A natural product derived from Trichoderma harzianum and its preparation method and application

Abstract

The present invention belongs to the field of Trichoderma secondary metabolites, and discloses a natural product derived from Trichoderma harzianum, a preparation method and an application thereof. The natural product has a structure shown in Formula I. The present invention separates and purifies the Trichoderma harzianum secondary metabolites to obtain the Trichoderma harzianum source natural product LU-11, which can be used to inhibit the growth of Fusarium graminearum PH-1, a wheat head blight disease, and induce and improve the disease resistance of wheat, providing a certain theoretical basis for the development of biological pesticides.

Description

Technical Field

[0001] The invention belongs to the field of Trichoderma secondary metabolites, and more specifically, relates to a natural product derived from Trichoderma harzianum, a preparation method and an application thereof. Background Art

[0002] As an important biocontrol factor, Trichoderma spp. has a significant inhibitory effect on a variety of plant pathogenic fungi such as Fusarium oxysporum cucumis, Xanthomonas oryzae pv.oryzae, Botrytis cinerea, and Fusarium graminearum, and can improve the disease resistance of plants. It is understood that Trichoderma mainly inhibits pathogens through competition, antibiotics, and antagonism, among which the antibiotic effect has attracted the attention of many researchers in recent years. During the growth and metabolism of Trichoderma, a series of secondary metabolites such as polypeptides, terpenes, trichoderma, steroids, alkaloids, and polyketides are produced, and these products contain a large number of antibiotic substances.

[0003] The pyrone substance 5,6-dihydro-6-pentyl-2H-pyran-2-one obtained by separation and purification from the secondary metabolites of Trichoderma viride LTR-2 has a certain inhibitory effect on 11 plant pathogens such as Rhizoctonia solani, Fusarium oxysporum, and Verticillium dahliae. 6PP is a common active metabolite secreted by Trichoderma with a coconut aroma. It has a good inhibitory effect on the mycelial growth of pathogens such as Macrophomina phaseolina, Rhizoctonia solani, Sclerotium rolfsii, Fusarium oxysporum, Phytophthora capsica, and Phytophthora melonis.

[0004] The secondary metabolites of Trichoderma can not only antagonize plant pathogens, but also induce and enhance plant disease resistance and promote plant growth.

[0005] Regarding antagonism of plant pathogens: ElHasan et al. found that 6-PAP can control seedling wilt caused by Fusarium moniliforme in corn, and increase the activity of resistance enzymes POD, PPO and β-1,3-glucanase in corn plants, inducing the defense response of corn plants. Marra et al. found that the combined use of harzianic acid (HA), 6-PP (6-pentyl-a-pyrone) and Trichoderma live bacteria can significantly promote plant growth and increase minerals in plants. Luo et al. isolated Trichokonins from T. pseudokoningii, an antimicrobial peptide. Under the stress of tobacco mosaic virus (TMV), it can increase the activity of antioxidant enzymes PAL and POD in tobacco, upregulate the expression of multiple plant defense genes, and improve the disease resistance of tobacco. Malmierca et al. found that trichodiene (TD) secreted by Trichoderma arundinaceum can induce the expression of tomato salicylic acid (SA) defense genes and reduce the development of lateral roots. Pascale et al. found that harzianic acid (HA) and 6-PP (6-pentyl-a-pyrone) can inhibit the incidence of grape powdery mildew, and 6-PP can increase the total polyphenol content and antioxidant activity in grapes as well as grape yield and quality.

[0006] However, there are few studies on the effect of Trichoderma secondary metabolites on the induction of disease resistance in plants. Summary of the invention

[0007] The purpose of the present invention is to provide a natural product derived from Trichoderma harzianum, a preparation method and an application thereof in view of the deficiencies of the prior art. The present invention separates and purifies the secondary metabolites of Trichoderma harzianum to obtain the natural product LU-11 derived from Trichoderma harzianum, which can be used to inhibit the growth of Fusarium graminearum PH-1, a wheat head blight pathogen, and induce and improve the disease resistance of wheat, thus providing a certain theoretical basis for the development of biological pesticides.

[0008] In order to achieve the above object, the first aspect of the present invention provides a natural product derived from Trichoderma harzianum, which has a structure shown in Formula I:

[0009]

[0010] The second aspect of the present invention provides a method for preparing a natural product derived from Trichoderma harzianum. The method comprises sequentially subjecting Trichoderma harzianum to rice fermentation culture and extraction, separation and purification to obtain the natural product derived from Trichoderma harzianum according to claim 1.

[0011] According to the present invention, preferably, the temperature of the rice fermentation culture is 25-30° C. and the time is 30-40 days.

[0012] According to the present invention, preferably, the extraction separation and purification comprises ethyl acetate extraction, normal phase silica gel column chromatography, reverse phase silica gel column chromatography and liquid chromatography separation performed in sequence.

[0013] According to the present invention, preferably, the ethyl acetate extraction is performed 3-4 times.

[0014] According to the present invention, preferably, the reduced pressure gradient elution system used in the normal phase silica gel column chromatography is a petroleum ether-dichloromethane-methanol system.

[0015] According to the present invention, preferably, the reduced pressure gradient elution system used in the reversed-phase silica gel column chromatography is a methanol-water system.

[0016] In the present invention, as a preferred embodiment, the preparation method of the Trichoderma harzianum-derived natural product, namely, the Trichoderma harzianum-derived LU-11, comprises the following steps:

[0017] 1) fermenting the spore liquid of Trichoderma harzianum with rice to obtain a fermented product;

[0018] 2) mixing the fermented product with ethyl acetate and subjecting the mixture to ultrasonic treatment for 3-4 times, and then subjecting the ethyl acetate containing the fermented product to vacuum rotary evaporation at 45° C. to obtain an ethyl acetate total extract;

[0019] 3) performing normal phase silica gel column chromatography on the total ethyl acetate extract to obtain a normal phase column chromatography fraction containing Trichoderma harzianum-derived LU-11;

[0020] 4) performing reverse phase silica gel column chromatography on the normal phase column chromatography fraction containing Trichoderma harzianum-derived LU-11 to obtain reverse phase column chromatography fractions containing Trichoderma harzianum-derived LU-11;

[0021] 5) The reverse phase column chromatography fraction containing Trichoderma harzianum-derived LU-11 was subjected to liquid chromatography separation and liquid chromatography detection analysis, and Trichoderma harzianum-derived LU-11 was detected in the fraction.

[0022] In the present invention, the preparation method of the spore liquid of Trichoderma harzianum comprises: on a PDA plate containing a colony of Trichoderma harzianum T-aloe, a bacterial cake is cut with a 9mm puncher, and the cake is placed in a conical flask containing 150mL PDB culture medium, and the conical flask is placed in a constant temperature shaker at 25°C and 180r / min for 4 days. The fermentation liquid of Trichoderma harzianum T-aloe cultured for 4 days is filtered with 4 layers of sterile gauze, 1mL of the filtered spore liquid is drawn, and the number of spores is observed and recorded under an optical microscope, and finally the spore liquid is placed in a 4°C refrigerator for standby use.

[0023] The third aspect of the present invention provides the application of the Trichoderma harzianum-derived natural product in the following aspects:

[0024] 1) Inhibit the growth of Fusarium graminearum, the pathogen of wheat fusarium wilt;

[0025] 2) Induce plant disease resistance;

[0026] 3) Prevent and control ergot disease in plant seedlings.

[0027] According to the present invention, preferably, the application of the Trichoderma harzianum-derived natural product in inhibiting the growth of Fusarium graminearum caused by wheat fusarium scab is carried out in the form of a solution, and the concentration of the Trichoderma harzianum-derived natural product solution is 5-100 mg / L. In the present invention, as a preferred embodiment, the concentration of the Trichoderma harzianum-derived natural product solution used to inhibit the growth of Fusarium graminearum caused by wheat fusarium scab is 5, 10, 20, 40, 60, 80 and 100 mg / L.

[0028] According to the present invention, preferably, the way in which the natural product derived from Trichoderma harzianum induces plant disease resistance includes increasing plant antioxidant enzymes, reducing plant cell membrane lipid peroxidation and reducing plant cell damage;

[0029] According to the present invention, preferably, the application of the Trichoderma harzianum-derived natural product in inducing plant disease resistance is carried out in the form of a solution, and the concentration of the Trichoderma harzianum-derived natural product solution is 0.1-100 mg / L. In the present invention, as a preferred embodiment, the concentration of the Trichoderma harzianum-derived natural product solution used to induce plant disease resistance is 0.1, 1, 10 and 100 mg / L.

[0030] According to the present invention, preferably, the application of the Trichoderma harzianum-derived natural product in the prevention and treatment of fusarium scab in plant seedlings is carried out in the form of a solution, and the concentration of the Trichoderma harzianum-derived natural product solution is 0.1-100 mg / L. In the present invention, as a preferred embodiment, the concentration of the Trichoderma harzianum-derived natural product solution used for the prevention and treatment of fusarium scab in plant seedlings is 0.1, 1, 10 and 100 mg / L.

[0031] The beneficial effects of the technical solution of the present invention are as follows:

[0032] The Trichoderma harzianum-derived natural product LU-11 separated and purified from the secondary metabolites of Trichoderma harzianum in the present invention is a newly discovered new natural product derived from Trichoderma harzianum, which can be used to inhibit the growth of Fusarium graminearum PH-1, the pathogen of wheat fusarium rust, and induce and improve the disease resistance of wheat, thus providing a certain theoretical basis for the development of biological pesticides.

[0033] The Trichoderma harzianum-derived natural product of the invention can be produced on a large scale and is naturally safe.

[0034] Other features and advantages of the present invention will be described in detail in the following detailed description. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] The above and other objects, features and advantages of the present invention will become more apparent through a more detailed description of exemplary embodiments of the present invention in conjunction with the accompanying drawings, wherein like reference numerals generally represent like components throughout the exemplary embodiments of the present invention.

[0036] Figure 1 The results show that the Trichoderma harzianum source LU-11 obtained in Example 1 of the present invention 1 H-NMR spectrum.

[0037] Figure 2 The results show that the Trichoderma harzianum source LU-11 obtained in Example 1 of the present invention 13 C-NMR spectrum.

[0038] Figure 3 The results of the test example 1 of the present invention on the inhibition of the growth of Fusarium graminearum (PH-1) caused by wheat fusarium rust by LU-11 from Trichoderma harzianum obtained in Example 1 are shown (where a, b, c, d, and e indicate significant differences at the p<0.05 level).

[0039] Figure 4 (A)-(D) show the results of the test example 2 of the present invention on the induction of disease resistance of wheat by the Trichoderma harzianum source LU-11 obtained in Example 1 (wherein "MDA" represents malondialdehyde, "POD" represents peroxidase, "CAT" represents catalase, "SOD" represents superoxide dismutase, "activity" represents activity, "content" represents content, and a, b, c, and d represent significant differences at the p<0.05 level).

[0040] Figure 5 The graph shows the effect of the Trichoderma harzianum-derived LU-11 obtained in Example 1 on the prevention and treatment of wheat seedling fusarium scab in Test Example 3 of the present invention. DETAILED DESCRIPTION

[0041] The preferred embodiments of the present invention will be described in more detail below. Although the preferred embodiments of the present invention are described below, it should be understood that the present invention can be implemented in various forms and should not be limited by the embodiments set forth herein. On the contrary, these embodiments are provided to make the present invention more thorough and complete, and to fully convey the scope of the present invention to those skilled in the art.

[0042] In the following embodiments,

[0043] The reagents or instruments used without indicating the manufacturer are all common products that can be purchased on the market.

[0044] The Gene Bank accession number of the Trichoderma harzianum strain is KC753766, which was isolated and preserved in the Microbiology Laboratory of Zhoukou Normal University.

[0045] Example 1

[0046] This embodiment provides a method for preparing a natural product derived from Trichoderma harzianum, the preparation method comprising:

[0047] S1: Preparation of spore solution of Trichoderma harzianum

[0048] On the PDA plate containing the T-aloe colony of Trichoderma harzianum, use a 9mm hole puncher to cut the bacterial cake, inoculate it into a conical flask containing 150mL PDB culture medium, and place the conical flask in a constant temperature shaker at 25°C and 180r / min for 4 days. Filter the 4-day cultured T-aloe fermentation liquid of Trichoderma harzianum with 4 layers of sterile gauze, absorb 1mL of the filtered spore liquid, place it under an optical microscope to observe and record the spore count, and finally place the spore liquid in a 4°C refrigerator for later use;

[0049] S2: Rice fermentation culture

[0050] Add 1 × 10 spores to the wide-mouth bottle containing rice medium. 7 cfu / mL of Trichoderma harzianum spore solution was placed in a constant temperature incubator at 28°C for 35 days to obtain a fermentation product;

[0051] S3: Ethyl acetate extraction

[0052] Put equal volumes of the fermented product and ethyl acetate into a wide-mouth bottle, sonicate with ultrasound for 30 min, repeat this operation 3-4 times, then perform vacuum rotary evaporation of the ethyl acetate containing the fermented product at 45°C to obtain 92 g of crude extract, which is placed in a 4°C refrigerator for later use;

[0053] S4: Normal phase silica gel column chromatography

[0054] 92 g of the crude extract was first dissolved in a mixed solution of dichloromethane and methanol (1:1), silica gel powder was added, the sample was mixed, and after vacuum evaporation at 45°C, it was added to a pre-loaded silica gel column and vacuum gradient eluted with a petroleum ether-dichloromethane-methanol system to obtain 22 groups of fractions. The 22 groups of fractions were vacuum rotary evaporated at 45°C and placed in a 4°C refrigerator for standby use;

[0055] S5: Reverse phase silica gel column chromatography

[0056] The 11th fraction of the 22 fractions separated by normal phase was dissolved in a mixed solution of dichloromethane and methanol (1:1), ODS reverse phase silica gel powder was added, the sample was mixed, and after vacuum evaporation at 45°C, it was added to a pre-loaded reverse phase silica gel column, and vacuum gradient elution was performed with a methanol-water system to obtain 10 fractions. The 10 fractions were vacuum rotary evaporated at 45°C and placed in a 4°C refrigerator for standby use;

[0057] S6: Liquid chromatography separation and detection analysis

[0058] The 10 groups of fractions obtained by reverse phase separation were detected and analyzed by high performance liquid chromatography, and Trichoderma harzianum-derived LU-11 was found in fractions 4 and 5. The elution system was 80% MeOH and the mobile phase was MeOH-H 2 O, elution time was 20 min, flow rate was 10 mL / min.

[0059] The Trichoderma harzianum source LU-11 has a structure shown in Formula I:

[0060]

[0061] The LU-11 spectral data of the Trichoderma harzianum source are: the measured value of positive ion HR-ESI-MS m / z [M+H] + is 314.1400, and the theoretical value [M+H] + (C18H20NO4) is 314.1392, giving the molecular formula of the compound as C 18 H 19 NO 4 . 1H NMR (500MHz, Methanol-d4) δ7.47(1H,s,H-6),7.27(2H,d,J=8.5Hz,H-15,19),6.81(2H,d,J=8.6Hz,H-16,18),5.51–5.39(2H,m,H -10,11),4.22(1H,h,J=6.7Hz,H-8),2.49–2.42(1H,m,Ha-9),2.03(1H,m,Hb-9),1.62(3H,d,J=5.1Hz,H-12),1.12(3H,d,J=6.8Hz, H-13).13CNMR(MeOD,126MHz): δ16.6(C-13),18.1(C-12),37.4(C-9),44.5(C-8),107.2(C-3),115.9(C-5),116.1(2C,C-16,18), 125.2(C-14),127.9(C-11),130.0(C-10),131.4(2C,C-15,19),140.6(C-6),158.3(C-17),163.9(C-4),177.4(C-2),213.3(C-7).

[0062] Test Example 1

[0063] This test example detects the effect of the Trichoderma harzianum-derived LU-11 obtained in Example 1 on the growth inhibition of Fusarium graminearum (PH-1), the pathogen of wheat fusarium rust.

[0064] Using the 96-well plate microplate reader method, Trichoderma harzianum LU-11 was added to the spore count of 1×10 5 cfu / mLPH-1 spore suspension, in which the concentration of Trichoderma harzianum-derived LU-11 was set to 5, 10, 20, 40, 60, 80 and 100 mg / L.

[0065] Setting: positive control 250 mg / L carbendazim, negative control PH-1 spore solution, blank control clean water.

[0066] The cells were cultured in a 28°C constant temperature incubator for 36 h, and the OD values ​​were measured using an enzyme marker. Three replicates were set up for each group.

[0067] Depend on Figure 3It can be seen that the concentration of LU-11 from Trichoderma harzianum is 5-100 mg / L, which has an inhibitory effect on PH-1. When the concentration of LU-11 from Trichoderma harzianum is 80 and 100 mg / L, the inhibition rate reaches 86.74% and 90.50%, which is 55.96% and 62.72% higher than the positive control of benomyl, with significant differences (p<0.05). However, there is no significant difference between 80 mg / L and 100 mg / L. Therefore, the optimal inhibitory concentration of LU-11 from Trichoderma harzianum to inhibit Fusarium graminearum PH-1 is 80 mg / L.

[0068] Test Example 2

[0069] This test example detects the ability of the Trichoderma harzianum-derived LU-11 obtained in Example 1 to induce disease resistance in wheat.

[0070] Sowing: Soak the wheat seeds in 45℃ warm water for 3h, then sow the wheat seeds in small square boxes, 20 seeds in each box. After the wheat seedlings grow to 10cm, spray the wheat leaves with 0.1mg / L, 1mg / L, 10mg / L and 100mg / L of Trichoderma harzianum source LU-11, spray 10mL for every 3 boxes, and repeat for 3 groups in total.

[0071] Inoculation: 24 hours after the above sowing treatment, the seedlings were inoculated with diseased wheat kernels.

[0072] Setting: Negative control: clean water, positive control: carbendazim.

[0073] Finally, on the 6th day after the diseased wheat kernels were inoculated, the MDA, CAT, POD and SOD of the wheat seedlings were measured.

[0074] Depend on Figure 4 (A)-(D) shows that:

[0075] When the seedlings of small surface were treated with different concentrations of LU-11 from Trichoderma harzianum, the MDA content decreased with the increase of concentration, and was lower than that of the carbendazim group and CK group, with significant differences.

[0076] Under the treatment of different concentrations of LU-11 from Trichoderma harzianum, the activities of SOD and CAT increased with the increase of concentration, and both were higher than those of the oxadipate group and the CK group, with significant differences. This shows that different concentrations of LU-11 from Trichoderma harzianum can improve the disease resistance of wheat, and the disease resistance effect is most significant in the range of 10-100 mg / L. This shows that LU-11 from Trichoderma harzianum can increase the antioxidant enzyme activity of wheat seedlings and reduce the degree of cell damage.

[0077] Test Example 3

[0078] This test example detects the effect of the Trichoderma harzianum-derived LU-11 obtained in Example 1 on the prevention and treatment of wheat seedling fusarium scab.

[0079] The same sowing and inoculation methods as in Test Example 2 were used.

[0080] Setting: Negative control: clean water, positive control: carbendazim.

[0081] After 6 days of receiving the disease, if Figure 5 As shown, the incidence of wheat was observed and the disease index of wheat seedling fusarium was graded according to Table 1, and the prevention effect and disease index were calculated (the calculation formulas of the disease index and prevention effect are shown in Formula 1). The calculation results are shown in Table 2.

[0082] From Table 2, we can see that:

[0083] As the concentration of LU-11 from Trichoderma harzianum increases, its ability to prevent and control wheat scab increases. When the concentration of LU-11 from Trichoderma harzianum is 0.1, 1, 10, and 100 mg / L, its control efficiency is 53.06%, 65.31%, 75.51%, and 81.63%, respectively, and its control efficiency is higher than that of the positive control oxadipamide (34.69%), which is 34.62%, 48.37%, 54.06%, and 57.50% higher, respectively. Therefore, LU-11 from Trichoderma harzianum can prevent and control the occurrence of wheat seedling scab.

[0084]

[0085]

[0086] Table 1

[0087]

[0088] Table 2

[0089]

[0090]

[0091] Note: a, b, c, d, e, and f indicate significant differences at the p<0.05 level

[0092] The embodiments of the present invention have been described above, and the above description is exemplary, not exhaustive, and is not limited to the disclosed embodiments. Many modifications and changes will be apparent to those skilled in the art without departing from the scope and spirit of the described embodiments.

Claims

1. A natural product derived from Trichoderma harzianum, It is characterized in that The natural product has the structure shown in Formula I:

2. A method for preparing a natural product derived from Trichoderma harzianum, It is characterized in that The preparation method comprises the steps of sequentially subjecting Trichoderma harzianum to rice fermentation culture and extraction, separation and purification to obtain the Trichoderma harzianum-derived natural product according to claim 1.

3. The method for preparing the natural product derived from Trichoderma harzianum according to claim 2, in, The temperature of the rice fermentation culture is 25-30° C. and the time is 30-40 days.

4. The method for preparing the natural product derived from Trichoderma harzianum according to claim 2, in, The extraction, separation and purification comprises ethyl acetate extraction, normal phase silica gel column chromatography, reverse phase silica gel column chromatography and liquid chromatography separation performed in sequence.

5. The method for preparing the natural product derived from Trichoderma harzianum according to claim 4, in, The ethyl acetate extraction is performed 3-4 times; The normal phase silica gel column chromatography uses a reduced pressure gradient elution system of petroleum ether-dichloromethane-methanol system; The reduced pressure gradient elution system used in the reversed-phase silica gel column chromatography is a methanol-water system.

6. Application of the natural product derived from Trichoderma harzianum according to claim 1 in the following aspects: 1) Inhibit the growth of Fusarium graminearum, the pathogen of wheat fusarium wilt; 2) Induce plant disease resistance; 3) Prevent and control ergot disease in plant seedlings.

7. The use according to claim 6, in, The application of the natural product derived from Trichoderma harzianum in inhibiting the growth of Fusarium graminearum causing wheat fusarium rust is carried out in the form of a solution, and the concentration of the natural product solution derived from Trichoderma harzianum is 5-100 mg / L.

8. The use according to claim 6, in, The natural products derived from Trichoderma harzianum induce plant disease resistance by increasing plant antioxidant enzymes, reducing plant cell membrane lipid peroxidation and reducing plant cell damage.

9. The use according to claim 8, in, The application of the natural product derived from Trichoderma harzianum in inducing plant disease resistance is carried out in the form of a solution, and the concentration of the natural product solution derived from Trichoderma harzianum is 0.1-100 mg / L.

10. The use according to claim 6, in, The application of the natural product derived from Trichoderma harzianum in preventing and treating fusarium scab of plant seedlings is carried out in the form of a solution, and the concentration of the natural product solution derived from Trichoderma harzianum is 0.1-100 mg / L.

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