Trichoderma invading high-yield auxin culture medium as well as culture method and application thereof

Abstract

The invention discloses a culture medium for high yield of auxin from trichoderma invading as well as a culture method and application of the culture medium, and belongs to the technical field of microbial fermentation. According to the invention, a special fermentation culture medium and culture conditions for high-yield IAA are designed and optimized aiming at the physiological characteristics of a specific strain Trichoderma invading HD2112, and the problem that efficient synthesis of the strain IAA cannot be realized by the existing general fermentation technology is effectively solved. The core of the technical scheme is that the optimal proportion of glucose, yeast extract powder and specific inorganic salt is determined, and precise pH, temperature and rotating speed parameters are matched, so that strain growth and IAA anabolic flow are synergistically regulated and controlled, the IAA yield in fermentation liquor is increased to 57.32 mg / L which is remarkably higher than that of a control strain, and directional activation of the high-yield potential of the strain is realized.

Description

Technical Field

[0001] This invention relates to the field of microbial fermentation technology, and in particular to a culture medium that invades Trichoderma and produces high levels of auxin, as well as its culture method and application. Background Technology

[0002] Trichoderma strains can produce indole-3-acetic acid (IAA), which not only directly promotes plant growth but may also further regulate plant growth and development through interactions with other plant hormones (such as gibberellins and cytokinins), particularly synergistic effects. This comprehensive effect makes Trichoderma strains a highly effective plant growth promoter, helping to improve crop growth rate, yield, and quality. Furthermore, the IAA produced by Trichoderma strains may also influence plant metabolic pathways by regulating gene expression, thereby enhancing the plant's adaptability to environmental stresses. For example, under adverse conditions such as drought and salinity, IAA can regulate the plant's antioxidant system, reduce oxidative damage, and enhance the plant's resistance. Auxin-producing Trichoderma strains promote plant growth and development comprehensively through multiple pathways and targets. The inventors previously screened an invading Trichoderma strain producing auxin IAA from the rhizosphere soil of Zhengdan 958 maize plants in Handan, Hebei Province, and disclosed it in patent CN115261241B, providing important microbial resources for agricultural production.

[0003] However, the core challenge in applying this strain to actual production lies in the difficulty of achieving efficient IAA synthesis and accumulation using existing fermentation techniques. Conventional Trichoderma fermentation methods are mostly based on general fungal culture media and standard culture conditions, without optimization for the physiological characteristics and IAA synthesis pathways of this specific invading Trichoderma strain. This leads to a mismatch between cell biomass growth and IAA yield during fermentation, resulting in low and unstable product yields that cannot meet the needs of large-scale agricultural applications. Although this strain has high yield potential, its IAA synthesis capacity cannot be fully utilized under current technological conditions, severely limiting its practical effectiveness and economic benefits as a bio-promoter.

[0004] Further analysis reveals that the existing technical problems stem primarily from three factors: first, a lack of precise regulation of key nutrients for IAA synthesis in this strain, such as insufficient compatibility of carbon sources, nitrogen sources, and precursor substances; second, the failure to effectively synchronize fermentation environmental parameters (such as pH, temperature, and dissolved oxygen) with the strain's growth and secondary metabolic cycle; and third, the difficulty in balancing the competitive relationship between mycelial growth and product synthesis in traditional fermentation processes, leading to a deviation of metabolic flow from efficient IAA accumulation. Solving these problems requires systematic optimization of the fermentation culture system. However, previous studies have largely focused on strain screening and functional verification, leaving a gap in the exploration of specific fermentation processes, thus preventing the application potential of this high-yield strain from being translated into practical production technological advantages.

[0005] Therefore, developing an efficient, stable, and scalable fermentation culture method for this specific invading Trichoderma strain has become an urgent need to overcome the bottleneck of its industrial application. Summary of the Invention

[0006] The purpose of this invention is to provide a culture medium for high auxin production by *Trichoderma* invasive species, its cultivation method, and its application, thereby addressing the problems existing in the prior art. This invention optimizes the culture medium and cultivation conditions to directionally activate the synthetic metabolism of *Trichoderma* invasive species HD2112, increasing its IAA yield to 57.32 mg / L, thus solving the problem of low yield in the prior art.

[0007] To achieve the above objectives, the present invention provides the following solution:

[0008] This invention provides a culture medium for Trichoderma to produce high levels of auxin IAA, wherein the culture medium uses glucose as a carbon source, yeast extract as a nitrogen source, and K2HPO4 and MgSO4·7H2O as inorganic salts.

[0009] The preservation number of the invading Trichoderma is CGMCC No. 40239.

[0010] Optionally, the components of the culture medium include 22.5g yeast extract, 5g glucose, 1.5g K2HPO4, 1.5g MgSO4·7H2O, and 1L water.

[0011] Optionally, the culture medium contains 0.1 wt% L-tryptophan.

[0012] This invention also provides a fermentation culture method for high-yield plant growth hormone IAA from Trichoderma, comprising the step of inoculating the Trichoderma into the culture medium for fermentation culture; wherein the Trichoderma is Trichoderma invasiveii, with preservation number CGMCCNo.40239.

[0013] Optionally, the Trichoderma is inoculated into the culture medium in the form of a spore suspension.

[0014] Optionally, the concentration of the spore suspension is 1×10⁻⁶. 7 The inoculum size is 1% (1 / mL).

[0015] Optionally, the pH of the fermentation medium is 6.

[0016] Optionally, the fermentation conditions are: shaking culture at 210 r / min at 28°C.

[0017] The present invention also provides the application of the culture medium described above in improving the production efficiency of the biocontrol agent that invades Trichoderma.

[0018] The present invention discloses the following technical effects:

[0019] This invention, by targeting the physiological characteristics of a specific strain invading Trichoderma HD2112, designed and optimized a dedicated fermentation medium and culture conditions for high IAA production, effectively solving the problem that existing general fermentation technologies cannot achieve efficient IAA synthesis by this strain. The core of the technical solution lies in determining the optimal ratio of glucose, yeast extract, and specific inorganic salts, and combining this with precise pH, temperature, and rotation speed parameters. This synergistically regulates the metabolic flux of strain growth and IAA synthesis, increasing the IAA yield in the fermentation broth to 57.32 mg / L, significantly higher than the control strain, thus achieving targeted activation of the strain's high-yield potential.

[0020] Furthermore, the high-yield IAA fermentation product prepared by the method described in this invention exhibits a significant growth-promoting effect in practical applications on tomatoes, verifying the effectiveness and practicality of this technical solution. By providing a complete, stable, and reproducible optimized system from culture medium formulation to fermentation process, this invention successfully transforms laboratory microbial resources into biological agents with clear application value, providing reliable technical support for the development of efficient and environmentally friendly plant growth promoters. Attached Figure Description

[0021] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0022] Figure 1 The graph shows the results of single-factor screening of the basal culture medium;

[0023] Figure 2 The graph shows the results of single-factor screening of carbon sources;

[0024] Figure 3 This is a graph showing the results of single-factor screening of nitrogen sources;

[0025] Figure 4 This is a graph showing the results of single-factor screening for inorganic salts.

[0026] Figure 5 The diagrams are 3D surface plots of the response surface; where A is the response surface plot of glucose and yeast extract powder to IAA production at a fixed disodium hydrogen phosphate concentration; B is the response surface plot of glucose and disodium hydrogen phosphate to IAA production at a fixed yeast extract powder concentration; and C is the response surface plot of yeast extract powder and disodium hydrogen phosphate to IAA production at a fixed glucose concentration.

[0027] Figure 6This is a graph showing the screening results for pH values;

[0028] Figure 7 This is a graph showing the results of the temperature screening process.

[0029] Figure 8 The result chart shows the rotational speed selection.

[0030] Figure 9 To construct a phylogenetic tree of strain HD2113 and its related strains based on ITS sequences;

[0031] Figure 10 A comparison chart of the yields of strain HD2113 and invading Trichoderma HD2112;

[0032] Figure 11 Phenotypic diagrams of tomato plants after 7 days of different treatments;

[0033] Figure 12 Phenotypic diagrams of tomato plants after 14 days for different treatments;

[0034] Figure 13 Phenotypic diagrams of tomato plants after 28 days of different treatments. Detailed Implementation

[0035] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0036] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0037] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0038] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0039] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0040] The Trichoderma HD2112 used in this embodiment of the invention is deposited at the China General Microbiological Culture Collection Center, accession number: CGMCC No. 40239, and has been disclosed in patent CN115261241B.

[0041] Example 1: A specific fermentation method for high-yield plant growth regulator production by invading Trichoderma HD2112

[0042] Preparation of Trichoderma HD2112 spore suspension:

[0043] (1) Activation of Trichoderma HD2112 strain: Take the Trichoderma HD2112 strain stored at -80℃ and place it on PDA medium. Incubate statically at 25℃ for 7 days until the mycelium is fully grown and a large number of conidia are produced.

[0044] (2) Scrape the above hyphae, transfer them to a 50 mL centrifuge tube and add 30 mL of deionized water and vortex. Collect the spore suspension by filtering with two layers of lens paper.

[0045] 1. Screening and optimization methods for high-yield plant growth regulator fermentation of Trichoderma HD2112

[0046] 1.1 Detection of auxin IAA

[0047] Qualitative and quantitative detection of auxin was performed using the Salkowski colorimetric method. The method is as follows:

[0048] (1) Preparation of Salkowski colorimetric solution: Take 4.5g FeCl3 in a beaker, add 300mL of deionized water to dissolve it, then add 587.4mL of 98% H2SO4, and after the temperature drops to room temperature, accurately dilute to 1L.

[0049] (2) Preparation of auxin IAA standard solution: Dissolve 200mg IAA in 2mL of anhydrous ethanol, mix and shake well to prepare 100mg / mL IAA stock solution. Dilute the IAA stock solution with anhydrous ethanol stepwise to prepare gradient working solutions of 0, 10, 20, 30, 50 and 100mg / mL.

[0050] (3) Preparation of L-tryptophan solution: Weigh 1g of L-tryptophan accurately using an analytical balance, pour it into a 100mL beaker, adjust the pH value to 1 with dilute hydrochloric acid, shake gently until completely dissolved, and filter with a 0.22μm bacterial filter to obtain the mother liquor.

[0051] (4) The above-prepared Trichoderma HD2112 spore suspension was inoculated into liquid King'B medium containing L-tryptophan solution. After shaking culture at 25℃ for 5 days, 100 μL of supernatant was centrifuged and an equal volume of Salkowski colorimetric solution was added. After standing in the dark for 30 min, the absorbance value was measured at a wavelength of 530 nm using a spectrophotometer. The uninoculated blank culture medium was used as a control. The amount of IAA produced by the strain was calculated by combining the standard curve.

[0052] 1.2 Effect of culture medium on the IAA production capacity of the strain

[0053] The above-prepared Trichoderma HD2112 spore suspension was used at a spore concentration of 1×10⁻⁶. 7 Standard auxin at a concentration of 0.1% ( / mL) was inoculated into King's B medium (20g peptone, 20mL glycerol, 1.5g K₂HPO₄, 1.5g MgSO₄·7H₂O, 1L water), Sabouraud dextrose medium (10g peptone, 40g glucose, 1L water), and Richard's medium (50g sucrose, 2g KNO₃, 1g K₂HPO₄, 0.5g FeSO₄, 0.5g KCl, 1L water) containing 0.1wt% L-tryptophan solution. These inoculated media were then incubated at 25°C with shaking for 9 days, with samples taken every 24 hours. Auxin was qualitatively and quantitatively detected using the Salkowski colorimetric method.

[0054] 1.3 Effect of carbon source on IAA production by strain

[0055] Carbon source screening was conducted based on the optimal culture medium identified in section 1.2. Glucose, sucrose, starch, and beet molasses were used as carbon sources, with an initial concentration of 20 g·L⁻¹. -1 At 20 g·L -1 Peptone was used as a nitrogen source, 1.5 g·L -1 K2HPO4 and 1.5 g·L -1 Using MgSO4·7H2O as an inorganic salt, with an L-tryptophan concentration of 0.1%, and 1 L of water as the solvent, the bacterial culture was inoculated at a ratio of 1%, and the corresponding IAA yield was calculated to determine the optimal carbon source.

[0056] 1.4 Effect of nitrogen source on the strain's ability to produce IAA

[0057] Nitrogen source screening was conducted based on the optimal culture medium identified in section 1.2. Tryptone, yeast extract, yeast extract, and ammonium sulfate were selected as nitrogen sources, with an initial concentration of 20 g·L⁻¹. -1 The optimal carbon source selected in section 1.3, and 1.5 g·L⁻¹, were used. -1 K2HPO4 and 1.5 g·L -1 Using MgSO4·7H2O as an inorganic salt, with an L-tryptophan concentration of 0.1%, and 1 L of water as the solvent, the bacterial culture was inoculated at a ratio of 1%, and the corresponding IAA yield was calculated to determine the optimal nitrogen source.

[0058] 1.5 Effects of inorganic salts on the IAA production capacity of the strain

[0059] Based on the optimal culture medium identified in section 1.2, inorganic salt screening was conducted. Potassium dihydrogen phosphate, magnesium chloride, disodium hydrogen phosphate, and sodium chloride were selected as inorganic salts, with their initial concentrations set at 1.5 g·L⁻¹. -1 Using the optimal carbon source selected in 1.3 and the optimal nitrogen source selected in 1.4, with an L-tryptophan concentration of 0.1% and 1 L of water as the solvent, the bacterial culture was inoculated at a ratio of 1%, and the corresponding IAA yield was calculated to determine the optimal inorganic salt.

[0060] 1.6 Optimization of IAA Production Capacity of Strains Using Response Surface Methodology

[0061] The optimal value was determined by referring to the value corresponding to the maximum IAA production of this factor obtained from single-factor experiments involving carbon source, nitrogen source, and inorganic salts. A three-factor, three-level Box-Behnken response surface methodology was employed, selecting carbon source concentration, nitrogen source concentration, and inorganic salt concentration as the three factors. High and low levels were set based on the optimal IAA production concentration from the single-factor experiments to investigate the significance of each factor on the IAA content of the response value and the optimal combination of each component.

[0062] 1.7 Effect of pH on the IAA production capacity of the strain

[0063] Based on the optimized culture medium, the pH was adjusted to 5, 6, 7, and 8 using 1M HCl and 1M NaOH, respectively, and then sterilized for later use. The seed culture was inoculated into the culture medium and cultured in a shaking incubator for 7 days. Three replicates were set up for each group to calculate the corresponding IAA yield and determine the optimal pH value for the strain.

[0064] 1.8 Effect of temperature on the IAA production capacity of the strain

[0065] Based on the optimized culture medium, the seed culture was inoculated into the medium and cultured in a shaking incubator for 7 days. The temperature of the shaking incubator was set at 16-32℃, with a gradient of 4℃, for a total of 5 temperature gradients. Three parallel studies were set up for each group to calculate the corresponding IAA yield and determine the optimal temperature for the strain.

[0066] 1.9 Effect of rotation speed on the IAA production capacity of the strain

[0067] Based on the optimized culture medium, the seed culture was inoculated into the culture medium and placed in a shaking incubator for 7 days. The shaking speed was adjusted to 150 r / min, 180 r / min, 210 r / min and 240 r / min, and the corresponding IAA yield was calculated. Three parallel groups were set up for each group to determine the optimal shaking speed for the strain.

[0068] 2. Screening and optimization results of fermentation methods for high-yield plant growth regulator production by invading Trichoderma HD2112

[0069] 2.1 Growth curve determination

[0070] The highest IAA yield of Trichoderma HD2112 was observed on day 7 of culture in King's B medium, reaching 9.7 mg / L. Figure 1 Therefore, King'B medium was chosen as the basic culture medium for the IAA production fermentation of Trichoderma HD2112.

[0071] 2.2 Carbon source screening

[0072] Several other single carbon sources were used to replace the original carbon source in the basic sporulation medium of *Trichoderma* HD2112 to prepare culture media, and the effect of carbon source type on the indoleacetic acid (IAA) yield of *Trichoderma* HD2112 was investigated. After 7 days of fermentation, the IAA yield of *Trichoderma* HD2112 in each single carbon source medium was statistically analyzed. The results showed that when sucrose was used as the single carbon source, the IAA yield of *Trichoderma* HD2112 was extremely low; in contrast, when glucose was used as the carbon source, it significantly promoted the increase of IAA yield of *Trichoderma* HD2112, reaching the highest value of 12.11 mg / L. Figure 2 Therefore, glucose was selected as the carbon source for the culture medium that promotes the production of IAA by Trichoderma HD2112 to carry out subsequent optimization work.

[0073] 2.3 Nitrogen source screening

[0074] The nitrogen source in the optimized medium for indoleacetic acid (IAA) production by *Trichoderma* HD2112 was replaced with other single nitrogen sources to prepare the medium, and the effect of nitrogen source on IAA production by *Trichoderma* HD2112 was investigated. After 7 days of fermentation, the IAA production of *Trichoderma* HD2112 in each single nitrogen source medium was statistically analyzed. The results showed that the fermentation broth using yeast extract as the single nitrogen source produced the highest IAA production of *Trichoderma* HD2112, reaching 15.5 mg / L. Figure 3 Therefore, yeast extract was selected as the nitrogen source for the IAA-producing medium of Trichoderma HD2112 to carry out further optimization work.

[0075] 2.4 Inorganic Salt Screening

[0076] The inorganic salts in the IAA-producing medium of *Trichoderma* HD2112, which had undergone carbon and nitrogen source optimization in the previous step, were replaced with other types of inorganic salts to prepare the medium. The effect of inorganic salts on the IAA production of *Trichoderma* HD2112 was investigated. After 7 days of fermentation, the IAA production of *Trichoderma* HD2112 in each inorganic salt medium was statistically analyzed. The results showed that when the inorganic salt was Na2HPO4, the IAA production reached 21.3 mg / L (… Figure 4 Based on this, Na2HPO4 was selected as the inorganic salt for invading the IAA-producing medium of Trichoderma HD2112 to carry out further optimization work.

[0077] 2.5 Response Surface Optimization of Carbon and Nitrogen Inorganic Salts

[0078] Based on the single-factor experiments testing the effects of culture conditions on IAA production by *Trichoderma* HD2112, a three-level response surface methodology (RSM) experiment was designed according to the Box-Behnken central composite design principle, considering three factors: carbon source, nitrogen source, and inorganic salt. The RSM design scheme and experimental results are shown in Table 1, and the variance analysis of the response surface quadratic regression prediction model is shown in Table 2. Table 1 shows that different carbon sources, nitrogen sources, and inorganic salts significantly affected IAA production by *Trichoderma* HD2112, with variations in IAA yield after culture under different conditions. The highest IAA yield (41.8 mg / mL) was achieved when the carbon source was 5 g / L glucose, the nitrogen source was 22.5 g / L yeast extract, and the inorganic salt was 0.5 g / L disodium hydrogen phosphate. The yield of Trichoderma HD2112 IAA was minimized to 16 mg / mL when the carbon source glucose was 17.5 g / L, the nitrogen source yeast extract was 22.5 g / L, and the inorganic salt disodium hydrogen phosphate was 1.75 g / L.

[0079] Table 1. Experimental design and results of Box-Benhnken central composite assay for Trichoderma HD2112 invasion.

[0080]

[0081] The experimental data were fitted using Design-Expert 8.0.6 Trial software, and a quadratic regression model was obtained to predict Trichoderma HD2112 on factors A, B, and C.

[0082] Y=17.28-6.55*A+2.96*B-1.86*C+0.1750*AB+1.63*AC-1.0*BC+8.41*A 2 +3.29B 2 +2.99C 2 .

[0083] Where Y represents the IAA yield of *Trichoderma* HD2112, A represents the glucose concentration, B represents the yeast extract concentration, and C represents the disodium hydrogen phosphate concentration. Analysis of variance and significance tests were performed on the model, and the results are shown in Table 2. The model's F=14.84, p<0.1, indicating the model is significant. The coefficient of determination R0 is... 2 =0.9502, p=0.0512, the lack-of-fit term is not significant, indicating that the equation has a high good fit and the model can predict the optimal conditions for co-culture of combined bacteria well. In the model, A, B, and A 2 B 2 C 2 The F-value significantly affected the IAA yield of Trichoderma HD2112, while C, AB, AC, and BC had no significant effect. Based on the F-values, the order of influence of the three factors on the IAA yield of Trichoderma HD2112 was: glucose (A) > yeast extract (L) > disodium hydrogen phosphate (C).

[0084] Table 2. Analysis of variance of the quadratic regression prediction model for Trichoderma HD2112 infestation.

[0085]

[0086] The shape of the 3D surface plot and contour lines of the response surface can intuitively reflect the magnitude of the interaction. In the surface effect of this model on the yield of Trichoderma HD2112 IAA, the slope of the response surface plot shows that when the values ​​of A and B are small, the curve of the response surface is steeper, indicating that A and B have a more significant impact on the yield of Trichoderma HD2112 IAA. When A and B increase, the curve of the response surface becomes flatter, indicating a smaller impact on the yield of Trichoderma HD2112 IAA. Figure 5 The shape of the contour lines being elliptical indicates a stronger interaction between the two factors. Figure 5 It can be seen that the contour lines of the interaction between the two factors are not obviously elliptical, indicating that the interaction between the two factors is not significant.

[0087] 2.6 pH screening

[0088] After culturing at different pH values ​​and 180 r / min for 7 days, the IAA yield of Trichoderma HD2112de was measured, and the results are as follows: Figure 6 As shown, the IAA yield of Trichoderma HD2112 reached its maximum value of 49.45 mg / L at pH=6. Therefore, pH=6 was selected as the optimal pH value for strain culture for subsequent experiments.

[0089] 2.7 Temperature Screening

[0090] After culturing at different temperatures and 180 r / min for 7 days, the IAA yield of Trichoderma HD2112 was measured, and the results are as follows: Figure 7 As shown, the IAA yield of Trichoderma HD2112 reached its maximum value of 49.45 mg / L at 28℃. Therefore, 28℃ was selected as the optimal temperature for strain culture for subsequent experiments.

[0091] 2.8 Rotational Speed ​​Screening

[0092] After incubation at 28℃ for 7 days at different rotation speeds, the IAA yield of Trichoderma HD2112 was measured, and the results are as follows: Figure 8 As shown, at 210 r / min, the IAA production of Trichoderma HD2112 reached its maximum value of 57.32 mg / L.

[0093] The above experiments show that the specific fermentation conditions for high IAA production by Trichoderma HD2112 are as follows: spore concentration of 1×10⁻⁶. 7 The standard inoculum of 0.1% L-tryptophan was inoculated into a liquid culture medium containing 0.1 wt% L-tryptophan solution (carbon source: 5 g / L glucose, nitrogen source: 22.5 g / L yeast extract, inorganic salt: 0.5 g / L disodium hydrogen phosphate). The pH was set to 6, the culture temperature was maintained at 28℃, and the culture was shaken at 210 r / min.

[0094] Example 2: Effect of fermentation method on the IAA production capacity of different Trichoderma species

[0095] The control strain was a *Trichoderma asperellum* strain isolated from a fresh soil sample of summer maize rhizosphere in Handan City, Hebei Province. The isolation, screening, and identification process is as follows:

[0096] Isolation of the bacterial strain: Weigh 1g of fresh rhizosphere soil sample from summer maize in Handan City, Hebei Province, add it to an Erlenmeyer flask containing 9 mL of sterile water, shake at 220 r / min for 30 min, and let stand at room temperature for 10 min. Take 1 mL of the supernatant and perform a 10-fold serial dilution to prepare 10...-2 ~10 -5 Bacterial suspension. Three suitable serial dilutions were selected, and 0.1 mL of each bacterial suspension was spread onto Bengal Red agar plates, with three replicates for each dilution. After inverting the plates, they were incubated at 25°C for 60 h. Single colonies were picked according to the typical colony morphology of Trichoderma and transferred to PDA medium. After 3-4 streak purifications, a pure culture was obtained and designated as strain HD2113. The pure strain was inoculated onto PDA slant and incubated at 25°C for 5 days, then sealed and stored at 4°C for later use.

[0097] Molecular biological identification: Based on morphological identification, aerial hyphae of *Trichoderma* cultured on PDA plates were scraped off and collected into 1.5 mL EP tubes. Genomic DNA was extracted using a fungal genomic DNA extraction kit. PCR amplification of the ITS fragment was performed using the *Trichoderma* genomic DNA as a template. Universal primers for fungal ITS sequences, ITS1 (5′-TCCGTAGGTGAACCTGCGG-3′) and ITS4 (5′-TCCTCCGCTTATTGATATGC-3′), were synthesized by Sangon Biotech (Shanghai) Co., Ltd. The 20 μL reaction system consisted of 7 μL ddH2O, 10 μL 2×PCR Master Mix, 1 μL DNA template, and 1 μL each of 10 μmol / L ITS1 and ITS4 primers. The reaction program was: 95℃ pre-denaturation for 5 min; 94℃ denaturation for 30 s, 55℃ annealing for 30 s, 72℃ extension for 60 s, for a total of 34 cycles, followed by incubation at 72℃ for 5 min. The amplified products were sent to Sangon Biotech (Shanghai) Co., Ltd. for sequencing. The obtained sequences were compared with the Nucleotide BLAST database in GenBank, and a phylogenetic tree was constructed using the maximum likelihood method with MAGE 7.0 software to determine the taxonomic position of the strain.

[0098] The ITS fragment of strain HD2113 is 578 bp in length. A phylogenetic tree was constructed using MAGE 7.0 software, as follows: Figure 9 As shown, strain HD2113 was found to be located on the same branch of the genus *Trichoderma* in the phylogenetic tree, and also belongs to a subbranch of *Trichoderma asperellum* OQ727299. The sequence alignment showed 100% consistency. Therefore, strain HD2113 was identified as *Trichoderma asperellum*.

[0099] Controlled experiment:

[0100] (1) The control strain Trichoderma hyaline HD2113 and the experimental strain Trichoderma hyaline HD2112 were fermented under the specific fermentation conditions finally determined in Example 1, i.e., the spore concentration was 1×10⁻⁶. 7The standard inoculum of 0.1% at a concentration of 0.1% was inoculated into a liquid culture medium containing 0.1 wt% L-tryptophan solution (carbon source: 5 g / L glucose, nitrogen source: 22.5 g / L yeast extract, inorganic salt: 0.5 g / L disodium hydrogen phosphate). The pH was set at 6, the culture temperature was maintained at 28℃, and the medium was shaken at 210 rpm for 7 days. Under the Salkowski colorimetric method, the auxin IAA yield of *Trichoderma echinococcus* HD2113 was 33.6 mg / L. Under the same conditions, the auxin IAA yield of *Trichoderma echinococcus* HD2112 was 57.32 mg / L, which was 1.7 times that of *Trichoderma echinococcus* HD2113. Figure 10 This indicates that the conditions are favorable for the efficient synthesis of IAA from Trichoderma HD2112.

[0101] (2) Adjust the composition of the liquid culture medium while keeping the culture conditions unchanged. The control strain Trichoderma hyacinth HD2113 and the experimental strain Trichoderma hyacinth HD2112 were fermented in different liquid culture media designed in Table 3 in the same way.

[0102] Table 3 Different Culture Medium Components

[0103]

[0104] After fermentation, the yield of IAA in the fermentation broth obtained from the control strain and the test strain was measured, and the results are shown in Table 4.

[0105] Table 4. IAA yield in fermentation broth of different control and test strains

[0106]

[0107] As shown in Table 4, after changing the composition of the liquid culture medium, the ability of Trichoderma HD2112 to produce IAA was significantly inhibited, even lower than that of the control Trichoderma hyaline HD2113.

[0108] Example 3: Practical application of high-yield plant growth regulator fermentation products from Trichoderma HD2112

[0109] Select plump and uniform tomato seeds. First, soak them in a 2% sodium hypochlorite (NaClO) solution for 3 minutes to disinfect them, then rinse them 3-5 times with sterile water to remove any residual disinfectant. Place the disinfected seeds in a petri dish lined with moistened filter paper and germinate them at a constant temperature of 25℃ in the dark for 24 hours, until most seeds show signs of germination. Sow one seed per hole in the pre-moistened cultivation substrate, covering it with about 0.5 cm of soil. After sowing, lightly spray water to moisten the surface of the substrate. Place the seedling tray in a light incubator at 25℃ and 85% humidity. One week after germination, transplant the uniformly growing tomato seedlings into pots 11 cm high and 12 cm in diameter.

[0110] One week after the seedlings have recovered, inoculant treatment was started. Two treatment groups were set up: (1) Inoculant treatment group: the leaves were sprayed with wettable powder of Trichoderma HD2112 until the leaves were evenly moistened but no droplets fell; (2) Blank control group (CK): an equal amount of sterile water was sprayed. Each treatment was replicated at least 10 times (i.e., 10 pots).

[0111] Preparation steps of wettable powder of Trichoderma HD2112: Trichoderma HD2112 was fermented under the specific fermentation conditions determined in Example 1. After fermentation, solid-liquid separation, drying, pulverization, and the addition of a carrier and adjuvants were performed to prepare a wettable powder. The powder composition (mass percentage) is: a suspension of Trichoderma spores loaded with Trichoderma spores (concentration 1×10⁻⁶). 9 The carrier silica (number of particles per mL) accounts for 89.7%, the dispersant sodium naphthalene sulfonate formaldehyde condensate (NNO) accounts for 5%, the wetting agent (sodium dodecyl sulfate) accounts for 5%, and the protective agent dextrin accounts for 0.3%.

[0112] Fourteen days after the first application, the inoculant treatment group was sprayed with the same amount of inoculant a second time, while the blank control group was sprayed with the same amount of sterile water at the same time. On days 7, 14, and 28 after the first treatment, the growth indicators of tomato plants, such as plant height, stem diameter, and leaf width, were measured.

[0113] The effects of microbial inoculant treatment on the growth of tomato seedlings, such as Figure 11 (7 days after treatment) Figure 12 (14 days after treatment) and Figure 13 As shown in Table 5, the average values ​​of the specific growth index measurements are summarized in Table 28 after treatment.

[0114] Table 5. Effects of Trichoderma HD2112 inoculant treatment on growth indicators of tomato seedlings.

[0115]

[0116] The results showed that, compared with the blank control group, spraying with Trichoderma HD2112 wettable powder significantly promoted the growth of tomato seedlings at all stages. Twenty-eight days after treatment, the plant height, stem diameter, leaf width, and leaf length of the inoculant-treated group increased by 46.8%, 32.4%, 105.3%, and 73.1% respectively compared with the control group, and the promoting effect became more pronounced with the extension of treatment time.

[0117] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A culture medium for invading Trichoderma that produces a high level of auxin IAA, characterized in that, The culture medium uses glucose as the carbon source, yeast extract as the nitrogen source, and K2HPO4 and MgSO4·7H2O as inorganic salts. The preservation number of the invading Trichoderma is CGMCC No. 40239.

2. The culture medium according to claim 1, characterized in that, The culture medium consists of 22.5g yeast extract, 5g glucose, 1.5g K2HPO4, 1.5g MgSO4·7H2O, and 1L water.

3. The culture medium according to claim 2, characterized in that, The culture medium contains 0.1 wt% L-tryptophan.

4. A fermentation culture method for high-yield plant growth regulator IAA by Trichoderma, characterized in that, The method includes the step of inoculating the Trichoderma in the culture medium according to any one of claims 1-3 for fermentation culture; wherein the Trichoderma is Trichoderma invasiveis, with the preservation number CGMCC No. 40239.

5. The fermentation culture method according to claim 4, characterized in that, The Trichoderma was inoculated into the culture medium in the form of a spore suspension.

6. The fermentation culture method according to claim 5, characterized in that, The concentration of the spore suspension was 1×10⁻⁶. 7 The inoculum size is 1% (1 / mL).

7. The fermentation culture method according to claim 4, characterized in that, The pH value of the fermentation medium is 6.

8. The fermentation culture method according to claim 4, characterized in that, The fermentation conditions were as follows: cultured at 28°C with shaking at a speed of 210 r / min.

9. The use of the culture medium according to any one of claims 1-3 in improving the production efficiency of the biocontrol agent that invades Trichoderma.

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