Application of Trichoderma acicularis TCS007 solid fermentation crude extract as a plant immune activator

Abstract

This invention relates to the application of *Trichoderma echinococcus* TCS007 solid-state fermentation crude extract as a plant immune activator. *Trichoderma echinococcus* TCS007 solid-state fermentation crude extract can significantly increase the chlorophyll content in sunflower leaves, promote photosynthesis, and enhance resistance. After inoculation with pathogens, *Trichoderma echinococcus* TCS007 solid-state fermentation crude extract can increase the activity of antioxidant enzymes and disease-related proteases, significantly reduce the content of reactive oxygen species in sunflower leaves, alleviate the degree of lipid peroxidation in leaf cell membranes, and protect plants from oxidative damage.

Description

Technical Field

[0001] This invention relates to the field of plant immune activation, specifically the application of Trichoderma echinosporum TCS007 solid fermentation crude extract as a plant immune activator, and particularly the application of Trichoderma echinosporum TCS007 solid fermentation crude extract inducing sunflower immunity against sclerotinia stem rot. Background Technology

[0002] *Trichoderma asperellum* is a novel biocontrol fungus capable of effectively colonizing the rhizosphere of plants. This fungus establishes a strong and stable symbiotic relationship with the host plant, indirectly inhibiting pathogen infection by inducing local or systemic defense mechanisms. In its interaction with plants, this fungus secretes secondary metabolites with antibacterial and plant-growth-promoting effects, and also regulates root structure, thereby improving nutrient absorption efficiency. *Trichoderma asperellum* is widely distributed and can survive in adverse environments such as drought and salinity. Related products are currently scarce, indicating significant development potential.

[0003] A novel *Trichoderma asperellum* TCS007 was isolated from Antarctic marine sediments, stabilized through mutagenesis, and identified and named *Trichoderma asperellum* TCS007. Currently, this strain is preserved at the China General Microbiological Culture Collection Center (CGMCC) under accession number CGMCC No. 15677. This strain was first disclosed in patents ZL201810935616.6 and ZL201810936028.4. *Trichoderma asperellum* TCS007 grows rapidly on PDA medium; at 26°C, the hyphae cover a 9 cm culture dish in approximately 3 days. The hyphae are of moderate thickness, exhibiting a felt-like texture, initially white and smooth, later forming dark green concentric conidia. On CMD medium, the strain forms clusters of conidiophores. The main branches of the conidiophores are tree-like, the flask pedicels are short, gradually tapering at the base and swelling in the middle, resembling an ampoule. The conidia of *Trichoderma echinosporum* TCS007 are 3.4-5.0 μm long and 2.8-4.0 μm wide, mostly spherical, subspherical, or oval in shape, with a rough surface covered with tiny spines.

[0004] In previous research, the research group of the inventors of this application discovered that a specific rice culture medium (80g rice + 120mL distilled water) on which Trichoderma echinococcus TCS007 produces secondary metabolites, after fermentation, yielded a solid crude extract by ethyl acetate extraction with an inhibition rate of 95.8% against Sclerotinia sclerotiorum (see Zheng Kebin, Antibacterial, Growth-promoting and Stress-resistant effects of Trichoderma echinococcus TCS007 in marine habitats, Master's thesis of Zhejiang Agriculture and Forestry University, published online from January 16, 2021 to February 15, 2021). Based on this, the inventors further studied the culture medium for industrial production of Trichoderma echinosporum TCS007 conidia (see patent CN202410872878.8). The patent selected a culture medium (25g wheat bran, 5g soybean flour, 0.6g each of MgSO4 and MnSO4, 39.7mL distilled water, initial pH 5.0), which can not only reduce the fermentation production cost, but also increase the number of Trichoderma echinosporum TCS007 conidia. The conidia can meet the requirements of industrial production and market application.

[0005] Secondary metabolites produced by *Trichoderma echinococcus* TCS007 during its stable growth period have a good inhibitory effect on *Sclerotinia sclerotiorum*. Based on this, a pot experiment was designed to study the control effect of the crude extract of *Trichoderma echinococcus* TCS007 solid-state fermentation on sunflower sclerotinia rot caused by *Sclerotinia sclerotiorum* and to explore its disease resistance induction mechanism. Summary of the Invention

[0006] The technical problem to be solved by this invention is that the crude extract of Trichoderma echinosporum TCS007 solid fermentation can stimulate the sunflower immune system and induce a defensive response, thereby improving the plant's resistance to Sclerotinia sclerotiorum and providing technical support for the development and application of biocontrol Trichoderma agents against sunflower Sclerotinia sclerotiorum disease.

[0007] One of the objectives of this invention is to apply the crude extract of Trichoderma hygroscopicum TCS007 solid fermentation as a plant immune activator.

[0008] Another objective of this invention is the application of the crude extract of Trichoderma echinosporum TCS007 solid fermentation as an induction of sunflower immunity against sclerotinia stem rot.

[0009] Preferably, the solid fermentation crude extract of Trichoderma echinosporum TCS007 can increase the chlorophyll content in sunflower leaves.

[0010] Preferably, the crude extract of Trichoderma echinosporum TCS007 solid fermentation can increase the activity of POD, SOD, CAT, APX, PAL and PPO enzymes in sunflower leaves.

[0011] Preferably, the crude extract of Trichoderma echinosporum TCS007 solid fermentation can increase the activity of GLU enzyme in sunflower leaves.

[0012] Preferably, the crude extract of Trichoderma echinosporum TCS007 solid fermentation can reduce the H2O2 content in sunflower leaves.

[0013] Preferably, the crude extract of Trichoderma echinosporum TCS007 solid-state fermentation can reduce O in sunflower leaves. 2- Generation rate.

[0014] The method for preparing the crude extract of *Trichoderma echinococcus* TCS007 solid-state fermentation involves extracting the *Trichoderma echinococcus* TCS007 solid-state fermentation product with an equal volume of ethyl acetate (1:1, V / V) three times. After extraction, the mixture is filtered and dried with anhydrous sodium sulfate. The dried liquid is then concentrated to dryness using a rotary evaporator under reduced pressure. Finally, the residue is redissolved in ethyl acetate and collected in a centrifuge tube. After the solvent evaporates, the crude extract of *Trichoderma echinococcus* TCS007 solid-state fermentation is obtained.

[0015] The method for preparing the solid-state fermentation product of *Trichoderma echinococcus* TCS007 involves weighing 25g of wheat bran, 5g of soybean flour, 0.6g each of MgSO4 and MnSO4, adding 39.7mL of water, setting the initial pH to 5.0, and using 2mL of *Trichoderma echinococcus* TCS007 inoculum (spore concentration 1×10⁻⁶). 7 The fungus was cultured in a mold incubator at 28℃ for 15 days with a light cycle of 21 hours per mL.

[0016] The concentration of the crude extract of Trichoderma echinosporum TCS007 solid fermentation is 1 mg / L-200 mg / L, preferably 10 mg / L, 20 mg / L, 30 mg / L, 40 mg / L, 50 mg / L, 80 mg / L, 100 mg / L, or 150 mg / L; the solvent used is N,N-dimethylformamide; the spraying amount is 1 mL-10 mL, preferably 2 mg / L, 4 mg / L, 6 mg / L, or 8 mg / L; the number of sprayings is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 times; preferably 3 or 5 times.

[0017] Beneficial technical effects of the present invention

[0018] This invention has found that using the crude extract of Trichoderma echinococcus TCS007 solid fermentation can induce sunflower plants to resist sclerotinia rot infection, and the effect of inducing sunflower plants to resist sclerotinia rot increases with the number of treatments.

[0019] This invention has found that the crude extract of Trichoderma echinosporum TCS007 solid fermentation can significantly increase the chlorophyll content of sunflower leaves, promote photosynthesis in sunflowers, and thus enhance their resistance.

[0020] This invention found that, 48-96 hours after inoculation with pathogens, the crude extract of Trichoderma echinosporum TCS007 solid fermentation improved the activity of antioxidant enzymes and disease-related proteases in sunflowers. Among them, the activities of CAT, PAL and APX enzymes were significantly increased and were proportional to the number of treatments.

[0021] This invention has found that the crude extract of Trichoderma echinosporum TCS007 solid fermentation significantly reduces the content of reactive oxygen species in sunflower leaves, alleviates the degree of lipid peroxidation in leaf cell membranes, and protects plants from oxidative damage.

[0022] Instruction manual illustrations

[0023] Figure 1 Chlorophyll content of sunflower leaves in different treatment groups;

[0024] Figure 2 The activities of POD (peroxidase), SOD (superoxide dismutase), CAT (catalase), APX (ascorbate peroxidase), PAL (phenylalanine ammonia-lyase), and PPO (polyphenol oxidase) in sunflower leaves under different treatment groups.

[0025] Figure 3 Protease activity in sunflower leaves under different treatment groups;

[0026] Figure 4 H2O2 content in sunflower leaves in different treatment groups;

[0027] Figure 5 Superoxide anion (O3) levels in sunflower leaves of different treatment groups 2- Generation rate; Detailed Implementation

[0028] The test culture medium for preparing the crude solid fermentation extract of Trichoderma hygroscopicum TCS007 described in this invention is as follows:

[0029] Patent application CN202410872878.8 describes an optimized wheat bran culture medium for sporulation: 25g wheat bran, 5g soybean flour, 0.6g MgSO4, 0.6g MnSO4, 39.7mL distilled water, initial pH 5.0, inoculum size 2mL (spore concentration 1×10⁻⁶). 7 The photoperiod was 21 h, the culture temperature was 28 ℃, and the culture time was 15 d;

[0030] PDA medium: anhydrous glucose 20.0 g / L, potato 200.0 g / L, agar powder 18.0 g / L, distilled water 1 L, pH 7.0;

[0031] Activation process of Trichoderma echinococcus TCS007: Under aseptic conditions, 10 μL of Trichoderma echinococcus TCS007 bacterial suspension was taken from a cryopreserved glycerol tube and evenly spread onto a PDA plate. The plate was then placed in an incubator at 28°C and incubated upside down in the dark for 5 days. Mycelial cakes were cut using an 8 mm diameter punch and placed in the center of a new PDA plate using an inoculation needle. The plate was then incubated upside down in the dark at 28°C for 5 days until ready for use.

[0032] Preparation steps of Trichoderma echinococcus TCS007 seed culture: Under aseptic conditions, the PDA medium containing Trichoderma echinococcus TCS007 grown to 2 / 3 capacity in a petri dish was rinsed with sterile water. Mycelia were removed by filtration through sterile gauze to obtain a suspension of Trichoderma echinococcus TCS007 spores. The spore concentration was determined under an optical microscope using a hemocytometer method, and the suspension was diluted with sterile water to a spore concentration of 1×10⁻⁶. 7 Seed culture per mL, ready for use.

[0033] Preparation of solid-state fermentation product of *Trichoderma echinococcus* TCS007: 25g wheat bran, 5g soybean flour, 0.6g MgSO4, 0.6g MnSO4, 39.7mL water, initial pH 5.0, and 2mL *Trichoderma echinococcus* TCS007 seed culture inoculation (spore concentration 1×10⁻⁶). 7 The sample was incubated in a mold culture chamber at 28℃ for 15 days with a light cycle of 21 hours per mL.

[0034] Preparation of crude extract from solid-state fermentation of *Trichoderma echinococcus* TCS007: The solid-state fermentation product of *Trichoderma echinococcus* TCS007 was extracted with an equal volume of ethyl acetate (1:1, V / V) three times. After extraction, the mixture was filtered and dried with anhydrous sodium sulfate. The dried liquid was concentrated to dryness using a rotary evaporator under reduced pressure. Finally, the residue was redissolved with ethyl acetate and collected in a centrifuge tube. After the solvent evaporated, the crude extract of solid-state fermentation of *Trichoderma echinococcus* TCS007 was obtained. This crude extract was then prepared into a 1% (w / w) stock solution with N,N-dimethylformamide (DMF) and serially diluted to 100 mg / L for later use.

[0035] Induction of immune assay methods

[0036] After sterilization, sunflower seeds were placed in 9cm petri dishes and germinated at 26℃ for 24 hours. Once the seeds showed signs of germination, they were planted in 9.5cm×7.5cm pots (containing a 1:1 weight ratio of substrate soil and loess), with 2 seeds sown per pot. The pots were then placed in a greenhouse for routine management. The experiment was designed with 5 treatment groups, each containing 15 pots.

[0037] CK treatment group: Each plant was sprayed with 2mL of sterile water once.

[0038] CKJ treatment group: Each plant was sprayed with 2 mL of sterile water once and then inoculated with Sclerotinia sclerotiorum.

[0039] M1 treatment group: Each plant was sprayed once with 2 mL of 100 mg / L Trichoderma acicularis TCS007 solid fermentation crude extract.

[0040] M3 treatment group: each potted plant was sprayed 3 times with 2 mL of 100 mg / L Trichoderma acicularis TCS007 solid fermentation crude extract.

[0041] M5 treatment group: each potted plant was sprayed 5 times with 2 mL of 100 mg / L Trichoderma acicularis TCS007 solid fermentation crude extract.

[0042] Treatment groups M1, M3, and M5 were sprayed once every 2 days. 24 hours after the last spraying, Sclerotinia sclerotiorum was inoculated onto the first true leaf of the sunflower.

[0043] The effect of Trichoderma echinosporum TCS007 solid-state fermentation crude extract on chlorophyll content in sunflower leaves was investigated without inoculating with Sclerotinia sclerotiorum.

[0044] Example 1

[0045] Effects of Trichoderma hyacinth TCS007 solid-state fermentation crude extract on chlorophyll content in sunflower leaves.

[0046] The chlorophyll content of leaves in different treatment groups (CK, M1, M3, M5) was determined by spectrophotometry. Second true leaves of sunflowers were collected from the same location. After removing the midrib, the leaves were cut into small pieces. 0.1g of leaf sample was weighed and placed in a mortar, along with 0.5g of quartz sand and an appropriate amount of calcium carbonate powder. Then, 5mL of 95% ethanol was added, and the mixture was ground until the leaf tissue turned white. The mixture was allowed to stand for 3-5 minutes to allow for complete precipitation. The precipitate was then filtered through filter paper into a volumetric flask and diluted to 50mL with 95% ethanol. Finally, the extracted sunflower chloroplast pigment solution was poured into a cuvette with a 1cm optical path. Using 95% ethanol as a control group, the absorbance was measured at different wavelengths (665nm, 649nm, and 470nm) using a spectrophotometer. The chlorophyll content was calculated using the following formula:

[0047] Ca = 13.95A 663 -6.88A 645 (1)

[0048] Cb = 24.96A 645 -7.32A 663 (2)

[0049] C T =C a +C b (3)

[0050] C hl = (C×V) / (0.5×50) (4)

[0051] In the formula: Ca: concentration of chlorophyll a, mg / g; Cb: concentration of chlorophyll b, mg / g; C T Total chlorophyll concentration, mg / g; C hl : Chlorophyll content, mg / g; V: Total volume of extract, mL.

[0052] Data Statistical Analysis

[0053] One-way ANOVA was performed using SPSS 26.0 software, and multiple tests were conducted on the ANOVA results (P < 0.05). Graphpad Prism 9.0 software was used to plot the data. (The same applies below.)

[0054] Chlorophyll is a key pigment in photosynthesis, and its content in plants is closely related to the photosynthetic rate. Higher plants mainly have chlorophyll a and chlorophyll b, and changes in their content are important indicators for assessing the intensity of photosynthesis. Figure 1 The results showed that, compared with the control group (CK), the M1, M3, and M5 treatment groups using the crude extract from Trichoderma echinococcus TCS007 solid-state fermentation all increased the content of chlorophyll a and chlorophyll b in sunflower leaves. Furthermore, the content of chlorophyll a and chlorophyll b showed an increasing trend with the number of treatments. Specifically, the chlorophyll content in the M3 treatment group increased by 46.2% compared to the CK treatment group, and the M5 treatment group increased by 51.7% compared to CK.

[0055] Example 2

[0056] Effects of Trichoderma echinosporum TCS007 solid-state fermentation crude extract on the activities of POD, SOD, CAT, APX, PAL, and PPO enzymes in sunflower leaves.

[0057] Stimulating the antioxidant system is an important adaptive mechanism for reducing ROS accumulation and minimizing oxidative damage in plants under stress. After sunflower plants are inoculated with pathogens, the crude extract from the solid-state fermentation of *Trichoderma echinococcus* TCS007 induces a rapid initiation of a defense response, producing various resistance-related defense enzymes and antioxidant enzymes to maintain ROS balance. Plant defense enzymes include POD, SOD, CAT, APX, PAL, PPO, and GLU. Among these, PAL is a key rate-limiting enzyme in the phenylpropanoid metabolic pathway, responsible for catalyzing reactions in this pathway to generate disease-resistant secondary metabolites such as phytoalexins, lignin, and phenolic compounds. PPO participates in the polymerization of phenolic substances, which is crucial for lignin formation. Their combined action helps enhance cell wall and tissue lignification, thereby helping plants resist pathogen invasion. SOD and CAT are important regulatory enzymes of intracellular reactive oxygen species (ROS) metabolism, capable of decomposing and scavenging H2O2 produced by ROS metabolism, preventing superoxide free radicals from damaging cell membranes. As one of the key enzymes in lignin synthesis, changes in the activity of POD are also an important indicator of the degree of cell damage.

[0058] Sclerotinia sclerotiorum was inoculated into the second true leaf of sunflower at 48h, 72h, and 96h, respectively. 0.1g of sample was taken using a punch and placed into a 2mL sterile EP tube. The EP tube was then frozen in liquid nitrogen, followed by grinding for 1min. 1mL of enzyme extraction buffer (containing phosphate buffer, sodium chloride, reducing agent, protease inhibitor, surfactant, and deionized water) was added. After thorough mixing, the mixture was centrifuged at 10000rpm for 20min at 4°C using a high-speed refrigerated centrifuge. The supernatant obtained was the test liquid and was used for later use.

[0059] The activities of POD (peroxidase), SOD (superoxide dismutase), CAT (catalase), APX (ascorbate peroxidase), PAL (phenylalanine ammonia-lyase), and PPO (polyphenol oxidase) were measured using kit methods. The specific enzyme activity units are defined as follows:

[0060] POD: OD per gram of tissue per minute 470 A change of 0.01 corresponds to one unit of enzyme activity.

[0061] SOD: In the xanthine oxidase coupling reaction, the enzyme activity at which the inhibition rate reaches 50% is 1 unit.

[0062] CAT: One unit is defined as the catalytic degradation of 1 μmol H2O2 per gram of tissue per minute.

[0063] APX: 1 unit is defined as 1 μmol of ascorbic acid oxidized per gram of tissue per minute.

[0064] PAL: One unit is defined as a change of 0.1 in absorbance at 290 nm per gram of tissue per milliliter of reaction system per minute.

[0065] PPO: One unit is defined as a change of 0.1 in absorbance at 410 nm per gram of tissue per milliliter of reaction system per minute.

[0066] Depend on Figure 2 It was found that, 48h, 72h, and 96h after sunflower inoculation with *Sclerotinia sclerotiorum*, the activities of POD, SOD, CAT, APX, PAL, and PPO enzymes in the leaves of the M3 and M5 treatment groups treated with *Trichoderma spp.* TCS007 solid-state fermentation crude extract showed consistent trends. Furthermore, the activities of POD, SOD, APX, PAL, and PPO enzymes significantly increased with increasing treatment frequency with *Trichoderma spp.* TCS007 solid-state fermentation crude extract. However, CAT enzyme activity decreased slightly with increasing application frequency of *Trichoderma spp.* TCS007 solid-state fermentation crude extract, possibly due to an enzyme complementarity mechanism that regulates the expression of other sunflower enzymes to more effectively manage oxidative stress. For example, increased activity of defensive enzymes such as SOD, POD, and APX can compensate for insufficient CAT activity.

[0067] When the M1 treatment group was inoculated with Sclerotinia sclerotiorum, at 48 h and 72 h, the POD enzyme activity increased by 158.6% and 26.8% respectively compared with the CKJ treatment group. Figure 2 A), SOD enzyme activity increased by 3.9% and 3.2%, respectively. Figure 2 B). At 48 h, 72 h, and 96 h after inoculation with *Sclerotinia sclerotiorum*, the CAT enzyme activity in the M1 treatment group was increased by 100.0%, 154.7%, and 394.5% compared to the CKJ treatment group, respectively. Figure 2 C), APX enzyme activity increased by 80.0%, 111.2%, and 42.9%, respectively. Figure 2 D), PAL enzyme activity increased by 21.6%, 22.1%, and 41.6%, respectively. Figure 2 E), PPO enzyme activity increased by 9.9%, 19.2%, and 27.1%, respectively. Figure 2 F).

[0068] Compared with the CKJ treatment group, the POD enzyme activity in the M3 treatment group increased by 211.0%, 59.8%, and 13.4%, respectively. Figure 2 A), SOD enzyme activity increased by 14.7%, 8.6%, and 8.8%, respectively. Figure 2 B), CAT enzyme activities increased by 92.8%, 128.8%, and 328.1%, respectively. Figure 2 C), APX enzyme activity increased by 246.3%, 228.6%, and 176.7%, respectively. Figure 2D), PAL enzyme activity increased by 33.5%, 25.1%, and 59.2%, respectively. Figure 2 E), PPO enzyme activity increased by 16.8%, 38.8%, and 74.3%, respectively. Figure 2 F).

[0069] Compared with the CKJ treatment group, the POD enzyme activity in the M5 treatment group increased by 255.7%, 162.3%, and 87.4%, respectively. Figure 2 A), SOD enzyme activity increased by 20.6%, 22.1%, and 17.6%, respectively. Figure 2 B), CAT enzyme activity increased by 69.9%, 105.1%, and 275.6%, respectively. Figure 2 C), APX enzyme activity increased by 492.5%, 422.4%, and 397.0%, respectively. Figure 2 D), PAL enzyme activity increased by 90.0%, 86.0%, and 146.8%, respectively. Figure 2 E), PPO enzyme activity increased by 137.5%, 168.5%, and 240.0%, respectively. Figure 2 F).

[0070] Example 3

[0071] Effects of solid-state fermentation crude extract of Trichoderma hyacinthus TCS007 on GLU enzyme activity in sunflower leaves.

[0072] The sampling time and sampling method are the same as in Example 2.

[0073] GLU activity assay follows the kit method. Enzyme activity unit definition: 1 mg of reducing sugar produced per gram of tissue per hour is defined as 1 enzyme activity unit.

[0074] Depend on Figure 3 It was found that at 48h, 72h, and 96h after inoculation with *Sclerotinia sclerotiorum*, the GLU enzyme activity in sunflower leaves of the CKJ treatment group decreased over time. After treatment with crude extract from *Trichoderma echinococcus* TCS007 solid-state fermentation, the GLU enzyme activity gradually increased with the number of treatments. Compared with the CKJ treatment group, the GLU enzyme activity in the M3 treatment group increased by 10.3%, 97.1%, and 186.9%, respectively, while the GLU enzyme activity in the M5 treatment group increased significantly by 49.3%, 215.7%, and 419.6%. Figure 3 ).

[0075] Example 4

[0076] The effect of solid-state fermentation crude extract of Trichoderma echinosporum TCS007 on H2O2 content in sunflower leaves was determined.

[0077] The sampling time and sampling method are the same as in Example 2.

[0078] The H2O2 content was determined according to the kit method, and the unit was expressed in μmol / g.

[0079] Depend on Figure 4 It was found that at 48h, 72h, and 96h after inoculation with *Sclerotinia sclerotiorum*, the H2O2 content in sunflower leaves of the CKJ treatment group increased significantly with increasing time. However, after treatment with the crude extract of *Trichoderma echinococcus* TCS007 solid-state fermentation, the H2O2 content decreased. The reduction effect was significant with increasing treatment frequency. Compared to the CKJ treatment group, the H2O2 content of the M3 treatment group decreased by 17.1%, 37.9%, and 46.4%, respectively, while the H2O2 content of the M5 treatment group decreased significantly by 8.4%, 38.4%, and 56.9%. Figure 4 ).

[0080] Example 5

[0081] Determination of the effect of Trichoderma acicularis TCS007 solid fermentation crude extract on O in sunflower leaves 2- The effect of the rate of generation.

[0082] The sampling time and sampling method are the same as in Example 2.

[0083] Superoxide anion (O 2- The production rate was determined according to the kit method, and the unit was expressed in μmol / min / g.

[0084] Depend on Figure 5 It can be seen that at 48h, 72h and 96h after inoculation with Sclerotinia sclerotiorum, the superoxide anion (O2) concentration in the CKJ treatment group was significantly higher than that in the control group. 2- The production rate of superoxide anions (O3) increased significantly. After treatment with the crude extract from Trichoderma echinosporum TCS007 solid-state fermentation, the production rate of superoxide anions (O3) in sunflower leaves increased significantly. 2- The production rate of superoxide anions (O3) showed a decreasing trend, and the decrease became more significant with each additional treatment. Compared with the CKJ treatment group, the M3 treatment group showed a higher rate of superoxide anion (O3) production. 2- The superoxide anion production rates decreased by 3.2%, 7.0%, and 14.0%, respectively, with the M5 treatment group showing a significant decrease in superoxide anion production rates of 10.1%, 19.8%, and 36.2%. Figure 5 ).

[0085] The above specific embodiments further illustrate the purpose, technical solution, and beneficial effects of the present invention. It should be understood that the above are merely specific embodiments of the present invention and are not intended to limit the scope of protection of the present invention. Any modifications, equivalent substitutions, improvements, etc., made on the basis of the technical solution of the present invention should be included within the scope of protection of the present invention.

Claims

1. Application of Trichoderma echinosporum TCS007 solid-state fermentation ethyl acetate crude extract inducing sunflower immunity against sclerotinia stem rot.

2. The application according to claim 1, characterized in that, The crude ethyl acetate extract of Trichoderma echinosporum TCS007 solid fermentation can increase the enzyme activity in sunflower leaves. The increased enzyme activity in sunflower leaves includes one or more of the following enzyme activities: POD, SOD, CAT, APX, PAL, PPO, and GLU.

3. The application according to claim 1, characterized in that, The crude ethyl acetate extract from solid-state fermentation of Trichoderma echinosporum TCS007 can reduce the H2O2 content in sunflower leaves.

4. The application according to claim 1, characterized in that, The crude ethyl acetate extract of Trichoderma acicularis TCS007, obtained from solid-state fermentation, can reduce O2 in sunflower leaves. 2- Generation rate.

5. The application according to any one of claims 1-4, characterized in that, The crude ethyl acetate extract from solid-state fermentation of Trichoderma echinococcus TCS007 was obtained by extracting the solid-state fermentation product of Trichoderma echinococcus TCS007 with ethyl acetate. After extraction, the product was filtered and dried with anhydrous sodium sulfate. The dried liquid was concentrated and evaporated to dryness under reduced pressure. The residue was redissolved with ethyl acetate and collected in a centrifuge tube. After the solvent evaporated, the crude ethyl acetate extract from solid-state fermentation of Trichoderma echinococcus TCS007 was obtained.

6. The application according to claim 5, characterized in that, A crude ethyl acetate extract solution of Trichoderma acicularis TCS007 solid fermentation was prepared using N,N-dimethylformamide as a solvent.

7. The application according to claim 5, characterized in that, The method for preparing the solid-state fermentation product of *Trichoderma echinosporum* TCS007 involves weighing 25g of wheat bran, 5g of soybean flour, 0.6g of MgSO4, and 0.6g of MnSO4, adding 39.7mL of water, setting the initial pH to 5.0, and the spore concentration to 1×10⁻⁶. 7 The inoculum size of Trichoderma hygroscopicum TCS007 was 2 mL, the photoperiod was 21 h, and the inoculum was placed in a mold incubator at 28 ℃ and cultured in the dark for 15 days.

Images