Eutectic extraction method of trichoderma harzianum secondary metabolite and application of trichoderma harzianum secondary metabolite in prevention and treatment of fungal diseases of red beauty

Abstract

The invention relates to the technical field of biological prevention and control, in particular to a eutectic extraction method of trichoderma harzianum secondary metabolites and application of the trichoderma harzianum secondary metabolites in prevention and control of fungal diseases of red beauty. Step 2, fermentation liquor treatment; step 3, DES extraction: mixing the hydrogen bond donor and the acceptor in proportion, heating and stirring at 50-90 DEG C until a uniform transparent liquid is formed, cooling to obtain DES, mixing the clarified fermentation liquor and DES to obtain a mixed liquor, performing ultrasonic-assisted extraction on the mixed liquor at the temperature lower than 50 DEG C, centrifuging, and collecting supernate to obtain a crude extract; and 4, purifying and concentrating. According to the method, the secondary metabolite of the trichoderma harzianum is extracted through DES, and activity retention and safety of the secondary metabolite are effectively considered.

Description

Technical Field

[0001] The present application relates to the field of biological control technology, and in particular to a low-melting eutectic extraction method for secondary metabolites of Trichoderma harzianum and its application in the prevention and control of fungal diseases of red beauty. Background Art

[0002] Hong Mei Ren (Citrus reticulata'Hong Mei Ren') belongs to the genus Citrus of the Rutaceae family. It is popular among consumers for its deep orange fruit, high pulp pulp rate, excellent sugar content and sweet orange aroma. However, it is easily infected by pathogens such as Diaporthe spp. during its growth, causing fungal diseases (such as gummosis), which manifests as the secretion of gelatinous substances from the trunk or branches, resulting in tree weakness and a reduction in fruit production of more than 30%.

[0003] Currently, the prevention and control of citrus gummosis mainly relies on chemical fungicides (such as carbendazim and prochloraz), but their residues in the fruit (0.2-0.8 mg / kg) far exceed the food safety standard (GB 2763-2021), and the resistance of pathogens is significantly enhanced after long-term use (the inhibition rate drops by more than 50%). Therefore, it is urgent to develop green and safe biological control technology.

[0004] As a biocontrol fungus, Trichoderma harzianum's secondary metabolites (such as antimicrobial peptides, Trichoderma harzianum acid, and diterpenoids) have broad-spectrum antibacterial activity against a variety of plant pathogens (such as Aspergillus, Fusarium, and Colletotrichum). Generally, the traditional extraction method of Trichoderma harzianum secondary metabolites relies on organic solvents such as methanol and chloroform, which has problems such as low extraction efficiency, high solvent toxicity, and environmental pollution.

[0005] As a green solvent, deep eutectic solvent (DES) has the advantages of low toxicity and high efficiency in the field of natural product extraction, but its application in the extraction of secondary metabolites of Trichoderma harzianum and the prevention and control of citrus diseases is still blank. There is no public report on the use of DES for the extraction of secondary metabolites of Trichoderma harzianum (such as polyketides, antimicrobial peptides, and diterpenes), and the extraction process parameters and selective extraction mechanism for such components have not yet been clarified. At the same time, there are problems such as lack of adaptability for field applications.

[0006] In response to the above problems, the present application proposes a DES-based method for the efficient extraction of secondary metabolites of Trichoderma harzianum, and verifies its application in the prevention and control of fungal diseases of Red Beauty. Summary of the invention

[0007] In order to provide an environmentally friendly fungicide, the present application provides a low-melting extraction method for secondary metabolites of Trichoderma harzianum and its application in the prevention and treatment of fungal diseases of Red Beauty.

[0008] The present application provides a eutectic extraction method for secondary metabolites of Trichoderma harzianum, comprising the following steps: A eutectic extraction method for secondary metabolites of Trichoderma harzianum comprises the following steps: (1) Activation and fermentation of strains: Activate the mycelium of Trichoderma harzianum on PDA medium, place it in a constant temperature incubator at 28° C. for activation culture for 5-7 days, take the mycelium block and inoculate it into PDB medium containing inducer, and culture it under shaking conditions of 28° C. and pH 5.5-7.0 to obtain Trichoderma harzianum fermentation liquid; (2) fermentation broth treatment: centrifuging the Trichoderma harzianum fermentation broth to remove mycelium, and taking the supernatant to obtain clarified fermentation broth; (3) DES extraction: The hydrogen bond donor and the acceptor are mixed in proportion, heated and stirred at 50-90°C until a uniform transparent liquid is formed, and then cooled to obtain DES. The clarified fermentation broth is mixed with DES to obtain a mixed solution, and the mixed solution is subjected to ultrasound-assisted extraction at a temperature below 50°C. After centrifugation, the supernatant is collected as the crude extract; (4) Purification and concentration: The crude extract is first subjected to vacuum distillation to recover most of the low-boiling point component DES, and then the active ingredients in the residue after vacuum distillation are extracted with ethyl acetate. The extract is concentrated at low temperature to an extract and dried to obtain the secondary metabolites of Trichoderma harzianum.

[0009] Preferably, the DES is a hydrophobic DES, the hydrogen bond donor is selected from at least one of caprylic acid, capric acid, and lauric acid, the hydrogen bond acceptor is selected from at least one of menthol, thymol, and carvacrol, and the molar ratio of the hydrogen bond donor to the hydrogen bond acceptor is 1:0.5-3.

[0010] Preferably, the hydrogen bond donor is octanoic acid, the hydrogen bond acceptor is menthol, and the molar ratio of octanoic acid to menthol is 2:1.

[0011] Preferably, in step 3, the mixing ratio of DES to clarified fermentation broth is 1:5-30.

[0012] Preferably, in step 1, the inducer comprises at least one of a copper salt, an iron salt, and a zinc salt.

[0013] Preferably, the concentration of the copper salt is 0.005-0.1 mM.

[0014] Preferably, in step 3, the extraction temperature is 30-70° C., and the extraction time is 40-90 min.

[0015] Preferably, the DES recovered in step 4 is reused for more than 3 times, and the extraction efficiency after each recovery is not less than 80% of the initial value.

[0016] An application of a secondary metabolite of Trichoderma harzianum, comprising diluting the secondary metabolite of Trichoderma harzianum extracted by the extraction method described in any one of claims 1 to 8 with sterile water to 4-10 μg / mL and applying the product by spraying on plants or ground root irrigation.

[0017] In summary, the present application includes at least one of the following beneficial technical effects: 1. This application adopts a hydrophobic low eutectic solvent (DES) system to replace traditional organic solvents (such as methanol and chloroform), and achieves efficient extraction of secondary metabolites of Trichoderma harzianum under low temperature conditions of 40-60°C by optimizing DES components (such as the molar ratio of hydrogen bond donors to acceptors is 1:0.3-3). Compared with traditional ultrasonic extraction, solvent reflux and other methods, the extraction efficiency of target products (antimicrobial peptides, Trichoderma harzianum acid) is increased by 2-4 times, and the antibacterial performance of active ingredients (such as the inhibition rate against Aspergillus niger) is ≥90%. DES solvent can be recycled through simple physical separation (such as stratification and distillation), with a solvent recovery rate of ≥80%, and the residual amount meets international agricultural product safety standards, avoiding environmental pollution and health risks of traditional solvents; 2. This application introduces a composite induction strategy during the fermentation stage by adding low concentrations of metal salts (such as 0.005-1.0 mM CuSO 4 ), activating the oxidative stress pathway of Trichoderma harzianum, increasing the total production of secondary metabolites by 2-4 times. Combined with the DES extraction process, a technical closed loop of "efficient induction → targeted release → green recycling" is formed; 3. This application scheme is applicable to large-scale fermentation systems (such as liquid fermentation) of various Trichoderma harzianum strains, and the cost of DES solvent is lower than that of traditional reagents, so it has industrialization potential and significant economic and environmental benefits. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 It is a graph showing the inhibitory effect of the chemical antibacterial agents of Example 1 and Comparative Examples 9-13 of the present application on Aspergillus niger.

[0019] Figure 2 This is a graph showing the changes in the Red Beauty citrus plants before and after root irrigation with the extract of the secondary metabolites of Trichoderma harzianum in Example 1 for 30 days.

[0020] Figure 3 This is a graph showing changes in leaves of Red Beauty citrus plants before and after using the extract of the secondary metabolites of Trichoderma harzianum in Example 1 for 30 days. DETAILED DESCRIPTION

[0021] The raw materials in this application include the following parts: Trichoderma harzianum: Different types of Trichoderma harzianum strains may be used. In the present application, only the strain T-22 with the deposit number ATCC 20847 is used as an example for illustration.

[0022] The present application is further described in detail below with reference to examples and comparative examples.

[0023] Example 1 A eutectic extraction method for secondary metabolites of Trichoderma harzianum comprises the following steps: (1) Activation and fermentation of strains: Activate the mycelium of Trichoderma harzianum on PDA medium, place it in a constant temperature incubator at 28°C for activation culture for 5-7 days, take a 5-8 mm mycelium block and inoculate it into PDB medium containing inducer, and culture it under shaking conditions of 28°C and pH 6.0 to obtain Trichoderma harzianum fermentation liquid; (2) Fermentation broth treatment: The fermentation broth of Trichoderma harzianum was passed through a 200-mesh nylon filter to remove large mycelial aggregates and culture medium residues. The filtrate was collected and centrifuged at 8000 × g for 20 min at 4°C to remove mycelium. The supernatant was taken to obtain clarified fermentation broth; (3) DES extraction: The hydrogen bond donor and the acceptor are mixed in proportion, heated and stirred at 70°C until a uniform transparent liquid is formed, and then cooled to obtain DES. The clarified fermentation broth and DES are mixed in proportion to obtain a mixed solution. The mixed solution is subjected to ultrasound-assisted extraction at a temperature below 50°C. After centrifugation, the supernatant is collected as the crude extract. (4) Purification and concentration: The crude extract is first subjected to vacuum distillation to recover most of the low-boiling point component DES, and then the active ingredients in the residue after vacuum distillation are extracted with ethyl acetate. The extract is concentrated at low temperature to an extract and dried to obtain the secondary metabolites of Trichoderma harzianum.

[0024] Wherein, DES is hydrophobic DES, the hydrogen bond donor in DES is octanoic acid, the hydrogen bond acceptor is menthol, and the molar ratio of octanoic acid to menthol is 2:1. In step 3, the mixing ratio of DES to clarified fermentation broth is 1:15, the extraction temperature of the mixed solution is 45°C, and the extraction time is 60 minutes. In step 1, copper sulfate with a concentration of 0.05 mM is also added as an inducer.

[0025] Example 2-3 Example 2-3 Based on the preparation method of Example 1, the components of DES were adjusted. The specific adjustments are shown in Table 1.

[0026] Comparative Example 1 Comparative Example 1 Based on the preparation method of Example 1, DES was replaced by ethyl acetate.

[0027] Detection of extraction quantity and antibacterial effect of secondary metabolites of Trichoderma harzianum The specific detection methods of the yields and antibacterial effects of the secondary metabolites of Trichoderma harzianum in the above Examples 1-3 and Comparative Example 1 are as follows: 1. Extraction volume 1L of Trichoderma harzianum fermentation broth was weighed and the mycelium was removed by centrifugation, and the supernatant was mixed with DES, and the mixed solution was subjected to ultrasonic-assisted extraction at a temperature below 50°C, and the supernatant was collected after centrifugation to obtain a crude extract. Subsequently, the crude extract was subjected to vacuum distillation to recover most of the low-boiling point components of DES, and the active ingredients in the residue after vacuum distillation were extracted with a small amount of ethyl acetate. The extract was concentrated at low temperature to an extract and dried to obtain the secondary metabolites of Trichoderma harzianum, and the secondary metabolites of Trichoderma harzianum obtained were weighed to obtain the extraction amount of the secondary metabolites of Trichoderma harzianum in 1L of Trichoderma harzianum fermentation broth.

[0028] 2.EC 50 Determination of value The inhibition rate of the growth of each strain by the different mass concentrations of the agent was calculated based on the net mycelial growth. The calculation formula is as follows: Inhibition rate (%) = (control colony diameter - treated colony diameter) / (control colony diameter - cake diameter) × 100%. The measured data is taken as the logarithm of the natural logarithm e of the concentration as the horizontal axis. By looking up the biostatistical probability value conversion table, the probability value corresponding to the inhibition rate is used as the vertical axis to obtain the regression equation and correlation coefficient R of each test concentration, and the EC is calculated using the probability value corresponding to the inhibition rate of 50%. 50 The value of .

[0029] According to the above detection method, the test results of Examples 1-3 and Comparative Example 1 were obtained, as shown in Table 1 below.

[0030] Table 1 DES components and secondary metabolites extracted from Examples 1-3 and Comparative Example 1 Referring to Table 1, by comparing Examples 1-3 with Comparative Example 1, it can be seen that compared with the extraction effect of ethyl acetate on the secondary metabolites of Trichoderma harzianum, the extraction effect of DES is better. Among them, when menthol in DES is used as a hydrogen bond acceptor and octanoic acid is used as a hydrogen bond donor, the two form a hydrophobic solvent environment through a hydrogen bond network, which can not only dissolve fat-soluble secondary metabolites (such as terpenes and polyketides) through hydrophobic action, but also combine with products with polar groups (such as antimicrobial peptides) through hydrogen bonds to achieve broad-spectrum extraction, thereby increasing the extraction amount of secondary metabolites of Trichoderma harzianum.

[0031] Embodiment 4-5 Example 4-5 Based on the preparation method of Example 1, the mixing ratio of DES and clarified fermentation broth was adjusted. The specific adjustment is shown in Table 2.

[0032] Comparative Examples 2-3 Comparative Example 2-3 Based on the preparation method of Example 1, the mixing ratio of DES and clarified fermentation broth was adjusted. The specific adjustment is shown in Table 2.

[0033] The extracts of the secondary metabolites of Trichoderma harzianum of Examples 4-5 and Comparative Examples 2-3 were subjected to the above performance tests. The test results are shown in Table 2.

[0034] Table 2 Mixing ratio of DES and clarified fermentation broth and extraction amount of secondary metabolites in Example 1, Examples 4-5 and Comparative Examples 2-3 Referring to Table 2, it can be seen from the comparison of Example 1, Examples 4-5 and Comparative Examples 2-3 that when the mixing ratio of DES to the clarified fermentation broth is 1:5-30, especially when the mixing ratio of DES to the clarified fermentation broth is 1:15, the extraction amount of the secondary metabolites of Trichoderma harzianum obtained is the highest. When the mixing ratio of DES to the clarified fermentation broth is too high or too low, it is not conducive to the extraction of the secondary metabolites in the clarified fermentation broth.

[0035] Embodiment 6-7 In Example 6-7, based on the preparation method of Example 1, the extraction temperature in step 3 was adjusted, and the specific adjustment is shown in Table 3.

[0036] Comparative Examples 4-5 Comparative Example 4-5 Based on the preparation method of Example 1, the extraction temperature in step 3 was adjusted, and the specific adjustment is shown in Table 3.

[0037] The extracts of the secondary metabolites of Trichoderma harzianum of Examples 6-7 and Comparative Examples 4-5 were subjected to the above performance tests. The test results are shown in Table 3.

[0038] Table 3 Extraction temperature and secondary metabolite extraction amount of Example 1, Example 6-7 and Comparative Examples 4-5 Referring to Table 3, it can be seen from the comparison of Example 1, Examples 6-7 and Comparative Examples 4-5 that when the extraction temperature is 30-70°C, especially when the extraction temperature is 45°C, the extraction amount of the secondary metabolites of Trichoderma harzianum is ideal. When the extraction temperature is too low, the fluidity of DES is poor, which affects the extraction amount of the secondary metabolites of Trichoderma harzianum; when the extraction temperature is too high, the secondary metabolites of Trichoderma harzianum in DES are easily destroyed and degraded, which affects the extraction amount of the secondary metabolites of Trichoderma harzianum.

[0039] Embodiment 8-9 In Example 8-9, based on the preparation method of Example 1, the extraction time in step 3 was adjusted, and the specific adjustment is shown in Table 4.

[0040] Comparative Examples 6-7 Comparative Example 6-7 is based on the preparation method of Example 1, and the extraction time in step 3 is adjusted. The specific adjustment is shown in Table 4.

[0041] The extracts of the secondary metabolites of Trichoderma harzianum of Examples 8-9 and Comparative Examples 6-7 were subjected to the above performance tests. The test results are shown in Table 4.

[0042] Table 4 Extraction time and secondary metabolite extraction amount of Example 1, Example 8-9 and Comparative Examples 6-7 Referring to Table 4, by comparing Example 1, Examples 8-9 and Comparative Examples 6-7, it can be seen that when the extraction time is 40-90 min, especially when the extraction time is 60 min, the extraction amount of the secondary metabolites of Trichoderma harzianum obtained is the most ideal. When the extraction time is too short, DES cannot fully extract the secondary metabolites of Trichoderma harzianum, resulting in too low an extraction amount of the secondary metabolites of Trichoderma harzianum. When the extraction time is too long, the content of the secondary metabolites of Trichoderma harzianum is not greatly increased. Considering from the perspective of energy consumption, 60 min is the most suitable.

[0043] Embodiment 10-11 In Example 10-11, based on the preparation method of Example 1, the components of the inducer were adjusted, and the specific adjustments are shown in Table 5.

[0044] Comparative Example 8 Comparative Example 8 is based on the preparation method of Example 1, but no inducer is added.

[0045] The extracts of the secondary metabolites of Trichoderma harzianum in Examples 10-11 and Comparative Example 8 were subjected to the above performance tests. The test results are shown in Table 5.

[0046] Table 5 Inducer components and secondary metabolite extraction amounts of Example 1, Examples 10-11 and Comparative Example 8 Referring to Table 5, by comparing Example 1, Examples 10-11 and Comparative Example 8, it can be seen that the addition of the inducer effectively increases the extraction amount of the secondary metabolites of Trichoderma harzianum, and when the component of the inducer is copper sulfate, the extraction amount of the secondary metabolites of Trichoderma harzianum obtained is the highest.

[0047] Comparative Examples 9-13 The materials of Comparative Examples 9-13 are specifically shown in Table 6.

[0048] The bactericides of Comparative Examples 9-13 were subjected to the above performance tests, and the test results are shown in Table 6.

[0049] Table 6 Bactericide names and performance test table of Example 1 and Comparative Examples 9-13 Referring to Table 6, it can be seen from the comparison of Example 1 and Comparative Examples 9-13 that the EC50 value of the secondary metabolites of Trichoderma harzianum is the lowest compared with the commonly used chemical fungicides, which indicates that the secondary metabolites of Trichoderma harzianum can inhibit the growth or activity of pathogens by 50% under lower concentration conditions. Therefore, the dosage of the secondary metabolites of Trichoderma harzianum required in the sterilization process is less. Compared with chemical fungicides, it is not only environmentally friendly, but also has the characteristics of higher sterilization efficiency.

[0050] See attached Figure 1 , af respectively reflect the inhibitory effects of the secondary metabolites of Trichoderma harzianum, 50% methylthiophanate, 80% mancozeb, 60% kasugastatin, 25% azoxystrobin, and 80% Bordeaux mixture on Aspergillus niger. Among them, the colony diameter in a is significantly smaller than that in the other culture media, which effectively reflects that the antibacterial effect of the secondary metabolites of Trichoderma harzianum has an ideal antibacterial effect compared with the above commonly used chemical fungicides.

[0051] Application Example 1 See also Figure 2 Before the use of fungicides, the entire main branch of the Red Beauty citrus suffering from gummosis showed water-soaked colloids, accompanied by yellowing and wilting of leaves. The extract of the secondary metabolites of Trichoderma harzianum in Example 1 diluted to 1 μg / mL was used to irrigate the roots of the Red Beauty plants in an integrated water and fertilizer manner, with 5L per tree. After 30 days, the diseased parts of the plants were effectively controlled, no new gum exudation appeared on the main branches, and the leaves returned to normal color and state.

[0052] Application Example 2 See also Figure 3 Before the use of fungicides, the leaves of the red beauty plants with gummosis also showed different degrees of morbidity, with large areas of leaves yellowing and withering. By using the extract of the secondary metabolites of Trichoderma harzianum of Example 1 diluted to 2 μg / mL, the leaves and branches were evenly sprayed in an atomized form until the liquid dripped. After 30 days, the growth state of the leaves returned to normal.

[0053] This specific embodiment is merely an explanation of the present application and is not a limitation of the present application. After reading this specification, those skilled in the art may make modifications to the present embodiment without any creative contribution as needed, but such modifications are protected by the patent law as long as they are within the scope of the claims of the present application.

Claims

1. A eutectic extraction method for secondary metabolites of Trichoderma harzianum, characterized in that: The following steps are involved: (1) Activation and fermentation of strains: Activate the mycelium of Trichoderma harzianum on PDA medium and place it in a constant temperature incubator at 28°C for 5-7 days. Take the mycelium block and inoculate it into PDB medium containing inducer. Shake and culture it at 28°C and pH 5.5-7.0 to obtain Trichoderma harzianum fermentation liquid. (2) Fermentation broth treatment: centrifuge the Trichoderma harzianum fermentation broth to remove mycelium, and take the supernatant to obtain clarified fermentation broth; (3) DES extraction: The hydrogen bond donor and the acceptor are mixed in proportion, heated and stirred at 50-90°C until a uniform transparent liquid is formed, and then cooled to obtain DES. The clarified fermentation broth is mixed with DES to obtain a mixed solution, and the mixed solution is subjected to ultrasound-assisted extraction at a temperature below 50°C. After centrifugation, the supernatant is collected as the crude extract. (4) Purification and concentration: The crude extract is first subjected to vacuum distillation to recover most of the low-boiling point component DES, and then the active ingredients in the residue after vacuum distillation are extracted with ethyl acetate. The extract is concentrated to an extract form at low temperature and dried to obtain the secondary metabolites of Trichoderma harzianum.

2. The method for eutectic extraction of secondary metabolites of Trichoderma harzianum according to claim 1, characterized in that: The DES is a hydrophobic DES, the hydrogen bond donor is selected from at least one of caprylic acid, capric acid, and lauric acid, the hydrogen bond acceptor is selected from at least one of menthol, thymol, and carvacrol, and the molar ratio of the hydrogen bond donor to the hydrogen bond acceptor is 1:0.3-3.

3. The eutectic extraction method of secondary metabolites of Trichoderma harzianum according to claim 2, characterized in that: The hydrogen bond donor is octanoic acid, the hydrogen bond acceptor is menthol, and the molar ratio of octanoic acid to menthol is 2:

1.

4. The method for eutectic extraction of secondary metabolites of Trichoderma harzianum according to claim 1, characterized in that: In step 3, the mixing ratio of DES to clarified fermentation broth is 1:5-30.

5. The method for eutectic extraction of secondary metabolites of Trichoderma harzianum according to claim 1, characterized in that: In step 1, the inducer comprises at least one of a copper salt, an iron salt, and a zinc salt.

6. The method for eutectic extraction of secondary metabolites of Trichoderma harzianum according to claim 5, characterized in that: The concentration of the copper salt is 0.005-0.1 mM.

7. The method for eutectic extraction of secondary metabolites of Trichoderma harzianum according to claim 1, characterized in that: In step 3, the extraction temperature is 30-70°C, and the extraction time is 40-90 minutes.

8. The method for eutectic extraction of secondary metabolites of Trichoderma harzianum according to claim 1, characterized in that: The DES recovered in step 4 is reused for more than 3 times, and the extraction efficiency after each recovery is not less than 80% of the initial value.

9. An application of secondary metabolites of Trichoderma harzianum, characterized in that: The secondary metabolites of Trichoderma harzianum extracted by the extraction method according to any one of claims 1 to 8 are diluted with sterile water to 0.5-10 μg / mL and applied by spraying on plants or ground root irrigation.

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