Marine fungus and application thereof in prevention and treatment of pepper phytophthora blight

Abstract

The invention belongs to the field of marine natural product chemistry, and particularly relates to marine fungi and application thereof in prevention and treatment of pepper phytophthora blight. The marine fungus crude extract disclosed by the invention shows very strong antifungal activity on phytophthora capsici under the concentration of 300 mu g / mL. 13 monomeric compounds are separated and identified, Territrem B shows a remarkable antifungal effect on phytophthora capsici, the EC50 value is 22.96 mu g / mL, mycelial growth, sporangium formation and zoospore generation of the phytophthora capsici can be effectively inhibited, and the defense capacity of pepper plants is enhanced; the form of the phytophthora capsici mycelium is changed, and the integrity of a cell membrane is destroyed. In addition, Territrem B inhibits the activity of antioxidant enzyme, reduces the content of soluble protein and increases the MDA level, thereby inhibiting the normal growth of phytophthora capsici.

Description

TECHNICAL FIELD

[0001] The application belongs to the field of marine natural product chemistry, and particularly relates to a marine fungus and application of the marine fungus in prevention and treatment of pepper blight. BACKGROUND

[0002] Pepper blight, commonly known as black rod, is a kind of devastating plant disease caused by infection of pepper Phytophthora capsici, which is widely distributed in global pepper planting areas and can occur at seedling stage and adult stage, and mainly occurs at adult stage. The pathogen can infect roots, stems, leaves and fruits, and can often cause absolute loss when serious. Traditional chemical control mainly relies on metalaxyl, azoxystrobin and other fungicides, but due to the rapid development of drug resistance of the pathogen, the control effect of these fungicides is decreasing year by year. The present application studies the crude extract and secondary metabolites of marine fungus Aspergillus sp. 20230605, and finds a series of secondary metabolites with strong inhibitory activity on pepper Phytophthora capsici. SUMMARY

[0003] The present application provides a marine fungus Aspergillus sp. 20230605, characterized in that the strain preservation information is as follows: preservation unit name: Guangdong Microbial Culture Collection Center; preservation unit address: 5th floor, No. 59 Building, Guangzhou Xianlie Middle Road 100 Courtyard, Guangdong Academy of Microbiology; preservation date: April 25, 2025; preservation number: GDMCC No. 66211; classification and naming: Aspergillus sp. The marine fungus is isolated from the stem of Acacia confusa.

[0004] Another embodiment of the present application provides a preparation method of compounds 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 and / or 13, characterized in that the method comprises the following steps:

[0005] (1) inoculate the marine fungus Aspergillus sp. 20230605 into rice solid culture medium, and culture at room temperature for 40-45 days to obtain a fermentation product;

[0006] (2) extract the fermentation product obtained in step (1) with an equal volume of ethyl acetate for 2-4 times, combine the extract and then concentrate under reduced pressure to obtain a crude extract;

[0007] (3) The crude extract obtained in step (2) is subjected to column chromatography on silica gel under reduced pressure, and gradient elution is performed using petroleum ether-ethyl acetate as the eluent, with the elution gradient being 100:0, 90:10, 80:20, 70:30, 60:40, 50:50, 40:60, 30:70, 20:80, 10:90, and 0:100, respectively, and two column volumes are collected for each gradient. The eluent obtained with a gradient of petroleum ether: ethyl acetate being 30:70 is concentrated, and then gradient elution is performed on an ODS column using MeOH-H2O as the eluent, with the elution gradient being 30:70, 20:80, 10:90, and 0:100, respectively, and two column volumes are collected for each gradient. The eluent obtained with a gradient of MeOH:H2O being 10:90 is concentrated, and then high-performance liquid chromatography (HPLC) is performed, with the chromatographic column being an Agilent C18, 9.4*250mm, 7μm, the flow rate being 2mL / min, and the mobile phase being MeOH:H2O=66:34, to obtain compounds 9 and 10; the mobile phase being MeOH:H2O=58:42, to obtain compounds 7 and 8; the mobile phase being MeOH:H2O=51:49, to obtain compounds 11, 12, and 13; the mobile phase being MeOH:H2O=20:80, to obtain compounds 1, 2, 3, and 5; and the mobile phase being MeOH:H2O=25:75, to obtain compounds 4 and 6.

[0008] Compounds 1-13 have the following structures:

[0009]

[0010] The proportions of the eluent or the mobile phase are all volume ratios; and the formula of the rice solid culture medium preferably contains 50g of rice, 50g of water, and 0.5g of sea salt per 1L of an Erlenmeyer flask.

[0011] Another embodiment of the present application provides a crude extract of a marine fungus Aspergillus sp. 20230605, characterized by a preparation method comprising the following steps:

[0012] (1) The marine fungus Aspergillus sp. 20230605 is inoculated into a rice solid culture medium, and the culture is incubated at room temperature for 40-45 days to obtain a fermentation product;

[0013] (2) The fermentation product obtained in step (1) is extracted with an equal volume of ethyl acetate for 2-4 times, and the extract is concentrated under reduced pressure to obtain a crude extract.

[0014] Another embodiment of the present application provides the use of the above-mentioned marine fungus Aspergillus sp. 20230605 in the preparation of compounds 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, and / or 13.

[0015] Another embodiment of the present application provides the use of the above-mentioned marine fungus Aspergillus sp. 20230605 in the preparation of the above-mentioned crude extract of marine fungus Aspergillus sp. 20230605.

[0016] Another embodiment of the present application provides the use of one or more of the above-mentioned compounds 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or a pesticidally acceptable salt thereof or the above-mentioned crude extract of marine fungus Aspergillus sp. 20230605 in the control of pepper blight. The pepper blight is caused by Phytophthora capsici.

[0017] Another embodiment of the present application provides a pesticidal composition for controlling pepper blight, characterized in that the pesticidal composition comprises one or more of the above-mentioned compounds 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or a pesticidally acceptable salt thereof or the above-mentioned crude extract of marine fungus Aspergillus sp. 20230605 as an active ingredient. The pesticidal composition optionally further comprises a pesticidally acceptable adjuvant. The pesticidal composition optionally further comprises other active ingredients for controlling Phytophthora capsici.

[0018] Compared with the prior art, the crude extract of marine fungus Aspergillus sp. 20230605 of the present application exhibits strong antifungal activity against Phytophthora capsici at a concentration of 300 μg / mL. Through bioactivity-guided isolation, 13 monomeric compounds were identified using 1D NMR and HR-ESI-MS data, among which Territrem B (compound 1) exhibited significant antifungal effect against Phytophthora capsici with an EC 50 value of 22.96 μg / mL. In vitro and in vivo experiments showed that Territrem B effectively inhibited the mycelial growth, sporangium formation and zoospore production of Phytophthora capsici, and enhanced the defense ability of pepper plants. According to SEM and TEM observations, Territrem B changed the morphology of the mycelium of Phytophthora capsici and damaged the integrity of the cell membrane. In addition, Territrem B inhibited the activity of antioxidant enzymes, reduced the soluble protein content and increased the MDA level, thereby inhibiting the normal growth of Phytophthora capsici. These results indicate that Territrem B has the potential to be used as a new agricultural fungicide and provides a scientific basis for development. BRIEF DESCRIPTION OF DRAWINGS

[0019] Figure 1 is a strain morphological chart of the marine fungus Aspergillus sp. 20230605 of the present application;

[0020] Figure 2is the antifungal activity EC of compound 1 (Territrem B) 50 value test chart;

[0021] Figure 3 is the inhibition activity chart of compound 1 (Territrem B) on sporangium formation (A) and zoospore production (B) of P. capsici at different concentrations;

[0022] Figure 4 is the effect chart of compound 1 (Territrem B) on the prevention and treatment of P. capsici in the pot experiment;

[0023] Figure 5 is the scanning electron microscope observation chart of the influence of compound 1 (Territrem B) on the morphology of P. capsici, from left to right, the scale is 50 μm, 10 μm and 5 μm, respectively;

[0024] Figure 6 is the transmission electron microscope observation chart of the influence of compound 1 (Territrem B) on the ultrastructure of P. capsici, the scale is 2.0 μm; wherein CW: cell wall; PM: plasma membrane; N: nucleus; M: mitochondria; V: vacuole;

[0025] Figure 7 is the influence chart of compound 1 (Territrem B) on the MDA content (A), SOD (B), CAT (C), POD (D) and GSH-PX (E) activity and soluble protein content (F) in pepper. DETAILED DESCRIPTION

[0026] In order to facilitate further understanding of the present application, the following examples are provided to make a more detailed description. However, these examples are only for better understanding of the application and do not limit the scope or implementation principles of the application, and the implementation of the application is not limited to the following.

[0027] Example 1

[0028] (1) The marine fungus Aspergillus sp. 20230605 was inoculated into a rice solid culture medium (210 bottles, formula: 50 g of rice, 50 g of water, 0.5 g of sea salt per 1 L of conical flask), and incubated at room temperature for 45 days to obtain a fermentation product;

[0029] (2) The fermentation product obtained in step (1) was extracted with an equal volume of ethyl acetate for 3 times, and the combined extract was concentrated under reduced pressure to obtain a crude extract (80.6 g).

[0030] Example 2

[0031] The crude extract obtained from Example 1 was subjected to column chromatography on silica gel (100-200 mesh) under reduced pressure, and gradient elution was performed using petroleum ether-ethyl acetate as the eluent, with elution gradients of 100:0, 90:10, 80:20, 70:30, 60:40, 50:50, 40:60, 30:70, 20:80, 10:90, and 0:100, and two column volumes were collected for each gradient. The eluate obtained with a gradient of petroleum ether: ethyl acetate of 30:70 was concentrated, and then gradient elution was performed on an ODS column using MeOH-H2O as the eluent, with elution gradients of 30:70, 20:80, 10:90, and 0:100, and two column volumes were collected for each gradient. The eluate obtained with a gradient of MeOH:H2O of 10:90 was concentrated, and then subjected to high-performance liquid chromatography (HPLC) preparation, with an Agilent C18 column (9.4 x 250 mm, 7 μm), a flow rate of 2 mL / min, and a mobile phase of MeOH:H2O = 66:34, to obtain compounds 9 (15.9 mg) and 10 (73.8 mg); a mobile phase of MeOH:H2O = 58:42, to obtain compounds 7 (21.4 mg) and 8 (33.7 mg); a mobile phase of MeOH:H2O = 51:49, to obtain compounds 11 (18.1 mg), 12 (26.9 mg), and 13 (21.9 mg); a mobile phase of MeOH:H2O = 20:80, to obtain compounds 1 (153.2 mg), 2 (13.2 mg), 3 (11.9 mg), and 5 (26.1 mg); and a mobile phase of MeOH:H2O = 25:75, to obtain compounds 4 (16.3 mg) and 6 (17.0 mg).

[0032]

[0033] Territrem B (1): HR-ESI-MS m / z: 549.2089 [M+Na] + ,(C 29 H 34 O9Na + , calcd. for 549.2095). 1 H NMR (CD3OD, 400 MHz) δ H7.16 (2H, s, H-2', H-6'), 6.73 (1H, s, H-8), 6.35 (1H, d, J = 10.0 Hz, H-3), 5.74 (1H, d, J = 10.0 Hz, H-2), 3.91 (6H, s, 3'-OCH3, 5'-OCH3), 3.81 (3H, s, 4'-OCH3), 3.65 (1H, d, J = 17.6 Hz, H-12a), 2.85 (1H, d, J = 17.6 Hz, H-12b), 2.42 (1H, m, H-6a), 2.08 (1H, m, H-6b), 1.89 (1H, m, H-5a), 1, 78 (1H, m, H-5b), 1.50 (3H, s, 6a-CH3), 1.49 (3H, s, 12b-CH3), 1.29 (3H, s, 4a-CH3), 1.16 (3H, s, 4b-CH3); 13 C NMR (CD3OD, 100 MHz): 203.3 (C, C-l), 166.9 (C, C-l l), 165.4 (C, C-7a), 159.4 (C, C-9), 155.0 (C, C-3' / C-5'), 154.2 (CH, C-3), 141.5 (C, C-4'), 128.2 (C, C-l'), 124.6 (CH, C-2), 104.0 (CH, C-2' / C-6'), 99.4 (CH, C-8), 98.9 (C, C-l la), 82.4 (C, C-6a), 81.2 (C, C-4a), 76.7 (C, C-12a), 61.2 (CH3, 4'-OCH3), 57.2 (C, C-12b), 56.8 (CH3, 3'-OCH3 / 5'-OCH3), 43.5 (C, C-4), 29.8 (CH2, C-6), 27.8 (CH2, C-12), 26.4 (CH2, C-5), 25.8 (CH3, 4a-CH3), 24.2 (CH3, 4b-Me), 24.1 (CH3, 6a-CH3), 22.7 (CH3, 12b-CH3) [1] .

[0034] Arisugacin A (2): HR-ESI-MS m / z: 519.1986 [M + Na] + ,(C 28 H 32 O8Na + , calcd. for 519.1989). 1 H NMR (DMSO-d6, 400 MHz) δ H7.44 (1H, dd, J = 8.4, 2.4 Hz, H-2'), 7.38 (1H, d, J = 2.4 Hz, H-6'), 7.06 (1H, d, J = 8.4 Hz, H-3'), 6.84 (1H, s, H-8), 6.34 (1H, d, J = 10.4 Hz, H-3), 5.65 (1H, d, J = 10.4 Hz, H-2), 3.83 (3H, s, 5'-OCH3), 3.81 (3H, s, 4'-OCH3), 3.47 (1H, d, J = 17.6 Hz, H-12a), 2.73 (1H, d, J = 17.6 Hz, H-12b), 2.28 (1H, m, H-6a), 1.97 (1H, m, H-6b), 1.74 (1H, m, H-5a), 1.66 (1H, m, H-5b), 1.39 (3H, s, 6a-CH3), 1.36 (3H, s, 12b-CH3), 1.20 (3H, s, 4a-CH3), 1.07 (3H, s, 4b-CH3); 13 C NMR (100 MHz, DMSO-d6) δ C : 200.9 (C, C-l), 163.3 (C, C-ll), 162.4 (C, C-7a), 156.6 (C, C-9), 153.2 (C, C-5'), 152.7 (CH, C-3), 149.0 (C, C-4'), 126.3 (C, C-l'), 123.2 (CH, C-2), 118.2 (CH, C-6'), 111.7 (CH, C-3'), 108.2 (CH, C-2'), 97.7 (CH, C-8), 97.1 (C, C-lla), 80.6 (C, C-6a), 79.1 (C, C-4a), 74.9 (C, C-12a), 55.7 (CH3, 4'-OCH3), 55.6 (CH3, 5'-OCH3), 55.3 (C, C-12b), 42.1 (C, C-4), 28.5 (CH2, C-6), 26.5 (CH2, C-12), 26.4 (CH2, C-5), 25.2 (CH3, 4a-CH3), 23.5 (CH3, 4b-CH3), 23.3 (CH3, 6a-CH3), 21.5 (CH3, 12b-CH3) [2]

[0035] Territrem C (3): HR-ESI-MS m / z: 535.1933 [M+Na] + ,(C 28 H 32 O9Na + , calcd. for 535.1939). 1 ​H NMR (CD3OD, 400 MHz) δ H : 7.15 (2H, s, H-2', H-6'), 6.63 (1H, s, H-8), 6.35 (1H, d, J = 10.0 Hz, H-3), 5.73 (1H, d, J = 10.0 Hz, H-2), 3.91 (6H, s, 3'-OCH3, 5'-OCH3), 3.63 (1H, d, J = 17.6 Hz, H-12a), 2.84 (1H, d, J = 17.6 Hz, H-12b), 2.42 (1H, m, H-6a), 2.06 (1H, m, H-6b), 1.88 (1H, m, H-5a), 1.78 (1H, m, H-5b), 1.50 (3H, s, 6a-CH3), 1.48 (3H, s, 12b-CH3), 1.29 (3H, s, 4a-CH3), 1.16 (3H, s, 4b-CH3); 13 C NMR (CD3OD, 100 MHz) δ C : 203.4 (C, C-1), 167.1 (C, C-11), 165.7 (C, C-7a), 160.1 (C, C-9), 154.2 (CH, C-3), 149.6 (C, C-3' / C-5'), 139.9 (C, C-4'), 124.6 (CH, C-2), 104.2 (CH, C-2' / C-6'), 98.1 (CH, C-8), 98.1 (C, C-11a), 82.2 (C, C-6a), 81.2 (C, C-4a), 76.8 (C, C-12a), 57.2 (C, C-12b), 57.0 (CH3, 3'-OCH3 / 5'-OCH3), 43.5 (C, C-4), 29.8 (CH2, C-6), 27.8 (CH2, C-12), 26.4 (CH2, C-5), 25.8 (CH3, 4a-CH3), 24.2 (CH3, 4b-CH3), 24.1 (CH3, 6a-CH3), 22.7 (CH3, 12b-CH3) [1] .

[0036]

[0037] 12a-dehydroxyisoterreulactone A (4): HR-ESI-MS m / z: 469.2234 [M+H] + ,(C 27 H 33 O7 + , calcd. for 469.2221). 1 H-NMR (400 MHz, DMSO-d6): δH 7.80 (2H, d, J = 8.8 Hz, H-2' and H-6'), 7.03 (2H, d, J = 8.8 Hz, H-3' and H-5'), 6.71 (1H, s, H-7), 4.98 (1H, s, 3a-OH), 3.81 (3H, s, 4'-OCH3), 2.62 (1H, m, H-12a), 2.43 (1H, m, H-13a), 2.35 (1H, m, H-11a), 2.29 (1H, m, H-13b), 2.18 (1H, m, H-11b), 2.01 (1H, m, H-11a), 1.92 (1H, m, H-4b), 1.87 (2H, m, H-5a and H-5b), 1.78 (1H, m, H-4a), 1.66 (1H, m, H-12b), 1.33 (3H, s, 3b-CH3), 1.29 (3H, s, 5a-CH3), 1.23 (3H, s, 3a-CH3), 1.18 (3H, s, 11b-CH3); 13 C-NMR (100 MHz, DMSO-d6): δ C 171.5 (C, C-1), 162.8 (C, C-10), 162.5 (C, C-6a), 161.1 (C, C-4'), 157.3 (C, C-8), 126.8 (CH, C-2' / C-6'), 123.5 (C, C-1'), 114.4 (CH, C-3' / C-5'), 98.2 (C, C-10a), 96.4 (CH, C-7), 89.9 (C, C-3a), 79.6 (C, C-5a), 77.9 (C, C-3), 55.4 (CH3, 4'-OCH3), 39.9 (CH, C-11a), 39.0 (C, C-11b), 33.4 (CH2, C-5), 29.5 (CH3, 3a-CH3), 29.1 (CH2, C-12), 28.4 (CH3, 3b-CH3), 26.9 (CH2, C-13), 26.2 (CH2, C-4), 20.6 (CH3, 11b-CH3), 19.7 (CH3, 5a-CH3), 16.4 (CH2, C-11) [3] .

[0038]

[0039] Arisugacin J (5): HR-ESI-MS m / z: 493.2187 [M+Na] + ,(C 27 H 34 O7Na +Calcd. for 493.2187). 1 H NMR (600 MHz, CD3OD) δ H : 7.80 (2H, d, J = 9.0 Hz, H-2' and H-6'), 7.02 (2H, d, J = 9.0 Hz, H-3' and H-5'), 6.57 (1H, s, H-8), 3.85 (3H, s, 4'-OCH3), 3.54 (1H, t, J = 3.0 Hz, H-3), 2.72 (1H, d, J = 16.8 Hz, H-12a), 2.47 (1H, m, H-6a), 2.39 (1H, d, J = 16.8 Hz, H-12b), 2.23 (1H, m, H-5a), 1.92 (1H, m, H-6b), 1.82 (2H, m, H-la / H-2a), 1.70 (1H, m, H-5b), 1.48 (3H, s, 6a-CH3), 1.29 (2H, m, H-lb / H-2b), 1.23 (3H, s, 12b-CH3), 1.12 (3H, s, 4a-CH3), 1.06 (3H, s, 4b-CH3); 13 C NMR (150 MHz, CD3OD) δ C : 167.4 (C, C-11), 166.1 (C, C-7a), 163.3 (C, C-4'), 159.8 (C, C-9), 128.1 (CH, C-2' / C-6'), 125.0 (C, C-l'), 115.4 (CH, C-3' / C-5'), 98.3 (C, C-lla), 98.2 (CH, C-8), 83.5 (C, C-6a), 83.2 (C, C-4a), 78.2 (C, C-12a), 77.7 (CH, C-3), 55.9 (CH3, 4'-OCH3), 44.4 (C, C-12b), 42.5 (C, C-4), 30.1 (CH2, C-6), 26.3 (CH2, C-12), 26.2 (CH2, C-5), 26.1 (CH2, C-2), 25.0 (CH3, 6a-CH3), 24.6 (CH3, 4a-CH3), 24.1 (CH3, 4b-CH3), 22.6 (CH2, C-l), 21.8 (CH3, 12b-CH3) [4] .

[0040]

[0041] Arisugacin D (6), HR-ESI-MS m / z: 511.2342 [M-H] - ,(C 29 H 35O8 - , calcd. for

[0042] 511.2326). 1 H NMR(CD3OD,400MHz)δ H :7.73(2H,d,J=8.4Hz,H-2',H-6'),7.01(2H,d,J=8.4Hz,H-3',H-5'),6.55(1H,s,H-8),3.84(3H,s,4'-OCH3),2.70(1H,d,J=16.4Hz,H-12α),2.44(1H,m,1α),2.37(1H,d,J=16.4Hz,H-12β),2.22(1H,m,H-6α),2.10(3H,s,H-14),1.96(1H,m,H-2β),1.81(2H,m,H-5),1.77(1H,m,H-2α),1.75(1H,m,H-6β),1.48(3H,s,6a-CH3),1.32(1H,m,H-1β),1.24(3H,s,12b-CH3),1.14(3H,s,4β-CH3),0.99(3H,s,4α-CH3); 13 C NMR(CD3OD,100MHz)δ C :171.5(C,C-13),167.2(C,C-11),166.0(C,C-7a),163.3(C,C-4'),159.8(C,C-9),128.1(CH,C-2' / C-6'),125.0(C,C-1'),115.5(CH,C-3' / C-5'),98.4(C,C-11a),98.2(CH,C-8),83.3(C,C-6a),82.1(C,C-4a),79.4(CH,C-3),78.0(C,C-12a),56.0(CH3,4'-OCH3),44.4(C,C-12b),43.2(C,C-4),30.3(CH2,C-6),26.5(CH2,C-12),26.3(CH2,C-5),25.2(CH3,6a-CH3),25.1(CH3,4α-CH3),23.9(CH3,4β-CH3),23.0(CH2,C-2),22.9(CH2,C-1),21.5(CH3,C-14),21.1(CH3,12b-CH3) [5] .

[0043]

[0044] Asperbutyrolactone A (7), HRESIMS at m / z: 463.1354 [M+Na] + , 463.1363 (calcd. for C 24 H 25 O8Na + , 463.1363 (calcd. for C 1 H NMR (600 MHz, CD3OD) δ H : 7.58 (2H, d, J = 9.0 Hz, H-2', H-6'), 6.87 (2H, d, J = 9.0 Hz, H-3', H-5'), 6.54 (1H, dd, J = 8.4, 2.4 Hz, H-6”), 6.45 (1H, d, J = 8.4 Hz, H-5”), 6.44 (1H, d, 2.4 Hz, H-2”), 6.11 (1H, d, J = 10.2 Hz, H-7”), 5.59 (1H, d, J = 9.8 Hz, H-8”), 3.79 (3H, s, H-7), 3.45 (2H, s, H-5), 1.33 (6H, s, H-10”, H-11”); 13 C NMR (150 MHz, CD3OD) δ C : 171.5 (C, C-6), 170.3 (C, C-1), 159.4 (C, C-4'), 153.3 (C, C-4”), 139.8 (C, C-2), 132.0 (CH, C-8”), 131.8 (CH, C-2”), 130.4 (CH, C-2' / C-6'), 129.5 (C, C-3), 129.2 (CH, C-6”), 126.7 (C, C-3”), 123.2 (C, C-1”), 123.1 (CH, C-7”), 122.0 (C, C-1'), 116.7 (CH, C-3' / C-5'), 116.5 (CH, C-5”), 86.7 (C, C-4), 77.2 (C, C-9”), 53.9 (CH3, C-7), 39.5 (CH2, C-5), 28.1 (CH3, C-10” / C-11”) [6] .

[0045]

[0046] (+)-3',3'-di-(dimethylallyl)-Butyrolactone II (8), HRESIMS at m / z: 491.2075 [M-H] - , 491.2086 (calcd. for C 29 H 31 O7 -,491.2064); 1 H NMR (400 MHz, CD3OD) δ H : 7.51 (1H, dd, J = 8.4, 2.4 Hz, H-6’), 7.39 (1H, d, J = 2.4, H-2’), 6.83 (1H, d, J = 8.4 Hz, H-5’), 6.53 (1H, dd, J = 8.0, 2.0, H-6”), 6.49 (1H, d, J = 8.0 Hz, H-5”), 6.38 (1H, d, J = 2.0 Hz, H-2”), 5.35 (1H, m, H-8’), 5.05 (1H, m, H-8”), 3.76 (3H, s, 6-OCH3), 3.41 (2H, d, J = 3.2 Hz, H-5), 3.33 (2H, d, J = 7.6 Hz, H-7’), 3.06 (2H, d, J = 7.6 Hz, H-7”), 1.80 (3H, s, H-10’), 1.73 (3H, s, H-11’), 1.65 (3H, s, H-10”), 1.56 (3H, s, H-11”); 13 C NMR (100 MHz, CD3OD) δ C : 171.6 (C, C-6), 170.6 (C, C-1), 157.1 (C, C-4’), 155.1 (C, C-4”), 139.5 (C, C-2), 134.2 (C, C-9’), 133.0 (C, C-9”), 132.4 (CH, C-2”), 129.7 (CH, C-6” / C-2’), 129.4 (C, C-3 / C-1’), 128.4 (C, C-3”), 128.2 (CH, C-6’), 125.2 (C, C-1”), 123.5 (CH, C-8”), 123.2 (CH, C-3’), 116.0 (CH, C-5’), 115.0 (CH, C-5”), 86.8 (C, C-4), 53.8 (CH3, C-7), 39.7 (CH2, C-5), 28.8 (CH2, C-7’), 28.7 (CH2, C-7”), 26.0 (CH3, C-10’), 25.9 (CH3, C-10”), 17.9 (CH3, C-11’), 17.8 (CH3, C-11”) [7] .

[0047]

[0048] Asperbutyrolactone B (9): HRESIMS at m / z: 339.0872 [M-H] - ,(calcd for C 19 H15 O6 - ,339.0863); 1 H NMR(600MHz,CD3OD)δ H :7.59(2H,d,J=9.0Hz,H-2',H-6'),7.11(3H,m,H-3”,4”,5”),6.87(2H,d,J=9.0Hz,H-3',H-5'),6.85(2H,d,J=6.6Hz,H-2”,6”),3.80(3H,s,H-7),3.57(2H,s,H-5); 13 C NMR(150MHz,CD3OD)δ C :171.5(C,C-6),170.2(C,C-1),159.4(C,C-4'),139.9(C,C-2),134.7(C,C-1”),131.5(CH,C-2” / C-6”),130.4(CH,C-2' / C-6'),129.0(C,C-3),128.8(CH,C-3” / C-5”),128.1(CH,C-4”),123.0(C,C-1'),116.7(CH,C-3' / C-5'),86.5(C,C-4),53.9(CH3,C-7),40.2(CH2,C-5) [6] .

[0049]

[0050] Butyrolactone I(10): HRESIMS at m / z: 447.1404 [M+Na] + ,(calcd.forC 24 H 24 O7Na + ,447.1414); 1 H NMR(400MHz,CD3OD)δ H :7.61(2H,d,J=8.8Hz,H-2',H-6'),6.89(2H,d,J=8.8Hz,H-3',H-5'),6.55(1H,dd,J=8.0,2.0Hz,H-6”),6.51(1H,d,J=8.0Hz,H-5”),6.43(1H,d,J=2.0Hz,H-2”),5.05(1H,t,J=7.2Hz,H-8”),3.75(3H,s,H-7),3.44(2H,d,J=4.4,H-5),3.09(2H,m,H-7”),1.66(3H,s,H-10”),1.57(3H,s,H-11”).13 C NMR (100 MHz, CD3OD) δ C : 171.5 (C, C-6), 170.3 (C, C-1), 159.1 (C, C-4'), 154.9 (C, C-4”), 139.5 (C, C-2), 133.0 (C, C-9”), 132.3 (CH, C-2”), 130.3 (CH, C-2' / C-6'), 129.7 (CH, C-6”), 129.3 (C, C-3), 128.4 (C, C-3”), 125.0 (C, C-1”), 123.4 (CH, C-8”), 123.0 (C, C-1'), 116.6 (CH, C-3' / C-5'), 115.0 (CH, C-5”), 86.8 (C, C-4), 53.8 (CH3, C-7), 39.5 (CH2, C-5), 28.6 (CH2, C-7”), 25.9 (CH3, C-10”), 17.8 (CH3, C-11”) [8] .

[0051]

[0052] Butyrolactone V (11): HRESIMS at m / z: 463.1351 [M+Na] + ,463.1363). 24 H 24 O8Na + ,463.1363). 1 H NMR (400 MHz, CD3OD) δ H : 7.56 (2H, d, J = 8.4 Hz, H-2', H-6'), 7.87 (2H, d, J = 8.4 Hz, H-3', H-5'), 6.53 (1H, d, J = 8.4, H-5”), 6.48 (1H, s, H-2”), 6.47 (1H, d, J = 8.4, H-6”), 3.78 (3H, s, H-7), 3.66 (1H, m, H-8”), 3.44 (2H, s, H-5), 2.79 (1H, td, J = 16.8, 5.6, H-7”a), 2.52 (1H, dd, J = 16.8, 7.2 Hz, H-7”b), 1.26 (3H, s, H-11”), 1.16 (3H, s, H-10”); 13 C NMR (100 MHz, CD3OD) δ C: 171.5 (C, C-6), 170.4 (C, C-1), 159.4 (C, C-4'), 153.4 (C, C-4"), 139.8 (C, C-2), 132.9 (CH, C-2"), 130.4 (CH, C-6"), 130.3 (CH, C-2' / C-6'), 129.2 (C, C-3), 126.1 (C, C-1"), 123.1 (C, C-1'), 120.6 (C, C-3"), 117.2 (C-5"), 116.6 (CH, C-3' / C-5'), 86.8 (C, C-4), 78.0 (C, C-9"), 70.4 (CH, C-8"), 53.9 (CH3, C-7), 39.5 (CH2, C-5), 32.0 (CH2, C-7"), 25.7 (CH3, C-10"), 20.9 (CH3, C-11"). [9] .

[0053]

[0054] Butyrolactone VI(12): HRESIMS at m / z: 481.1455 [M+Na] + ,(calcd.forC 24 H 25 O9Na + ,481.1469); 1 H NMR (400 MHz, CD3OD) δ H : 7.58 (2H, d, J = 8.8 Hz, H-2', H-6'), 6.87 (2H, d, J = 8.8 Hz, H-3', H-5'), 6.62 (1H, d, J = 1.6, H-2"), 6.52 (1H, d, J = 8.0, H-5"), 6.49 (1H, dd, J = 8.0, 1.6, H-6"), 3.79 (3H, s, H-7), 3.45 (2H, s, H-5), 3.44 (1H, m, H-8"), 2.69 (1H, dd, J = 14.0, 2.0 Hz, H-7"a), 2.54 (1H, dd, J = 14.0, 10.0 Hz, H-7"b), 1.18 (6H, s, H-10", H-11"). 13 C NMR (100 MHz, CD3OD) δ C: 171.6 (C, C-6), 170.4 (C, C-l), 159.4 (C, C-4'), 155.9 (C, C-4"), 139.8 (C, C-2), 134.3 (CH, C-2"), 130.5 (C, C-3), 130.4 (CH, C-2' / C-5'), 129.3 (CH, C-6"), 127.3 (C, C-3"), 125.5 (C, C-l"), 123.1 (C, C-l'), 116.6 (CH, C-3' / C-5'), 115.9 (CH, C-5"), 86.7 (C, C-4), 80.6 (C, C-9"), 73.8 (CH, C-8"), 53.9 (CH3, C-7), 39.5 (CH2, C-5), 34.0 (CH2, C-7"), 25.5 (CH3, C-10"), 25.1 (CH3, C-l l")

[10] .

[0055]

[0056] Aspernolide B (13): HRESIMS at m / z: 441.1552 [M-H] - ,(calcd for C 24 H 25 O8 - ,441.1544). 1 H NMR (400 MHz, CD3OD) δ H : 7.58 (2H, d, J = 8.8 Hz, H-2', H-6'), 6.87 (1H, d, J = 8.8, H-3', H-5'), 6.50 (1H, dd, J = 8.8, 2.0, H-6"), 6.49 (1H, d, J = 8.8, H-5"), 6.45 (1H, d, J = 2.0, H-2"), 3.79 (3H, s, H-7), 3.42 (2H, s, H-5), 2.44 (2H, m, H-7"), 1.53 (2H, t, J = 7.6, H-8"), 1.20 (6H, s, H-10", H-l l"); 13 C NMR (100 MHz, CD3OD) δ C: 171.6 (C, C-6), 170.4 (C, C-1), 159.4 (C, C-4'), 155.4 (C, C-4"), 139.8 (C, C-2), 132.9 (CH, C-2"), 130.4 (CH, C-2' / C-6'), 129.7 (CH, C-6"), 129.2 (C, C-3), 127.9 (C, C-1"), 125.2 (C, C-3"), 123.2 (C, C-1'), 116.6 (CH, C-3' / C-5'), 115.2 (CH, C-5"), 86.9 (C, C-4), 71.5 (C, C-9"), 53.9 (CH3, C-7), 44.7 (CH2, C-8"), 39.5 (CH2, C-5), 29.3 (CH3, C-11"), 29.0 (CH3, C-10"), 25.8 (CH2, C-7")

[11] .

[0057] Example 3 Activity Test

[0058] 1. Anti-fungal activity test:

[0059] (1). Experimental instruments and materials: constant temperature incubator, pipette, electronic balance, 0.5 cm diameter puncher, ruler, sealing film, tweezers, sterilization pot, PDA medium, dimethomorph, analytical pure methanol, pepper Phytophthora capsici pathogen.

[0060] (2). Mycelial growth rate inhibition method was used to detect the mycelial inhibition rate in vitro

[12] . 10 mg of the sample to be tested was completely dissolved with a small amount of methanol, and the sample to be tested was mixed with 50 mL of PDA medium to a final concentration of 10, 15, 20, 30, and 40 μg / mL, respectively, and uniformly poured into the sterilized flat plate; then the activated plant pathogen was punched into a fungus cake with a sterilized puncher (diameter 0.5 cm), and the fungus cake with mycelium was inoculated into the center of the sterile PDA flat plate, and the same volume of methanol without compound was used as a blank control, and dimethomorph was used as a positive control. The treatment and control were placed in an incubator at (27±1) °C in the dark for 2-6 days, and the experiment was ended when the control group of pathogenic fungi grew to a colony diameter of more than 5.5 cm. All treatments were repeated 3 times. The diameter of the inhibition zone (cm) was measured using a standard scale and a cross method, and the average value was taken, the mycelial growth inhibition rate = (colony diameter of the control group - colony diameter of the treatment group) / (colony diameter of the control - 0.5) x 100%; then the EC 50 value and independent regression equation were calculated using SPSS software.

[0061] (3). Experimental results: Compounds 1-13 were evaluated for their antifungal efficiency against P. capsici at a concentration of 40 pg / mL. Based on the activity results, it was found that Territrem B (1) showed the highest inhibitory activity at a concentration of 40 pg / mL with an inhibition rate of 71.67%; the inhibitory effects of other compounds are shown in Table 1, and the EC 50 value of Territrem B was determined to be 22.96 pg / mL Figure 2 ).

[0062] Table 1. Effect of compounds 1-13 on P. capsici mycelial proliferation (40 pg / mL)

[0063]

[0064]

[0065] 2. Evaluation of sporangium formation and zoospore production of P. capsici.

[0066] (1). Experimental instruments and materials: The instruments used in the experiment are consistent with those in the antifungal activity part test.

[0067] (2). Sporangium formation: Territrem B was dissolved in methanol and then diluted with sterile water containing 1% Tween-80 to prepare solutions with concentrations of 0, 5, 10, 20, 30, and 40 pg / mL. Fresh P. capsici mycelial plugs were incubated in 50 mL V8 liquid medium at 25°C in the dark for 2 days. Then the mycelial plugs were transferred to different concentrations of Territrem B prepared solutions, and the culture was incubated for another day. The mycelial plugs were washed with sterile water three times, and methanol solvent was used as a blank control. A small amount of mycelial plugs was collected from the edge of the colony, and 30 random fields were observed under a 40x magnification optical microscope to evaluate sporangium formation. Three repeated experiments were performed. The inhibition rate of Territrem B on sporangium formation of P. capsici

[13] was calculated. Sporangium formation inhibition rate = [(total number of sporangia in the control group - total amount of sporangia in the treatment group) / total number of sporangia in the control group] x 100%.

[0068] (3). Zoospore production: After inducing sporangium formation, the mycelial plugs were placed in sterile water containing 0, 10, 15, 20, 30, and 40 pg / mL of Territrem B, and the same experimental procedure was repeated three times. Then the culture was incubated at 4°C for 30 minutes to induce cold conditions, and then incubated at room temperature for 5 hours. The inhibition rate of Territrem B on zoospore release was observed and calculated under a microscope

[13] , and the EC 50The sporulation inhibition rate of animals = [(number of empty sporangia in the control group - number of empty sporangia in the treatment group) / number of empty sporangia in the control group] x 100%.

[0069] (4). The results of the experiment showed that Territrem B can effectively inhibit the growth of sporangia and zoospores. With the increase of concentration, the number of sporangia and zoospores produced decreased significantly, and the inhibition was proportional to the concentration, with EC 50 values of 18.44 and 22.33 μg / mL Figure 3 )

[0070] 3. In vivo protective effect of Territrem B on Phytophthora capsici

[0071] (1). Experimental instruments and materials: sprayer, analytical methanol, pipette, pepper pot, Tween-80 solution, conical flask, and Phytophthora capsici.

[0072] (2). Experimental steps: Pepper plants were cultivated in a greenhouse to the four to six leaf stage. Territrem B was dissolved in 100 μg / mL methanol, then added to 0.1% Tween-80 aqueous solution to prepare a test solution with a concentration of 200 μg / mL. A total of 15 mL of the prepared solution was evenly sprayed on the pepper plants using a sprayer. After 24 hours, a Phytophthora capsici spore suspension was sprayed on the plants at a concentration of 1 x 10 7 CFU / mL. An equal volume of methanol was added to the Tween-80 aqueous solution as a blank control, and 200 μg / mL dimethomorph was used as a positive control. Each treatment group had 10 replicates, and after 14 days of incubation, disease symptoms and disease control effects

[14] were evaluated. Disease severity was evaluated using a 0-5 point scale, as follows: 0: no obvious disease symptoms; 1: mild wilting of the heart leaves; 2: 2-3 leaf wilting, leaf stalk drooping; 3: 4-5 leaf wilting and drooping, stem tip wilting, and brown lesions appearing; 4: 6-7 leaf wilting and drooping, stem necrosis and browning; and 5: plant death or severe browning and lodging.

[0073] Disease index = ∑(number of each level of disease x level value of this level) / (total number of leaves surveyed x highest level value) x 100%; protection effect (%) = [(disease index of the treatment group - disease index of the control group) / disease index of the control group] x 100%

[0074] (3). Experimental results:

[0075] In addition, Territrem B (TEB) and Dimethomorph (DMM) at a concentration of 200 μg / mL showed protection effects of 90.33% and 93.03%, respectively Figure 4). The disease control effect of 200 pg / mL Territrem B was equivalent to that of 200 pg / mL DMM. The results showed that TEB had a significant protective effect on P. capsici in pepper, effectively reducing the infection of diseases and maintaining the health of plants.

[0076] 4. Scanning Electron Microscope

[0077] (1) Experimental materials: PBS buffer solution, P. capsici mycelium, electron microscope fixative, phosphate buffer, ethanol solution.

[0078] (2) Experimental steps: The fungal tissue was washed with PBS three times to clean the surface of the mycelium. Then the sample was fixed with electron microscope fixative at room temperature for 2 hours in the dark, and then transferred to 4°C storage. After fixation, the sample was rinsed with 0.1 mol / L phosphate buffer (PB, pH 7.4) three times, each time for 15 minutes, and then fixed with 1% osmium tetroxide solution at room temperature for 2 hours in the dark. After fixation, the sample was rinsed with 0.1M PB three times, each time for 15 minutes. Then the sample was dehydrated through a series of ethanol solutions (30%, 50%, 70%, 80%, 90%, 95% and 100%) for 15 minutes at each concentration, and then in isopropyl acetate for 15 minutes. After dehydration, the sample was dried in a critical point dryer and mounted on a conductive carbon-coated double-sided tape. Finally, the sample was gold-coated for about 30 seconds using an ion sputtering coater. Then, the prepared sample was examined under SEM to observe the ultrastructural details of the fungal tissue

[15] .

[0079] (3) Experimental results: According to the observation of P. capsici mycelium morphology by scanning electron microscope (SEM), the surface of untreated mycelium was smooth, normal in shape and complete in outer wall. After treatment with 23 pg / mL Territrem B, the mycelium surface showed obvious wrinkles, irregular shape Figure 5 ). Contact with 46 pg / mL Territrem B caused severe mycelium wrinkling, leading to severe morphological deformation, local rupture and obvious signs of apoptosis.

[0080] 5. Transmission Electron Microscope

[0081] (1) Experimental materials: PBS buffer solution, P. capsici mycelium, electron microscope fixative, phosphate buffer, ethanol solution and acetone solution, optical microscope, 812 resin, ultrasonic water, uranyl acetate stain.

[0082] (2) Experimental procedure: Fungal tissues were fixed with electron microscope fixative for 2 hours at room temperature in the dark, then stored at 4°C. Then fixed with 1% osmium tetroxide for 2 hours at room temperature, then washed with 0.1M PB for three times 15 minutes. Tissues were dehydrated in a series of graded ethanol (30%, 50%, 70%, 80%, 95%, 100%) for 20 minutes, then dehydrated with 100% acetone twice for 15 minutes each. The tissues were infiltrated with a 1:1 mixture of acetone and 812 resin at 37°C for 2-4 hours, then with a 1:2 mixture overnight at 37°C, and with pure 812 resin for 5-8 hours at 37°C. The samples were placed in embedding molds, polymerized overnight at 37°C, and then cured at 60°C for 48 hours. The resin blocks were trimmed into 1.5 μΜ semi-thin sections, stained with toluidine blue, and located under a light microscope. Ultrathin sections (60-80 nm) were cut, placed on 150 mesh copper grids, stained with 2% uranyl acetate for 8 minutes, and then washed in 70% ethanol and ultrapure water. The grids were stained with 2.6% lead citrate for 8 minutes, then washed and dried. The samples were observed and imaged for analysis using a transmission electron microscope (TEM)

[16] .

[0083] (3) Experimental results: Untreated mycelial cells had an intact structure, with organelles evenly distributed in the cytoplasm and no damage. After treatment with 23 μg / mL Territrem B, the structure of the mycelial cells was distorted, the vacuoles were destroyed, the organelles disappeared, and the internal cell structure became chaotic. Treatment with 46 μg / mL Territrem B caused significantly enhanced ultrastructural damage, including obvious separation of the cytoplasmic membrane, rupture of the cell membrane, dissolution and disappearance of most organelles, and gradual disintegration and apoptosis. These findings indicate that Territrem B disrupts the morphology and ultrastructure of P. capsici mycelium, ultimately leading to cell apoptosis Figure 6 ).

[0084] 2.6. Physiological parameter evaluation

[0085] 1. Preparation of homogenate: P. capsici mycelial disks were inoculated into PDB medium and incubated at 25°C with shaking at 180 rpm for 4 days. In the treatment group, Territrem B was added to a final concentration of 24 and 48 μg / mL, and incubation was continued under the same shaking conditions. Dimethorph was used as a positive control, and PDB medium without test sample was used as a blank control. Each experimental group was repeated three times. After 48 hours of incubation, the mycelium was collected by filtration and dried, then mixed with physiological saline at a ratio of 9:1. The mycelium was ground into a paste using liquid nitrogen. The homogenate was centrifuged at 5000 rpm for 10 minutes at 4°C, and the supernatant was carefully collected and stored at -20°C for subsequent testing.

[0086] 2. Determination of malondialdehyde (MDA) content: According to the aforementioned method, the MDA content was measured using a commercially available ELISA kit (TBA method, Nanjing Jiancheng Bioengineering Institute). Each experimental group was performed in triplicate. The reagents were added to the samples in the order specified in the kit instructions, and then mixed with a vortex mixer. The samples were incubated in a water bath at 95°C for 40 minutes and cooled under running water. The samples were centrifuged at 3000-4000 rpm for 10 minutes, and the supernatant was collected

[13] . The absorbance at 532 nm was measured using a microplate reader.

[0087] 3. Determination of CAT, SOD, POD, GSH-PX activity and soluble protein content in P. capsici mycelia: The activities of catalase (CAT), superoxide dismutase (SOD), peroxidase (POD), glutathione peroxidase (GSH-PX), and soluble protein content were measured using commercial detection kits provided by Nanjing Jiancheng Bioengineering Institute, according to the experimental procedures in the literature and the commercial instructions provided in the purchased kit

[13] .

[0088] 4. Experimental results: The experimental results showed that the increase in the concentration of Territrem B treatment was associated with an increase in MDA content Figure 7 ). After applying different concentrations of Territrem B (0, 23, and 46 μg / mL), the MDA content in the P. capsici mycelia increased to 1.59, 1.99, and 2.55 times that of the control group, respectively. The increase in antioxidant enzyme (SOD, CAT, POD, and GSH-PX) activity can reduce lipid peroxidation, remove excess active oxygen in P. capsici cells, and thus protect cells from oxidative damage. Compared with the control group, the activities of SOD, CAT, POD, and GSH-PX in the mycelia decreased in a concentration-dependent manner Figure 7 ) as the concentration of Territrem B increased. This indicates that Territrem B damages the antioxidant system of the mycelia, thereby hindering the normal growth of P. capsici mycelia; and different concentrations of Territrem B (0, 23, 46 μg / ml) reduced the soluble protein content in the P. capsici mycelia to 2.58, 2.66, and 3.40 times that of the control group, respectively Figure 7 . This indicates that Territrem B can affect the normal growth and development of P. capsici by inhibiting the production of soluble proteins and related metabolic processes.

[0089] References:

[0090] [1] Lee, S. S.; Peng, F. C; Chiou, C. M.; Ling, K. H. NMR assignment of territrem A, B, and C and the structure of MB2, the major metabolite of territrem B by rat liver microsomal fraction. Journal of Natural Products. 1992, 55(2): 251-255.

[0091] [2] Omura, S.; Kuno, F.; Otoguro, K.; Sunazuka, T.; Shiomi, K.; Masuma, R.; Iwai, Y. Arisugacin, a novel land selective inhibitor of acetylcholinesterase from Penicillium sp. FO-4259. Journal of Antibiotics. 1995, 48(7), 745-746.

[0092] [3] Nong, X. H.; Wang, Y. F.; Zhang, X. Y.; Zhou, M. P.; Xu, Xi. Y.; Qi, S. H. Territrem and butyrolactone derivatives from a marine-derived fungus Aspergillus terreus. Marine Drugs. 2014, 12(12), 6113-6124.

[0093] [4] Sun, X. H.; Kong, X. L.; Gao, H. Q; Zhu, T. J.; Wu, G. W.; Gu, Q. Q; Li, D. H. Two new meroterpenoids produced by the endophytic fungus Penicillium sp. SXH-65. Archives of Pharmacal Research. 2014, 37(8), 978-982.

[0094] [5] Otoguro, K.; Shiomi, K.; Yamaguchi, Y.; Aral, N.; Sunazuka, T.; Masuma, R.; Iwai, Y.; Omura S. Arisugacins C and D, novel acetylcholinesterase inhibitors and their related novel metabolites produced by Penicillium sp. FO-4259-11. Journal of Antibiotics. 2000, 53(1), 50-57.

[0095] [6] Zhou, Y. W.; Chen, X. C; Tang, Y. Q.; Liu, Y. N.; Zhao, Z. X.; Cui, H. New butanolide derivatives from the marine derived fungus Aspergillus terreus GZU-31-1 by chemical epigenetic manipulation. Natural Product Research. 2024, 38(8), 1334-1340.

[0096] [7] Bao, J.; Li, X. X.; Zhu, K. K.; He, F.; Wang, Y. Y.; Yu, J. H.; Zhang, X. Y.; Zhang, H. Bioactive aromatic butenolides from a mangrove sediment originated fungal species, Aspergillus terreus SCAU011. Fitoterapia, 2021, 150, 104856.

[0097] [8] Kitagawa, M.; Okabe, T.; Ogino, H.; Matsumoto, H.; Suzuki-Takahashi, I.; Kokubo, T.; Higashi, H.; Saitoh, S.; Taya, Y. Butyrolactone I, a selective inhibitor of cdk2 and cdc2 kinase. Oncogene, 1993, 8(9), 2425-2432.

[0098] [9] Wang, J. F.; Lu, Z. Y.; Liu, P. P.; Wang, Y.; Li, J.; Hong, K.; Zhu, W. M. Cytotoxic polyphenols from the fungus Penicillium expansum 091006 endogenous with the mangrove plant Excoecaria agallocha. Planta Medica. 2012, 78(17), 1861-1866.

[0099]

[10] Ibrahim, S. R. M.; Elkhayat, E. S.; Mohamed, G. A.; Khedr, A. I. M.; Fouad, M. A.; Kotb, M. H. R.; Ross, S. A. Phytochemistry Letters. 2015, 14, 84-90.

[0100]

[11] Parvatkar, R. R.; D'Souza, C; Tripathi, A.; Naik, C. G. Aspernolides A and B, butenolides from a marine-derived fungus Aspergillus terreus. Phytochemistry. 2009, 70(1), 128-132.

[0101]

[12] Wang, Y.; Sun, Y.; Zhan, Y.; Zhan, X.; Feng, J. T. Antifungal activity and biochemical response of cuminic acid against Phytophthora capsicileon. Molecules. 2016, 21(6), 756.

[0102]

[13] He, J. G.; Dou, M. L.; Xie, J.; Hou, S.; Liu, Q. F.; Hu, Z.; Zhang, B. J.; Zheng, S.; Yin, F. M.; Zhang, M.; Xie, C. P.; Lu, D. D.; Ding, X. F.; Zhu, C. H.; Sun, R. F. Discovery of zeylenone from Uvaria grandiflora as potential botanical fungicide. Pest Management Science. 2021, 77(12), 5407-5417.

[0103]

[14] Qiu S. X.; He, H.; ruan, H. C; Guan, X.; Hu, F. P. Biological control of pepper Phytophthora blight by endophytic TB2 (Bacillus sp.). Acta Phytopathologica Sinica, 2004, 34(2), 173-719.

[0104]

[15] Zhao, Z.; Li, F. Y.; Ning, J. W.; Peng, R.; Shang, J. M.; Liu, H.; Shang, M. Y.; Bao, X. Q.; Zhang, D. Novel compound FLZ alleviates rotenone-induced PD mouse model by suppressing TLR4 / MyD88 / NF-κB pathway through microbiota-gut-brain axis. Acta Pharmaceutica Sinica B. 2021, 11(9), 2859-2879.

[0105]

[16] He, W. B.; Sun, Z. C.; Tong, G.; Zeng, L.; He, W. L.; Chen, X. P.; Zhen, C. E.; Chen, P. Y; Tan, N.; He, P. C. FUNDC1 alleviates doxorubicin-induced cardiotoxicity by restoring mitochondrial-endoplasmic reticulum contacts and blocked autophagic flux. Theranostics. 2024, 14(9), 3719-3738.

Claims

1. A marine fungus Aspergillus sp. 20230605, characterized by The strain preservation number is GDMCC No.66211.

2. A crude extract of the marine fungus Aspergillus sp. 20230605 as claimed in claim 1, characterized in that The preparation method of the crude extract comprises the following steps: (1) inoculating the marine fungus Aspergillus sp.20230605 into a rice solid culture medium, and culturing at room temperature for 40-45 days to obtain a fermentation product; (2) extracting the fermentation product obtained in step (1) with an equal volume of ethyl acetate for 2-4 times, combining the extract liquid, and concentrating under reduced pressure to obtain the crude extract.

3. A process for the preparation of a compound 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 and / or 13 characterized in that The preparation method comprises the following steps: The crude extract of claim 2 is subjected to reduced pressure silica gel column chromatography, and petroleum ether-ethyl acetate is used as an eluent for gradient elution, and the elution gradient is 100:0, 90:10, 80:20, 70:30, 60:40, 50:50, 40:60, 30:70, 20:80, 10:90 and 0:100 respectively, two column volumes are collected for each gradient, the eluent obtained by using petroleum ether: ethyl acetate as 30:70 is concentrated, and then reversed-phase ODS column chromatography is carried out by using MeOH-H2O as an eluent for gradient elution, and the elution gradient is 30:70, 20:80, 10:90 and 0:100 respectively, two column volumes are collected for each gradient, the eluent obtained by using MeOH:H2O as 10:90 is concentrated, and then high performance liquid chromatography (HPLC) is carried out, the chromatographic column is Agilent C18, 9.4*250mm, 7μm, the flow rate is 2mL / min, the mobile phase is MeOH:H2O=66:34, to obtain compounds 9 and 10; the mobile phase is MeOH:H2O=58:42, to obtain compounds 7 and 8; the mobile phase is MeOH:H2O=51:49, to obtain compounds 11, 12 and 13; the mobile phase is MeOH:H2O=20:80, to obtain compounds 1, 2, 3 and 5; the mobile phase is MeOH:H2O=25:75, to obtain compounds 4 and 6; The structures of compounds 1-13 are as follows:

4. The marine fungus Aspergillus sp.20230605 of claim 1 is used for preparing the compounds 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 and / or 13 of claim 3.

5. The marine fungus Aspergillus sp.20230605 of claim 1 is used for preparing the crude extract of the marine fungus Aspergillus sp.20230605 of claim 2.

6. The compounds 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 of claim 3 or the crude extract of the marine fungus Aspergillus sp.20230605 of claim 2 or one or more thereof are used for preventing and treating pepper blight.

7. Use according to claim 6, characterized in that The pepper blight is caused by Phytophthora capsici Leonian.

8. A pesticidal composition for controlling Phytophthora capsici, characterized by The pesticide composition contains, as an active ingredient, one or more of the compounds 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 of claim 3 or a pesticidally acceptable salt thereof or the crude extract of the marine fungus Aspergillus sp. 20230605 of claim 2.

9. The pesticidal composition of claim 8, characterized in that The pesticide composition can optionally further contain a pesticidally acceptable adjuvant.

10. The pesticidal composition according to any one of claims 8 to 9, characterized in that The pesticide composition can optionally contain other active ingredients for controlling Phytophthora capsici.

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