trichoderma strain HN36-1 at 24 h and 48 h is... Figure 7 The colony diameter of Trichoderma strain HN2 at 24 h and 48 h is... Figure 2 The colony diameter of Trichoderma strain NJAU4742 at 24 h and 48 h is... Figure 8 The growth of seven *Trichoderma* strains under different treatments is shown in the figure. The results indicate that all seven strains could grow on the tested culture media, but significant differences existed between treatments. Compared to the control (CK) and glucose as a carbon source, low dilutions (50x and 100x) of stevia polysaccharide significantly increased the colony diameter of all seven *Trichoderma* strains, promoting their growth. NJAU4742 and TB2 showed the most significant effects; compared to the control (CK), at a 50x dilution of stevia polysaccharide, the colony diameter increased by 49.2% and 131.3%, respectively; at a 100x dilution, the colony diameter increased by 49.2% and 132.2%, respectively. Example 2

[0046] This embodiment studies the effect of stevia polysaccharide on sporulation ability of Trichoderma, as detailed below.

[0047] (1) Preparation of Trichoderma spore suspension: Seven Trichoderma strains, namely TB2, NJAU4742, WJG7, JR701, SY39-2, HN36-1 and HN2, were activated and inoculated on PDA plates. They were placed in a light incubator at 28°C and cultured for 72 h. 10 mL of sterile water was added in a sterile environment, and the spore suspension was obtained by scraping with a spreader and filtering through a filter cloth for later use.

[0048] (2) Shake flask liquid fermentation culture: Prepare carbon-free basal medium (MSM), add stevia polysaccharide to prepare a 50-fold and 100-fold dilution concentration, adjust the pH to 7, inoculate into the liquid medium at a 1% inoculation amount, and culture at 30°C and 170 rpm. After 60 h of culture, take samples for counting.

[0049] (3) Spore counting method: Use a pipette to draw 100 uL of Trichoderma fermentation broth that has been thoroughly shaken and mixed, and drop it into the groove of the counting plate. Place a coverslip on the counting chamber of the hemocytometer and allow the bacterial broth to seep into the counting chamber through the gap between the coverslip and the counting plate. Let it stand for a while until the bacteria settle and stabilize naturally. Then start counting the total number of conidia in the five large squares (80 small squares) of the counting chamber: "top left, bottom left, top right, bottom right, and middle". The concentration of Trichoderma conidia (conidia / mL) = total number of conidia in 80 small squares / 80 × 400 × 10000 × dilution factor. Perform three replicates for each experiment and take the average value.

[0050] The test results are as follows Figure 9 As shown: Except for Trichoderma NJAU4742 and TB2, the spore yield of other Trichoderma strains failed to reach 10. 7 The spore yield of Trichoderma NJAU4742 and TB2 can both reach 10 spores / mL. 7 pcs / mL

[0051] Microscopic images of the fermentation broths of Trichoderma NJAU4742 and TB2 are shown below. Figure 10 and Figure 11 As shown: Figure 10 It is Trichoderma NJAU4742. Figure 11 As shown in the figure, the spore yield of Trichoderma TB2 in a 50-fold dilution of stevia polysaccharide was significantly higher than that in a 100-fold dilution of stevia polysaccharide; and the spore yield of Trichoderma TB2 in a 100-fold dilution of stevia polysaccharide was significantly higher than that in a 50-fold dilution of stevia polysaccharide. Example 3

[0052] This embodiment studies the effect of stevia polysaccharide-TB2 fermented biological agent on cucumber growth promotion, as detailed below.

[0053] Fermentation broth for treatment groups T1-T6 was prepared as follows.

[0054] ①T1 treatment group: The purchased stevia polysaccharide was diluted 100 times with water and the pH was adjusted to 7.

[0055] ②T2 treatment group: Prepare carbon-free basal medium (MSM), add stevia polysaccharide to prepare a 100-fold dilution concentration, and adjust the pH to 7.

[0056] ③T3 treatment group: The purchased stevia polysaccharide was diluted 100 times with water, the pH was adjusted to 7, and then TB2 spore suspension was inoculated at a volume percentage of 1% and cultured at 30°C and 170 rpm for 120 hours. The preparation method of TB2 spore suspension was as follows: Trichoderma TB2 strain was activated, inoculated on PDA plates, placed in a light incubator at 28°C and cultured for 72 h. 10 mL of sterile water was added in a sterile environment, and the spore suspension was obtained by scraping with a spreader and filtering through a filter cloth.

[0057] ④T4 treatment group: The purchased stevia polysaccharide was diluted 100 times with water, the pH was adjusted to 7, and then TB2 spore suspension was inoculated at a volume percentage of 1% and cultured at 30°C and 170 rpm for 120 hours. The precipitate was removed by centrifugation and the supernatant was collected. The preparation method of TB2 spore suspension is as follows: Trichoderma TB2 strain was activated, inoculated on PDA plates, placed in a light incubator at 28°C and cultured for 72 h. 10 mL of sterile water was added in a sterile environment, and the spore suspension was obtained by scraping with a spreader and filtering through a filter cloth.

[0058] ⑤T5 treatment group: Trichoderma TB2 strain was activated and inoculated onto PDA plates. The plates were placed in a light incubator at 28°C and cultured for 72 h. 10 mL of sterile water was added in a sterile environment, and the spore suspension was obtained by scraping with a spreader and filtering through a filter cloth. Carbon-free basal medium (MSM) was prepared, and stevia polysaccharide was added to prepare a 100-fold dilution concentration. The pH was adjusted to 7, and then inoculated into the liquid medium at a volume percentage of 1%. The medium was cultured at 30°C and 170 rpm for 120 h.

[0059] ⑥T6 treatment group: Trichoderma TB2 strain was activated and inoculated onto PDA plates. The plates were placed in a light incubator at 28°C and cultured for 72 h. 10 mL of sterile water was added in a sterile environment, and the spore suspension was obtained by scraping with a spreader and filtering through a filter cloth. Carbon-free basal medium (MSM) was prepared, and stevia polysaccharide was added to prepare a 100-fold dilution concentration. The pH was adjusted to 7, and then inoculated into the liquid medium at a volume percentage of 1%. The medium was cultured at 30°C and 170 rpm for 120 h. The precipitate was removed by centrifugation, and the supernatant was collected.

[0060] 2. The effects of fermented biological agents on cucumber growth were studied, as detailed below.

[0061] (1) Disinfection and germination of cucumber seeds: Soak cucumber seeds in sterile deionized water at 50℃ for 30 min, then immediately transfer them to sterile water at 30℃ for 1 h. After draining the water, soak the seeds in 70% ethanol for 3-4 min, shaking continuously during the soaking process. Rinse three times with sterile water, then soak in 2% sodium hypochlorite for 3-4 min, shaking continuously as well. Finally, rinse multiple times with sterile deionized water until the sodium hypochlorite and the outer coating of the cucumber seeds are completely removed, thus completing the disinfection of the cucumber seeds. Place the disinfected cucumber seeds with a similar degree of plumpness evenly in a 90 mm petri dish lined with sterile filter paper. Moisten each filter paper with 3 ml of the liquid prepared in the above steps, keeping the filter paper moist. Incubate in the dark at 30℃ for 3-5 days until the cucumber seeds show signs of germination.

[0062] (2) Cucumber seedling raising: Select uniform and plump cucumber seeds with white tips and sow them into the seedling substrate. When the cucumbers in the substrate have grown to 2 true leaves, select seedlings with uniform growth and transplant them for subsequent experiments.

[0063] (3) Potted plant experimental treatment: The experimental setup was set up according to the 6 treatments in Table 2, with 6 replicates. One week after transplanting, the roots were irrigated. After 30 days of cultivation, the cucumber phenotypic traits were measured.

[0064]

[0065] Experimental results are as follows Figure 12 , Figure 13 and Figure 14 As shown: Figure 12 From left to right, the treatments are T1, T2, T3, T4, T5, and T6. As can be seen from the figure, the roots of the T6 treatment group are significantly thicker than those of the other treatment groups.

[0066] Figure 13 The three plants on the left are T6 treatment, and the three groups on the right are CK (T2 treatment). As can be seen from the figure, the roots of the T6 treatment plants have filled the nutrient pot, and the roots are significantly thicker than those of the CK treatment group.

[0067] Figure 14 The left side of the figure shows the experimental results of the aboveground dry weight of cucumber plants under different treatments, while the right side shows the experimental results of the underground dry weight of cucumber plants under different treatments. As can be seen from the figure, the aboveground dry weight of cucumber plants under treatment T6 was significantly better than that under treatments T1-T4, and the underground dry weight was significantly better than that under treatments T1-T5.

[0068] Although the T3 treatment contained the Trichoderma TB2 strain, the results showed no significant advantage in either the aboveground or underground dry weight of cucumbers. In fact, the root dry weight of this treatment was the lowest. This indicates that although the TB2 strain was reported to have certain growth-promoting functions in the applicant's aforementioned application: 202311753128.0, this study found that the strain itself does not promote cucumber growth. This may be related to the colonization ability of Trichoderma TB2 on cucumber roots, or it may also be related to the composition of its extracellular metabolites.

[0069] Treatment T4 consisted of stevia polysaccharide diluted 100 times and fermentation supernatant from Trichoderma TB2. The results showed no significant advantage in either the aboveground or underground dry weight of cucumber, with treatment T4 yielding the lowest aboveground dry weight. This indicates that while stevia polysaccharide diluted 100 times can promote the growth and sporulation of Trichoderma TB2, its extracellular components do not produce related metabolites that promote cucumber growth when only stevia polysaccharide is present.

[0070] The T6 group achieved the highest dry weight in both aboveground and underground parts, indicating that the fermentation of stevia polysaccharide diluted 100 times with Trichoderma TB2 in MSM medium produced relevant metabolites that promote cucumber growth. Calculations showed that the T6 treatment significantly increased the aboveground dry weight and underground dry weight (root dry weight) by 21.6% and 40.6%, respectively, compared to the control. Example 4

[0071] This embodiment details the determination and analysis of metabolic components in the liquid fermentation broth of the T6 treatment group and the CK group.

[0072] 1. Non-targeted metabolomics detection was performed on the liquid fermentation broth of the T5 treatment group and the CK (T2 treatment) using the GC-MS technology platform. A total of 44 metabolites were detected in the fermentation broth.

[0073] (1) An intergroup difference analysis was performed on the liquid fermentation broth of the T5 treatment group, specifically as follows: Figure 15 As shown, PCA analysis revealed that the TB2 group (stevia polysaccharide product fermented with TB2) and the CK group (T2, unfermented) were completely separated on principal component 1 (PC1). PC1 explained 87.1% of the variation, indicating that TB2 fermentation significantly altered the overall characteristics of the samples. The TB2 group samples clustered tightly, suggesting that its fermentation products exhibited good stability and consistency.

[0074] (2) Differential metabolite analysis, such as Figure 16 As shown, metabolomics analysis of TB2-treated and unfermented control (CK) samples revealed significant differences in their metabolomic profiles. Compared to CK, TB2 treatment significantly increased the abundance of various metabolites related to plant growth and signal regulation, including phytosterols (such as β-sitosterol, stigmasterol, and chickpea sterol), phenolic compounds (such as chlorogenic acid and caffeic acid), flavonoid precursors, and unsaturated fatty acids (such as linoleic acid and ethyl oleate). These upregulated metabolites are known to promote root growth, regulate hormone balance, enhance cell membrane fluidity, and have antioxidant effects in plants, and may promote root development by regulating secondary metabolism and signal transduction pathways.

[0075] (3) Enrichment analysis of differential metabolite metabolic pathways, such as Figure 17As shown, KEGG metabolic pathway enrichment analysis revealed that TB2 fermentation treatment significantly affected multiple core metabolic pathways. The most significant enrichment included key coenzyme synthesis pathways such as biotin metabolism, coenzyme F420 biosynthesis, and folic acid metabolism. The upregulation of these metabolites may enhance the overall activity and antioxidant capacity of the metabolic system. Furthermore, secondary metabolic pathways such as steroids, alkaloids, flavonoids, and amino acids also showed significant enrichment, potentially regulating root development by influencing plant hormone balance and signal transduction. Simultaneously, the enrichment of glutathione metabolism, nucleotide synthesis, and ABC transporter pathways indicates that TB2 fermentation products play a crucial role in carbon / nitrogen metabolism, energy balance, and rhizosphere signaling transport. These pathway changes further support the functional association between differentially metabolized metabolites and root growth-promoting effects.

[0076] In summary, this invention utilizes stevia polysaccharides to replace traditional carbon sources, demonstrating a significant promotion of Trichoderma colony growth, superior to traditional glucose carbon sources. This component exhibits a particularly pronounced effect on the growth and sporulation of strain TB2. Fermentation of this strain in MSM medium in the presence of stevia polysaccharides, followed by filtration, yields a fermented biological agent. This agent promotes plant growth, reduces agricultural dependence on synthetic fertilizers, and aligns with the requirements of green agriculture. Furthermore, this invention optimizes the Trichoderma liquid fermentation process, adjusting stevia polysaccharide concentration, pH, and culture conditions, thereby improving production efficiency. The sporulation yield during liquid fermentation can reach 10... 7 The output of active ingredients is stable at 1 / mL.

[0077] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are specific and detailed, they should not be construed as limiting the scope of the invention. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these modifications and improvements all fall within the scope of protection of the present invention. Therefore, the scope of protection of the present invention should be determined by the appended claims.

Claims

1. A fermentation biological agent based on stevia polysaccharides that promotes sporulation of Trichoderma, characterized in that, The fermented biological agent is produced by *Trichoderma brevis*. Trichoderma brevicompactum It was obtained by fermentation of strain TB2, stevia polysaccharide solution, and MSM medium; the specific method is as follows: (1) Preparation of spore suspension: Trichoderma brevicornu Trichoderma brevicompactum After the TB2 strain was activated, it was inoculated onto a PDA plate and placed in a 28°C light incubator for 72 h. Then, 10 mL of sterile water was added in a sterile environment, and the spore suspension was obtained by scraping with a spreader and filtering through a filter cloth. (2) Preparation of fermented biological agent: Prepare MSM medium, add stevia polysaccharide to prepare a 100-fold dilution concentration, adjust the pH to 7, and then inoculate the spore suspension of step (1) into the liquid medium at an inoculation amount of 1% by volume. Ferment in a shaker at 30°C and 170 rpm for 120 hours. Centrifuge to remove the precipitate and take the supernatant to obtain the fermented biological agent. The short-dense Trichoderma Trichoderma brevicompactum The preservation number of strain TB2 is CCTCC NO: M20231913; The MSM culture medium consists of the following components: 2.0 g of (NH4)2SO4, 0.2 g of MgSO4·7H2O, 0.01 g of CaCl2·2H2O, 0.001 g of FeSO4·7H2O, 1.5 g of Na2HPO4·12H2O, 41.5 g of KH2PO4, and 1000 mL of distilled water.

2. The application of the fermented biological agent according to claim 1 in promoting cucumber growth.

3. The application of the fermentation biological agent according to claim 1 in the preparation of bio-organic fertilizer.

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