Fungus Diaporthe unshiuensis H-111 and cytochalasin compounds produced therefrom, and applications thereof

Abstract

The invention belongs to the field of microbial metabolites and agricultural herbicides, and specifically discloses fungi Diaporthe unshiuensis H-111 and the cytochalasin compounds produced therefrom, and their applications. Diaporthe unshiuensis H-111 can be prepared by fermentation culture to obtain compounds H-111-1 to H-111-6. Activity test results show that these compounds have the activity of inhibiting weed growth and can be developed as herbicides. The cytochalasin compounds prepared by the present invention are produced by fermentation culture using microorganisms, and have the characteristics of simple operation, short production cycle, low cost, low residue, etc.

Description

Technical Field

[0001] The invention belongs to the field of microbial metabolites and agricultural herbicides, and specifically relates to fungus Diaportheunshiuensis H-111 and cytochalasin compounds produced by the fungus, and applications thereof. Background Art

[0002] The problem of herbicide resistance is becoming increasingly serious and has become one of the key issues threatening global food security. Using metabolites with herbicidal activity in natural organisms to develop new bio-source herbicides is one of the best ways to solve the problem of herbicide resistance, because such compounds often have novel chemical structures, unique targets, broad spectrum, low toxicity, low residue, and are less affected by the environment.

[0003] The applicant screened a Diaporthe unshiuensis H-111, and the activity test results showed that its fermentation liquid had a significant growth inhibitory effect on barnyard grass seedlings. It uses microorganisms for fermentation and cultivation, and has the characteristics of simple operation, short production cycle, low cost, and low residue. It can be developed into a new type of microbial herbicide and can also provide a reference for the bionic synthesis of herbicides. Summary of the invention

[0004] The object of the present invention is to provide the isolated fungus Diaporthe unshiuensis H-111, the deposit number of the fungus is: CCTCC NO: M20241273.

[0005] Another object of the present invention is to provide metabolites of the fungus, wherein the metabolites include 6 compounds, namely H-111-1 to H-6.

[0006] Another object of the present invention is to provide the use of the above-mentioned fungus or its metabolites in weed control.

[0007] In order to achieve the above object, the present invention adopts the following technical measures:

[0008] The applicant isolated a fungus from the leaves of naturally diseased Digitaria unshiuensis in Wulongquan, a suburb of Wuhan City, Hubei Province, and found that the fungus or its fermentation liquid has weed control function. The strain was sent to the China Center for Type Culture Collection for preservation on June 19, 2024, and the classification name is: Diaporthe unshiuensis H-111, the preservation number is CCTCC NO: M20241273, and the address is: Wuhan University, Wuhan, China.

[0009] The protection scope of the present invention also includes:

[0010] Fermentation broth of the fungus Diaporthe unshiuensis H-111.

[0011] The main active ingredients of the fermentation broth described above include compounds H-111-1, H-111-2, H-111-3, H-111-4, H-111-5 and / or H-111-6;

[0012] The structural formulas of H-111-1 to 6 are respectively:

[0013]

[0014] The preparation method of the main active ingredient of the fermentation broth comprises: freeze-drying the fermentation broth of the fungus Diaporthe unshiuensis H-111, extracting it with ethyl acetate to obtain a crude extract of the fermentation broth; subjecting the crude extract of the fermentation broth to silica gel column chromatography, gradient eluting it with a mixture of petroleum ether and ethyl acetate in different volume ratios to obtain multiple groups of eluents, and combining the components containing similar compounds according to UPLC-MS analysis, and finally merging them into four parts: Fr.1, Fr.2, Fr.3 and Fr.4;

[0015] Fr.4 was separated and purified by preparative HPLC to obtain H-111-1 and H-111-2, Fr.3 was separated and purified by preparative HPLC to obtain H-111-3, Fr.2 was separated and purified by preparative HPLC to obtain H-111-4, and Fr.2 was separated and purified by preparative HPLC to obtain H-111-5 and H-111-6.

[0016] The above-mentioned preparative HPLC is preferably a Waters 2767 high performance liquid phase preparation instrument, using a Sunfire C18 OBD preparative column, 5 μm, 19×250 mm, and adopting a 40%-100% acetonitrile / water gradient elution.

[0017] The fungus Diaporthe unshiuensis H-111, or the fermentation liquid, or the main effective component of the fermentation liquid described above is used in weed control.

[0018] The use of the fungus Diaporthe unshiuensis H-111, or the fermentation liquid, or the main active component of the fermentation liquid in the preparation of herbicides.

[0019] In the above application, preferably, the grass is Amaranthus retroflexus, Erigeron cantoniensis, Chicory, Echinochloa crusgalli, Crabgrass and / or Setaria viridis.

[0020] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0021] 1. The present invention screened out a strain of Diaporthe unshiuensis H-111 fungus for the first time, and its fermentation liquid has a weed control function. The results of the potted stem and leaf spraying experiment showed that the inhibition rate of the strain fermentation liquid on the fresh weight of barnyard grass was 64.3%.

[0022] 2. The present invention obtains 6 new structural cytochalasin compounds H-111-1 to 6 by fermenting, extracting, separating and purifying the Diaporthe unshiuensis H-111 strain in large quantities, covering three different skeletons and showing different degrees of herbicidal activity, among which H-111-5 has an inhibition rate of 69.42% and 85.35% on the roots and stems of Amaranthus retroflexus at 400 μM, and an inhibition rate of 88.11% and 100% on the roots and stems of Echinochloa crusgalli. The herbicidal activity of H-111-5 on Echinochloa crusgalli is stronger than that of the commercial product 2,4-D. The cytochalasin compounds H-111-1 to 6 have herbicidal activity on both monocotyledonous weeds and dicotyledonous weeds. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 This is a schematic diagram of the Diaporthe unshiuensis H-111 strain plate. DETAILED DESCRIPTION

[0024] The present invention is further described in detail below in conjunction with specific examples, which are only used to explain the present invention and are not used to limit the scope of the present invention. The test methods used in the following examples are conventional methods unless otherwise specified; the materials, reagents, etc. used are reagents and materials that can be obtained from commercial channels unless otherwise specified.

[0025] Embodiment 1:

[0026] Acquisition of Diaporthe unshiuensis fungus:

[0027] 1) The strain was isolated from leaves of naturally infected crabgrass in Wulongquan, a suburb of Wuhan, Hubei Province. Tissue blocks were cut from the junction of diseased and healthy parts, and the surface was disinfected in a 0.1% mercuric chloride solution for 3 minutes and rinsed with sterile water 3 times;

[0028] 2) placing the tissue block obtained after washing in step 1) on a PDA culture medium for constant temperature culture at a temperature of 28° C.;

[0029] 3) After the colonies grow out of the constant temperature culture in step 2), the hyphae are picked from the edge of the colony and transferred to a new PDA medium for culture. After separation and purification, the Diaporthe unshiuensis H-111 strain ( Figure 1 ).

[0030] Embodiment 2:

[0031] The fermentation method of Diaporthe unshiuensis H-111 fungus comprises the following steps:

[0032] The strain Diaporthe unshiuensis H-111 was activated on a PDA plate and cultured at 28°C for 7 days. A 4 mm hole puncher was used to make colony discs, which were inoculated into 200 bottles of fermentation medium, with 4 colony discs per bottle. The culture was shaken at 28°C and 120 r / min for 7 days to obtain 2×10 7 CFU / ml fermentation broth.

[0033] Fermentation medium: maltose 6.25 g / L, malt extract 6.25 g / L, yeast extract 1 g / L, peptone 0.625 g / L, potassium dihydrogen phosphate 1.25 g / L, magnesium sulfate 0.625 g / L, the rest is water, natural pH value, 500 mL conical bottle with a liquid volume of 100 mL.

[0034] Embodiment 3:

[0035] The herbicidal effect of Diaporthe unshiuensis H-111 fermentation liquid:

[0036] Select barnyard grass with the same nutritional status and 4 leaves, take an appropriate amount of Diaporthe unshiuensis H-111 fermentation liquid obtained in Example 2 and spray it on barnyard grass seedlings by stem-leaf spraying method (90 mL / m 2 ), sterile water containing 0.1% Tween-80 was used as blank control, three replicates were set for each test, and each test was repeated three times, 28°C, light intensity 8000lx. After 14 days, the fresh weight was weighed and the inhibition rate was calculated. Inhibition rate = [(average fresh weight of control - average fresh weight of treatment) / average fresh weight of control] × 100%.

[0037] Experimental results: Spraying the fermentation liquid of strain Diaporthe unshiuensis H-111 in potted plants for 14 days had a significant inhibitory effect on the fresh weight of barnyard grass seedlings, with an inhibition rate of 64.3%.

[0038] Embodiment 4:

[0039] Isolation and identification of effective substances in the fermentation broth of Diaporthe unshiuensis H-111:

[0040] 1) Separation of compounds:

[0041] Extraction of metabolites: After freeze-drying the fermentation broth, 500 mL of methanol / water was added to moisten it, and then 20 L of ethyl acetate was used for stirring and extraction three times. The extracts were combined and vacuum concentrated to obtain a crude extract of the fermentation broth;

[0042] Isolation and purification of compounds H-111-1 to 6: The crude extract of the fermentation broth was dissolved in a small amount of methanol, and mixed evenly with 100-200 mesh silica gel in a mass ratio of 1:1. After the methanol was evaporated, 200-300 mesh silica gel with a mass 10 times that of the extract was loaded on a column (60×400mm glass column). Dry loading was adopted, and gradient elution was performed with petroleum ether-ethyl acetate as the eluent (volume ratios of 7:1, 5:1, 3:1, 1:1, and 0:1, respectively). The volume of each gradient elution was 1L, and each elution component was 100mL. The obtained components were subjected to UPLC-MS analysis (Waters Xevo TQD ultra-high performance liquid chromatography-mass spectrometry analyzer, ACQUITY BEH C18 analytical column, 2.1×100 mm, 1.7 μm, 5%-100% acetonitrile / water), and the components containing similar compounds were combined and finally merged into four parts: Fr.1, Fr.2, Fr.3 and Fr.4.

[0043] Fr.4 was separated and purified by preparative HPLC to obtain H-111-1 and H-111-2, Fr.3 was separated and purified by preparative HPLC to obtain H-111-3, Fr.2 was separated and purified by preparative HPLC to obtain H-111-4, and Fr.2 was separated and purified by preparative HPLC to obtain H-111-5 and H-111-6.

[0044] The above-mentioned preparative HPLC was a Waters 2767 high performance liquid phase preparation instrument, using a Sunfire C18OBD preparative column, 5 μm, 19×250 mm, and adopting a 40%-100% acetonitrile / water gradient elution.

[0045] 2) Structural identification of compounds

[0046] The structural formula of the cytochalasin compounds isolated from the fermentation product of Diaporthe unshiuensis H-111 is as follows:

[0047]

[0048] Compounds H-111-1 to 6 are all white solids. The HR-ESI-MS, 1D-NMR and 2D-NMR spectral data for structural identification are as follows:

[0049] HR-ESI-MS data: H-111-1 (m / z 476.2797, [M+H] + ) It is inferred that its molecular formula is C30 H 37 NO4. Compound H-111-2 (m / z 595.3008, [M+H] + ), and its molecular formula is inferred to be C 33 H 42 N2O8; H-111-3(m / z 476.2793,[M+H] + ), and its molecular formula is inferred to be C 30 H 37 NO4;H-111-4(m / z 508.3066,[M+H] + ), and its molecular formula is inferred to be C 31 H 41 NO5;H-111-5(m / z 508.3057,[M+H] + ), and its molecular formula is inferred to be C 31 H 41 NO5;H-111-6(m / z476.2805,[M+H] + ), and its molecular formula is inferred to be C 30 H 37 NO4.

[0050] The NMR data are shown in Tables 1 and 2:

[0051] Table 1. Compound H-111-1-6 (CDCl3) 1 H-NMR (500 MHz) data (δ H ,J Hz)

[0052]

[0053]

[0054] Table 2. Compound H-111-1-6 (CDCl3) 13 C-NMR data (125MHz)

[0055]

[0056]

[0057] Embodiment 5:

[0058] Herbicidal activity test of compounds H-111-1 to 6:

[0059] The herbicidal activity was tested using the petri dish bioassay method.

[0060] Test seeds: Amaranthus retroflexus seeds collected in the field, Echinochloa crus-galli seeds collected in the field. Due to space limitations, this example only shows the inhibition of these two weeds, and the fermentation liquid or compounds H-111-1 to 6 provided by the present invention have different degrees of inhibition effects on the remaining dicotyledonous or monocotyledonous weeds, such as (Amaranthus retroflexus, Erigeron sphaerocephala, Chicory, Digitaria, Setaria viridis).

[0061] Experimental steps: Soak the above seeds in 0.5% KMnO4 for 20 minutes, then wash with pure water until colorless, and dry for use. Weigh 0.6 mg of each compound H-111-1~6, dissolve in methanol to 3 mL (concentration of about 400 μM); weigh 0.27 mg of positive control 2,4-D, dissolve in methanol to 3 mL (concentration of about 400 μM). Put 2 layers of filter paper with a diameter of 3.3 cm in each well of a 6-well plate, add 500 μL of sample to each well, add 500 μL of methanol to CK, and repeat 3 times for each sample. After the methanol is fully evaporated, add 500 μL of sterile water to each well, and add 9 seeds of the same size to each well, evenly distributed. Then put it in a 25°C greenhouse, light for 12 hours, dark for 12 hours, measure the root length and stem length after 4 days, and calculate the inhibition rate of roots and stems respectively by comparing with the blank control.

[0062] Experimental results: The herbicidal activities of compounds H-111-1 to 6 are listed in Table 3. Compounds H-111-1 to 6 showed different degrees of herbicidal activity against barnyard grass and Amaranthus retroflexus, among which H-111-5 had an inhibition rate of 69.42% and 85.35% on the roots and stems of Amaranthus retroflexus at 400 μM, and an inhibition rate of 88.11% and 100% on the roots and stems of barnyard grass, respectively. The herbicidal activity of H-111-5 against barnyard grass was stronger than that of commercial 2,4-D.

[0063] Table 3 Inhibitory effects of compounds H-111-1 to 6 on Amaranthus retroflexus and Echinochloa crusgalli (inhibition rate %)

[0064]

[0065] The above embodiments are only for illustration and are not intended to limit the protection scope of the present invention. Those skilled in the art may make various changes or modifications to the present invention, and these equivalent changes are also within the scope defined by the claims of the present invention.

Claims

1. An isolated fungus Diaporthe unshiuensis H-111, the fungus Diaporthe unshiuensis The deposit number of H-111 is CCTCC NO: M20241273.

2. The fungus according to claim 1 Diaporthe unshiuensis Fermentation broth of H-111.

3. The fermentation broth according to claim 2, characterized in that: The main active ingredients of the fermentation broth include: compound H-111-1, the structural formula of which is as follows: 、 Compound H-111-2 has the following structural formula: 、 Compound H-111-3, the structural formula is as follows: 、 Compound H-111-4 has the following structural formula: 、 Compound H-111-5 has the following structural formula: 、 And compound H-111-6, the structural formula is as follows: 。 4. Use of the strain according to claim 1 or the fermentation liquid according to claim 2 in preparing a herbicide.

5. The use according to claim 4, wherein the herbicide is used to kill weeds including Amaranthus retroflexus, Erigeron sphaerocephalus, Chicory, Echinochloa crus-galli, Crabgrass and / or Setaria viridis.

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