Strain z25 of longibrachiatum with broad-spectrum anti-disease of strawberry and application thereof

Abstract

This invention belongs to the field of agricultural microbiology technology and relates to the longibrachiatum strain ZN25, which exhibits broad-spectrum resistance to strawberry diseases, and its applications. The longibrachiatum strain ZN25 is classified as *Trichoderma longibrachiatum* and has the accession number CGMCC No. 42426. This strain, isolated from the strawberry endophytic environment, exhibits broad-spectrum resistance to the pathogens of strawberry anthracnose (*Anthracnose sianna*) and root rot (*Fusarium oxysporum*). It inhibits pathogen growth through multiple mechanisms and simultaneously produces a series of volatile gaseous substances with antibacterial activity, enabling green and efficient control of strawberry anthracnose and root rot.

Description

Technical Field

[0001] This invention belongs to the field of agricultural microbial technology and relates to the Trichoderma longifolia strain ZN25, which has broad-spectrum resistance to strawberry diseases, and its applications. Background Technology

[0002] Strawberries are an important fruit crop with a wide planting area and significant economic benefits. However, in the process of large-scale strawberry cultivation, continuous cropping obstacles are prominent, and the resulting frequent outbreaks of diseases seriously restrict the high-quality development of the strawberry industry. Among them, anthracnose (mainly caused by *Colletotrichum siamense*) and root rot (mainly caused by *Fusarium oxysporum*) are the two most serious fungal diseases in strawberry production.

[0003] Strawberry anthracnose mainly affects the petioles, leaves, runners, and shortened stems of strawberries. After infection, it easily leads to wilting of plants, and in severe cases, it can cause large-scale seedling death, with yield reductions reaching 30%-50%. Strawberry root rot mainly infects the petioles, runners, and roots, causing browning and rotting of the roots, affecting the plant's absorption of water and nutrients. It manifests as yellowing of the above-ground parts, weakened growth, and eventually death of the entire plant. In continuously cropped fields, the incidence rate can be as high as 50% or more.

[0004] Currently, the control of strawberry anthracnose and root rot still mainly relies on chemical pesticides. However, long-term and excessive use of chemical pesticides can easily lead to pesticide resistance in pathogens, reducing the effectiveness of control. At the same time, pesticide residues pollute the soil, water bodies, and other ecological environments, and also affect the quality and safety of strawberry fruits, which does not meet the development needs of green and organic agriculture. Therefore, developing safe, efficient, and environmentally friendly biological control products has become an urgent need to solve the problem of strawberry diseases.

[0005] Trichoderma fungi are an important class of biocontrol microorganisms, widely distributed in nature. Due to their multiple biocontrol mechanisms, including competing for nutrients and space, parasitizing pathogens, inducing systemic resistance in plants, and secreting antimicrobial metabolites, they are widely used in the biological control of plant diseases. However, in existing technologies, most Trichoderma strains used for strawberry disease control are isolated from the soil environment, such as the invention patent application with publication number CN 120464502A. Trichoderma resources derived from the endophytic environment of strawberry plants have not yet been fully developed and utilized.

[0006] Plant endophytic microorganisms have undergone long-term co-evolution with host plants, resulting in stronger host adaptability and niche advantages. When used as biocontrol strains to control host diseases, they are more likely to colonize and play a role. Summary of the Invention

[0007] To address the shortcomings of insufficient biocontrol resources of existing Trichoderma strains and the lagging development of endophytic Trichoderma, this invention provides a long-branch Trichoderma strain ZN25 with broad-spectrum resistance to strawberry diseases. This strain was isolated from the strawberry endophytic environment and has broad-spectrum resistance to the pathogens of strawberry anthracnose (Anthracnose sianna) and root rot (Fusarium oxysporum). It can inhibit the growth of pathogens through multiple mechanisms and at the same time produce a series of volatile gaseous substances with antibacterial activity, enabling green and efficient control of strawberry anthracnose and root rot.

[0008] The present invention specifically adopts the following technical solution:

[0009] In a first aspect, the present invention provides a longibrachiatum strain ZN25 with broad-spectrum resistance to strawberry diseases, which is classified as Trichoderma longibrachiatum and has the accession number CGMCC No.42426.

[0010] Secondly, the present invention provides a microbial inoculant containing the aforementioned Trichoderma longifolia strain ZN25.

[0011] In a further embodiment, the microbial agent contains conidia, mycelium, and / or metabolites of the *Trichoderma longicornis* strain ZN25. Furthermore, the metabolites are volatile metabolites, such as (+)-β-cedrene, spiro[4.5]dec-8-en-7-ol, γ-methylcamphene, 1,3,5,6,7-pentamethylbicyclo[3.2.0]hept-2,6-diene, longicorene-(V4), benzaldehyde, 1-octanol, and naphthalene.

[0012] In a further embodiment, the microbial agent is formulated as a wettable powder, suspension, or volatile antibacterial packet.

[0013] The preparation method of the volatile antibacterial pack includes:

[0014] (1) Culture of strains

[0015] Trichoderma longifolia was inoculated into a solid-state fermentation medium and fermented at 25°C for 7 days to obtain the fermentation product. The mass ratio of wheat bran to corn flour in the solid-state fermentation medium was 3:1, and the amount of water added was 60% of the dry weight of the medium.

[0016] (2) Formulation preparation

[0017] The fermentation product and adsorbent are mixed, ground evenly, and then placed into a breathable bag to obtain the final product. The adsorbent is zeolite powder. The mass ratio of the fermentation product to the adsorbent is 1:3.

[0018] The microbial inoculant can be applied by foliar spraying, root dipping, root irrigation, or by placing it in the strawberry growing environment.

[0019] This invention also provides the application of one or more of the following components in the prevention and control of strawberry diseases:

[0020] (1) The Trichoderma longifolia strain ZN25;

[0021] (2) The conidia, mycelium and / or metabolites of the Trichoderma longifolia strain ZN25;

[0022] (3) The microbial inoculant. When the microbial inoculant contains conidia of the *Trichoderma longicornis* strain ZN25, the concentration of conidia of *Trichoderma longicornis* strain ZN25 in the microbial inoculant is not less than 1 × 10⁻⁶. 6 cfu / mL, at this point the microbial agent is a liquid preparation.

[0023] This invention also provides a strawberry disease control agent, which contains one or more of the following active ingredients:

[0024] (1) The Trichoderma longifolia strain ZN25;

[0025] (2) The conidia, mycelium and / or metabolites of the Trichoderma longifolia strain ZN25;

[0026] (3) The microbial agent mentioned above.

[0027] The strawberry diseases mentioned are strawberry anthracnose caused by Anthracnose sicca and / or strawberry root rot caused by Fusarium oxysporum.

[0028] The beneficial effects of this invention are as follows:

[0029] The *Trichoderma longicornis* of this invention exhibits significant inhibitory effects on *Anthracis siamensis* in three aspects: competition for nutrients and space, hyperparasitism, and volatile metabolism, with a particularly significant effect on hyperparasitism. In controlling *Fusarium oxysporum*, the *Trichoderma longicornis* of this invention also shows significant antibacterial effects on non-volatile metabolites in addition to the above three aspects.

[0030] This invention achieves a 72% control efficacy against anthracnose by foliar spraying of Trichoderma longifolia on strawberry plants, and a 58% control efficacy against root rot by root dipping treatment. Attached Figure Description

[0031] Figure 1 Colony morphology of strain ZN25 on PDA medium (incubated at 25 ℃ for 7 days);

[0032] Figure 2 Morphological images of conidia and conidiophores of strain ZN25 (optical microscope, 1000×).

[0033] Figure 3Phylogenetic tree constructed based on TEF1-RBP2 gene sequences;

[0034] Figure 4 Figure 1: Plate confrontation test results of strain ZN25 against strawberry pathogens; A: Inhibitory effect of strain ZN25 on *Anthracis sicca*; B: Inhibitory effect of strain ZN25 on *Fusarium oxysporum*; C: *Anthracis sicca*; D: *Fusarium oxysporum*.

[0035] Figure 5 A: Inhibitory effect of volatile substances from strain ZN25 on strawberry pathogens; B: Inhibitory effect of volatile substances from strain ZN25 on *Anthracis sicca*; C: *Anthracis sicca*; D: *Fusarium oxysporum*.

[0036] Figure 6 A: Inhibitory effect of fermentation metabolites of strain ZN25 on strawberry pathogens; B: Inhibitory effect of fermentation metabolites of strain ZN25 on *Anthracis sicca*; C: *Anthracis sicca*; D: *Fusarium oxysporum*.

[0037] Figure 7 A: Hyperparasitic effect of strain ZN25 on strawberry pathogens; B: Hyperparasitic effect of strain ZN25 on *Anthracis sicca*; C: Hyperparasitic effect of strain ZN25 on *Fusarium oxysporum*. Arrows indicate that *Trichoderma longipes* hyphae entwine with pathogen hyphae, while *Anthracis sicca* hyphae are broken.

[0038] Figure 8 : Pot experiment results of strain ZN25 in controlling strawberry anthracnose. (First row: ZN25(5d) + Colletotrichum siamense, Second row: H2O(5d) + H2O, Third row: H2O(5d) + Colletotrichum siamense); Arrows indicate strawberry anthracnose lesions.

[0039] Preservation Information

[0040] Preserved biological material: Trichoderma longifolia ZN25;

[0041] Accession number: CGMCC No. 42426;

[0042] Classification and naming: Trichoderma longibrachiatum;

[0043] Preservation institution: China General Microbiological Culture Collection Center, China Committee on the Preservation and Management of Microbial Culture Collections;

[0044] Address of the depository: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing;

[0045] Preservation date: January 9, 2026. Detailed Implementation

[0046] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions in the embodiments of this invention will be clearly described below. Obviously, the described embodiments are only some, not all, of the embodiments of this invention. Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this invention.

[0047] The strawberry pathogens used in the examples and their sources: Fusarium oxysporum and Colletotrichum siamense were both isolated from the Zhengzhou Municipal Crop Variety Testing Center and provided by the Strawberry Research Group of Zhengzhou Academy of Agricultural Sciences.

[0048] The strawberry variety 'Ninglu' used in this example was provided by the Strawberry Research Group of Zhengzhou Academy of Agricultural Sciences.

[0049] The PDA culture medium used in the examples consisted of 200 g potato, 20 g glucose, 20 g agar, and 1000 mL distilled water.

[0050] PD medium: 200 g potato, 20 g glucose, 1000 mL distilled water.

[0051] CMA medium: 20 g corn flour, 20 g agar, 1000 mL distilled water.

[0052] Example 1

[0053] I. Isolation and Identification of Strains

[0054] 1. Strains Isolation

[0055] (1) Sample collection

[0056] Petiole tissues of the main strawberry variety 'Ninglu' cultivated in Zhengzhou were collected and rinsed three times with sterile water to remove surface impurities.

[0057] (2) Surface disinfection

[0058] Cut strawberry petiole tissue into 3-5 mm long segments, soak them in 75% ethanol solution for 1-2 min, disinfect them in 0.1% mercuric chloride solution for 3-4 min, and finally rinse them with sterile water 2-3 times to remove surface disinfectant residue.

[0059] (3) Isolation and culture

[0060] Disinfected strawberry petiole tissue segments were inoculated onto PDA medium plates and cultured in the dark at 25 ℃ for 3-5 days. After mycelia grew on the plates, edge mycelia were picked and transferred to new PDA plates for purification culture to obtain a purified strain, which was designated as ZN25.

[0061] 2. Strain identification

[0062] (1) Morphological characteristics

[0063] Purified strain ZN25 with a diameter of 5 mm was inoculated onto PDA medium and cultured in the dark at a constant temperature of 25 °C. Observations showed that purified strain ZN25 grew rapidly in a radial pattern on PDA medium. After 24 h of inoculation, ZN25 colonies reached a diameter of 6 cm and began to conjugate, appearing dark green. The surface of the ZN25 colonies was velvety, without obvious alternating concentric rings. The conjugation zone expanded outward from the inoculation point, surrounded by a mycelial growth zone, gradually spreading to the entire culture dish. A distinct yellow pigment was observed on the reverse side of the medium. Figure 1 The mycelium is colorless and transparent, septate, and grows rapidly; conidia are elliptical, pale green, and 3.2-4.5 μm × 1.8-2.5 μm in size; conidiophores are ampoule-shaped; chlamydospores can be produced during culture (…). Figure 2 ).

[0064] (2) Molecular biological identification

[0065] The ZN25 mycelium blocks to be identified were inoculated into PD medium and cultured at 25 ℃ and 150 r / min for 48 h. Mycelia approximately the size of mung beans were collected, frozen in liquid nitrogen, and ground into powder. Total DNA was extracted using a fungal DNA genomic extraction kit (OmegaBio-Tek). The TEF1 and RBP2 gene sequences were amplified by PCR using primer pairs TEF1-728F / TEF1-LLErev and fRPB2-5f / fRPB2-7cr, respectively. The primer sequences used are as follows:

[0066] TEF1-728F: 5'-CATCGAGAAGTTCGAGAAGG-3' (SEQ ID NO: 1);

[0067] TEF1-LLErev: 5'-AACTTGCAGGCAATGTGG-3' (SEQ ID NO: 2);

[0068] fRPB2-5f: 5'-GAYGAYMGWGAYCAYTTYGG-3' (SEQ ID NO: 3);

[0069] fRPB2-7cr: 5'-CCCATRGCTTGYTTRCCCAT-3' (SEQ ID NO: 4).

[0070] The PCR products were purified and sent to BGI Genomics Zhengzhou Branch for sequencing. The TEF1 and RBP2 gene sequences of strain ZN25 were obtained, as shown in SEQ ID NO:5 and SEQ ID NO:6, respectively. BLAST alignment of the sequencing results in GenBank showed that the TEF1 and RBP2 gene sequences had 99.58% (1174bp / 1179bp) and 99.82% (1122bp / 1124bp) homology with *Trichoderma longibrachiatum* strain HL167, respectively. A phylogenetic tree was constructed using the maximum likelihood method based on the TEF1-RBP2 DNA tandem sequences in MEGA 10.2.6 software. Figure 3 The results showed that strain ZN25 aggregated with Trichoderma longibrachiatum HL167.

[0071] In summary, strain ZN25 was identified as *Trichoderma longibrachiatum*.

[0072] II. Flat Plate Standoff Test

[0073] The plate confrontation method was used. Three-day-old (5 mm diameter) Trichoderma longifolia (ZN25) mycelial discs and seven-day-old (5 mm diameter) strawberry pathogen mycelial discs (Fusarium oxysporum and Anthracnoseus sicca) mycelial discs were transferred to opposite sides of 9 cm diameter PDA plates, with a straight-line distance of 4 cm. Plates inoculated only with pathogen mycelial discs served as controls. After incubation at 25 ℃ for 7 days, the radius of the pathogens in the treated and control groups was measured to calculate the inhibition rate. The experiment was conducted in triplicate. Inhibition rate (%) = (Control colony radius - Treated colony radius) / Control colony radius × 100%.

[0074] The results showed that *Trichoderma longicornis* had an inhibition rate of 78% against *Anthracis sinensis* and 81% against *Fusarium oxysporum*. Figure 4 It can be seen that the hyphae of *Trichoderma longicornis* in the treatment group wrapped around and intertwined with the *Anthracis siamense* colony, gradually spreading towards the center of the *Anthracis siamense* colony, and producing a large number of conidia on the *Anthracis siamense* colony. Looking at the reverse side of the culture medium, a dark green hyphal disintegration zone was also formed at the junction of the two colonies, indicating that the hyphae of *Trichoderma longicornis* had a certain destructive effect on the hyphae of *Anthracis siamense*. Figure 4 It can be seen that during the confrontation between Trichoderma longifolia and Fusarium oxysporum colonies, Trichoderma longifolia wraps around and produces a large number of conidia at the edge of the Fusarium oxysporum colony, but does not spread towards the center of the colony.

[0075] III. Analysis of Volatile Gases

[0076] 1. Detection of the antibacterial effect of volatile metabolites of Trichoderma longifolia

[0077] Three-day-old (5 mm diameter) Trichoderma longifolia (ZN25) mycelial cakes and seven-day-old (5 mm diameter) mycelial cakes of strawberry pathogens (Fusarium oxysporum and Anthracnoseus sicca) were inoculated into the center of 9 cm diameter PDA plates. The edges of the Trichoderma longifolia and pathogen plates were aligned and the openings were joined together, with Trichoderma longifolia in the lower layer and pathogens in the upper layer. The plates were then sealed with triple-layer sealing film and incubated at 25 ℃ without being moved. A plate inoculated solely with pathogens served as a control. After 7 days of incubation, the sealing film was opened, and the radius of the pathogens in both the treatment and control groups was measured to calculate the inhibition rate. The experiment was conducted in triplicate. Inhibition rate (%) = (Control colony radius - Treatment colony radius) / Control colony radius × 100%.

[0078] The results showed that the volatile metabolites of *Trichoderma longicornis* in this embodiment could inhibit *Fusarium oxysporum* and *Anthracis sicca*. The average inhibition rates of *Trichoderma longicornis* against *Anthracis sicca* and *Fusarium oxysporum* were 35% and 53%, respectively. Figure 5 It can be seen that the volatile compounds in the treated Trichoderma longifolia caused extensive pigment deposition in the *Anthracis siamensis* colonies, abnormal mycelial growth, decreased activity, and the inability to produce conidia. Additionally, from Figure 5 It can be seen that the volatile compounds of Trichoderma longifolia in the treatment group can cause Fusarium oxysporum hyphae to not spread to the surrounding area, the hyphae at the edge are sparse, and the growth rate is extremely slow.

[0079] 2. Detection of the antibacterial effect of non-volatile metabolites of Trichoderma longifolia

[0080] Fresh *Trichoderma longicornis* (ZN25) mycelial cakes were inoculated into 200 mL of PD medium and cultured at 25 ℃ with shaking at 150 r / min for 4 days. After centrifuging the *Trichoderma longicornis* fermentation broth at high speed for 2 min, the supernatant was collected and filtered through a 0.22 μm microporous membrane to obtain sterile fermentation broth. The sterile fermentation broth was mixed with PDA medium at a ratio of 10% and then poured onto plates. Fresh strawberry pathogens (*Fusarium oxysporum* and *Anthracis siamba*) mycelial cakes with a diameter of 5 mm were inoculated into the center of each plate and cultured at 25 ℃. A control was prepared by inoculating pathogens onto plates mixed with sterile distilled water and PDA medium. After 7 days of culture, the sealing film was opened, and the radius of the pathogens in the treatment and control groups was measured to calculate the inhibition rate. The experiment was set up with 3 replicates. Inhibition rate (%) = (control colony radius - treatment colony radius) / control colony radius × 100%.

[0081] The results showed that the fermentation metabolites of *Trichoderma longicornis* in the treatment group had a very weak inhibitory effect on *Anthracis sinensis*, with an average inhibition rate of approximately 8%. The fermentation metabolites of *Trichoderma longicornis* in the treatment group had a better inhibitory effect on *Fusarium oxysporum*. Figure 6 The average inhibition rate was 49%.

[0082] 3. HS-SPME / GC-MS analysis of volatile metabolites of Trichoderma longifolia

[0083] Volatile metabolites produced by *Trichoderma longiflorum* (ZN25) after culturing on PDA medium at 25 °C for 5 days were isolated and identified using headspace solid-phase microextraction combined with gas chromatography-mass spectrometry (HS-SPME / GC-MS). Specifically, 5 mL of heated PDA medium was poured into a 20 mL sterile headspace extraction flask and cooled. *Trichoderma longiflorum* was then inoculated onto the slant and incubated at 25 °C for 5 days. The headspace extraction flask was then placed in a 50 °C water bath for 20 min. The solid-phase extraction head was then inserted into the headspace extraction flask for extraction and adsorption. After adsorption, the extraction head was then placed in a GC-MS system for desorption. The specific analytical conditions are as follows: the chromatographic column is an HP-5MS capillary column (30 m × 0.25 mm × 0.25 μm); the injection port temperature is 250 ℃; the carrier gas is helium, and the flow rate is 1.0 mL / min; the temperature program is as follows: initial temperature 50 ℃, hold for 3 min, increase to 200 ℃ at 5 ℃ / min, hold for 5 min, then increase to 280 ℃ at 10 ℃ / min, hold for 3 min; the injection volume is 1 μL, the split ratio is 10:1; the mass spectrometer detector is an EI source with an electron energy of 70 eV and a scan range of 35-500 m / z.

[0084] The results showed that a total of 60 chromatographic peaks were detected in the volatile metabolites of Trichoderma longifolia, of which 59 compounds were identifiable. After screening, approximately 36 classes of major active VOCs were identified, among which 8 substances were closely related to antibacterial activity. Specific information is shown in Table 1 below:

[0085] Table 1. Eight volatile gaseous substances produced by Trichoderma longifolia

[0086]

[0087] IV. Hyperparasitic phenomenon of Trichoderma longifolia

[0088] A 7-day-old strawberry pathogen mycelium with a diameter of 5 mm was inoculated onto one side of a 9 cm diameter CMA plate and incubated at 25 ℃ for 36 h. Then, a 3-day-old Trichoderma longifolia (ZN25) mycelium with a diameter of 5 mm was inoculated onto the same CMA plate 4 cm away from the inoculation point of the strawberry pathogen. The plate was then incubated at 25 ℃. When the distance between the two colonies was 5 mm, a 5 mm * 5 mm colony was taken from the middle of the intersection. The colony sample was fixed to the sample holder with cryo-glue and rapidly frozen in supercooled liquid nitrogen snow. Under vacuum conditions, the sample was transferred to the cold stage of the preparation chamber installed at the port of the scanning electron microscope sample chamber. After cryo-sublimation etching and sputtering coating, the sample was finally transferred to the cold stage in the scanning electron microscope sample chamber for ultrastructural observation.

[0089] From scanning electron microscope images ( Figure 7 As can be seen, there is extensive adhesion and entanglement at the contact points between *Trichoderma longicornis* and the pathogen, indicating that *Trichoderma longicornis* exhibits hyperparasitic activity against both pathogens. Numerous instances of *Anthracis sicca* cell wall and cell membrane disruption and hyphal breakage were observed on the co-cultured CMA plates, while these phenomena were not observed on other CMA plates. This demonstrates that *Trichoderma longicornis* possesses a strong hyperparasitic ability against *Anthracis sicca*.

[0090] V. Pot Experiment

[0091] 1. Preparation of spore suspension

[0092] Trichoderma longifolia (ZN25) was inoculated onto PDA medium and cultured at 25 °C for 7 days. The spores were then washed away with sterile water to form a spore suspension, which was adjusted to a concentration of 1 × 10⁻⁶. 6 CFU / mL available for use.

[0093] Anthrax mebacillus was inoculated into 150 mL of PD liquid medium and cultured for 7 days at 25 °C and 150 r / min in a shaker. The spores were then filtered through four layers of sterile gauze and adjusted to a concentration of 1×10⁻⁶. 6 A spore suspension of CFU / mL was prepared for subsequent use. Fusarium oxysporum mycelium was inoculated into 150 mL of PD medium and cultured for 7 days at 25 °C and 150 r / min in a shaker. Spores were filtered through four layers of sterile gauze and adjusted to a concentration of 1×10⁻⁶. 6 A spore suspension of cfu / mL is provided for subsequent use.

[0094] 2. Detection of the biocontrol ability of Trichoderma longifolia (ZN25) against strawberry diseases.

[0095] (1) Prevention and control of anthrax

[0096] Healthy 'Ninglu' strawberry tissue culture seedlings with similar growth and height of about 10-15 cm were selected and transplanted into a cultivation substrate of peat moss:vermiculite:perlite = 3:1:1. After the seedlings were stabilized, the following three experimental treatments were carried out by foliar spraying: (1) inoculated with distilled water for 5 days and then inoculated with distilled water again; (2) inoculated with distilled water for 5 days and then inoculated with anthracnose spore suspension; (3) inoculated with Trichoderma longifolia spore suspension for 5 days and then inoculated with anthracnose spore suspension. The seedlings were then cultured in light and dark conditions (16 h / 8 h) at 28 ℃ and humidity above 85%. After 10 days, the disease status of the leaves was graded. Five replicates were set up for each group, and the experiment was repeated 3 times. A grading standard was established based on the number of leaf lesions: Grade 0, 0 leaf lesions; Grade 1, 0-10 leaf lesions; Grade 2, 10-20 leaf lesions; Grade 3, 20-30 leaf lesions; Grade 4, 30 or more leaf lesions. Disease index = ∑(number of diseased leaves at each level × number of disease levels) / (total number of leaves surveyed × 4) × 100. Control effect (%) = (disease index of control area - disease index of treatment area) / disease index of control area × 100%.

[0097] The incidence of anthrax was observed in each group 7 days after inoculation. The results showed that ( Figure 8 Strawberry plants treated with distilled water showed no black spots on their leaves. However, strawberry plants sprayed with *Anthracnose spores* suspension five days after distilled water treatment all developed anthracnose symptoms, with 3-40 black spots appearing on their leaves. The disease index was as high as 37.558, significantly higher than other treatments. Strawberry plants sprayed with *Trichoderma longicornis* spore suspension five days after treatment showed significantly reduced anthracnose symptoms. The disease index after treatment with *Trichoderma longicornis* spore suspension was 10.341, achieving a control effect of 72.257%.

[0098] (2) Prevention and control of root rot

[0099] Healthy 'Ninglu' strawberry tissue culture seedlings with similar growth and a height of about 10-15 cm were selected. The following three experimental treatments were carried out using root dipping: (1) Tissue culture strawberry seedlings were dipped in distilled water for 5 min before transplanting, and 10 mL of distilled water was applied to the shortened stem area after 7 days; (2) Tissue culture strawberry seedlings were dipped in distilled water for 5 min before transplanting, and 10 mL of Fusarium oxysporum spore suspension was applied to the shortened stem area after 7 days; (3) Tissue culture strawberry seedlings were dipped in Trichoderma longifolium spore suspension for 5 min before transplanting, and 10 mL of Fusarium oxysporum spore suspension was applied to the shortened stem area after 7 days. The substrate used for transplanting was a cultivation substrate of peat moss, vermiculite and perlite in a 3:1:1 ratio. After transplanting, the seedlings were placed at 25 ℃ and under light and dark conditions (16 h / 8 h). After 30 days, the disease status of the strawberry seedlings was observed. Each treatment consisted of 5 pots, with 3 replicates. The degree of necrosis of the vascular bundles in the shortened stem is classified into four levels: Level 0 is when the vascular bundles are normal in color and there are no external symptoms; Level 1 is when less than 1 / 4 of the vascular bundles are discolored; Level 2 is when 1 / 4 to 1 / 2 of the vascular bundles are discolored; Level 3 is when 1 / 2 to 3 / 4 of the vascular bundles are discolored; and Level 4 is when more than 3 / 4 of the vascular bundles within the stem are discolored. Disease index = ∑(number of roots at each level × vascular bundle necrosis level) / (total number of roots surveyed × 4) × 100. Control effect (%) = (disease index of control area - disease index of treatment area) / disease index of control area × 100%.

[0100] Thirty days after inoculation, the disease incidence of strawberry seedlings in each group was observed. The results showed that strawberry plants treated with distilled water did not exhibit yellowing or wilting of leaves, and the vascular bundles of the roots showed a slightly yellowish-white color on the cross-section. Strawberry plants treated with distilled water and then inoculated with *Fusarium oxysporum* spore suspension mostly showed slower growth, withered leaf edges, wilting of some older leaves, browning of vascular bundle edges, and reddish-brown patches on some cross-sections. The disease index was as high as 36.667, significantly higher than other treatments. Strawberry plants treated with *Trichoderma longicornis* spore suspension and then inoculated with *Fusarium oxysporum* spore suspension showed significantly reduced root rot symptoms, and some even exhibited vigorous growth. The disease index after treatment with *Trichoderma longicornis* spore suspension was 15.000, with a control effect of 58.333%.

[0101] VI. Preparation and application of antibacterial agents based on the volatile gases of Trichoderma longifolia (ZN25)

[0102] (1) Culture of strains

[0103] Trichoderma longifolia was inoculated into a solid fermentation medium (wheat bran: corn flour = 3:1, with water added at 60% of the dry weight of the medium) and fermented at 25 °C for 7 days to obtain the fermentation product.

[0104] (2) Formulation preparation

[0105] The fermentation product and the adsorbent (zeolite powder) are mixed at a mass ratio of 1:3, ground thoroughly and evenly, and then packed into a breathable cloth bag to make a volatile gas antibacterial pack.

[0106] (3) How to use

[0107] Place the antibacterial packs inside the strawberry greenhouse, every 10 m... 2 Place one container and replace it regularly (every 15 days). It inhibits the growth of pathogens by releasing volatile gases, thus helping to prevent and control strawberry anthracnose and root rot.

[0108] 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 of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A *Trichoderma longicornis* strain ZN25 exhibiting broad-spectrum resistance to strawberry diseases, characterized in that... The longibrachiatum strain ZN25 is classified as Trichoderma longibrachiatum and has the accession number CGMCC No. 42426.

2. A microbial inoculant, characterized in that, The microbial agent contains the Trichoderma longifolia strain ZN25.

3. The microbial inoculant according to claim 2, characterized in that, The microbial agent contains conidia, mycelium and / or metabolites of the Trichoderma longifolia strain ZN25.

4. The microbial inoculant according to claim 3, characterized in that, The metabolites are volatile metabolites.

5. The microbial inoculant according to claim 2, characterized in that, The microbial agent is in the form of a wettable powder, suspension, or volatile antibacterial packet.

6. A microbial inoculant according to claim 5, characterized in that, The preparation method of the volatile antibacterial pack includes: Step 1: Inoculate *Trichoderma longifolia* onto a solid-state fermentation medium and ferment at 25 °C for 7 days to obtain the fermentation product; the mass ratio of wheat bran to corn flour in the solid-state fermentation medium is 3:1, and the amount of water added is 60% of the dry weight of the medium; Step 2: Mix the fermentation product with the adsorbent, grind them evenly, and then put them into a breathable bag to obtain the final product.

7. A microbial inoculant according to claim 6, characterized in that, The adsorbent mentioned in step 2 is zeolite powder.

8. The application of one or more of the following ingredients in the prevention and control of strawberry diseases, characterized in that, (1) The Trichoderma longifolia strain ZN25 according to claim 1; (2) Conidia, mycelium and / or metabolites of the Trichoderma longifolia strain ZN25 as described in claim 1; (3) The microbial agent according to any one of claims 2-7; the strawberry disease is strawberry anthracnose caused by Anthracnose sicca and / or strawberry root rot caused by Fusarium oxysporum.

9. The application according to claim 8, characterized in that, When the microbial inoculant contains conidia of the *Trichoderma longicornis* strain ZN25, the concentration of conidia of *Trichoderma longicornis* strain ZN25 in the microbial inoculant is not less than 1 × 10⁻⁶. 6 cfu / mL.

10. A strawberry disease control agent, characterized in that, The drug contains one or more of the following active ingredients: (1) The Trichoderma longifolia strain ZN25 according to claim 1; (2) Conidia, mycelium and / or metabolites of the Trichoderma longifolia strain ZN25 as described in claim 1; (3) The microbial agent according to any one of claims 2-7; the strawberry disease is strawberry anthracnose caused by Anthracnose sicca and / or strawberry root rot caused by Fusarium oxysporum.

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