Trichoderma asperellum and its application in promoting growth and resisting anthracnose of tea tree

Abstract

The application discloses a Trichoderma asperellum and application of the Trichoderma asperellum in promoting growth of tea trees and resisting anthracnose, relates to the field of biological control and agricultural microbial technology, and proposes a brand-new Trichoderma asperellum, named SWUSL-3, which has a natural colonization advantage for a microecological environment of tea garden soil and leaves, still has high activity under a NaCl stress of up to 7%, breaks the conventional Trichoderma salt limit of 2% to 3%, and has great potential to be developed into an environment-friendly anti-stress biological pesticide or microbial fertilizer. The Trichoderma asperellum SWUSL-3 has hyperparasitism, metabolic product antibacterial activity and iron source competition activity on two core pathogenic bacteria of tea tree anthracnose; in a living body inoculation test, the relative prevention efficiency on Colletotrichum gloeosporioides is as high as 92.95%, and a disease spot disease index is as low as 1.40%, and the prevention efficiency is significant. In addition, the Trichoderma asperellum SWUSL-3 has the abilities of nitrogen fixation, phosphorus dissolution, IAA production and iron carrier production, and can significantly promote the growth of tea tree seedlings.

Description

Technical Field

[0001] This invention relates to the field of biological control and agricultural microbial technology, specifically to a strain of Trichoderma echinosporum and its application in promoting growth and resisting anthracnose in tea trees. Background Technology

[0002] Crops are highly susceptible to fungal pathogens during their growth, leading to various soil-borne diseases. Common plant fungal pathogens are tolerant of acidic environments and can grow in acidic soils. In recent years, modern agriculture has faced serious problems such as secondary soil salinization and the sequestration and loss of nutrients such as phosphorus and nitrogen. In a slightly acidic microecological environment accompanied by salt stress, crop growth is weakened, immunity is low, and they are extremely vulnerable to *Colletotrichum gloeosporioides* (Colletotrichum gloeosporioides). Colletotrichum gloeosporioides ) and Camellia thorn discus ( C. camelliae The combined infection of pathogens such as _____. However, acidic and high-osmotic salinity environments often significantly weaken the colonization and control effects of conventional microbial biocontrol agents. Therefore, it is necessary to screen for a microorganism that can colonize in complex adverse environments of slightly acidic and saline conditions, while simultaneously enhancing plant nutrient absorption, in order to more stably and effectively control soil-borne diseases.

[0003] Trichoderma ( Trichoderma Trichoderma spp. is a widely distributed filamentous fungus commonly used as a biocontrol agent. However, the efficacy of most existing strains decreases significantly in soils with salinity as high as 3% or nutrient-poor conditions. Currently, there are few Trichoderma strains that can tolerate high salt levels and possess both disease resistance and growth-promoting abilities such as phosphorus solubilization, nitrogen fixation, and IAA production. Therefore, there is an urgent need to develop a biocontrol strain that combines stress-resistant colonization, nutrient competition with pathogens, and crop growth promotion and disease resistance to fundamentally solve the problem of anthracnose control in plants under complex adverse conditions. Summary of the Invention

[0004] To address the aforementioned shortcomings of the prior art, this invention provides a Trichoderma acicularis and its application in promoting growth and resisting anthracnose in tea trees.

[0005] To achieve the above-mentioned objectives, the technical solution adopted by this invention is as follows: Provides a Trichoderma acicularis ( T. asperellum SWUSL-3, the Trichoderma asperellum described therein was deposited on December 23, 2024 at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 41730 and classified as Trichoderma asperellum.

[0006] The present invention also provides a biocontrol agent comprising the above-mentioned Trichoderma hydatids SWUSL-3.

[0007] Furthermore, the biocontrol agent is in the form of a liquid formulation; the spore concentration of *Trichoderma hygroscopica* SWUSL-3 in the liquid formulation is 1 × 10⁻⁶. 6 ~10 8 CFU / mL.

[0008] The present invention also provides the application of the above-mentioned Trichoderma spp. SWUSL-3 or the above-mentioned biocontrol agent in the prevention and control of anthracnose in tea trees.

[0009] Furthermore, when tea trees are infected with anthracnose pathogens, the above-mentioned biocontrol agent is sprayed on the leaves, and before spraying, 2 wt.% of Tween-80 is added to the biocontrol agent and mixed well.

[0010] Furthermore, the pathogens causing anthracnose in tea trees include *Colletotrichum camelliatum* (…). C. camelliae CS 1-1 or Colloidal anthrax ( C. gloeosporioides )CS 05.

[0011] Furthermore, the hyphae of Trichoderma SWUSL-3 have the ability to reparasitize and produce siderophores to capture free iron ions, which restricts the growth of pathogens due to the lack of essential iron, thereby inhibiting the growth of Colletotrichum camelliae CS 1-1 and / or Colletotrichum CS 05.

[0012] The present invention also provides the application of the volatile metabolites of Trichoderma SWUSL-3 mentioned above in the prevention and control of anthracnose in tea trees.

[0013] The present invention also provides the application of the above-mentioned Trichoderma spp. SWUSL-3 or the above-mentioned biocontrol agent in promoting the growth of tea seedlings.

[0014] Furthermore, a method to promote the growth of tea seedlings is to use a spore concentration of 1×10⁻⁶. 6 ~10 8 The bacterial solution of CFU / mL was used to infuse the roots of tea seedlings.

[0015] The beneficial effects of this invention are as follows: This invention proposes a novel Trichoderma acicularis strain, named SWUSL-3, which has a natural colonization advantage in the soil and leaf micro-ecological environment of tea gardens. It still maintains high vitality under stress of up to 7% NaCl, breaking the salt tolerance limit of conventional Trichoderma acicularis at 2%~3%. It has great potential to be developed into an environmentally friendly stress-resistant biological pesticide or microbial fertilizer.

[0016] Trichoderma echinosporum SWUSL-3 exhibits antibacterial activities against the two core pathogens of anthracnose in tea trees, including hyperparasitism, metabolite inhibition, and competition for iron sources. In in vivo inoculation tests, it showed a relative control efficacy of up to 92.95% against Colletotrichum gloeosporioides, with a lesion disease index as low as 1.40%, demonstrating significant control efficacy. Furthermore, Trichoderma echinosporum SWUSL-3 possesses nitrogen-fixing, phosphorus-solubilizing, IAA-producing, and siderophore-producing abilities, which can significantly promote the growth of tea seedlings.

[0017] It can significantly increase the biomass of mature tea seedlings and, as an elicitor, significantly enhance the activity of antioxidant defense enzymes such as SOD, POD, CAT, and APX in the host leaves. Attached Figure Description

[0018] Figure 1 This is the phylogenetic tree of the Trichoderma hygroscopicum strains screened in Example 1; Figure 2 Colony morphology characterization of Trichoderma hygroscopicum SWUSL-3 of this invention; Figure 3 This is a comparison of the growth and reproduction of Trichoderma hyacinthus SWUSL-3 under different environmental conditions in Example 2; Figure 4 This is a comparison of the growth and reproduction of Trichoderma hygroscopicum SWUSL-3 under different NaCl concentrations in Example 3; Figure 5 This is a comparison of the phosphate-solubilizing abilities of different strains in Example 4; Figure 6 This is a comparison of the IAA production capacity of different strains in Example 4; Figure 7 This is a comparison of the siderophore production capacity of different strains in Example 4; Figure 8 This is a comparison of the antibacterial ability of different strains against the anthracnose pathogen of tea trees in Example 5; Figure 9 This is a characterization of the hyperparasitic effect of Trichoderma hygroscopicum SWUSL-3 of the present invention on the anthracnose pathogen of tea trees in Example 5; Figure 10 This is a comparison of the increased activity of defense enzymes induced by different strains in Example 7. Detailed Implementation

[0019] The specific embodiments of the present invention are described below to enable those skilled in the art to understand the present invention. However, it should be understood that the present invention is not limited to the scope of the specific embodiments. For those skilled in the art, various changes are obvious as long as they are within the spirit and scope of the present invention as defined and determined by the appended claims. All inventions utilizing the concept of the present invention are protected.

[0020] Example 1: Screening and identification of Trichoderma hygroscopicum SWUSL-3 Rhizosphere soil samples were collected from healthy mature tea trees in a tea garden in Delong Town, Nanchuan District, Chongqing, and applied using a gradient dilution coating method. 100 μL of a 10-1 concentration was used... -3 10 -4 Soil suspensions were spread onto PDA agar plates containing antibiotics and incubated at 28°C. Colonies with typical Trichoderma characteristics were picked and purified multiple times. Repeat tests yielded three vigorous strains, named SHUSL-1, SHUSL-2, and SHUSL-3.

[0021] Molecular biological identification of the three strains was performed based on internal transcribed spacers (ITS) and translation elongation factor-1α (TEF-1α). Translation elongation factors-1α , TEF ) and the gene encoding the second largest subunit of RNA polymerase II ( RNA polymerase II subunit 2 , RPB2 The sequences were analyzed, and a joint phylogenetic tree was constructed using the maximum likelihood method with MEGA 11.0 software. The results are as follows: Figure 1 As shown. Based on the comparison results, all three strains were determined to be *Trichoderma hygroscopica* (…). T. asperellum ).

[0022] SBUSL-3 strain was taken and morphologically identified, such as... Figure 2 As shown, where Figure 2 In the diagram, A and B represent the colony morphology of this strain on PDA medium. Figure 2 The C in the figure represents the conidia of this strain; from Figure 2 This strain forms grayish-white colonies on PDA medium, with no visible pigment secretion and a strong coconut odor. After 3 days of culture, white conidial clusters first appear at the periphery of the colonies, then gradually spread towards the center and turn dark green, eventually covering the entire 90mm petri dish within 5 days. Mature colonies exhibit a unique "peripheral-dominant" radial sporulation pattern: dense dark green conidia at the periphery, while only sporulation points form in the central area. This is consistent with the morphological characteristics of *Trichoderma echinosporum*.

[0023] Example 2: Determination of optimal carbon source, nitrogen source and pH of Trichoderma spunlacea SWUSL-3 To reveal the nutritional adaptability of SFUSL-3 strain in the environment, its carbon and nitrogen source utilization preferences were determined using Czapek's medium as the basal medium. Using equicarbon equivalents of glucose, sucrose, soluble starch, mannitol, and malt extract as the sole carbon source, and equinicarbon equivalents of ammonium sulfate, peptone, yeast extract, and calcium nitrate as the sole nitrogen source, the mycelial growth rate and sporulation were observed.

[0024] Optimal pH determination was also performed: activated Trichoderma echinosporum SWUSL-3 mycelial blocks were inoculated onto PDA plates with different pH gradients (pH 4.0, 5.0, 6.0, 7.0, 8.0, 9.0) and incubated at 28°C. Colony diameter and sporulation were measured and recorded.

[0025] The results are as follows Figure 3 As shown, where, Figure 3 In the figure, A represents a comparison of the mycelial growth rate of Trichoderma hygroscopicum SWUSL-3 under different carbon sources. Figure 3 B in the figure represents a comparison of spore concentration (i.e., spore production) of Trichoderma spp. SWUSL-3 under different carbon sources; Figure 3 C in the figure represents a comparison of the mycelial growth rate of Trichoderma SWUSL-3 under different nitrogen sources. Figure 3 D in the figure represents a comparison of spore concentration (i.e. spore production) of Trichoderma spp. SWUSL-3 under different nitrogen sources; Figure 3 E in the figure represents a comparison of the mycelial growth rate of Trichoderma spunlaceum SWUSL-3 at different pH values. Figure 3 F in the figure represents the comparison of spore concentration (i.e. spore production) of Trichoderma spp. SWUSL-3 at different pH values.

[0026] Depend on Figure 3 As shown in A~D, *Trichoderma hygroscopicum* SWUSL-3 can grow and produce spores normally on all five carbon sources, with mannitol being its optimal carbon source. Since sugar alcohols are common in the rhizosphere exudates of tea trees, and nitrates are the main available nitrogen in tea garden soil, this nutrient preference of the strain provides physiological support for its rapid colonization around tea trees.

[0027] Depend on Figure 3 As shown in E and F, Trichoderma spunlacea-3 can grow and sporulate at pH 4–9. When the culture medium pH is 6.0, the colony growth rate of SWUSL-3 is the fastest and the sporulation rate is the highest, with an optimal growth pH of 6. This is consistent with the slightly acidic environment suitable for tea plants.

[0028] Example 3: Verification of salt tolerance of Trichoderma hyacinthus SWUSL-3 The *Trichoderma hydathoides* SWUSL-3 of this invention was inoculated onto PDA plates containing 1%, 3%, 5%, 7%, and 10% NaCl, respectively, and the mycelial growth rate and sporulation yield were statistically analyzed. The results are as follows: Figure 4 As shown, where Figure 4 In the figure, A represents a comparison of the mycelial growth rate of Trichoderma spunlaceum SWUSL-3 under different salt concentrations. Figure 4B in the figure represents a comparison of sporulation of Trichoderma SWUSL-3 under different salt concentrations. Under high osmotic salt stress of 5% NaCl, the strain Trichoderma SWUSL-3 disclosed in the prior art (CN119410494) was severely inhibited, while the SHUSL-3 of the present invention still has high mycelial growth and sporulation capacity under high salt stress of up to 7% NaCl, showing an unexpected high salt tolerance effect and ensuring its application potential in secondary salinized soils.

[0029] Example 4: Verification of the multiple growth-promoting functions of Trichoderma hygroscopicum SWUSL-3 The nitrogen-fixing ability of *Trichoderma echinosporum* SWUSL-3 was verified. Specifically, activated *Trichoderma echinosporum* SWUSL-3 was inoculated into Assoube nitrogen-free medium and incubated in the dark at 28°C. Its growth was observed. If it could grow in the medium (marked as "+"), it was subcultured five times consecutively, and its nitrogen-fixing ability was observed. Mycelial cakes were prepared from the edge of the activated *Trichoderma* colony using a 5mm diameter sterile punch. The mycelial cakes were inoculated into inorganic phosphorus liquid medium and incubated at 28°C and 220 rpm. -1 The culture was shaken and incubated for 4 days. The culture medium was centrifuged at 12000×g for 15 min to collect the supernatant. The phosphorus content of the supernatant was detected by inductively coupled plasma optical emission spectrometry (ICP-OES). PDB liquid medium inoculated with the same volume of PDA culture medium block was used as a blank control. Each treatment was repeated in triplicate.

[0030] The growth of the strain in Assumption nitrogen-free medium is shown in Table 1 below; Table 1

[0031] As shown in Table 1, Trichoderma suvius L-3 can grow in Assab nitrogen-free medium after 5 consecutive subcultures, indicating that it has nitrogen-fixing ability.

[0032] The phosphorus-solubilizing ability of *Trichoderma echinosporum* SWUSL-3 was verified. Specifically, *Trichoderma* mycelial cakes were prepared using a 5 mm diameter sterile punch. The mycelial cakes were inoculated into an inorganic phosphorus liquid medium and incubated at 28°C and 220 rpm. -1 The culture was shaken and incubated for 4 days. The culture medium was centrifuged at 12000×g for 15 minutes to collect the supernatant. The phosphorus content of the supernatant was detected by inductively coupled plasma optical emission spectrometry (ICP-OES). PDB liquid medium inoculated with the same volume of PDA culture medium block was used as a blank control. Each treatment was repeated in triplicate.

[0033] The existing strain SWU B077R1 was also used for comparison, and the results were as follows: Figure 5 As shown, by Figure 5It can be seen that the phosphate-solubilizing ability of Trichoderma spunlaceum SWUSL-3 is significantly higher than that of the existing strain SWU B077R1.

[0034] The ability of Trichoderma hyacinthii SWUSL-3 to produce IAA was verified. Specifically, the target strain was inoculated into King's B medium and incubated at 28°C and 220 rpm. -1 After 48 hours of shaking culture, the culture medium was centrifuged at 12000×g for 10 minutes, and 200 μL of supernatant was collected and thoroughly mixed with 400 μL of Salkowski chromogenic reagent. At the same time, a blank control was set up with 200 μL of PDB medium added.

[0035] The concentration of IAA in the supernatant was also measured. Specifically, the supernatant was thoroughly mixed with Salkowski chromogenic reagent at a volume ratio of 1:2, allowed to stand at room temperature in the dark for 30 minutes, and then the absorbance was measured at a wavelength of 530 nm. An equal volume of sterile PDB culture medium was added as a blank control to zero the reaction. A linear regression equation (R²) between absorbance and concentration was established by preparing a series of IAA standard solutions. 2 ≥0.99), and calculate the IAA concentration; We also compared it with the existing strain SWU B077R1, and the results were as follows: Figure 6 As shown, where, Figure 6 In the figure, A represents the macroscopic characterization of the two strains after reacting at room temperature in the dark for 30 minutes. Figure 6 B in the figure represents a comparison of IAA concentrations after cultivation of the two strains.

[0036] Depend on Figure 6 The presence of a red characteristic reaction in the solution of SWUSL-3 indicates that *Trichoderma hygroscopicum* SWUSL-3 has the ability to synthesize indoleacetic acid (IAA). Furthermore, the IAA yield of *Trichoderma hygroscopicum* SWUSL-3 reaches 19.5 mg / L.

[0037] The siderophore-producing capacity of *Trichoderma hydathoides* SHUSL-3 was compared and verified with that of the existing strain SWU B077R1. Specifically, the activated *Trichoderma* strain was inoculated into the center of CAS solid medium and incubated in the dark at 28°C for 5 days. The siderophore production capacity was determined by observing whether an orange halo formed around the colony. Siderophore yield was measured by using a 5mm diameter sterile punch to create mycelial cakes from the edge of the activated *Trichoderma* colony, inoculating them into iron-free Czapek liquid medium, and incubating at 28°C and 220 rpm. -1After shaking culture for 3-5 days, collect the culture medium into 50 mL centrifuge tubes and centrifuge at 12000×g for 15 min. Take the supernatant and mix it with the CAS detection solution at a 1:1 volume ratio. Use an equal volume of sterile PDB medium as a blank control to zero the sample. Measure the absorbance at 680 nm. Siderophore yield is expressed as siderophore activity units (SU), calculated using the formula: SU(%) = (Ar-As) / Ar×100; where Ar is the absorbance of the siderophore detection solution and As is the absorbance of the mixture of siderophore detection solution and fungal fermentation broth.

[0038] The results are as follows Figure 7 As shown, where, Figure 7 In the diagram, A represents the macroscopic characterization of the two strains. Figure 7 In the figure, B represents a comparison of the siderogenic vectors of the two strains; by Figure 7 It can be seen that the presence of orange-red inhibition zones in both strains indicates that both *Trichoderma hyacinthus* SHUSL-3 and the existing strain SWU B077R1 possess siderophore-producing capabilities. However, the siderophore-producing capability of *Trichoderma hyacinthus* SHUSL-3 of this invention is significantly greater than that of strain SWU B077R1.

[0039] The effect of promoting the growth of tea seedlings in pots was verified. Specifically, healthy seedlings from mature tea trees with uniform growth were selected and treated with a concentration of 1×10⁻⁶. 7 Trichoderma spore suspension (CFU / mL) was used to treat the roots of *Tetratympanum var. *mairei* seedlings by drenching them with a puncture wound, with water treatment serving as a control. Each treatment group was replicated in triplicate. Biomass parameters were measured after 30 days, and the results are shown in Table 2 below.

[0040] Table 2

[0041] Lowercase letters indicate significant differences in data within the same column (p<0.05).

[0042] As shown in Table 2, compared with the control group and strain SWU B077R1, the Trichoderma hygroscopicum SHUSL-3 treatment of the present invention significantly increased the seedling height, root length and aboveground and underground biomass.

[0043] In summary, the *Trichoderma echinococcus* SHUSL-3 of this invention possesses nitrogen-fixing, phosphorus-solubilizing, IAA-producing, and siderophore-producing abilities that promote the growth of tea seedlings. While its nitrogen-fixing and IAA-producing abilities are not significantly different from the existing strain SWUB077R1, *Trichoderma echinococcus* SHUSL-3 of this invention is significantly superior to strain SWUB077R1 in phosphorus-solubilizing and siderophore-producing abilities. In vivo verification on tea seedlings also demonstrates that *Trichoderma echinococcus* SHUSL-3 has a significantly better overall growth-promoting effect on tea seedlings than the existing strain SWUB077R1; its phosphorus-solubilizing and siderophore-producing abilities have a greater impact on the growth-promoting effect of the strain on tea seedlings.

[0044] Example 5: In vitro antagonistic and reparasitic effects of SWUSL-3 on anthracnose fungus of tea tree. This embodiment uses the main pathogen of anthracnose in tea trees, *Colletotrichum camelliae* (… C. camelliae CS 1-1 and Colloidal anthracnose ( C. gloeosporioides CS 05 was used as the target for evaluation. The existing strain SWU B077R1 was also used for comparison.

[0045] The study included a plate confrontation test: The competitive effect of *Trichoderma* on pathogens was observed using the plate confrontation method. *Trichoderma echinococcus* SWUSL-3 and anthracnose fungal discs were prepared using a 5mm diameter sterile punch. Using PDA medium of equal size as a control, *Trichoderma echinococcus* SWUSL-3 and the two anthracnose fungi were inoculated on opposite sides of the center line of the PDA medium, spaced 60mm apart, and cultured in triplicate for each treatment. After incubation at 28℃ for 4 days, the growth of the pathogens was observed, and the diameter was measured using the cross-cross method. The inhibition rate was calculated using the formula. ×100%; The results are as follows Figure 8 As shown in Table 3, Figure 8 In the figure, A represents the macroscopic characterization of the antibacterial activity of the two strains against Colletotrichum gloeosporioides. Figure 8 B in the figure represents the macroscopic characterization of the antibacterial activity of two strains against *Colletotrichum camelliae*; [The remaining text appears to be a fragment and requires further context for accurate translation.] Figure 8 As shown in Table 3, the inhibition rate of Trichoderma spunlacea SWUSL-3 against the two anthracnose fungi of tea trees is higher than that of the existing strain SWU B077R1.

[0046] Table 3

[0047] The hyperparasitic effect of *Trichoderma hydathoides* SWUSL-3 was also observed. Specifically, using a slide culture microscopy method, *Trichoderma hydathoides* SWUSL-3 and *Anthracis chinensis*, the pathogen of tea tree anthracnose, were inoculated on opposite sides of a PDA membrane and cultured in a confrontational manner. Figure 9 As shown, where Figure 9 In the figure, A represents the hyphal microstructure of SWUSL-3 and Camellia thorn CS 1-1. Figure 9 In the diagram, B represents the hyphal microstructure of SWUSL-3 and Colletotrichum CS 05; from Figure 9 It is evident that SWUSL-3 exhibits a strong reparasitic ability; its hyphae actively seek out and tightly entwine with the hyphae of *Colletotrichum gloeosporioides*, causing malformation of the pathogenic hyphae. Combined with its siderophore activity as described in Example 4, SWUSL-3 can rapidly bind free iron ions by secreting large amounts of siderophores, thus limiting the growth of the pathogen due to a lack of essential iron, demonstrating a significant competitive antibacterial advantage.

[0048] The antibacterial properties of metabolites were also tested: the antibacterial rate of volatile substances was determined using the double-plate inverted method; the antibacterial rate of non-volatile substances was determined using the sterile fermentation filtrate. The results are shown in Table 4 below.

[0049] Table 4

[0050] As shown in Table 4, the antibacterial rates of both volatile and non-volatile metabolites of SWUSL-3 of the present invention are significantly better than those of existing strains, and the antibacterial rate of volatile metabolites of SWUSL-3 is better than that of non-volatile metabolites.

[0051] Example 6: In vivo control effect of SWUSL-3 formulation against anthracnose in tea trees. The control efficacy of *Trichoderma hygroscopicum* SWUSL-3 and *Trichoderma hygroscopicum* SWU B077R1 against anthracnose in tea trees was determined using an in vitro leaf inoculation method. SWUSL-3 was inoculated into a fermentation medium, and spores were collected and diluted with sterile water to a concentration of 1×10⁻⁶. 7 CFU / mL spore suspension. The Trichoderma spore suspension was mixed with 2% Tween-80 as a surfactant and then evenly sprayed onto the surface of one-year-old tea tree leaves (after pre-treating with an inoculation needle). The standard was that the mixed solution existed in the form of non-shedding droplets. The tea seedlings were inoculated with the pathogen 24 hours after planting, with a water treatment as a control. Each treatment was repeated three times. The seedlings were incubated at a constant temperature of 25℃ for 5 days. The growth of the tea seedlings was observed daily, and the disease index and control effect of each treatment group were statistically analyzed. The results are shown in Table 5 below.

[0052] Table 5

[0053] As shown in Table 5, the relative control efficacy of the SHUSL-3 pretreatment group against *Colletotrichum camelliatum* CS 1-1 and *Colletotrichum gloeosporioides* CS 05 reached 73.58% and 92.65%, respectively, which were significantly better than *Trichoderma hygroscopicum* SWU B077R1, demonstrating extremely high potential for field application.

[0054] Example 7: Effect of SWU SL-3 on the activity of related defense enzymes in old-growth tea leaves Tea leaves from large-leaf trees treated with *Trichoderma hygroscopicum* SWUSL-3 and *Trichoderma hygroscopicum* SWU B077R1 and a water control in Example 6 were ground and centrifuged to prepare crude enzyme solutions. The activities of superoxide dismutase (SOD), peroxidase (POD), catalase (CAT), and ascorbate peroxidase (APX) were then measured. The results are as follows: Figure 10 As shown, where, Figure 10 In the figure, A represents a comparison of ascorbate peroxidase activity after treatment with different strains. Figure 10 In the figure, B represents a comparison of catalase activity after treatment with different strains. Figure 10 In the figure, C represents a comparison of peroxidase activity after treatment with different strains. Figure 10 In the figure, D represents a comparison of superoxide dismutase activity after treatment with different strains. Figure 10 It was found that the activities of the four antioxidant defense enzymes mentioned above were significantly higher in tea leaves treated with SWUSL-3 than in the control group and the control strain treatment group. This demonstrates that Trichoderma echinosporum SWUSL-3 can not only directly inhibit bacteria, but also act as an immune elicitor to activate the systemic defense response of tea plants.

Claims

1. A strain of Trichoderma acicularis ( Trichoderma asperellum SWUSL-3, characterized in that, The *Trichoderma hygroscopica* species was deposited on December 23, 2024, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 41730.

2. A biocontrol agent, characterized in that, It contains Trichoderma hygroscopicum SWUSL-3 as described in claim 1.

3. The biocontrol agent according to claim 2, characterized in that, The biocontrol agent is in liquid form; the spore concentration of Trichoderma spp. SWUSL-3 in the liquid formulation is 1×10⁻⁶. 6 ~10 8 CFU / mL.

4. The application of Trichoderma hygroscopicum SWUSL-3 as described in claim 1, or the biocontrol agent as described in claim 2 or 3, in the control of anthracnose in tea trees.

5. The application according to claim 4, characterized in that, When tea trees are infected with anthracnose pathogens, the biocontrol agent described in claim 2 or 3 is sprayed on the leaves, and before spraying, 2 wt.% of Tween-80 is added to the biocontrol agent and mixed well.

6. The application according to claim 4, characterized in that, The pathogens causing anthracnose in tea trees include *Colletotrichum camelliatum* (…). C. camelliae CS 1-1 and / or Colloidal anthrax ( C. gloeosporioides )CS 05.

7. The application according to claim 6, characterized in that, The hyphae of Trichoderma SWUSL-3 have the ability to reparasitize and can produce siderophores to competitively bind free iron ions, thus limiting the growth of pathogens due to the lack of essential iron elements, thereby inhibiting the growth of Colletotrichum camelliae CS 1-1 and Colletotrichum collodion CS 05.

8. The application of the volatile metabolites of Trichoderma SWUSL-3 as described in claim 1 in the prevention and control of anthracnose in tea trees.

9. The application of Trichoderma hygroscopicum SWUSL-3 as described in claim 1 or the biocontrol agent as described in claim 2 or 3 in promoting the growth of tea seedlings.

10. The application according to claim 9, characterized in that, The method to promote the growth of tea seedlings is as follows: using a spore concentration of 1×10 6 ~10 8 The bacterial solution of CFU / mL was used to infuse the roots of tea seedlings.

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