Trichoderma strain and application thereof in degrading mycotoxins and inhibiting toxin-producing fungi

Abstract

The application discloses a strain of Trichoderma and application of the strain in degrading mycotoxins and inhibiting toxin-producing fungi, wherein the strain is a Trichoderma (Trichoderma harzianum) strain GG-T40 with a preservation number of CCTCC NO: M20231843. Trichoderma The strain can simultaneously and efficiently degrade eight kinds of mycotoxins including AFB1, ZEN, T-2, ST, OTA, AOH, AME and TeA, and the removal rates of the eight kinds of mycotoxins are all greater than or equal to 60% after the strain is co-cultured with the mycotoxins for 7 days. The strain also has strong fungistatic and toxin-inhibiting effects, and the toxin production inhibition rates of the strain on Aspergillus flavus and Fusarium graminearum are 62.5% and 74.4% respectively. The strain can be applied to degrade mycotoxins in agricultural products and food and inhibit moldy toxin production of the agricultural products and food.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of mycotoxin biodegradation and agricultural biological control, and particularly relates to a Trichoderma strain and application of the Trichoderma strain in degrading mycotoxins and inhibiting toxin-producing bacteria. BACKGROUND

[0002] Mycotoxin contamination in agricultural products, food and feed poses a serious threat to global health and agricultural economy. Mycotoxins are mainly toxic secondary metabolites produced by fungi such as Aspergillus, Fusarium and Penicillium, and have strong carcinogenicity, immunosuppression and neurotoxicity. The most common mycotoxins include Aflatoxin B1 (AFB1), Zearalenone (ZEN), Deoxynivalenol (DON), Fumonisin (FBs), Ochratoxin (OTA), T-2 Toxin (T-2), Sterigmatocystin (ST), Alternariol (AOH), Alternariol monomethyl ether (AME), Tenuazonic acid (TeA) and Deoxynivalenol derivatives (3A-DON, 15A-DON).

[0003] These mycotoxins often do not exist alone in actual pollution, but show obvious co-contamination characteristics. Their coexistence can bring synergistic toxicity risk, complicating risk assessment and prevention and control work. Traditional physical and chemical detoxification methods are low in efficiency, high in cost or destroy nutritional quality, and it is urgent to develop effective, sustainable and multi-target control strategies for mycotoxin management.

[0004] Biological detoxification technology (utilizing microorganisms or their enzyme preparations) possesses high specificity, environmental friendliness, and biosafety. Trichoderma, widely distributed in soil and plant rhizosphere, is a recognized safe fungus genus and has a long history of use as a biofertilizer, soil conditioner, and biocontrol agent. Studies have shown that most Trichoderma species can degrade fungal toxins through secreted metabolites, and due to their rapid growth rate, they can also control harmful fungi through mechanisms such as parasitism, competition, and metabolite-based antifungal activity. Although many Trichoderma species capable of degrading fungal toxins or inhibiting toxin-producing fungi have been reported, such as CN120399963A disclosing a new species of Streptomyces antagonizing a broad spectrum of plant pathogenic fungi and its application; CN120249153A disclosing Streptomyces Knowlesi, a polyfungin B-producing fungus, and its application; and CN120399943A disclosing a strain of Streptomyces alogie and its application in the biocontrol of toxin-producing fungi, a Trichoderma species with high degradation efficiency, broad-spectrum activity, and combined antifungal and detoxifying properties is still lacking. Summary of the Invention

[0005] Therefore, the object of the present invention is to provide a Trichoderma strain ( Trichoderma This study describes the application of strain GG-T40 (Trichoderma spp.) in degrading mycotoxins and inhibiting toxin-producing fungi. This strain, screened from 65 Trichoderma strains, can simultaneously degrade eight mycotoxins and inhibit the mycelial growth and toxin production of toxin-producing fungi such as Aspergillus flavus and Fusarium graminearum. This strain can be used to degrade mycotoxins in agricultural products and food, and also to inhibit toxin production from mold growth in agricultural products and food.

[0006] To achieve the above objectives, the present invention provides the following technical solution: The present invention first isolates Trichoderma from the rhizosphere soil of maize in Jinan City, Shandong Province. Trichoderma (spp.) GG-T40. This strain was deposited on October 9, 2023, at the China Center for Type Culture Collection (address: Wuhan University, Wuhan, China), with accession number: CCTCC NO: M20231843. This strain has the ability to degrade fungal toxins and inhibit toxin-producing bacteria.

[0007] The fermentation method of the above-mentioned Trichoderma strain GG-T40 is characterized by culturing in PDB medium under dark conditions for 3-7 days, with a culture temperature of 25-30℃ (preferably 28℃) and a culture humidity of 85-90% (preferably 90%), and the culture supernatant is filtered to obtain the fermentation broth.

[0008] This invention also provides a biological agent with Trichoderma strain GG-T40 as the active ingredient. The active ingredient includes the Trichoderma strain GG-T40, its fermentation broth, intracellular components, and active bacterial cells. The fermentation broth of strain GG-T40 is the primary component in the biological agent that degrades mycotoxins.

[0009] The application also provides an enzyme preparation for degrading mycotoxins, wherein the active ingredient of the enzyme preparation is an extracellular enzyme secreted by the Trichoderma strain GG-T40, and the enzyme preparation is prepared from a fermentation broth of the strain GG-T40.

[0010] The application provides an application of the Trichoderma strain GG-T40 or a biological microbial agent in simultaneously degrading multiple mycotoxins, wherein the mycotoxins are AFB1, ZEN, T-2, ST, OTA, AOH, AME, TeA and the like.

[0011] The application also provides an application of the Trichoderma strain GG-T40 or a biological microbial agent in inhibiting the mycelium growth of toxin-producing fungi (Aspergillus flavus, Fusarium graminearum and the like) and inhibiting toxin production (e.g., AFB1 production by Aspergillus flavus and DON production by Fusarium graminearum).

[0012] The application has the following technical effects:

[0013] 1. Compared with the prior art, the application provides a Trichoderma strain GG-T40, which can secrete high-activity extracellular enzymes and efficiently degrade eight kinds of mycotoxins including AFB1, ZEN, T-2, ST, OTA, AOH, AME and TeA; after the strain is co-cultured with the mycotoxins for 7 days, the removal rate of the fermentation broth to the eight kinds of toxins with a concentration of 125 ppb is all greater than or equal to 60%, the removal rate of the fermentation broth to AFB1 with a concentration of 125 ppb is 100%, and the removal rate of the fermentation broth to AFB1 with a concentration of 15 ppm is still 90%. The strain has high degradation efficiency and strong broad-spectrum property to the mycotoxins, and has great application potential.

[0014] 2. The Trichoderma strain GG-T40 has strong inhibition on bacteria and toxin production, and the inhibition rates of the strain to the mycelium growth of Aspergillus flavus and Fusarium graminearum are 23.4% and 27.9% respectively, and the inhibition rates of the strain to the toxin production of Aspergillus flavus and Fusarium graminearum are 62.5% and 74.4% respectively. It is shown that the strain can be applied to degrade mycotoxins in agricultural products and food, and also can inhibit the mold and toxin production of agricultural products and food. BRIEF DESCRIPTION OF DRAWINGS

[0015] Figure 1 It is a colony picture of the strain GG-T40 after being cultured on a PDA plate for 7 days;

[0016] Figure 2 It is a degradation rate of the strain GG-T40 to different mycotoxins, wherein A is a degradation rate of the strain GG-T40 to AFB1, ZEN, DON, 3A-DON, 15A-DON, T-2, FB1, FB2, ST, OTA, AOH, AME and TeA with a concentration of 125 ppb, and B is a degradation rate of the strain GG-T40 to AFB1 with different concentrations (50 ppb, 500 ppb, 10 ppm, 15 ppm);

[0017] Figure 3 Figure 4 is a chart showing the AFB1 degradation rate of fermentation liquor of strain GG-T40 after high-temperature high-pressure treatment, proteinase K treatment, and proteinase K + SDS treatment, respectively;

[0018] Figure 4 Figure 5 is a picture of colonies of Aspergillus flavus and Fusarium graminearum grown in PDA medium containing fermentation liquor of strain GG-T40 for 7 days. DETAILED DESCRIPTION

[0019] The terms used in the present application have meanings generally understood by those of ordinary skill in the art, unless otherwise defined. The present application is described in further detail below in conjunction with specific examples and with reference to the data. The following examples are merely for the purpose of illustrating the present application and are not intended to limit the scope of the present application in any way.

[0020] Example 1: Isolation, purification, and identification of the strain

[0021] 1. Isolation and purification of the strain

[0022] The strain of the present application is isolated from corn rhizosphere soil in Jinan, Shandong Province, and the specific isolation steps are as follows:

[0023] (1) Take 10 g of plant rhizosphere soil sample and dissolve in a conical flask containing 90 mL of purified water, oscillate at 30 ℃ and 180 r / min for 15 min, and then incubate in a 90 ℃ water bath for 10 min. After cooling to room temperature, take the soil solution and dilute it in a test tube to prepare a suspension.

[0024] (2) Take 1 mL of the suspension and transfer it into 9 mL of sterile water as the first dilution, and continuously perform 5 dilution gradients. Using the spread plate method, take 100 μL of the suspension of each dilution gradient and evenly spread the dilution liquid on the Bengal red medium plate using an L-shaped spreader. The Bengal red medium contains chloramphenicol and has an inhibitory effect on bacteria, which is beneficial for the isolation of fungi. Seal the plate with a sterilized sealing film and place it in a 28 ℃ incubator for 3 d. Observe the plate every day and check for the growth of new colonies.

[0025] (3) According to the differences in colony morphology, color, and size, use an inoculation loop to pick out strains similar to Trichoderma and purify them on potato dextrose agar medium (PDA). If necessary, purify multiple times until single strains appear. Subsequently, perform molecular biology identification to further determine the type of the strain.

[0026] Bengal red medium formula (containing chloramphenicol): 5.0 g of proteose peptone, 10.0 g of glucose, 1.0 g of potassium dihydrogen phosphate, 0.5 g of magnesium sulfate, 15 g of agar, 0.033 g of Bengal red, 0.1 g of chloramphenicol per liter of medium.

[0027] 2. Strain identification

[0028] After the strain GG-T40 was grown on PDA medium for 7 days, the mycelium growth showed a radial expansion mode, and formed a circular colony of 8-9 cm on the agar medium. The colony was initially white and fluffy, with a regular edge, and gradually turned light yellow with the extension of the culture time, and formed a dense layer of conidia on the surface, and showed a typical powdery appearance after maturation. Figure 1 ).

[0029] Molecular biology strain identification was performed by Shenzhen Huada Gene Company Limited (https: / / en.genomics.cn / ) according to the internal transcribed spacer (ITS) sequence. The total DNA was extracted by the fungal genome extraction kit of Shenzhen Huada Gene Company Limited, and was used for amplification of the target gene ITS. The amplification was performed by using universal primers ITS1 / ITS4, and the amplification program was as follows: 96℃ for 5 min; 96℃ for 30 s, 56℃ for 30 s, 72℃ for 1 min, 35 cycles; 72℃ for 5 min. 3 μL of the PCR product was subjected to 1% agarose electrophoresis for product detection, and the band characteristics were observed. The amplification product was sent to Shenzhen Huada Gene Company Limited for sequencing, and the gene sequence obtained by determination was subjected to sequence alignment analysis with the NCBI database by using BLAST software. The ITS sequence of the strain GG-T40 is shown as SEQ ID No. 1.

[0030] ITS sequence of GG-T40 (SEQ ID No. 1):

[0031] .

[0032] Based on morphological characteristics and gene sequence comparison analysis, strain GG-T40 was identified as Trichoderma ( Trichoderma This strain was deposited on October 9, 2023, at the China Center for Type Culture Collection (address: Wuhan University, Wuhan, China), with accession number CCTCC NO: M20231843.

[0033] Example 2: Degradation effect of Trichoderma strain GG-T40 on various mycotoxins

[0034] The AFB1(or ZEN, DON, 3A-DON, 15A-DON, FB1, FB2, T-2, ST, OTA, AOH, AME, TeA) toxin is added to the sterilized PDB culture medium to make the final concentration 125 ppb; the PDB culture medium containing the mycotoxin is added to the 12-hole cell culture plate, 2 ml per hole; the puncher is used to take the circular mycelium block (about 6 mm in diameter) from the edge of the activated Trichoderma strain GG-T40 colony, which is transferred to the 12-hole cell culture plate with a toothpick, one mycelium block per hole, and placed in the incubator (28°C, dark condition) for 7 days. The PDB without Trichoderma is used as a blank control, and three repeats are performed. After 7 days, the mycelium liquid is taken out with a disposable plastic rubber dropper and added to a 5-ml centrifuge tube. 0.2 mL of the mycelium liquid is mixed with 0.8 mL of acetonitrile solution (or methanol water 1:1), filtered through a 0.22 μM disposable filter membrane, and then detected by liquid chromatography-mass spectrometry to detect the content of AFB1(or ZEN, DON, 3A-DON, 15A-DON, FB1, FB2, T-2, ST, OTA, AOH, AME, TeA) toxin in the mycelium liquid, and the degradation rate of the strain to the mycotoxin is calculated.

[0035] Degradation rate (%) = 100 x (A1 - A2) / A1

[0036] Wherein, A1 is the average content of AFB1(or ZEN, DON, 3A-DON, 15A-DON, T-2, FB1, FB2, ST, OTA, AOH, AME, TeA) in the blank control group; A2 is the average content of AFB1(or ZEN, DON, 3A-DON, 15A-DON, FB1, FB2, T-2, ST, OTA, AOH, AME, TeA) in the experimental group.

[0037] The removal rate of strain GG-T40 to AFB1, ZEN, DON, 3A-DON, 15A-DON, FB1, FB2, T-2, ST, OTA, AOH, AME, TeA with a concentration of 125 ppb is shown in Figure 2 A (the AFB1 degradation rate is 100%, the ZEN degradation rate is 84.1%, the DON degradation rate is 4.15%, the 3A-DON degradation rate is 0.45%, the 15A-DON degradation rate is 42.77%, the FB1 degradation rate is -2.63%, the FB2 degradation rate is 6.08%, the T-2 degradation rate is 59.8%, the ST degradation rate is 99.4%, the OTA degradation rate is 73.5%, the AOH degradation rate is 76.5%, the AME degradation rate is 83.6%, and the TeA degradation rate is 70.0%); Figure 2B is the degradation rate of different concentrations of AFB1 (50 ppb, 500 ppb, 10 ppm, 15 ppm) at different culture times. After 7 days of culture, the degradation rates of AFB1 with concentrations of 50 ppb, 500 ppb, 10 ppm and 15 ppm were 100%, 97.7%, 93.5% and 90.2%, respectively. The degradation rates of the strain on AFB1, ZEN, T-2, ST, OTA, AOH, AME, TeA and other eight mycotoxins were all ≥60%, indicating that the strain can simultaneously and efficiently degrade these eight mycotoxins, and the degradation rate on high-concentration AFB1 (15 ppm) still reached 90%, indicating that it has high toxin tolerance.

[0038] Example 3: Analysis of the degradation differences of different active components of Trichoderma strain GG-T40 on AFB1 and ZEN

[0039] (1) Extracellular metabolite component: GG-T40 strain was cultured in PDA medium in the dark for 5 days (culture temperature 28°C, culture humidity 90%), and 8 pieces of 6.0 mm diameter plugs were punched along the edge of the colony with a puncher and transferred into 100 mL of PDB medium. It was placed in a constant temperature shaker and cultured at 28°C with a shaking speed of 125 rpm for 7 days; after 10 days, the culture supernatant was collected, filtered with filter paper first, and then filtered with a 0.22 μM filter membrane, and the filtrate was used as the extracellular metabolite component (Extracellular Component, EC) of the strain;

[0040] (2) Active mycelium: After filtration, the mycelium was collected and washed twice with PBS buffer; then, a part of the mycelium was resuspended in 100 mL of PBS buffer as the active cell component (Active Component, AC);

[0041] (3) Intracellular metabolite component: the other part of the mycelium was freeze-dried, immediately ground in a mortar under liquid nitrogen environment after freeze-drying, then resuspended in sterilized PBS buffer (about 2.0 mL of PBS was used for resuspension per gram of mycelium), and ultrasonicated for 30 minutes under ice bath condition. After centrifugation (4500 × g, 10 min, 4 °C), the supernatant was filtered with a 0.22 μM filter membrane, and the filtrate was used as the intracellular metabolite component (Intracelluar Component, IC);

[0042] (4) AFB1 and ZEN standard were added into each component EC, AC, IC and control group PBS buffer, the final concentration of AFB1 and ZEN was 500 ppb, and the mixture was incubated at 37°C for 36 h. Then 1.0 mL of each component was filtered through 0.22 μM filter membrane, and the concentration of AFB1 was determined by HPLC-FLD.

[0043] As shown in Table 1, the removal rates of extracellular metabolic components of strain GG-T40 on AFB1 and ZEN were 91.5% and 92.5%, respectively, the removal rates of active bacteria on AFB1 and ZEN were 8.6% and 16.2%, respectively, and the removal rates of intracellular metabolic components on AFB1 and ZEN were 12.7% and 18.8%, respectively. The removal rates of extracellular metabolic components on AFB1 and ZEN were significantly higher than those of active bacteria and intracellular metabolic components, indicating that extracellular metabolic components played a major role in the removal of AFB1 and ZEN by strain GG-T40.

[0044] Table 1 Removal rates of different cell components of Trichoderma GG-T40 on AFB1 and ZEN

[0045]

[0046] Note: * Different letters represent extremely significant differences, according to the Turkey ± Kramer multiple comparison analysis of one-way ANOVA, p < 0.001.

[0047] Example 4: Analysis of active components of extracellular metabolic components

[0048] To investigate whether the degradation of mycotoxins by Trichoderma strain GG-T40 is enzymatic, its extracellular metabolites were subjected to heating and treatment with proteinase K (>30 U / mg). Extracellular metabolites of strain GG-T40 were obtained according to the method described in Example 3. 5 mL of the extracellular metabolite solution was autoclaved at 121°C for 30 min. Another 5 mL of the extracellular metabolite solution was treated with proteinase K to a final concentration of 1 mg / mL and incubated at 37°C for 2 h. Finally, 5 mL of the extracellular metabolite solution was treated with proteinase K and sodium dodecyl sulfate (SDS) to final concentrations of 1 mg / mL and 1% (w / v), respectively, and incubated at 37°C for 5 h. The control group was treated with PBS buffer in the same manner as the previous experiments. Add AFB1 standard to the above-prepared solution to a final concentration of 500 ppb, and react at 37°C in the dark for 24 h. Collect the culture medium with a syringe, filter through a 0.22 μM filter membrane, and determine the AFB1 concentration in the sample by HPLC-FLD. Calculate the degradation rate of AFB1 by different treatment solutions according to the following formula.

[0049] Degradation rate (%) = 100 × (C - S) / C

[0050] Where C is the AFB1 concentration in the control group and S is the AFB1 concentration in the experimental group.

[0051] like Figure 3 After heat treatment at 121 °C, the degradation rate of AFB1 by extracellular metabolites decreased from 92% to 85%. Treatment with proteinase K reduced the degradation rate to 65%, and treatment with proteinase K+SDS reduced it to 24%. Analysis of variance showed that heat treatment had no significant effect on the degradation rate of AFB1, while treatment with proteinase K and proteinase K+SDS both had significant effects. This indicates that proteases play a major role in the degradation of AFB1 by strain GG-T40, suggesting that the active component in the extracellular metabolites is a protease, specifically a thermostable protease.

[0052] Example 5: Effect of Trichoderma strain GG-T40 on inhibiting the growth and toxin production of Aspergillus flavus and Fusarium graminearum.

[0053] (1) Preparation of fermentation broth of strain GG-T40: Fermentation broth of strain GG-T40 was obtained by culturing in PDB medium under dark conditions for 5 days at a temperature of 28℃ and a humidity of 90%.

[0054] (2) Preparation of PDA medium containing GG-T40 fermentation broth: PDA medium was prepared and sterilized at 121°C for 30 min, then cooled to 55°C. 15 mL of liquid PDA medium and 5 mL of Trichoderma fermentation filtrate were mixed at 55°C, then quickly poured into a flat plate, and 10 μL of Aspergillus flavus spore solution (1 × 10 7 spores / mL) or Fusarium graminearum mycelium (6 mm in diameter) was added to the center of the flat plate after it was cooled and solidified. Aspergillus flavus or Fusarium graminearum was inoculated on the flat plate without GG-T40 fermentation broth as a blank control, and each group was repeated 3 times. After incubation at 28°C in the dark for 7 days, the colony diameter of the toxin-producing fungus was measured, and the inhibition rate of strain GG-T40 on the toxin-producing fungus was calculated using the following formula.

[0055] Inhibition rate (%) = 100 × (R1-R2) / R1

[0056] wherein R1 is the colony diameter of the control group of toxin-producing fungus, and R2 is the colony diameter of the experimental group of toxin-producing fungus.

[0057] (3) After measuring the colony diameter of the toxin-producing fungus, a puncher with a diameter of 8 mm was used to punch the mycelium block from the inoculation point of the toxin-producing fungus at a regular distance along the radius of the colony growth. The mycelium (about 1 g) was transferred to a 15 mL centrifuge tube and accurately weighed. 5 mL of methanol-water (80:20, v / v) solution was added to each tube, and the sample was shaken in a shaker at room temperature at a speed of 250 rpm for 1 h. Then the sample was centrifuged, double-layer filter paper was used for filtration, and the content of mycotoxin in the filtrate was determined by ultra-high performance liquid chromatography. The content of AFB1 was determined for Aspergillus flavus group, and the content of DON was determined for Fusarium graminearum group. The inhibition rate of strain GG-T40 on toxin production was calculated using the following formula:

[0058] Inhibition rate (%) = 100 × (M1 – M2) / M1

[0059] wherein M1 is the content of mycotoxin produced by the growth of toxin-producing fungus in the control group, and M2 is the content of mycotoxin produced by the growth of toxin-producing fungus in the experimental group.

[0060] Figure 4 As shown in Table 2, the metabolic products secreted by strain GG-T40 can inhibit the production of AFB1 by Aspergillus flavus, with an inhibition rate of 62.5%, and can also inhibit the production of DON by Fusarium graminearum, with an inhibition rate of 74.4%.

[0061] Table 2 Effect of metabolites of Trichoderma GG-T40 on AFB1 production by Aspergillus flavus and DON production by Fusarium graminearum

[0062]

Claims

1. A Trichoderma ( Trichoderma strain GG-T40 (spp.) is characterized by, The preservation number of the strain GG-T40 is CCTCC NO: M20231843.

2. The fermentation method of Trichoderma strain GG-T40 according to claim 1, characterized in that, The Trichoderma strain GG-T40 was cultured in PDB medium under dark conditions for 3-7 days at a temperature of 25-30℃ and a humidity of 85-90%. The culture supernatant was filtered to obtain the fermentation broth.

3. A biological agent with the Trichoderma strain GG-T40 of claim 1 as the active ingredient, wherein the active ingredient comprises the Trichoderma strain GG-T40 and the fermentation broth of the strain.

4. The application of the Trichoderma strain GG-T40 of claim 1 or the biological agent of claim 3 in the simultaneous degradation of multiple mycotoxins, wherein the mycotoxins are aflatoxin B1 (AFB1), zearalenone (ZEN), T-2 toxin (T-2), chromophore toxin (ST), ochratoxin (OTA), albiflorin (AOH), albiflorin monomethyl ether (AME), and albiflorin ketone acid (TeA).

5. The application of the Trichoderma strain GG-T40 of claim 1 or the biological agent of claim 3 in inhibiting the mycelial growth and toxin production of toxin-producing fungi, wherein the toxin-producing fungi are Aspergillus flavus and / or Fusarium graminearum.

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