Trichoderma hedgehog JK1-1 and application of Trichoderma hedgehog JK1-1 as biocontrol bacterium in prevention and treatment of plant diseases

Abstract

The invention discloses a Trichoderma hedgehog JK1-1 strain and an application of the Trichoderma hedgehog JK1-1 strain as a biocontrol bacterium in prevention and treatment of plant diseases. The Trichoderma erinaceum JK1-1 provided by the invention is separated from field soil, is easy to culture and strong in adaptability, and is a very safe biological control mediator; experiments prove that the trichoderma hedgehog JK1-1 and the volatile substances thereof have broad-spectrum antibacterial ability, have good prevention and treatment effects on main diseases of various crops, and particularly have outstanding inhibition effects on in-vitro botrytis cinerea and prevention and treatment effects on in-vivo tomato botrytis cinerea. The strain is used as a biocontrol fungus for preventing and treating plant diseases, a new biocontrol choice can be provided for preventing and treating the plant diseases, the use of chemical pesticides is reduced, and volatile metabolites of the strain are expected to play a role in preventing and treating botrytis cinerea after harvesting and storing and preserving fruits and vegetables after harvesting to replace compounds harmful to the environment and human bodies.

Description

Technical Field

[0001] The present invention relates to the technical field of microorganism and plant disease prevention and control, and in particular to a Trichoderma ergifer JK1-1 and its application as a biocontrol bacterium in preventing and controlling plant diseases. Background Art

[0002] Gray mold (Botrytis cinerea), also known as Botrytis cinerea, is a broad-host pathogen that causes a variety of plant diseases. It primarily targets crops such as tomatoes, citrus fruits, peppers, eggplants, cucumbers, grapes, strawberries, and rapeseed. Botrytis cinerea is widely distributed in the air and can not only infect field crops but also cause significant losses in the postharvest stage. As a globally cultivated and important crop, tomatoes occupy a crucial position in the vegetable market. Their rich supply of vitamins, minerals, and antioxidants positively impacts human health. Postharvest tomatoes are highly susceptible to gray mold. This fungal disease, caused by Botrytis cinerea, can rapidly spread across the leaves, stems, flowers, and fruits of tomatoes under favorable conditions of temperature and high humidity. The diseased areas manifest as water-soaked lesions and a gray mold layer, significantly affecting the appearance of tomatoes and causing fruit rot. It also degrades their nutritional content, significantly reducing their edible and economic value.

[0003] Traditional chemical pest control methods pose challenges such as pathogen resistance, pesticide residues, and potential harm to the environment and human health. Biological control, as a green, environmentally friendly, and sustainable disease control strategy, has garnered increasing attention. In recent years, a wealth of biocontrol resources have been discovered, and numerous strains have been shown to exhibit significant inhibitory effects against gray mold. Trichoderma (Trichoderma sp.) is a versatile biocontrol agent (BCA) that exerts its biocontrol effects through both direct and indirect pathways. Trichoderma can directly inhibit the growth of pathogens and disrupt their cell structure through parasitism, niche competition, and the production of secondary metabolites. It can also indirectly promote plant growth, stimulate plant immunity, and modulate rhizosphere soil microbial communities. Trichoderma possesses enormous biocontrol potential due to its strong adaptability, wide range of effects, and diverse mechanisms of action. VOCs, due to their low molecular weight, high volatility, rapid diffusion, and ability to control a wide range of pathogens, have become a research hotspot in biocontrol. The production of volatile and non-volatile secondary metabolites is a key characteristic of Trichoderma, playing an important and effective role in inhibiting plant pathogens and promoting plant growth. Utilizing fungal volatile organic compounds (VOCs) to inhibit pathogen growth is an environmentally friendly strategy. Therefore, identifying highly effective Trichoderma strains and developing products that leverage their unique characteristics is crucial. Summary of the Invention

[0004] The purpose of the present invention is to address the above problems and provide a strain of Trichoderma ergillus JK1-1 and its use as a biocontrol bacterium in preventing and controlling plant diseases.

[0005] In order to achieve its purpose, the present invention adopts the following technical solutions:

[0006] The first aspect of the present invention provides a strain of Trichoderma erinaceum JK1-1, which is deposited in the General Microbiology Center of China Culture Collection Administration of Microorganisms with a deposit registration number of CGMCC No.41830.

[0007] A second aspect of the present invention provides a bacterial agent, the active ingredient of which comprises the above-mentioned Trichoderma juganata JK1-1 or its fermentation broth.

[0008] The activation culture method of Trichoderma jugendig JK1-1 is as follows: culturing in a culture medium at 15° C. to 33° C.

[0009] Preferably, the culture medium is PDA culture medium; preferably, the culture temperature is 25° C. to 33° C., and the culture is carried out on a plate for 1 to 7 days or 3 to 5 days.

[0010] A third aspect of the present invention provides a product for inhibiting plant pathogens or preventing and controlling plant diseases, wherein the active ingredient comprises the volatile organic compounds produced by the Trichoderma juganata JK1-1 according to claim 1.

[0011] The volatile organic compounds include one or more of citral, 2-methylvaleric acid, 4-methylvaleric acid, phenyl acetate, acetophenone, angelica lactone, ethyl phenylacetate, methyl benzoate, 2,3-butanedithiol, (-)-4-terpineol, 2,6-dimethyl-2-heptanol, benzyl acetate, methyl heptanoate, 2-octanol, ethyl sorbate, and diisopropyl adipate.

[0012] A fourth aspect of the present invention provides the use of the Trichoderma erinaceum JK1-1, the bacterial agent, or the product in any of the following applications:

[0013] (1) Application in inhibiting Botrytis cinerea or preventing and treating plant gray mold;

[0014] (2) Application in the preparation of products for inhibiting Botrytis cinerea or preventing and treating plant gray mold.

[0015] In the application, the plants include tomatoes, citrus, peppers, eggplants, cucumbers, grapes, strawberries, and rapeseed.

[0016] A fifth aspect of the present invention provides the use of the Trichoderma erinaceum JK1-1, the bacterial agent, or the product in any of the following applications:

[0017] (1) Use in inhibiting plant pathogenic fungi or in preventing and controlling plant diseases caused by said plant pathogenic fungi;

[0018] (2) Use in the preparation of products for inhibiting plant pathogenic fungi or preventing and treating plant diseases caused by said plant pathogenic fungi.

[0019] The plant pathogenic fungi include Penicillium italicum, the pathogen of citrus anthracnose, Colletrichum litchi Trag, the pathogen of mango / pepper anthracnose, Colletotrichum acutatum, the pathogen of strawberry anthracnose, Colletotrichum fragariae, the pathogen of cucumber wilt, Fusarium oxysporum (Schl.) F.sp cucumerinum Owen.anamorph, the pathogen of wheat fusarium graminearum Sehw, and the pathogen of banana wilt, Fusarium oxysporum f.sp.cubense.

[0020] The beneficial effects of the present invention are as follows: the Trichoderma erinaceum JK1-1 of the present invention is isolated from field soil, is easy to culture, has strong adaptability, and has not undergone any genetic modification, so it will not cause any hidden dangers to the ecological environment when released into the natural world, and is a very safe biological control medium; research has confirmed that the Trichoderma erinaceum JK1-1 of the present invention and its volatile substances have broad-spectrum antibacterial capabilities, and at the same time have good preventive and control effects on a variety of major crop diseases, and the inhibitory effect on in vitro gray mold and the preventive effect on tomato gray mold in vivo are particularly outstanding. Using it as a biocontrol fungus for the prevention and control of plant diseases can provide a new biocontrol option for the prevention and control of plant diseases, reduce the use of chemical pesticides, and its volatile metabolites are expected to play a role in the prevention and control of post-harvest gray mold and the storage and preservation of fruits and vegetables after harvest, replacing compounds harmful to the environment and human body. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 These are the morphological characteristics of strain JK1-1 on PDA culture medium, where A and B represent colony morphology (front and back), C represents hyphae morphology, and D represents spore morphology.

[0022] Figure 2 This is the TEF1 gene sequence of strain JK1-1. The phylogenetic tree was constructed using the neighbor-joining method with Saccharomyces as the outgroup. A is the electrophoresis gel image of the fragment, and B is the phylogenetic tree.

[0023] Figure 3 The ANI values ​​of strain JK1-1 based on the sequence alignment of Trichoderma Scaffold and other similar species identified by KmerFinder were calculated (using the ANI tool OAT software to perform OrthoANI algorithm and BLAST calculation).

[0024] Figure 4These are the results of the salt and alkali tolerance experiment of strain JK1-1.

[0025] Figure 5 These are the results of the temperature and pH tolerance experiments of strain JK1-1, where A is the temperature tolerance phenotype, B is the pH tolerance phenotype, and C and D are the inhibition rates at different temperatures and pH values, respectively.

[0026] Figure 6 These are the results of the siderophore production test of strain JK1-1.

[0027] Figure 7 These are the results of the carbon and nitrogen source utilization ability experiment of strain JK1-1.

[0028] Figure 8 These are the results of the broad-spectrum antifungal ability of strain JK1-1 in confrontation culture with 9 pathogens including tomato gray mold. A is the phenotypic inhibition rate graph of the 8 pathogens, B is the phenotypic result graph of gray mold, and C is the inhibition rate of gray mold.

[0029] Figure 9 These are the experimental results of the inhibition of volatile substances of strain JK1-1 on the growth of mycelium of Botrytis cinerea by co-culture, where A is the phenotype, B is the inhibition rate, and C is the electron micrograph of mycelium.

[0030] Figure 10 Figure 1 is the control effect of strain JK1-1 on tomato fruit inoculated with gray mold, where A is the control phenotype, B is the lesion diameter, C is the soluble solids content, D is the VC content, and E is the titratable acid content.

[0031] Figure 11 This is a pie chart showing the classification percentages of volatile substances produced by strain JK1-1.

[0032] Figure 12 The inhibitory effects of 32 screened volatile single substances on Botrytis cinerea at concentrations of 500 μL / L, 200 μL / L, and 100 μL / L (arranged from left to right in the figure). DETAILED DESCRIPTION

[0033] The present invention will be further described below with reference to the embodiments, but the present invention is not limited thereto.

[0034] The experimental methods in the following examples are conventional methods unless otherwise specified.

[0035] The culture medium used in the examples is:

[0036] PDA medium: 200 g potatoes, 20 g glucose, 20 g agar, dilute to 1 L, and autoclave at 121°C for 30 min.

[0037] Example 1 Identification of strain JK1-1

[0038] 1. Isolation of strain JK1-1

[0039] In April 2023, rhizosphere soil from a cherry tomato plantation in Wulie Town, Dongfang City, Hainan Province, was selected from plots severely affected by gray mold. Soil was collected from the surrounding areas of the plant in relatively mildly affected fields. Surface soil was removed, and 10 g of soil from the root zone was collected. The collected soil was placed in a sterile Erlenmeyer flask, 100 mL of sterile water was added, and the mixture was shaken thoroughly and allowed to stand for 10 min. The upper liquid was aspirated for a concentration gradient dilution. 100 μL of each dilution was evenly spread on polydimethylsiloxane (PDA) solid medium and incubated at 26°C for 3 days until colonies emerged. Colonies were selected for expansion and purification three times. The pathogen of gray mold was screened using the plate standoff method. Botrytis cinerea was inoculated 1.5 cm from one side of a solid PDA medium. The strain to be screened was inoculated with a sterile toothpick on the other side. Simultaneously, the strain to be screened was inoculated on a fresh PDA medium for propagation and preservation. After 5 days, the effect of the confrontation culture was observed, and the antibacterial effects of the different strains screened were inferred based on the diameter of the inhibition zone. The most efficient control strain was obtained based on the antibacterial effect and was numbered as bacterial block JK1-1. It was stored in a -80°C refrigerator in 50% glycerol using the filter paper method.

[0040] 2. Morphological identification of strain JK1-1

[0041] The Trichoderma strain was inoculated onto a PDA plate and cultured at 25°C for 3 days. A mycelial agar block (5 mm in diameter) at the edge of the colony was taken with a borer and inoculated onto a PDA plate (20 mL / plate, plate diameter: 9 cm) to observe the colony morphology.

[0042] like Figure 1 Strain JK1-1, grown on PDA medium for 3-4 days, completely filled the plate. The colonies were white and radial, with flocculent aerial hyphae. They produced no pigment and a strong coconut aroma. Microscopic observation revealed conidiophores emerging from the side branches of the hyphae, and conidia were spherical. Based on their morphology, strain JK1-1 can be roughly classified as a Trichoderma genus.

[0043] 3. Molecular identification of strain JK1-1

[0044] JK1-1 cells that had grown normally for 4 days were collected, genomic DNA was extracted, and the TEF-1 gene was amplified using the primers in Table 1. The PCR reaction system was as shown in Table 1, and the amplification procedure was as shown in Table 3. The electrophoresis results of the PCR amplification products were as follows: Figure 2 A, The fragment size of the amplified product is between 500-750 bp, which is consistent with the normal fragment size of the TEF-1 sequence.

[0045] Table 1 PCR primers

[0046]

[0047] Table 2 PCR reaction system

[0048]

[0049] Table 3 PCR amplification program

[0050]

[0051] The PCR amplification product was sequenced, and the sequencing results showed that the TEF-1α gene sequence of strain JK1-1 (SEQ ID NO. 3) was as follows:

[0052]

[0053] The TEF-1α sequences of strain JK1-1 were compared for homology in NCBI, and the 10 sequences with the highest homology were downloaded. The phylogenetic tree was constructed using the neighbor-joining method using MEGA 11 software, as shown in Figure 2 B shows that strain JK1-1 and Trichoderma erinaceum CEN1420 are clustered together, indicating that strain JK1-1 may belong to the genus Trichoderma erinaceum. Combined with the bacterial morphology observation, the strain was preliminarily determined to be Trichoderma erinaceum.

[0054] 4. Whole-genome identification of strain JK1-1

[0055] The average nucleotide identity (ANI) of the whole genome sequencing of the strain JK1-1 from the Scaffold sequence alignment of Trichoderma was compared with other species of similar genera identified by KmerFinder, and a threshold of less than or equal to 95% was used as a reference for identification as a new species. Figure 3 It was found that the similarity threshold between JK1-1 and Trichoderma erinaceum BJ212-21 reached 97.85%, and it was identified as Trichoderma erinaceum.

[0056] In summary, strain JK1-1 was identified as Trichoderma erinaceum by polyphasic taxonomy.

[0057] The deposit information of strain JK1-1 is as follows:

[0058] The strain JK1-1 was sent to the General Microbiology Center of the China Microorganism Culture Collection Administration (CGMCC) for preservation in March 2025. The address of the depository unit is: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing; the deposit date is March 14, 2025, the deposit number is CGMCC No.41830, and the classification name is Trichoderma erinaceum.

[0059] Example 2 Growth and adaptability experiment of strain JK1-1

[0060] 1. Salt and alkali tolerance of strain JK1-1

[0061] 5mm cakes of strain JK1-1 were transferred to PDA culture medium containing NaCl or NaHCO3, respectively. The gradient concentration (w / v) of NaCl was set as: 0%, 2%, 4%, 6%, 8%, 10%; the gradient concentration (w / v) of NaHCO3 was set as: 0%, 0.1%, 0.2%, 0.4%, 0.8%, 1%. After inoculation, the culture dish was inverted and cultured in a 25℃ incubator for 3-5 days. The results are shown in Figure 2. Figure 4 , strain JK1-1 can grow at NaCl concentrations between 0% and 10%, and its growth ability gradually weakens with increasing concentrations ( Figure 4 A, B), the colony can grow when the NaHCO3 concentration is 0% to 0.4% ( Figure 4 A, C), strain JK1-1 has a wide range of saline-alkaline adaptability.

[0062] 2. Temperature and pH tolerance of strain JK1-1

[0063] 1. Temperature resistance

[0064] 5mm JK1-1 bacterial cake was transferred to PDA medium and inverted in the incubator at 4℃, 15℃, 20℃, 25℃, 28℃, 30℃, 33℃ and 37℃ for 3 days in the dark. Figure 5 A. Strain JK1-1 can grow at temperatures between 15°C and 33°C, with an optimum temperature between 25°C and 33°C. It does not grow at all at 4°C and 37°C, and has a wide temperature adaptability range.

[0065] 2. pH resistance

[0066] The pH values ​​of the culture medium were adjusted to 3, 4, 5, 6, 7, 8, 9, and 10 respectively with 0.1 mol / L HCl and NaOH solutions, and 5 mmJK1-1 bacterial cakes were transferred to PDA culture medium with different pH values ​​in sequence and inverted in a 25°C incubator for 3 to 5 days. Figure 5 B. JK1-1 can grow between pH = 4-10, and the colony grows fastest at pH = 5. Its pH adaptability range is also relatively wide.

[0067] 3. Iron production capacity of strain JK1-1

[0068] The strain JK1-1 was inoculated into CAS test medium, pH = 7.4, sterilized at 115℃ for 20 minutes, and cultured at 26℃. Each treatment group was repeated 3 times. During the culture process, the colonies were continuously observed for the presence of an orange-pink transparent light ring around them. If a transparent light ring is formed, it proves that the antagonistic bacteria have the ability to produce siderophores. Figure 6JK1-1 formed an orange-pink transparent circle on the CAS culture medium, which proved that strain JK1-1 had the ability to produce siderophores, which could chelate iron in the soil by producing siderophores, thereby inhibiting the growth of pathogens in the soil.

[0069] 4. Ability of strain JK1-1 to utilize carbon and nitrogen sources

[0070] Solid culture media were prepared by replacing the carbon and nitrogen sources in Czapek Dox Agar with six different carbon sources (D-fructose, maltose, D-galactose, sucrose, glucose, and soluble starch) and six different nitrogen sources (ammonium sulfate, urea, peptone, ammonium nitrate, sodium nitrate, and yeast extract). Well-growing strain JK1-1 was isolated. Mycelial fragments (5 mm in diameter) were punched from the edge of the colony and inoculated into the center of a 90 mm Petri dish. Three plates were inoculated for each treatment, representing three replicates. The plates were incubated inverted at 26°C in the dark for 7 days, and the colony diameters were measured.

[0071] The results are as follows Figure 7 JK1-1 can grow on all six carbon sources, including D-fructose, and grows fastest on soluble starch. It can grow on all five nitrogen sources, including yeast extract, sodium nitrate, ammonium sulfate, ammonium nitrate, and peptone, but not on urea. Yeast extract and peptone show the fastest growth.

[0072] Example 3: Plate confrontation inhibition experiment of strain JK1-1 and pathogens of nine crop diseases including gray mold

[0073] The preserved strain JK1-1 was inoculated on PDA medium and cultured at 26°C for 5 days to serve as the test strain. The pathogens listed in Table 4: Penicillium italicum, Colletotrichum acutatum, Colletotrichum fragariae, Fusarium oxysporum (Schl.) F.sp cucumerinum Owen.anamorph, Fusarium oxysporum Schlechtf.sp.cubense (EFSm.) Snyd.et Hans (Race 4)), Colletotrichum litchi Trag, Colletotrichum acutatum, FusaHum graminearum Sehw and Botrytis cinerea were used as the control targets, and the inhibitory effect of strain JK1-1 on the above plant diseases was detected by the plate colony confrontation culture method. The specific operation is as follows: each pathogen was inoculated in the center of a 90 mm plate, and JK1-1 bacterial cakes (5 mm) were inoculated 5 mm from the edge on both sides as the treatment group. The corresponding pathogen was inoculated in the center and blank PDA bacterial cakes were inoculated on both sides as the control group. The plates were sealed with parafilm to prevent the leakage of volatile substances. Each treatment was repeated 3 times. After culturing at 26°C for 7 days, the colony diameters (mm) of each control group and the colony diameters (mm) of the plate cultured in the treatment group were measured and the inhibition rate was calculated using the following formula:

[0074] Inhibition rate (%) = (colony diameter of control group - colony diameter of treated group) / colony diameter of control group × 100%

[0075] The results are shown in Table 4. Figure 8 A. Strain JK1-1 has a strong inhibitory ability against pathogenic bacteria of eight crops, including citrus, pepper, banana, strawberry, cucumber, lychee, mango and wheat, with an inhibition rate of over 67.77%. Strain JK1-1 almost covers the pathogen colonies. After being inoculated onto the plate, it grows rapidly, almost covering the entire culture dish. It can entangle the pathogenic hyphae of crops, causing them to rupture, and plays an effective role in preventing and controlling crop diseases.

[0076] like Figure 8 B, C, strain JK1-1 has a significant inhibitory effect on gray mold, with an inhibition rate of up to 84.24%.

[0077] Table 4 Pathogens

[0078]

[0079] Example 4 Experiment on fumigation of gray mold with volatile substances from strain JK1-1

[0080] In this embodiment, the plate-to-plate method was used to determine the inhibition rate of VOCs produced by strain JK1-1. The bacterial cake (5 mm) of strain JK1-1 was inoculated on a PDA plate and cultured continuously at 26 ° C for 7 days. The JK1-1 bacterial plates that had been cultured for different days were respectively placed on the plates that had just been inoculated with gray mold cake (5 mm) (the plate cover was removed, the plate inoculated with gray mold was placed on the top, the plate inoculated with strain JK1-1 was placed on the bottom, and the blank plate was placed under the control group). In order to prevent the leakage of volatiles, two layers of sealing film were used for sealing. The plates were then cultured at 26 ° C for 5 days until the gray mold in the control group was full of bacterial plates. The colony diameters (mm) of the control group and the colony diameters (mm) of the treatment group were measured and the inhibition rate was calculated. The formula is as follows:

[0081] Inhibition rate (%) = (control colony diameter - treatment group colony diameter) / control colony diameter × 100%

[0082] In JK1-1 interlocking plates cultured for different days ( Figure 9 (0 day means just after inoculation, 1 day means 1 day after inoculation, and so on). The VOCs produced by strain JK1-1 showed a very significant inhibitory effect on the growth of Botrytis cinerea. When the control group was full of Botrytis cinerea, except for a small amount of Botrytis cinerea growing on the JK1-1 plate with a culture time of 0 day, no colonies grew on the Botrytis cinerea plates with the other culture days. Figure 9 A), the antibacterial rate is as high as 100% ( Figure 9 B).

[0083] To investigate the effects of VOCs produced by JK1-1 on Botrytis cinerea hyphae, Botrytis cinerea, cultured at 26°C for 3 days, was co-cultured with the biocontrol fungus JK1-1, cultured at 26°C for 3 days. Two days later, the Botrytis cinerea hyphae were fixed with 2.5% glutaraldehyde for 2 hours, and then observed under a scanning electron microscope for morphological characteristics. Scanning electron microscopy (scale bar = 10 μm) revealed that, compared to the normal control hyphae, the Botrytis cinerea hyphae fumigated with VOCs produced by JK1-1 exhibited significant shrinkage, distortion, and damage. Figure 9 C).

[0084] Example 5: Effect of fumigation with volatile substances from strain JK1-1 on the prevention and control of tomato fruits inoculated with gray mold

[0085] To investigate whether VOCs fumigation treatment of strain JK1-1 has antifungal activity against Botrytis cinerea in tomatoes, uniform-sized and pest-free cherry tomato fruits were washed with 0.1% NaClO solution and air-dried. The tomato epidermis (center of the equator) was gently punctured with a 1 ml sterile syringe needle, and then a Botrytis cinerea mycelium cake (5 mm) was inoculated on the punctured area of ​​the tomato for later use. Five plates of strain JK1-1 that had been inoculated and cultured for 3 days were placed at the bottom of a fumigation pot (inner diameter 21 cm, height 5.5 cm, volume 7 L) (with the lid removed). Fifteen tomato fruits inoculated with Botrytis cinerea were placed on the ceramic plate of the fumigation pot and covered. The fumigation pot lid and the joints between the lid and the pot body were coated with medical vaseline and wrapped with sealing plastic film to prevent the escape of VOCs produced by strain JK1-1. Five blank PDA plates not inoculated with the biocontrol fungus JK1-1 served as controls. Each treatment consisted of 12 large fumigation pots, kept at 26°C. Tomatoes were monitored for disease and sampled 0, 2, 4, 6, and 8 days after treatment. For sampling, hyphae were removed from the tomato surface, and fruit tissue around the inoculation point was collected using a 1.5 cm diameter borer. Tissue from 15 fruits was pooled, immediately immersed in liquid nitrogen, and stored in a –80°C refrigerator for further analysis. This experiment was repeated three times.

[0086] like Figure 10 A. Two days after inoculation, gray mold quickly infected the fruit from the inoculation point on the tomato fruit in the control group, and lesions appeared. As the inoculation time increased, gray mold hyphae gradually covered the fruit surface in the control group, and the fruit lesions gradually expanded. By the eighth day after inoculation, the diameter of the fruit lesions had reached 31.609 mm ( Figure 10 B), whereas the JK1-1-treated fruits did not show signs of disease until the sixth day, and the fruits maintained a good appearance until the end of treatment. These results indicate that VOC fumigation with JK1-1 significantly inhibits the progression of gray mold caused by Botrytis cinerea.

[0087] To analyze the effects of VOC fumigation treatment of JK1-1 on tomato fruit quality after inoculation with gray mold, physiological indicators such as titratable acid, vitamin C, and soluble solids were measured. Soluble solids (TSS) content was determined using a handheld refractometer (Atago, Japan). Titratable acid (TA) content was determined using a 0.01 M NaOH solution (Kavitha et al., 2014). 2 g of homogenized tomato puree was thoroughly mixed with 100 mL of distilled water. 5 mL of the homogenate filtrate was titrated with 0.01 mol L⁻¹ NaOH. Phenolphthalein was used as an indicator, and the acidity was calculated using a malic acid conversion factor of 0.067 as a standard. The acidity was expressed as a percentage. Vitamin C content was determined using the 2,6-dichlorophenol indophenol (DPIP) method. 5 g of homogenate sample was weighed and diluted to 100 mL with 2% oxalic acid. 5 mL of the extracted homogenate filtrate was titrated with a standardized DPIP solution. 0.1 mg mL⁻¹ was used to titrate the filtrate. -1 The dye calibration value is calculated by titrating DPIP with standard ascorbic acid solution and the result is expressed as mg / 100g. -1 The fresh weight of fruit of FW was measured three times. The results showed that the titratable acid ( Figure 10 E) and soluble solids ( Figure 10 D) There was no significant difference between the control group and the treatment group in the two indicators. In addition, it is worth noting that in the late treatment period (6-8 days), the VC content in the treatment group was significantly higher than that in the control group ( Figure 10 C), indicating that VOC fumigation with strain JK1-1 may have increased the VC content of tomato fruit. In summary, VOC fumigation with strain JK1-1 can significantly inhibit the progression of gray mold caused by Botrytis cinerea and maintain or improve fruit quality to a certain extent.

[0088] Example 6 GC-MS Identification of Volatile Substances of Strain JK1-1 and the Control Effect of Its Single Substances on Botrytis Cinerea

[0089] Strain JK1-1 was inoculated into PDA medium and cultured at 25°C for 3 days. A blank PDA medium was used as a blank control and sent to Wuhan Maiwei Biotechnology Co., Ltd. for detection and identification of volatile metabolites. The detection process is as follows:

[0090] 1. Sample extraction process

[0091] (1) Take out the sample from the -80℃ freezer (solid fresh sample is assumed by default unless otherwise specified) and grind it with liquid nitrogen. Vortex mix it evenly and weigh about 0.2 g (0.2 mL) of each sample into a headspace bottle.

[0092] (2) Add 0.2 g of NaCl powder and 20 μL (10 μg / mL) of internal standard solution respectively;

[0093] (3) Fully automated headspace solid phase microextraction (HS-SPME) was used to extract samples for GC-MS analysis.

[0094] 2. Chromatographic mass spectrometry acquisition conditions

[0095] HS-SPME extraction conditions:

[0096] The sample was shaken at 60°C for 5 minutes, and a 120μm DVB / CWR / PDMS extraction tip was inserted into the sample headspace vial for 15 minutes of headspace extraction. The extraction tip was then desorbed at 250°C for 5 minutes, followed by GC-MS separation and identification. Before sampling, the tip was conditioned at 250°C for 5 minutes in a Fiber Conditioning Station. Note: New tips are conditioned in the Fiber Conditioning Station for 2 hours before extraction. The SPMEArrow fiber was used, which offers up to 10 times the sensitivity of traditional SPME fibers.

[0097] Chromatographic conditions:

[0098] A DB-5MS capillary column (30 m × 0.25 mm × 0.25 μm, Agilent J&W Scientific, Folsom, CA, USA) was used. The carrier gas was high-purity helium (99.999% purity or higher). The injection port temperature was 250°C, and splitless injection was used with a solvent delay of 3.5 min. The temperature program was as follows: 40°C for 3.5 min, then increased to 100°C at 10°C / min, then to 180°C at 7°C / min, and finally to 280°C at 25°C / min and held for 5 min.

[0099] Mass spectrometry conditions:

[0100] The electron impact ion source (EI) was used with a source temperature of 230°C, a quadrupole temperature of 150°C, an mass spectrometer interface temperature of 280°C, an electron energy of 70 eV, and a scan mode of selected ion detection (SIM) with precise qualitative and quantitative ion scanning (GB 23200.8-2016).

[0101] A total of 1,225 metabolites were detected based on the GC-MS detection platform and the company's own database. The specific data are shown in Table 5 below, and the pie chart of the relative content of the substance classification is shown in Figure 11. Analysis of the chart data shows that the volatile substances produced by strain JK1-1 are mostly terpenes (21.28%), esters (20.73%) and ketones (11.49%). The relative content of these three substances accounts for more than 50% of the total relative content. It is speculated that these three types of substances may play a major role in biocontrol and antibacterial activities. Based on this, by consulting literature and materials, and considering that volatile substances will be used as post-harvest fumigants for fruits, they must meet the practical requirements of low cost, trace efficiency and safety to the human body. We screened 32 volatile metabolites classified mainly as terpenes, esters and ketones for antibacterial activity tests.

[0102] Table 5 Metabolite detection data of strain JK1-1

[0103] Primary classification of substances Quantity (kind) Relative content Terpenes 263 0.212816079 ester 241 0.207307149 ketone 137 0.114857207 hydrocarbons 109 0.09056614 alcohol 101 0.08825918 aldehyde 70 0.071939186 Nitrogen-containing compounds 69 0.056411144 Heterocyclic compounds 65 0.049989289 acid 54 0.044024559 phenol 41 0.039951666 amine 33 0.014850059 ether 20 0.006996384 Aromatics 12 0.001310145 Halogenated hydrocarbons 6 0.000599497 Sulfur compounds 3 0.000122316 (blank) 1 0

[0104] *Note: The (blank) column in Table 5 represents substances with a CAS number of 0-0-0, meaning that the substance is not included in the NIST database.

[0105] Table 6 shows the inhibition rate data of 32 volatile metabolites after screening at concentrations of 500μL / L, 200μL / L, and 100μL / L. As shown in Table 6, all 32 volatile substances produced by strain JK1-1 have an inhibitory effect on Botrytis cinerea. Among them, 14 volatile substances showed extremely strong inhibitory effects, and the inhibition rate was still as high as 100% at a low concentration of 100μL / L. The remaining 18 volatile substances showed a dose-dependent effect, that is, the higher the concentration, the higher the inhibition rate. The lowest inhibition rate at a low concentration of 100μL / L can reach 21.21%. There are significant differences between different concentrations. The inhibition results are shown in Table 6. Figure 12 .

[0106] Table 6 Inhibition rate of 32 volatile substances produced by strain JK1-1 on Botrytis cinerea

[0107]

[0108]

[0109] *Note: a, b, and c after the data in Table 6 represent significant differences at the 0.05 level (P<0.05).

Claims

1. A strain of Trichoderma erinaceum JK1-1 was deposited in the General Microbiology Center of China Culture Collection Administration of Microorganisms with the deposit registration number of CGMCC No.41830.

2. A bacterial agent, the active ingredient of which comprises the Trichoderma juganata JK1-1 according to claim 1 or its fermentation liquid.

3. The microbial agent according to claim 2, wherein: The activation culture method of Trichoderma jugendig JK1-1 is as follows: culturing in a culture medium at 15° C. to 33° C.

4. The microbial agent according to claim 3, wherein: The culture medium is PDA culture medium; preferably, the culture temperature is 25° C. to 33° C., and the culture is carried out on a plate for 1 to 7 days or 3 to 5 days.

5. A product for inhibiting plant pathogens or preventing and controlling plant diseases, characterized in that: The active ingredients thereof comprise volatile metabolic organic compounds produced by the Trichoderma juganata JK1-1 according to claim 1.

6. The product according to claim 5, characterized in that: The volatile metabolic organic compounds include one or more of citral, 2-methylvaleric acid, 4-methylvaleric acid, phenyl acetate, acetophenone, angelica lactone, ethyl phenylacetate, methyl benzoate, 2,3-butanedithiol, (-)-4-terpineol, 2,6-dimethyl-2-heptanol, benzyl acetate, methyl heptanoate, 2-octanol, ethyl sorbate, and diisopropyl adipate.

7. Use of the Trichoderma erinaceum JK1-1 according to claim 1, the bacterial agent according to claim 2, or the product according to claim 5 in any of the following: (1) Application in inhibiting Botrytis cinerea or preventing and treating plant gray mold; (2) Application in the preparation of products for inhibiting Botrytis cinerea or preventing and treating plant gray mold.

8. The use according to claim 7, characterized in that: The plants include tomatoes, citrus, peppers, eggplants, cucumbers, grapes, strawberries, and rapeseed.

9. Use of the Trichoderma erinaceum JK1-1 according to claim 1, the bacterial agent according to claim 2, or the product according to claim 5 in any of the following: (1) Use in inhibiting plant pathogenic fungi or in preventing and controlling plant diseases caused by said plant pathogenic fungi; (2) Use in the preparation of products for inhibiting plant pathogenic fungi or preventing and treating plant diseases caused by said plant pathogenic fungi.

10. The use according to claim 9, characterized in that: The plant pathogenic fungi include Penicillium italicum, the pathogen of citrus anthracnose, Colletrichum litchi Trag, the pathogen of mango / pepper anthracnose, Colletotrichum acutatum, the pathogen of strawberry anthracnose, Colletotrichum fragariae, the pathogen of cucumber wilt, Fusarium oxysporum (Schl.) F.sp cucumerinum Owen.anamorph, the pathogen of wheat fusarium graminearum Sehw, and the pathogen of banana wilt, Fusarium oxysporum f.sp.cubense.

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