Trichoderma asperellum bbr-a and its application in preventing and treating blueberry fungal diseases

Abstract

This invention discloses a *Trichoderma echinococcus* strain BBR-A and its application in the control of fungal diseases in blueberries, belonging to the field of biological control technology for blueberry fungal diseases. A *Trichoderma echinococcus* strain BBR-A was isolated from the rhizosphere soil of healthy blueberries and deposited at the China Center for Type Culture Collection (CCTCC) on June 4, 2025, with accession number CCTCC NO: M20251239. The *Trichoderma echinococcus* strain BBR-A of this invention significantly inhibits *Botrytis cinerea*, *Alternaria alternata*, and *Fusarium oxysporum*. Microscopic and electron microscopic examination revealed that *Trichoderma echinococcus* hyphae exhibited hyperparasitic phenomena such as entanglement, penetration, and dissolution upon contact with the hyphae of these three pathogens. The incidence of disease in blueberry roots and leaves treated with *Trichoderma echinococcus* was significantly reduced. These experimental results demonstrate that *Trichoderma echinococcus* BBR-A has a significant inhibitory effect on the growth and pathogenicity of blueberry fungal pathogens and can be used as a biological control agent for the control of blueberry fungal diseases, showing good application prospects.

Description

Technical Field

[0001] This invention belongs to the field of biological control technology for blueberry fungal diseases, specifically relating to a Trichoderma hygroscopica BBR-A and its application in the control of blueberry fungal diseases. Background Technology

[0002] Blueberries are small berries with high nutritional value, rich in anthocyanins, polyphenols, vitamins, pectin, amino acids, minerals, and other nutrients. They have strong antioxidant capabilities and are low in sugar and fat, making them beneficial to human health. The Food and Agriculture Organization of the United Nations (FAO) lists them as one of the five healthiest foods for humans. With in-depth research into the functional components of blueberries, it has been discovered that they have effects such as protecting eyesight, relieving eye fatigue, enhancing immunity, preventing cardiovascular disease, scavenging free radicals, and anti-tumor properties. In recent years, with the rapid development of the blueberry industry, diseases have also increased year by year, causing serious damage. Common blueberry diseases include gray mold, leaf spot, and root rot, which greatly harm the healthy growth, industrial development, and economy of blueberries, leading to yield reductions of 20%-50%, and have become a bottleneck for the industry. Currently, the control of blueberry fungal diseases both domestically and internationally mainly relies on traditional chemical fungicides, such as mancozeb, chlorothalonil, and pyraclostrobin. However, long-term use can lead to environmental pollution, pesticide residues, and increased drug resistance in pathogens. Therefore, developing green and environmentally friendly biological agents to control fungal diseases in blueberries is of great significance to the blueberry industry.

[0003] Trichoderma acicularis ( Trichoderma asperellum Trichoderma harzianum is a recognized highly effective biocontrol bacterium, but its antibacterial spectrum and growth-promoting mechanism vary greatly depending on the strain. In biological control, invention patent CN118638647A discloses a strain of Trichoderma harzianum HBTG3306, which has a control efficacy of 64.18% against tea leaf spot disease, but it uses a solid-state fermentation and drying process to prepare spore powder. Invention patent CN118995429A discloses Trichoderma harzianum HM-8, used to control sweet potato root rot and ginger stem base rot, with a control efficacy of over 80%, but it is also prepared as a wettable powder. Direct application of high-concentration spore solution or mycelial fermentation broth can maximize the preservation of strain activity and the antibacterial active substances produced by metabolism (e.g., Trichoderma harzianum TAZ61 in CN119081891A was treated with conidial solution irrigation), confirming the effectiveness of direct application of spore solution. However, current technologies lack a specific *Trichoderma* strain that targets the combined occurrence of blueberry gray mold, leaf spot, and root rot, and can be applied directly via spore suspension. Therefore, screening for a dedicated strain that can efficiently colonize the blueberry rhizosphere, exhibit strong antagonistic effects against multiple blueberry pathogens, and provide excellent control upon direct application is of significant practical value. Summary of the Invention

[0004] To address the shortcomings of the existing technology, the purpose of this invention is to provide an endophytic fungus—Trichoderma hygroscopicum—isolated from blueberry roots. Trichoderma asperellum This invention relates to BBR-A and applies it to suppress blueberry fungal diseases. It effectively solves the technical defects of existing technologies, such as the scarcity of biological control resources and the limited control methods for blueberry fungal diseases, and provides a new and efficient biological control strain and its application method for the green control of blueberry fungal diseases.

[0005] To achieve the above objectives, the present invention provides the following technical solution:

[0006] In a first aspect, the present invention provides a healthy blueberry rhizosphere soil containing Trichoderma hygroscopicum ( Trichoderma asperellum BBR-A was deposited at the China Center for Type Culture Collection on June 4, 2025, with accession number CCTCC NO: M20251239.

[0007] Based on the above technical solution, the strain was further isolated from the rhizosphere soil of healthy blueberries in a blueberry planting base in Dalian, Liaoning Province. Its morphological characteristics are as follows: initially, it is a white flocculent hyphae on PDA medium, and after 3 days, the conidia are dark green with concentric rings; the conidiophores are opposite or whorled, and the spores are nearly spherical; the ITS sequence has 100% homology with Trichoderma echinosporum in GenBank.

[0008] Secondly, the present invention provides a biological agent, wherein the active ingredient in the biological agent is Trichoderma echinosporum BBR-A and / or Trichoderma echinosporum BBR-A spores.

[0009] Based on the above technical solution, the preparation process of the biological agent further includes the following steps: inoculating the Trichoderma hygroscopicum BBR-A into a fermentation medium and culturing it to obtain the agent.

[0010] Based on the above technical solution, the fermentation medium further includes PDA liquid medium.

[0011] Based on the above technical solution, the cultivation conditions are further specified as 25-35℃ and the cultivation time is 10-72h.

[0012] Thirdly, the present invention provides the application of the above-mentioned Trichoderma hygroscopicum BBR-A and biological agents in the prevention and control of fungal diseases in blueberries.

[0013] Based on the above technical solutions, blueberry fungal diseases further include blueberry gray mold, blueberry leaf spot, and blueberry root rot.

[0014] Based on the above technical solution, further, the pathogens of blueberry gray mold include Botrytis cinerea (… Botrytis cinereaThe pathogens causing blueberry leaf spot include Alternaria (…). Alternaria Alternate The pathogens causing blueberry root rot include Fusarium oxysporum (…). Fusarium oxysporum ).

[0015] Based on the above technical solution, the specific application process is as follows: the spore liquid, mycelial liquid or biological agent of Trichoderma echinosporum BBR-A is applied to the blueberry plants by soil irrigation (root irrigation), root dipping, foliar spraying or trench application.

[0016] Based on the above technical solution, the concentration of the spore solution of Trichoderma hydatids BBR-A is further 1×10⁻⁶. 5 CFU / mL ~ 1×10 7 CFU / mL.

[0017] Compared with the prior art, the present invention has the following beneficial effects: The *Trichoderma hygroscopicum* BBR-A strain of the present invention can significantly inhibit *Botrytis cinerea* (…). Botrytis cinerea Alternaria ( Alternaria. Alternate ) and Fusarium oxysporum ( Fusarium oxysporum The antibacterial rate reached about 70%; microscopic and electron microscopic scanning revealed that Trichoderma echinosporum hyphae exhibited reparasitic phenomena such as entanglement, penetration, and dissolution after contact with the hyphae of the three pathogens; the incidence of disease in blueberry roots and leaves treated with Trichoderma echinosporum was significantly reduced; the above experimental results prove that Trichoderma echinosporum BBR-A has a significant inhibitory effect on the growth and pathogenicity of blueberry fungal diseases, and can be used as a biological control agent for the prevention and control of blueberry fungal diseases. Attached Figure Description

[0018] To more clearly illustrate the embodiments of the present invention, the accompanying drawings involved in the embodiments will be briefly described below.

[0019] Figure 1 Microscopic images of spores and hyphae of Trichoderma hygroscopicum BBR-A in Example 1, as well as front and back views of colonies.

[0020] Figure 2 This is a phylogenetic tree diagram of Trichoderma hyacinthus BBR-A in Example 1.

[0021] Figure 3 The figures show the results of plate confrontation experiments between Trichoderma echinosporum BBR-A and Trichoderma harzianum in Example 2, and three pathogenic fungi: Botrytis cinerea, Alternaria alternata, or Fusarium oxysporum.

[0022] Figure 4 The images show the results of hyperparasitism microscopy and scanning electron microscopy of Trichoderma hygrostis BBR-A hyphae in Example 3, which are observed in relation to the hyphae of three pathogenic fungi: Botrytis cinerea (A), Alternaria alternata (B), or Fusarium oxysporum (C).

[0023] Figure 5 The graph shows the results of Trichoderma hygroscopicum BBR-A spores inhibiting the pathogenicity of three pathogens: Botrytis cinerea (A), Alternaria alternata (B), and Fusarium oxysporum (C) in Example 4. Detailed Implementation

[0024] The present invention will be described in detail below with reference to the embodiments. However, the implementation of the present invention is not limited thereto. Obviously, the embodiments described below are only some embodiments of the present invention. For those skilled in the art, other similar embodiments can be obtained without creative effort and all fall within the protection scope of the present invention.

[0025] Example 1: Isolation and Identification of Trichoderma echinosporum strain BBR-A Ten rhizosphere soil samples were collected from healthy blueberry plants at a blueberry plantation in Dalian, Liaoning Province. After mixing, 20g of each sample was placed in 180mL of sterile water and vortexed for 30 minutes. The mixture was then serially diluted to 10⁻⁶. -3 10 -4 10 -5 Spread 0.1 mL of each culture onto a PDA plate containing streptomycin sulfate and incubate in the dark at 28°C for 3-5 days. Select single colonies with typical Trichoderma colony characteristics, and after repeated purification, obtain pure culture strains, designated BBR-A.

[0026] Morphological observation: After incubation at 28℃ for 72 hours on PDA plates, the colonies initially appeared as white flocculents, gradually turning dark green, with well-developed aerial hyphae; under a microscope, conidiophores were opposite or whorled, phialides were ampoule-shaped, and conidia were nearly spherical with fine warts on the surface. Figure 1 ).

[0027] Molecular identification: Genomic DNA was extracted using the CTAB method, and PCR amplification was performed using primers ITS1 (5'-TCCGTAGGTGAACCTGCGG-3') and ITS4 (5'-TCCTCCGCTTATTGATATGC-3'). The products were sequenced to obtain the ITS region sequence of this strain (nucleotides as shown in SEQ ID No. 1). The sequence was aligned with NCBI BLAST and compared with *Trichoderma hydatids* (…). Trichoderma asperellum The sequence homology reached 100%, and a phylogenetic tree was constructed using MEGA10.0 as follows: Figure 2 As shown; based on morphological characteristics, it was identified as Trichoderma echinosporum, named BBR-A, and deposited at the China Center for Type Culture Collection (CCTCC) on June 4, 2025, with accession number CCTCC NO: M20251239.

[0028] The ITS region sequence of this strain is as follows (SEQ ID No. 1): .

[0029] Example 2: In vitro antibacterial activity assay Test pathogen: Botrytis cinerea ( Botrytis cinerea Alternaria ( Alternaria alternate Fusarium oxysporum ( Fusarium oxysporum ).

[0030] Trichoderma: Trichoderma echinosporum BBR-A ( T. asperellum Trichoderma harzianum ( T. harzianum ‌) (CCTCC DF2008158); Using the plate confrontation method, a 5 mm diameter pathogenic fungal disc (Botrytis cinerea, Alternaria alternata, or Fusarium oxysporum) was inoculated on one side of the edge of a PDA plate, and a Trichoderma disc of the same size (Trichoderma echinosporum BBR-A or Trichoderma harzianum) was inoculated 3 cm away on the opposite side. After incubation at 25°C in the dark for 3 days, the colony radius of the pathogen pointing towards Trichoderma was measured. Plates inoculated only with the pathogen served as controls. The inhibition rate was calculated using the formula: Inhibition rate (%) = (Control colony diameter) / (Inhibition rate of pathogens) = ... Treatment colony diameter / (control colony diameter) The inhibition rate was calculated by multiplying the diameter of the fungal cake by 100%, and the results are shown in Table 1. *Trichoderma echinococcus* BBA-R showed an inhibition rate of 68.7% against *Botrytis cinerea*, 61.8% against *Alternaria alternata*, and 55.7% against *Fusarium oxysporum*. The results indicate that different *Trichoderma* species have inhibitory effects on pathogens, but *Trichoderma echinococcus* BBR-A isolated from blueberry rhizosphere soil showed a significantly better inhibitory effect than *Trichoderma harzianum* (e.g., *Trichoderma echinococcus* diameter). Figure 3 As shown in the figure, this demonstrates that BBR-A can be used for the biological control of fungal diseases in blueberries.

[0031] Table 1. Statistics on the inhibition rate of Trichoderma against three pathogens.

[0032] Example 3: Hyperparasitic effect of Trichoderma echinosporum on pathogens Test pathogen: Botrytis cinerea ( B. cinerea Alternaria ( A. Alternate Fusarium oxysporum ( F. oxysporum ).

[0033] Trichoderma: Trichoderma echinosporum BBR-A ( T. asperellum ) The hyphae of Trichoderma echinosporum BBR-A and the hyphae of three pathogens were inoculated separately onto PDA plates with the same glass slide or silicon wafer. After the hyphae came into contact, the state of Trichoderma echinosporum BBR-A when in contact with the pathogen hyphae was observed under an optical microscope and a scanning electron microscope, respectively.

[0034] The results are as follows Figure 4 As shown, A represents the confrontation between *Trichoderma echinococcus* BBR-A and *Botrytis cinerea* hyphae; B represents the confrontation between *Trichoderma echinococcus* BBR-A and *Alternaria alternata* hyphae; and C represents the confrontation between *Trichoderma echinococcus* BBR-A and *Fusarium oxysporum* hyphae. The results indicate that *Trichoderma echinococcus* BBR-A hyphae can entangle, penetrate, and dissolve the hyphae of these three pathogens, proving that *Trichoderma echinococcus* BBR-A has a strong hyperparasitic phenomenon on these three pathogens.

[0035] Example 4: Potted Plant Control Efficacy Test Tested variety: Eurica Sunrise potted blueberry seedlings.

[0036] Test pathogen: Botrytis cinerea ( B. cinerea Alternaria ( A. Alternate Fusarium oxysporum ( F. oxysporum ).

[0037] Trichoderma: Trichoderma echinosporum BBR-A ( T. asperellum ) Dilute the Trichoderma bBR-A spore solution with water to 1×10⁻⁶. 6 CFU / mL. When inoculating blueberry leaves, spray the entire blueberry leaf with 2 mL of a diluted Trichoderma echinococcus BBR-A spore solution. Two days later, the leaves treated with Trichoderma echinococcus spores are then sprayed with 1×10⁻⁶ spores. 6 2 mL of CFU / mL *Botrytis cinerea* and 1×10 6 2 mL of Alternaria champacli (CFU / mL) was administered. Simultaneously, 1×10⁻⁶ CFU / mL of Alternaria champacli was sprayed onto each of the following solutions. 6 2 mL of CFU / mL *Botrytis cinerea* and 1×10 6 A control treatment was prepared by adding 2 mL of *Alternaria alternata* (CFU / mL) to blueberry leaves. All treated blueberry seedlings were placed in an incubator at 25°C for 5 days while maintaining humidity, and the disease index was then recorded. For root inoculation, a root drenching method was used, similarly adding 1×10⁻⁶ CFU / mL of the above-mentioned fungicide. 6 Apply 20 mL of a CFU / mL Trichoderma spore dilution to the blueberry roots and treat for 2 days. Then apply a 1×10⁻⁶ CFU / mL solution of Trichoderma spores. 6 Apply 20 mL of CFU / mL Fusarium oxysporum spore solution to the blueberry roots. Simultaneously, use a root-drenching solution with a concentration of 1×10⁻⁶ CFU / mL. 6 A control experiment was conducted by applying 20 mL of CFU / mL Fusarium oxysporum spore solution to the roots of blueberries; all treated blueberry seedlings were then placed in an incubator with moisture and cultured at 25°C for 5 days, and the disease index was then recorded.

[0038] Leaf disease severity grading standards: Grade 0 - No lesions; Grade 1 - Lesions covering less than 10% of the leaf area; Grade 2 - Lesions covering 10-25% of the leaf area; Grade 3 - Lesions covering 25-50% of the leaf area; Grade 4 - Lesions covering more than 50% of the leaf area.

[0039] Grading standards for root diseases: Grade 0 - No lesions; Grade 1 - Some fibrous roots turn brown; Grade 2 - A few main roots show lesions; Grade 3 - Nearly half of the roots turn black and rot; Grade 4 - Most roots rot, and the plant wilts or dies.

[0040] Disease index = ∑(number of plants at each level × level) / (total number of plants × highest level) × 100 The results are as follows Figure 5 As shown, the diseased area of ​​blueberry leaves treated with Trichoderma echinosporum BBR-A was significantly less than that of leaves inoculated only with Botrytis cinerea. Figure 5 A), the diseased area of ​​blueberry leaves treated with Trichoderma echinosporum BBR-A was significantly less than that of leaves inoculated only with Alternaria alternata (A). Figure 5 B), blueberry roots treated with Trichoderma hygroscopicum BBR-A showed significantly fewer disease outbreaks than roots inoculated only with Fusarium oxysporum. Figure 5C). The statistical results of their disease index are shown in Table 2. The disease index of blueberries treated with Trichoderma echinosporum BBR-A was significantly lower than that of the control group. This shows that Trichoderma echinosporum BBR-A can effectively inhibit the pathogenicity of the fungus.

[0041] Table 2. Statistics of Disease Indices

[0042] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A strain of Trichoderma acicularis ( Trichoderma asperellum BBR-A, characterized in that, It was deposited at the China Center for Type Culture Collection on June 4, 2025, with accession number CCTCC NO: M20251239.

2. A biological agent, characterized in that, The active ingredient in the biological agent is Trichoderma hygroscopica BBR-A and / or the spores of Trichoderma hygroscopica BBR-A as described in claim 1.

3. The biological agent according to claim 2, characterized in that, The preparation process of the biological agent includes the following steps: inoculating the Trichoderma hygroscopicum BBR-A into a fermentation medium and culturing it to obtain the agent.

4. The biological agent according to claim 3, characterized in that, The fermentation medium includes PDA liquid medium.

5. The biological agent according to claim 3, characterized in that, The culture conditions are 25-35℃ and the culture time is 10-72h.

6. The application of Trichoderma hygroscopicum BBR-A as described in claim 1 and the biological agent as described in any one of claims 2-5 in the prevention and control of fungal diseases in blueberries.

7. The application according to claim 6, characterized in that, Blueberry fungal diseases include blueberry gray mold, blueberry leaf spot, and blueberry root rot.

8. The application according to claim 7, characterized in that, The pathogens causing blueberry gray mold include Botrytis cinerea (… Botrytis cinerea The pathogens causing blueberry leaf spot include Alternaria (…). Alternaria Alternate The pathogens causing blueberry root rot include Fusarium oxysporum (…). Fusarium oxysporum ).

9. The application according to claim 6, characterized in that, The specific application process is as follows: apply the spore liquid, mycelial liquid or biological agent of Trichoderma hygroscopicum BBR-A to the blueberry plants by soil irrigation (root irrigation), root dipping, foliar spraying or trench application.

10. The application according to claim 9, characterized in that, The concentration of the spore suspension of Trichoderma acicularis BBR-A was 1×10⁻⁶. 5 CFU / mL ~ 1×10 7 CFU / mL.

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