Fermentation biological agent for promoting trichoderma to produce spore based on stevia polysaccharide as well as preparation method and application of fermentation biological agent

Abstract

The invention relates to the technical field of microorganisms, in particular to a fermentation biological agent for promoting trichoderma to produce spores based on stevia polysaccharide and a preparation method and application thereof.According to the fermentation biological agent for promoting trichoderma to produce spores based on stevia polysaccharide, waste obtained after stevia rebaudiana processing, namely stevia polysaccharide, is subjected to secondary utilization to replace a traditional carbon source, and it is found that the stevia polysaccharide can remarkably promote trichoderma colony growth and is superior to a traditional glucose carbon source; the component has a particularly obvious effect of promoting growth and spore production of a strain TB2, the strain is fermented in an MSM culture medium in the presence of stevia polysaccharide and then filtered to prepare a fermented biological preparation, and the fermented biological preparation has a good growth promoting function on plants, reduces dependence of agricultural production on synthetic chemical fertilizers, and meets the requirements of green agriculture. In addition, by optimizing a trichoderma liquid fermentation process and adjusting the concentration, pH and culture conditions of stevia polysaccharide, the production efficiency is improved, the sporulation quantity of liquid fermentation can reach 107 / mL, and the output of active ingredients is stable.

Description

Technical Field

[0001] The present invention relates to the field of microbial technology, and in particular to a fermentation biological preparation based on stevia polysaccharide that promotes Trichoderma spore production, and a preparation method and application thereof. Background Art

[0002] Stevia polysaccharides are a byproduct of stevia leaves that are extracted, settled, and concentrated using membranes to produce a 50% solids content. They are considered industrial waste. Primarily composed of dietary fiber polysaccharides (crude polysaccharides), they are low in sugar and calories, combined with antioxidant properties, and have potential for functional applications.

[0003] Trichoderma is widely distributed in nature and holds significant biocontrol applications. Many species not only antagonize a variety of soil-borne fungal pathogens but also promote plant growth. Due to its complex and diverse biocontrol mechanisms, including competition, hyperparasitism, antibiosis, and induction of plant resistance, Trichoderma holds an irreplaceable position in the sustainable management of plant disease control.

[0004] Liquid fermentation is a commonly used modern fermentation technique, primarily utilizing the free liquid substrate in the fermentation broth as a feedstock, supplemented by a controlled rotational speed and ventilation rate. Compared to solid-state fermentation, submerged liquid fermentation ensures uniformity of nutrients in the culture medium and facilitates control of parameters such as temperature and pH, enabling efficient production of high-quality Trichoderma propagules. Liquid fermentation also boasts a high degree of automation, making it amenable to industrial production.

[0005] Currently, most Trichoderma products on the market are used in production as biological preparations. While these preparations offer numerous advantages, they also suffer from numerous drawbacks. For example, Trichoderma preparations are often solid-state cultures, resulting in low automation, high costs, and compromised product quality. Furthermore, stevia is widely used in food additives, sweeteners, and the pharmaceutical industry. The waste residue from stevia processing produces a large amount of stevia polysaccharides. If not properly utilized, this not only negatively impacts the environment but also wastes significant amounts of nutrient resources. To address these existing issues, the present invention optimizes Trichoderma culture efficiency by utilizing 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, develop their possibilities in microbial fermentation, effectively utilize stevia polysaccharides at a high value, and reduce pollution to the environment.

[0007] To achieve the above purpose, the technical solution adopted by the present invention is: a fermentation biological preparation based on stevia polysaccharide to promote Trichoderma spore production, the fermentation biological preparation is prepared from Trichoderma brevis Trichoderma brevicompactum strain TB2, stevia polysaccharide solution and MSM culture medium fermentation; the short dense Trichoderma Trichoderma brevicompactumTB2, whose classification is named as: Trichoderma brevicompactum TB2, Chinese classification name: Trichoderma brevis TB2, preservation number: CCTCC NO: M20231913; the strain is deposited in the China Center for Type Culture Collection, address: No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province, the preservation date is October 16, 2023, and the strain has been disclosed in patent application: 202311753128.0.

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

[0009] Furthermore, the MSM culture medium obtained by fermentation is composed 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 preparation, which is:

[0011] (1) Preparation of spore suspension: Trichoderma brevis Trichoderma brevicompactum After the strain TB2 was activated, it was inoculated on a PDA plate for culture, sterile water was added in a sterile environment, and the spore suspension was obtained by scraping with a coating rod and filtering through a filter cloth.

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

[0013] Trichoderma brevis Trichoderma brevicompactum The deposit 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: culture in a light incubator at 28°C for 72 h.

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

[0017] The present invention also includes the use of the fermented biological preparation or the fermented biological preparation prepared by the method in promoting the growth of cucumbers.

[0018] The present invention also includes the use of the fermented biological preparation or the fermented biological preparation prepared by the method in preparing biological organic fertilizer.

[0019] The present invention has the following beneficial effects: By reusing stevia polysaccharide, a waste product from stevia processing, experiments have shown that stevia polysaccharide can replace traditional carbon sources and significantly promote the growth of Trichoderma colonies when used as a carbon source, outperforming traditional glucose carbon sources. Furthermore, the utilization of stevia polysaccharide transforms waste into valuable resources, reducing environmental pollution and lowering costs. This component has a particularly significant effect on promoting the growth and spore production of the TB2 strain. Combined with the growth-promoting efficacy of this strain, verification has shown that fermentation of this strain in MSM medium in the presence of stevia polysaccharide followed by filtration can produce a fermented biopharmaceutical, which has a significant growth-promoting effect on cucumbers. However, experiments have shown that while the TB2 strain has growth-promoting properties, the strain itself does not significantly promote the growth of cucumbers. This demonstrates that the fermented biopharmaceutical of the present application effectively overcomes the technical drawback of the TB2 strain's lack of a significant growth-promoting effect on cucumbers, achieving efficient resource utilization of stevia polysaccharide. This fermented biopharmaceutical exhibits excellent plant growth-promoting properties, reduces agricultural production's reliance on synthetic fertilizers, and meets the needs of green agriculture. Potted plant experiments have shown that fermentation biological preparations can significantly increase seedling biomass, improve root development, and promote plant growth. The fermentation biological preparation prepared by the present invention optimizes the liquid fermentation process of Trichoderma and improves production efficiency by adjusting the concentration of stevia polysaccharides, pH and culture conditions. The spore production of liquid fermentation can reach 10 7 pcs / mL, and the output of active ingredients is stable. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 The colony diameters of Trichoderma strain TB2 are shown at 24 h and 48 h, with the left side showing 24 h and the right side showing 48 h.

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

[0022] Figure 3 The colony diameters of Trichoderma strain WJG7 at 24 h and 48 h are shown; the left side is 24 h and the right side is 48 h.

[0023] Figure 4 The colony diameters of Trichoderma strain JR701 at 24 h and 48 h are shown; the left side is at 24 h and the right side is at 48 h.

[0024] Figure 5The colony diameters of Trichoderma strain SY39-2 at 24 h and 48 h are shown; the left side is at 24 h and the right side is at 48 h.

[0025] Figure 6 The colony diameters of Trichoderma strain HN36-1 at 24 h and 48 h are shown; the left side is at 24 h and the right side is at 48 h.

[0026] Figure 7 The colony diameters of Trichoderma strain HN2 at 24 h and 48 h are shown; the left side is at 24 h and the right side is at 48 h.

[0027] Figure 8 The graph shows the growth results of 7 strains of Trichoderma under different treatments.

[0028] Figure 9 This is the result of conidia production of 7 strains of Trichoderma under different treatments.

[0029] Figure 10 These are microscopic images of Trichoderma NJAU4742 under different liquid fermentation treatments.

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

[0031] Figure 12 This is the result diagram of the effects of different treatments on the growth of cucumber; from left to right are T1, T2, T3, T4, T5, and T6.

[0032] Figure 13 This is a graph showing the effects of different treatments on the root growth of cucumbers; the three plants on the left are under T6 treatment, and the three plants on the right are under CK treatment.

[0033] Figure 14 This is a graph showing the effects of different treatments on the aboveground / underground dry weight of cucumber plants; the left picture shows the aboveground dry weight, and the right picture shows the underground dry weight.

[0034] Figure 15 PCA analysis of differences among different treatments.

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

[0036] Figure 17 This is the enrichment analysis diagram of differential metabolic pathways in different treatments. Biomaterial deposit information

[0037] The deposit information of the TB2 strain of this application is: Trichoderma brevis Trichoderma brevicompactum TB2, whose classification is named as: Trichoderma brevicompactumTB2, Chinese classification name: Trichoderma brevis TB2, preservation number: CCTCC NO: M20231913; the strain is deposited in the China Center for Type Culture Collection, address: No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province, the preservation date is October 16, 2023, and the strain has been disclosed in patent application: 202311753128.0. DETAILED DESCRIPTION

[0038] All features disclosed in this specification, or all steps in the disclosed methods or processes, except mutually exclusive features and / or steps, can be combined in any manner.

[0039] Any feature disclosed in this specification (including any accompanying claims and abstract), unless otherwise stated, is merely an example of a series of equivalent or similar features. Example 1

[0040] This example studies the effect of stevia polysaccharides on the growth of Trichoderma strains, as follows.

[0041] The stevia polysaccharides used in the examples of this application were purchased from Dongtai Haorui Biotechnology Co., Ltd., and their main indicators and properties are shown in Table 1.

[0042]

[0043] 2. Experimental method: Seven strains of Trichoderma, TB2, NJAU4742, WJG7, JR701, SY39-2, HN36-1, and HN2, were selected and carbon-free basal medium (MSM) was prepared. Stevia polysaccharide was added in proportion to prepare 50, 100, 500, and 1000 times dilution concentrations. Agar 20 g·L was added. -1 Adjust the pH to 7 and sterilize by autoclaving at 115°C for 30 minutes. A medium supplemented with the conventional carbon source glucose served as a positive control (Glu) to verify the strain's growth ability, and a medium without stevia served as a negative control (CK). Using a pipette, 5 μL of spores from seven Trichoderma strains were inoculated onto the surface of the solid culture medium and incubated at 30°C for 48 hours. The presence and growth of colonies on the plates at different stevia concentrations were observed and recorded at 24 and 48 hours, and their diameters were measured.

[0044] 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, Figure 2 For Trichoderma strain NJAU4742, the colony diameter at 24 h and 48 h, Figure 3 The colony diameter of Trichoderma strain WJG7 at 24 h and 48 h, Figure 4For Trichoderma strain JR701, the colony diameter at 24 h and 48 h, Figure 5 The colony diameter of Trichoderma strain SY39-2 at 24 h and 48 h, Figure 6 The colony diameter of Trichoderma strain HN36-1 at 24 h and 48 h, Figure 7 For Trichoderma strain HN2, the colony diameter at 24 h and 48 h, Figure 2 For Trichoderma strain NJAU4742, the colony diameter at 24 h and 48 h, Figure 8 The growth of seven Trichoderma strains under different treatments is shown in the figure. The results show that all seven strains were able to grow on the tested media, but there were significant differences between the treatments. Compared with CK and glucose as carbon sources, treatment with stevia polysaccharides at low dilutions (50 and 100x) significantly increased the colony diameters of all seven strains, promoting their growth. NJAU4742 and TB2 showed the most significant effects. Compared with CK, colony diameters increased by 49.2% and 131.3%, respectively, at a 50-fold dilution of stevia polysaccharides; and by 49.2% and 132.2%, respectively, at a 100-fold dilution. Example 2

[0045] This example studies the effect of stevia polysaccharide on the spore production ability of Trichoderma, as follows.

[0046] (1) Preparation of Trichoderma spore suspension: 7 strains of Trichoderma, TB2, NJAU4742, WJG7, JR701, SY39-2, HN36-1, and HN2, were activated and inoculated on PDA plates. The plates were placed in a light incubator at 28°C for 72 h. 10 mL of sterile water was added in a sterile environment. The plates were scraped with a spreading rod and filtered through a filter cloth to obtain a spore suspension for later use.

[0047] (2) Shake flask liquid fermentation culture: Prepare carbon-free basal medium (MSM), add stevia polysaccharide in proportion to make 50-fold and 100-fold dilution concentrations, adjust the pH to 7, inoculate 1% of the inoculum into the liquid culture medium, culture at 30°C and 170 rpm, and take samples and count them after 60 h of culture.

[0048] (3) Spore counting method: Use a pipette to draw 100 μL of thoroughly shaken Trichoderma fermentation liquid and drop it into the groove of the counting plate. Place a cover glass on the counting chamber of the hemocytometer to allow the bacterial liquid to seep into the counting chamber through the gap between the cover glass and the counting plate. Let it stand for a while. After the bacteria settle naturally and stabilize, start counting the total number of conidia in the five large squares (80 small squares) of the counting chamber: "upper left, lower left, upper right, lower right, and middle". The conidia concentration of Trichoderma (pieces / mL) = the total number of conidia in 80 small squares / 80×400×10000×dilution factor. Perform 3 replicates for each experiment and take the average value.

[0049] The test results are as follows Figure 9 As shown in the figure, except for Trichoderma NJAU4742 and TB2, the spore production of other Trichoderma strains failed to reach 10 7 / mL, while the spore yields of Trichoderma NJAU4742 and TB2 can reach 10 7 Pieces / mL.

[0050] Microscopic examination of Trichoderma NJAU4742 and TB2 fermentation broth Figure 10 and Figure 11 As shown: Figure 10 It is Trichoderma NJAU4742, Figure 11 It can be seen from the figure that the spore yield of Trichoderma NJAU4742 in 50-fold stevia polysaccharide solution is significantly higher than that in 100-fold stevia polysaccharide solution; the spore yield of Trichoderma TB2 in 100-fold stevia polysaccharide solution is significantly higher than that in 50-fold stevia polysaccharide solution. Example 3

[0051] This example studies the growth-promoting effect of stevia polysaccharide-TB2 fermentation biological preparation on cucumber, as follows.

[0052] The fermentation broths of the T1-T6 treatment groups were prepared as follows.

[0053] ① Treatment group T1: The purchased stevia polysaccharide was directly diluted 100 times with water and the pH was adjusted to 7.

[0054] ②T2 treatment group: Prepare carbon-free basal medium (MSM), add stevia polysaccharide to make a 100-fold dilution concentration, and adjust the pH to 7.

[0055] ③T3 treatment group: The purchased stevia polysaccharide was directly diluted 100 times with water, the pH was adjusted to 7, and then the TB2 spore suspension was inoculated at an inoculum rate of 1% by volume and cultured at 30°C and 170 rpm for 120 hours; wherein, the preparation method of the TB2 spore suspension is as follows: the Trichoderma TB2 strain was activated, inoculated on a PDA plate, placed in a light incubator at 28°C, and cultured for 72 hours. In a sterile environment, 10 mL of sterile water was added, and the spore suspension was obtained by scraping with a coating rod and filtering through a filter cloth.

[0056] ④T4 treatment group: The purchased stevia polysaccharide was directly diluted 100 times with water, the pH was adjusted to 7, and then the TB2 spore suspension was inoculated at an inoculum rate of 1% by volume 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 the TB2 spore suspension was as follows: the Trichoderma TB2 strain was activated, inoculated on a PDA plate, placed in a light incubator at 28°C, and cultured for 72 hours. 10 mL of sterile water was added in a sterile environment, and the spore suspension was obtained by scraping with a coating rod and filtering through a filter cloth.

[0057] ⑤T5 treatment group: The Trichoderma TB2 strain was activated and inoculated on a PDA plate. The plate was placed in a light incubator at 28°C for 72 h. 10 mL of sterile water was added in a sterile environment. The spore suspension was scraped with a spreader and filtered through a filter cloth to obtain a spore suspension. A carbon-free basal medium (MSM) was prepared, stevia polysaccharide was added to a 100-fold dilution concentration, the pH was adjusted to 7, and then the spore suspension was inoculated into the liquid culture medium at a volume percentage of 1%. The plate was cultured at 30°C and 170 rpm for 120 h.

[0058] ⑥T6 treatment group: The Trichoderma TB2 strain was activated and inoculated on a PDA plate. The plate was placed in a light incubator at 28°C for 72 h. 10 mL of sterile water was added in a sterile environment. The spore suspension was scraped with a spreader and filtered through a filter cloth to obtain a spore suspension. A carbon-free basal medium (MSM) was prepared, stevia polysaccharide was added to a 100-fold dilution concentration, the pH was adjusted to 7, and then the suspension was inoculated into the liquid culture medium at a volume percentage of 1%. The plate was incubated at 30°C and 170 rpm for 120 h. The precipitate was removed by centrifugation, and the supernatant was obtained.

[0059] 2. Study the effects of fermented biological preparations on cucumber growth, as follows.

[0060] (1) Disinfection and germination of cucumber seeds: Soak the cucumber seeds in sterile deionized water at 50°C for 30 minutes, then immediately transfer them to sterile water at 30°C and soak for 1 hour. After pouring out the water, soak them in 70% ethanol for 3-4 minutes, shaking them continuously. Rinse them with sterile water three times, then soak them in 2% sodium hypochlorite for 3-4 minutes, shaking them continuously. Finally, rinse them with sterile deionized water several times until the sodium hypochlorite and the outer coating of the cucumber seeds are rinsed clean. The disinfected cucumber seeds with basically the same plumpness are evenly placed in a 90mm culture dish lined with sterile filter paper. Soak the filter paper with 3ml of the liquid prepared in the above steps respectively, keep the filter paper moist, and place them in the dark at 30°C for germination for 3-5 days until the cucumber seeds turn white.

[0061] (2) Cucumber seedling: Select uniform and full-sized white cucumber seeds and sow them in the seedling substrate. When the cucumber in the substrate grows to 2 true leaves, select seedlings with consistent growth and transplant them for subsequent experiments.

[0062] (3) Pot experiment treatment: The experiment was set up according to the 6 treatments in Table 2, with 6 replicates. The seedlings were transplanted and treated with root irrigation after one week, and the samples were collected for determination of cucumber phenotype after 30 days of culture.

[0063]

[0064] The experimental results are shown in Figure 12 , Figure 13 and Figure 14 : Figure 12 From left to right in the figure are T1 treatment, T2 treatment, T3 treatment, T4 treatment, T5 treatment and T6 treatment. As can be seen from the figure, the root system of T6 treatment is significantly stronger than that of the other treatment groups.

[0065] Figure 13 In the figure, the left 3 plants are T6 treatment, and the right 3 groups are CK (T2 treatment). As can be seen from the figure, the root system of T6 treatment is full of nutrient pots, and is significantly stronger than that of the CK treatment group.

[0066] Figure 14 In the figure, the left is the experimental result of the aboveground dry weight of cucumber plants under different treatments, and the right is the experimental result 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 T6 treatment is significantly better than that under T1-T4 treatment, and the underground dry weight is significantly better than that under T1-T5 treatment.

[0067] Although T3 treatment contains Trichoderma TB2 strain, from the results, neither the aboveground dry weight nor the underground dry weight of cucumber is significantly better under T3 treatment, and the root dry weight under this treatment is the lowest in the underground dry weight results. Therefore, although TB2 strain has been reported to have certain growth-promoting function in the applicant's aforementioned application 202311753128.0, it is found in the present application that the strain itself does not promote the growth of cucumber, which may be related to the colonization ability of Trichoderma TB2 on cucumber roots, or may be related to the composition of its extracellular metabolites.

[0068] T4 treatment is the fermentation supernatant of Trichoderma TB2 diluted 100 times with stevioside. From the results, neither the aboveground dry weight nor the underground dry weight of cucumber is significantly better under T4 treatment, and the aboveground dry weight under this treatment is the lowest in the aboveground dry weight results. Therefore, although stevioside diluted 100 times can promote the growth and sporulation of Trichoderma TB2, the composition of its extracellular products does not produce related metabolites that promote the growth of cucumber in the presence of only stevioside.

[0069] The T6 treatment achieved the highest dry weights for both aboveground and belowground parts, indicating that fermentation of stevia polysaccharides diluted 100-fold with Trichoderma TB2 in MSM medium produced metabolites that promote cucumber growth. Calculations showed that the T6 treatment significantly increased aboveground and belowground dry weights (root dry weight) by 21.6% and 40.6%, respectively, compared to the control. Example 4

[0070] In this example, the metabolome composition of the liquid fermentation broth of the T6 treatment group and the CK group was determined and analyzed as follows.

[0071] 1. Non-targeted metabolomics analysis was performed on the liquid fermentation broth of the T5 treatment group and CK (T2 treatment) using the GC-MS technology platform, and a total of 44 metabolites were detected in the fermentation broth.

[0072] (1) Comparative analysis of the differences between the liquid fermentation broth of the T5 treatment group was performed, as follows: Figure 15 As shown: PCA analysis shows that the TB2 group (stevia polysaccharide products fermented with TB2) and the CK group (T2, unfermented) are completely separated on principal component 1 (PC1), with PC1 explaining 87.1% of the variation, indicating that TB2 fermentation significantly altered the overall characteristics of the samples. The TB2 group samples clustered closely, indicating that its fermentation products were stable and consistent.

[0073] (2) Differential metabolite analysis Figure 16 Metabolomic analysis of TB2-treated and unfermented control (CK) samples revealed significant differences in their metabolic profiles. Compared with CK, TB2 treatment significantly increased the abundance of several metabolites associated with plant growth and signal regulation, including phytosterols (e.g., β-sitosterol, stigmasterol, and chickpea sterol), phenolic compounds (e.g., chlorogenic acid and caffeic acid), flavonoid precursors, and unsaturated fatty acids (e.g., linoleic acid and ethyl oleate). These upregulated metabolites are known to promote root growth, regulate hormone balance, enhance cell membrane fluidity, and provide antioxidant benefits in plants. These metabolites may promote root development by regulating plant secondary metabolism and signal transduction pathways.

[0074] (3) Metabolic pathway enrichment analysis of differential metabolites such as Figure 17As shown: KEGG metabolic pathway enrichment analysis showed that TB2 fermentation treatment significantly affected multiple core metabolic pathways. The most significantly enriched pathways included key coenzyme synthesis pathways such as biotin metabolism, coenzyme F420 biosynthesis, and folic acid metabolism. The upregulation of these metabolites may have enhanced the overall activity and antioxidant capacity of the metabolic system. In addition, secondary metabolic pathways such as steroids, alkaloids, flavonoids, and amino acids also showed significant enrichment, which may regulate root development by affecting plant hormone balance and signal transduction. At the same time, the enrichment of glutathione metabolism, nucleotide synthesis, and ABC transporter pathways indicated that TB2 fermentation products played a key role in carbon / nitrogen metabolism, energy balance, and rhizosphere signal substance transport. These pathway changes further support the functional association between differential metabolites and root growth promotion effects.

[0075] In summary, the present invention, by reusing stevia polysaccharides to replace traditional carbon sources, found that it can significantly promote the growth of Trichoderma colonies, which is better than traditional glucose carbon sources. This component has a particularly significant effect on the growth and spore production of strain TB2. The strain is fermented in MSM culture medium in the presence of stevia polysaccharides and then filtered to prepare a fermentation biological preparation. The fermentation biological preparation has a good growth-promoting function for plants, reduces the dependence of agricultural production on synthetic fertilizers, and meets the needs of green agriculture. In addition, the present invention improves production efficiency by optimizing the Trichoderma liquid fermentation process, adjusting the stevia polysaccharide concentration, pH and culture conditions, and the liquid fermentation spore production can reach 10 7 pcs / mL, and the output of active ingredients is stable.

[0076] The above-described embodiments merely illustrate several embodiments of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the present invention. It should be noted that a person skilled in the art would be able to make various modifications and improvements without departing from the spirit of the present invention, all of which fall within the scope of protection of the present invention. Therefore, the scope of protection of the present invention shall be determined by the appended claims.

Claims

1. A fermentation biological preparation based on stevia polysaccharide to promote Trichoderma sporulation, characterized in that: The fermentation biological preparation is prepared from Trichoderma brevis Trichoderma brevicompactum strain TB2, stevia polysaccharide solution and MSM culture medium fermentation; the short dense Trichoderma Trichoderma brevicompactum The deposit number of strain TB2 is CCTCC NO: M20231913.

2. The fermented biological preparation according to claim 1, characterized in that The stevia polysaccharide solution is prepared by diluting the solution 50-100 times.

3. The fermented biological preparation according to claim 1, characterized in that 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.

4. A method for preparing a fermented biological preparation according to any one of claims 1 to 3, characterized in that: The method is: (1) Preparation of spore suspension: Trichoderma brevis Trichoderma brevicompactum After the TB2 strain was activated, it was inoculated on a PDA plate for culture, sterile water was added in a sterile environment, and the spore suspension was obtained by scraping with a coating rod and filtering through a filter cloth; (2) Preparation of fermentation biological preparation: prepare MSM culture medium, add stevia polysaccharide to make a 50-100 times dilution concentration, adjust the pH to 7, and then inoculate it into liquid culture medium at a volume percentage of 1%, ferment for 120 hours, centrifuge to remove the precipitate, and take the supernatant to obtain the fermentation biological preparation; Trichoderma brevis Trichoderma brevicompactum The deposit number of strain TB2 is CCTCC NO: M20231913.

5. The method according to claim 4, characterized in that The dilution concentration in step (2) is 100 times.

6. The method according to claim 4, characterized in that The PDA plate culture conditions of step (1) are: culture in a light incubator at 28°C for 72 h.

7. The method according to claim 4, characterized in that The liquid culture medium culture conditions of step (2) are: temperature 30°C, shaking on a 170 rpm platform for 120 hours.

8. Use of the fermented biological preparation according to any one of claims 1 to 3 or the fermented biological preparation prepared by the method according to any one of claims 4 to 7 in promoting cucumber growth.

9. Use of the fermented biological preparation according to any one of claims 1 to 3 or the fermented biological preparation prepared by the method according to any one of claims 4 to 7 in the preparation of bio-organic fertilizer.

Images