Method for preparing dmoa mixed terpene compound by sclerotium penicillium HY5, dmoa mixed terpene compound and application thereof in herbicide

Abstract

The application provides a method for preparing a DMOA mixed terpene compound by using Sclerotinia sclerotiorum HY5, the DMOA mixed terpene compound and application of the DMOA mixed terpene compound in herbicides, and belongs to the technical field of weed control. The DMOA mixed terpene compound prepared by using the Sclerotinia sclerotiorum HY5 can regulate the growth of weeds, can promote or inhibit the growth of weeds, has the development potential as a green pesticide lead compound, and can be used for developing a novel structure and unique natural product herbicide or plant growth regulator product.

Description

Technical Field

[0001] This invention belongs to the field of weed control technology, specifically relating to a method for preparing DMOA-type mixed-source terpenoid compounds from Penicillium sclerotiorum HY5, the DMOA-type mixed-source terpenoid compounds and their application in herbicides. Background Technology

[0002] Farmland weed infestation is a prominent problem facing sustainable agricultural development. The discovery and large-scale application of chemical herbicides have played an important role in reducing weed damage and freeing up agricultural labor. However, the extensive use of chemical herbicides has also led to a series of problems, such as increased weed resistance, aggravated farmland environmental pollution, and recurring crop damage from herbicides. These issues directly affect grain yield and food safety, and seriously hinder the adjustment of agricultural planting structure and the development of the green food industry.

[0003] Microbial secondary metabolites are an important source of natural herbicides, with successful commercialization precedents. Among them, herbicidal active substances from plant-derived fungi are the most widely reported and have become a hot topic in natural herbicide research. Alternaria and Coelomycetes genera have been the most studied, producing herbicidal active compounds mainly including non-proteinogenic amino acids, cytochalasin, nonenol, benzofuran, α-pyranone, and benzoquinone, etc. Their herbicidal mechanisms include inhibition of photosystem II, inhibition of the synthesis and metabolism of key substances, obstruction of energy transfer, disruption of cell membrane function, and interference with mitosis.

[0004] Mixed-origin terpenes are a class of compounds partially derived from the terpene pathway, typically possessing unique and complex chemical skeletons and remarkably diverse biological activities. Many plants, animals, bacteria, and fungi can produce mixed-origin terpenes, with fungi being the primary producers, particularly Penicillium and Aspergillus. Based on the biosynthetic pathway of the non-terpene portion, mixed-origin terpenes can be classified into 12 classes. Among these, mixed-origin terpenes using 3,5-dimethyl serotonin (DMOA) as a precursor represent a large class of natural products; since their first discovery in 1976, over 100 DMOA-type mixed-origin terpenes have been reported. Some reports indicate that DMOA-type mixed-origin terpenes possess potential insecticidal activity.

[0005] Currently, the herbicidal activity of this type of compound has not been reported, but it is expected to lead to the development of natural product herbicides with novel structures and unique mechanisms of action. Summary of the Invention

[0006] One of the objectives of this invention is to provide the application of Penicillium sclerotiorum HY5 in the preparation of DMOA-type mixed-source terpenoid compounds. This invention utilizes Penicillium sclerotiorum HY5 to prepare DMOA-type mixed-source terpenoid compounds.

[0007] The second objective of this invention is to provide DMOA-type mixed-source terpenoid compounds prepared by the aforementioned application, which can regulate the growth of herbaceous plants.

[0008] The third objective of this invention is to provide a method for preparing DMOA-type mixed-source terpenoid compounds using Penicillium sclerotiorum HY5.

[0009] The fourth objective of this invention is to provide the application of the DMOA-type mixed-source terpenoid compounds or the DMOA-type mixed-source terpenoid compounds prepared by the method in regulating the growth of herbaceous plants. These compounds can promote or inhibit the growth of herbaceous plants.

[0010] A fifth objective of this invention is to provide the application of the aforementioned DMOA-type mixed-source terpenoid compound or the DMOA-type mixed-source terpenoid compound prepared by the aforementioned method in the preparation of herbicides or plant growth regulators. This DMOA-type mixed-source terpenoid compound has the potential to be developed as a lead compound for agricultural-related products and can be used to develop novel natural product herbicides or plant growth regulators with unique structures and mechanisms of action.

[0011] To achieve the above objectives, the present invention provides the application of Penicillium sclerotiorum HY5 in the preparation of DMOA-type mixed-source terpenoid compounds.

[0012] Preferably, the DMOA-type mixed-source terpenoid compound includes any one of compounds 1 to 9: Compounds 1-9 are all biosynthetic precursors of 3,5-dimethyltetracycline, which has the structure shown in Formula I: Formula I.

[0013] The present invention also provides a method for preparing DMOA-type mixed-source terpenoid compounds using Penicillium sclerotiorum HY5, wherein the DMOA-type mixed-source terpenoid compounds include compounds 1 to 9; The preparation method of compounds 1-9 includes the following steps: (1) Fermentation products were obtained by fermenting Penicillium HY5; (2) The fermentation product is soaked in ethyl acetate and filtered to obtain filtrate 1 and filter residue. Filtrate 1 is concentrated and dried to obtain fermentation broth extract 1. The filter residue is soaked in dichloromethane-methanol mixed solvent and filtered to collect filtrate 2. After concentration, water is added to obtain concentrated solution. The concentrated solution is extracted with ethyl acetate, concentrated and dried to obtain fermentation broth extract 2. (3) Fermentation broth extract 1 and fermentation broth extract 2 were combined to obtain crude extract; (4) The crude extract was subjected to silica gel column chromatography and C24 chromatography in sequence. 18One or more of the following methods are used to obtain column chromatography fractions containing the compound: reverse silica gel column chromatography, Sephadex LH-20 column chromatography, thin-layer chromatography separation, and HPLC purification.

[0014] Preferably, in step (1), the culture medium for fermentation includes rice culture medium or corn culture medium.

[0015] Preferably, the rice culture medium formula includes: rice and sea salt water, wherein the mass-to-volume ratio of rice to sea salt water is 90 g: 110 mL; the corn culture medium formula includes: corn and sea salt water, wherein the mass-to-volume ratio of corn to sea salt water is 90 g: 110 mL.

[0016] Preferably, the fermentation culture time is 20 to 40 days, and the fermentation culture temperature is 25 to 30°C.

[0017] This invention also provides the application of DMOA-type mixed-source terpenoids in regulating the growth of herbaceous plants, wherein the DMOA-type mixed-source terpenoids are compounds 1-9 or DMOA-type mixed-source terpenoids prepared by the method.

[0018] Preferably, the regulation includes promoting or inhibiting the growth of herbaceous plants.

[0019] This invention also provides the application of DMOA-type mixed-source terpenoid compounds in the preparation of herbicides or plant growth regulators, wherein the DMOA-type mixed-source terpenoid compounds are compounds 1-9 or DMOA-type mixed-source terpenoid compounds prepared by the method.

[0020] Preferably, the application targets include dicotyledonous weeds and / or monocotyledonous weeds.

[0021] Preferably, the dicotyledonous weeds include amaranth and clover, and the monocotyledonous weeds include goosegrass and foxtail grass.

[0022] Compared with the prior art, the present invention has the following beneficial effects: This invention provides the application of Penicillium sclerotium HY5 in the preparation of DMOA-type mixed-source terpenoid compounds. The preservation number of Penicillium sclerotium HY5 is CGMCC No. 20263. Using Penicillium sclerotium HY5, DMOA-type mixed-source terpenoid compounds that can regulate the growth of herbaceous plants were prepared, including those that promote or inhibit the growth of herbaceous plants.

[0023] This invention provides the application of the DMOA-type mixed-source terpenoid compound or the DMOA-type mixed-source terpenoid compound prepared by the method in the preparation of herbicides or plant growth regulators. The DMOA-type mixed-source terpenoid compound has the potential to be developed as a lead compound for agricultural-related products and can be used to develop natural product herbicides or plant growth regulators with novel structures and unique mechanisms of action. Attached Figure Description

[0024] Figure 1 The diagram shows the structures of DMOA-type mixed-source terpenoid compounds 1-9 isolated in Example 1, where 1 represents compound 1, berkeleydione; 2 represents compound 2, preaustinoid A3; 3 represents compound 3, miniolutelide B; 4 represents compound 4, 22-epoxyberkeleydione; 5 represents compound 5, 22-deoxyminiolutelide B; 6 represents compound 6, miniolutelide C; 7 represents compound 7, berkeleyacteal; 8 represents compound 8, preaustinoid A2; and 9 represents compound 9, berkeleyacetal A.

[0025] Figure 2 The results show the inhibitory activity of the compound at a concentration of 200 μg / mL on the germination of four types of weed seeds in Example 1.

[0026] Figure 3 Example 1 illustrates the inhibitory effects of compounds 1, 3, 4, 5, 6, and 7 on seed germination, where A is Amaranthus retroflexus, B is Clover, C is Setaria viridis, and D is Eleusine indica.

[0027] Figure 4 The results show the inhibitory activity of compound 6 at different concentrations on the germination of Eleusine indica seeds in Example 1.

[0028] Figure 5 The transcriptomic and metabolomics analysis results of *Eleusine indica* seeds treated with compound 6 in Example 1 are shown below. A is a volcano plot of differentially expressed genes (DEGs), with red and blue dots representing upregulated and downregulated differentially expressed genes, respectively. B is the enrichment of the KEGG pathway by DEGs, with the x-axis and y-axis representing enrichment factors and pathway terms, respectively. The significance of DEGs is indicated by color and size. C is a volcano distribution map of differentially expressed metabolites (DEMs). D is the KEGG pathway enrichment analysis of DEMs. E is the principal component analysis (PCA) of CK and compound 6, with the x-axis and y-axis representing the first principal component (PC1) and the second principal component (PC2), respectively.

[0029] Figure 6The expression levels of selected genes were verified using real-time quantitative PCR in Example 1. Values ​​are expressed as mean ± standard deviation, with "****" indicating... P <0.0001. Detailed Implementation

[0030] This invention provides the application of *Penicillium sclerotium* HY5 in the preparation of DMOA-type mixed-source terpenoid compounds. The preservation number of *Penicillium sclerotium* HY5 is CGMCC No. 20263. See reference: Wang W, Wang M, Wang XB, Li YQ, Ding JL, Lan MX, Gao X, Zhao DL, Zhang CS and Wu GX (2022) Phytotoxic Azaphilones From the Mangrove-Derived Fungus Penicillium sclerotiorum HY5. This invention utilizes Penicillium sclerotium HY5 to prepare DMOA-type mixed-source terpenoid compounds that can regulate the growth of herbaceous plants, including DMOA-type mixed-source terpenoid compounds that promote the growth of herbaceous plants and DMOA-type mixed-source terpenoid compounds that inhibit the growth of herbaceous plants.

[0031] The present invention also provides that the DMOA-type mixed-source terpenoid compounds include any one of compounds 1 to 9: Compounds 1-9 are all biosynthetic precursors of 3,5-dimethyltetracycline, which has the structure shown in Formula I: Formula I.

[0032] In this invention, 3,5-dimethyl morphose acid mixed-source terpenoids are abbreviated as DMOA mixed-source terpenoids; the English name of compound 1 is berkeleydione, CAS: 681250-16-8; the English name of compound 2 is preaustinoid A3, CAS: 2987168-73-8; the English name of compound 3 is miniolutelide B, CAS: 1012080-80-6; the English name of compound 4 is 22-epoxyberkeleydione, CAS: 1011803-04-5; the English name of compound 5 is 22-deoxyminiolutelide B, CAS: 1799846-33-5; the English name of compound 6 is miniolutelide C, CAS: 1799846-34-6; the English name of compound 7 is berkeleyacteal, CAS: 3058700-24-3; and the English name of compound 8 is preaustinoid. A2, CAS: 679403-04-4; Compound 9 is named Berkeleyacetal A, CAS: 959772-65-7.

[0033] The present invention also provides a method for preparing DMOA-type mixed-source terpenoid compounds using Penicillium sclerotiorum HY5, wherein the DMOA-type mixed-source terpenoid compounds include compounds 1 to 9; The preparation method of compounds 1-9 includes the following steps: (1) Fermentation products were obtained by fermenting Penicillium HY5; (2) The fermentation product is soaked in ethyl acetate and filtered to obtain filtrate 1 and filter residue. Filtrate 1 is concentrated and dried to obtain fermentation broth extract 1. The filter residue is soaked in dichloromethane-methanol mixed solvent and filtered to collect filtrate 2. After concentration, water is added to obtain concentrated solution. The concentrated solution is extracted with ethyl acetate, concentrated and dried to obtain fermentation broth extract 2. (3) Fermentation broth extract 1 and fermentation broth extract 2 were combined to obtain crude extract; (4) The crude extract was subjected to silica gel column chromatography and C24 chromatography in sequence. 18 One or more of the following methods are used to obtain column chromatography fractions containing the compound: reverse silica gel column chromatography, Sephadex LH-20 column chromatography, thin-layer chromatography separation, and HPLC purification.

[0034] In step (1) above, when preparing the fermentation product, the mycelium of strain HY5 after activation is inoculated into PDW medium to obtain seed liquid; the preferred culture temperature is 25~32℃, more preferably 26~31℃, more preferably 27~30℃, and most preferably 28℃; the preferred culture speed is 150rpm~200rpm, more preferably 160rpm~190rpm, and more preferably 170rpm or 180rpm; the preferred culture time is 1~3 days, more preferably 2~3 days, and more preferably 3 days. The seed liquid is inoculated into corn medium or rice medium to obtain fermentation product; the rice medium formula includes: rice and sea salt water, the mass-volume ratio of rice to sea salt water is 90 g: 110 mL, wherein the sea salt water is prepared by adding 3% crude sea salt to 110 mL of deionized water. The specific preparation method of rice medium is as follows: 90g of rice is added to 110mL of sea salt water, and sterilized by high pressure at 121℃ for 20min. The corn culture medium formulation comprises corn and seawater brine, wherein the mass-to-volume ratio of corn to seawater brine is 90 g:110 mL, wherein the seawater brine is prepared by adding 3% crude sea salt to 100 mL of deionized water. The corn culture medium is prepared according to the above-described method for preparing rice culture medium. The fermentation time is 20-40 days, more preferably 25-35 days, and more preferably 30 days; the fermentation temperature is 25-30℃, more preferably 26-29℃, and more preferably 27℃ or 28℃.

[0035] In step (2) above, when soaking the fermentation product with ethyl acetate, the soaking time is preferably 24 hours and the soaking temperature is preferably 25°C; after filtration, filtrate 1 and filter residue are obtained, and filtrate 1 is concentrated and dried to obtain fermentation broth extract 1; the filter residue (i.e. the fermentation product soaked in ethyl acetate) is soaked again with dichloromethane-methanol mixed solvent, filtered, and filtrate 2 is collected. The soaking time is preferably 24 hours and the soaking temperature is preferably 25°C. Filtrate 2 is concentrated and dried to obtain concentrated product. Water is added to prepare concentrated solution. The volume ratio of concentrated product to water is 1:1 (v / v). The concentrated solution is extracted 3 times with an equal volume of ethyl acetate, and the extracts are combined, concentrated, and dried to obtain fermentation broth extract 2.

[0036] In step (3) above, fermentation extract 1 and fermentation extract 2 are mixed evenly when combined to obtain crude extract.

[0037] In this invention, when preparing compound 1, the culture medium for preparing the fermentation product in step (1) above is rice culture medium, the fermentation temperature is selected as 28℃, and the fermentation time is 30 days. The crude extract obtained is prepared using any solvent or combination of solvents such as ethyl acetate, methanol, and dichloromethane, more preferably dichloromethane-methanol solvent, and more preferably dichloromethane-methanol solvent with a volume fraction of 1:1 (v / v). In step (4), when using silica gel column chromatography for separation, gradient elution is employed. The eluent for gradient elution includes PE-EtOAc with volume fractions of 90% / 10%, 80% / 20%, 70% / 30%, 50% / 50%, 30% / 70%, and 0 / 100%, and MeOH-EtOAc with volume fractions of 10% / 90%, 20% / 80%, and 30% / 70%. The eluted fraction of 50% / 50% PE-EtOAc is collected as Fr.3. The Fr.3 fraction is then subjected to gradient elution using C18 reversed silica gel column chromatography, with the elution system consisting of methanol-EtOAc with volume fractions of 30%~100%. Aqueous solution; collect the eluent of 80% methanol-water solution as Fr.3.5; Fr.3.5 is separated by Sephadex LH-20 gel chromatography column, and thin-layer chromatography is performed using a PE:EtOAc developing system with a PE:EtOAc volume ratio of 1:1 (v / v). The chromatograms with the same color development are combined and further separated and purified by semi-preparative high-performance liquid chromatography. The compound with a final retention time of 84.02 min is collected to obtain compound 1; the eluent of the semi-preparative high-performance liquid chromatography is a MeOH:H2O solution with a volume fraction of 50:50 (v / v).

[0038] In this invention, when preparing compound 2, based on the overall preparation of compound 1 described above, the uncollected portion of compound 1 is separated and purified by semi-preparative high-performance liquid chromatography (HPLC), and further separated and purified by semi-preparative HPLC. The eluent for the semi-preparative HPLC is a solution with a volume fraction of MeOH:H2O=70:30 (v / v). The compound with a final retention time of 14.27 min is collected to obtain compound 2.

[0039] In this invention, when preparing compound 3, the culture medium for preparing the fermentation product in step (1) above is rice culture medium, the fermentation temperature is selected as 28℃, and the fermentation time is 30 days. The crude extract obtained is prepared using any solvent or combination of solvents such as ethyl acetate, methanol, and dichloromethane, more preferably dichloromethane-methanol solvent, and more preferably dichloromethane-methanol solvent with a volume fraction of 1:1 (v / v). In step (4), when using silica gel column chromatography for separation, gradient elution is employed. The eluent for gradient elution includes PE-EtOAc with volume fractions of 90% / 10%, 80% / 20%, 70% / 30%, 50% / 50%, 30% / 70%, and 0 / 100%, and MeOH-EtOAc with volume fractions of 10% / 90%, 20% / 80%, and 30% / 70%. The eluted fraction of 50% / 50% PE-EtOAc is collected as Fr.3. The Fr.3 fraction is then subjected to gradient elution using C18 reversed silica gel column chromatography with a volume fraction of 30%~100% methanol. - Aqueous solution; collect the eluent of 70% methanol-water solution as Fr.3.4; Fr.3.4 is separated by chromatography using a Sephadex LH-20 gel chromatography column, and thin-layer chromatography is performed using CH2Cl2-MeOH as the developing system, with a volume ratio of CH2Cl2:MeOH of 20:1 (v / v). The chromatographic solutions with the same color development are combined, and then preparative thin-layer chromatography separation and purification are performed using CH2Cl2-MeOH solvent to obtain compound 3; preferably, the volume ratio of CH2Cl2-MeOH solvent is CH2Cl2:MeOH = 20:1 (v / v).

[0040] In this invention, when preparing compound 4, based on the overall preparation of compound 3 described above, preparative thin-layer chromatography (TLC) is performed using CH2Cl2:MeOH = 20:1 to obtain compound 3. The impure portion is further separated using CH2Cl2:Acetone = 20:1 to obtain Fr.3.4.1. Fr.3.4.1 is then subjected to semi-preparative high-performance liquid chromatography (HPLC), with CH3CN-H2O solvent as the eluent. The compound with a final retention time of 42.02 min is collected to obtain compound 4. Preferably, the CH3CN-H2O is used with a volume fraction of 35:65 (v / v).

[0041] In this invention, when preparing compound 5, the culture medium for preparing the fermentation product in step (1) above is rice culture medium, the fermentation temperature is selected as 28℃, and the fermentation time is 30 days. The crude extract obtained is prepared using any solvent or combination of solvents such as ethyl acetate, methanol, and dichloromethane, more preferably dichloromethane-methanol solvent, and more preferably dichloromethane-methanol solvent with a volume fraction of 1:1 (v / v). In step (4), when using silica gel column chromatography for separation, gradient elution is employed. The eluent for gradient elution includes PE-EtOAc with volume fractions of 90% / 10%, 80% / 20%, 70% / 30%, 50% / 50%, 30% / 70%, and 0 / 100%, and MeOH-EtOAc with volume fractions of 10% / 90%, 20% / 80%, and 30% / 70%. The elution fraction of 30% / 70% PE-EtOAc is collected as Fr.4. The Fr.4 fraction is then subjected to gradient elution using C18 reversed silica gel column chromatography with a methanol-water solution of volume fractions of 30%~100%. The eluent of 40% methanol-water solution is collected as Fr.4.2. Fr.4.2 is then separated by chromatography using a Sephadex LH-20 gel chromatography column with CH2Cl2-MeOH solvent as the developing system for thin-layer chromatography, preferably. The volume ratio of CH2Cl2 to MeOH in the CH2Cl2-MeOH solvent was CH2Cl2:MeOH = 10:1 (v / v). The thin-layer chromatography solutions with the same color development were combined, concentrated and dried to obtain compound 5.

[0042] In this invention, when preparing compound 6, the culture medium for preparing the fermentation product in step (1) above is rice culture medium, the fermentation temperature is selected as 28℃, and the fermentation time is 30 days. The crude extract obtained is prepared using any solvent or combination of solvents such as ethyl acetate, methanol, and dichloromethane, more preferably dichloromethane-methanol solvent, and more preferably dichloromethane-methanol solvent with a volume fraction of 1:1 (v / v). In step (4), when using silica gel column chromatography for separation, gradient elution is employed. The eluent for gradient elution includes PE-EtOAc with volume fractions of 90% / 10%, 80% / 20%, 70% / 30%, 50% / 50%, 30% / 70%, and 0 / 100%, and MeOH-EtOAc with volume fractions of 10% / 90%, 20% / 80%, and 30% / 70%. The elution fraction of 30% / 70% PE-EtOAc is collected as Fr.4. The Fr.4 fraction is then subjected to gradient elution using C18 reversed silica gel column chromatography with a methanol-water solution of volume fractions of 30%~100%. The eluent of 50% methanol-water solution is collected as Fr.4.3. Fr.4.3 is then separated by chromatography using a Sephadex LH-20 gel chromatography column, preferably using CH2Cl2-MeOH solvent as the developing system. The volume ratio of CH2Cl2 to MeOH in the CH2Cl2-MeOH solvent was CH2Cl2:MeOH = 10:1 (v / v). The thin-layer chromatography solutions with the same color development were combined, concentrated and dried to obtain compound 6.

[0043] In this invention, the preparation of compound 7 is carried out based on the overall preparation of compound 6 described above. After obtaining compound 6 by preparative thin-layer chromatography using CH2Cl2:MeOH = 10:1 (v / v), the impure portion is designated as Fr.4.3. Fr.4.3 is then subjected to semi-preparative high-performance liquid chromatography (HPLC) separation. The eluent for the semi-preparative HPLC is CH3CN-H2O solvent. The compound with a final retention time of 19.76 min is collected to obtain compound 7. Preferably, the CH3CN-H2O is prepared with a volume fraction of 45:55 (v / v).

[0044] In this invention, when preparing compound 8, the culture medium for preparing the fermentation product in step (1) above is rice culture medium, the fermentation temperature is selected as 28℃, and the fermentation time is 30 days. The crude extract obtained is prepared using any solvent or combination of solvents such as ethyl acetate, methanol, and dichloromethane, more preferably dichloromethane-methanol solvent, and more preferably dichloromethane-methanol solvent with a volume fraction of 1:1 (v / v). In step (4), when using silica gel column chromatography for separation, gradient elution is employed. The eluent for gradient elution includes PE-EtOAc with volume fractions of 90% / 10%, 80% / 20%, 70% / 30%, 50% / 50%, 30% / 70%, and 0 / 100%, and MeOH-EtOAc with volume fractions of 10% / 90%, 20% / 80%, and 30% / 70%. The eluted fractions of 80% / 20% and 70% / 30% PE-EtOAc are collected as Fr.2. The Fr.2 fraction is then subjected to gradient elution using C18 reversed silica gel column chromatography with a methanol-water solution of volume fractions of 30%~100%. The eluted fraction of 40% methanol-water solution is collected as Fr.2.2. Fr.2.2 is then subjected to Sephadex chromatography. Chromatographic separation was performed using an LH-20 gel chromatography column. Thin-layer chromatography was performed using CH2Cl2-MeOH solvent as the developing system, with a volume ratio of CH2Cl2 to MeOH of 10:1 (v / v). Chromatographic solutions with the same color were combined and further separated and purified using semi-preparative high-performance liquid chromatography (SPLC). Compounds with a final retention time of 23.4 min were collected to obtain compound 8. The eluent for the semi-preparative SPLC was a MeOH-H2O solution with a volume fraction of 65:35 (v / v).

[0045] In this invention, when preparing compound 9, the culture medium for preparing the fermentation product in step (1) above is corn culture medium, the fermentation temperature is selected as 28℃, and the fermentation time is 30 days. The crude extract obtained is prepared using any solvent or combination of solvents such as ethyl acetate, methanol, and dichloromethane, more preferably dichloromethane-methanol solvent, and more preferably dichloromethane-methanol solvent with a volume fraction of 1:1 (v / v). In step (4), when using silica gel column chromatography for separation, gradient elution is employed. The eluent for gradient elution includes PE-EtOAc with volume fractions of 90% / 10%, 80% / 20%, 70% / 30%, 50% / 50%, 30% / 70%, and 0 / 100%, and MeOH-EtOAc with volume fractions of 10% / 90%, 20% / 80%, and 30% / 70%. The eluting fraction Hr.3 of 70% / 30% PE-EtOAc is collected. The Hr.3 fraction is then subjected to gradient elution using C18 reversed silica gel column chromatography. The elution system used a methanol-water solution with a volume fraction of 30%–80%; the eluents of 40% and 50% methanol-water solutions were collected as Hr.3.2; Hr.3.2 was separated by chromatography using a Sephadex LH-20 gel chromatography column, and thin-layer chromatography was performed using CH2Cl2-MeOH solvent as the developing system, with a volume ratio of CH2Cl2 to MeOH of 10:1 (v / v). The chromatograms with the same color development were combined and further separated and purified by semi-preparative high-performance liquid chromatography. The compound with a final retention time of 26.87 min was collected to obtain compound 9; the eluent for the semi-preparative high-performance liquid chromatography was a CH3CN-H2O solution with a volume fraction of 40:60 (v / v).

[0046] This invention also provides the application of DMOA-type mixed-source terpenoids in regulating the growth of herbaceous plants, wherein the DMOA-type mixed-source terpenoids are compounds 1-9 or DMOA-type mixed-source terpenoids prepared by the method described above. The regulation includes promoting or inhibiting the growth of herbaceous plants.

[0047] This invention also provides the application of DMOA-type mixed-source terpenoid compounds in the preparation of herbicides or plant growth regulators, wherein the DMOA-type mixed-source terpenoid compounds are compounds 1-9 or DMOA-type mixed-source terpenoid compounds prepared by the method described above. The application targets include dicotyledonous weeds and / or monocotyledonous weeds; preferably, the dicotyledonous weeds include amaranth and clover, and the monocotyledonous weeds include goosegrass and foxtail.

[0048] In this invention, plant growth regulators are used to promote plant growth, such as promoting the growth of corn, wheat, soybeans, etc., or promoting the growth of forage grass; herbicides are used to inhibit plant growth, including dicotyledonous weeds and monocotyledonous weeds. Preferably, the dicotyledonous weeds include amaranth and clover, and the monocotyledonous weeds include goosegrass and foxtail grass.

[0049] In this invention, compounds 1, 3, 4, and 5 promote the growth of the dicotyledonous weed *Amaranthus retroflexus*, and compound 4 also promotes the growth of the dicotyledonous weed *Clerodendrum trifolium*. After structural optimization, they may have the potential to be used as plant growth promoters.

[0050] In this invention, compounds 3, 4, and 5 inhibited the growth of the monocotyledonous weed *Eleusine indica*. Compound 6 (miniolutelide C), at a concentration of 200 μg / mL, exhibited herbicidal activity against clover embryo elongation with an inhibition rate of 66.4%, and against *Setaria viridis* embryo elongation with an inhibition rate as high as 95.3%. Its inhibitory effect on *Eleusine indica* radicle and embryo elongation was most prominent, with inhibition rates of 93.2% and 87.0%, respectively. This effect was superior to the positive controls glyphosate and acetochlor. EC on embryo and radicle elongation... 50 The concentration ranges from 17.9 to 19.1 μg / mL. Although its inhibitory activity is not as good as that of acetochlor, it is better than that of glyphosate. It has the potential to be developed as a lead compound for green pesticides, and its herbicidal activity against monocotyledonous weeds is much better than that against dicotyledonous weeds.

[0051] The references involved in this invention are as follows: [1] STIERLE DB, STIERLE AA, HOBBS JD, STOKKEN J, CLARDY J.Berkeleydione and berkeleytrione, new bioactive metabolites from an acid mineorganism. Organic Letters, 2004, 6: 1049-1052. [2] LO HC, ENTWISTLE R, GUO CJ, AHUJA M, SZEWCZYK E, HUNG JH,CHIANG YM, OAKLEY BR, WANG CC C. Two separate gene clusters encode the biosynthetic pathway for the meroterpenoids austinol and dehydroaustinolin Aspergillus nidulans. Journal of the American Chemical Society, 2012, 134:4709-4720. [3] IIDA M, OOI T, KITO K, YOSHIDA S, KANOH K, SHIZURI Y, KUSUMI T.Three new polyketide−terpenoid hybrids from Penicillium sp. Organic Letters,2008, 10: 845-848. [4] HOANG T P T, ROULLIER C, BOUMARD M C, du PONT T R, NAZIH H,GALLARD J F, POUCHUS Y F, BENIDDIR M A, GROVEL O. Metabolomics-drivendiscovery of meroterpenoids from a mussel-derived Penicillium ubiquitum .Journal of Natural Products, 2018, 81: 2501-2511. [5] LI J, YANG X, LIN Y Y, YUAN J, LU Y J, ZHU X, LI J, LI M F, LIN YC, HE J G, LIU L. Meroterpenes and azaphilones from marine mangroveendophytic fungus Penicillium 303#. Fitoterapia, 2014, 97: 241-246. [6] FENG Q M, YU Y, TANG M X, ZHANG T Y, ZHANG M Y, WANG H F, HAN YQ, ZHANG Y X, CHEN G, PEI Y H. Four new hybrid polyketide-terpenoidmetabolites from the Penicillium sp. SYPF7381 in the rhizosphere soilof Chinese Pulsatilla . Fitoterapia, 2018, 125: 249-257. [7] MATSUDA Y, AWAKAWA T, WAKIMOTO T, ABE I. Spiro-ring formation iscatalyzed by a multifunctional dioxygenase in austinol biosynthesis. Journal of the American Chemical Society, 2013, 135: 10962-10965. [8] STIERLE DB, STIERLE AA, PATACINI B. The berkeleyacetals, threemeroterpenes from a deep water acid mine waste Penicillium . Journal of NaturalProducts, 2007, 70: 1820-1823.

[0052] In the following embodiments of the present invention, HY5 is Penicillium sclerotiorum. Penicillium sclerotiorum The preservation number is CGMCC No. 20263. *Penicillium sclerotium* Penicillium sclerotiorum HY5 is referenced in: Wang W, Wang M, Wang XB, Li YQ, Ding JL, Lan MX, Gao X, Zhao DL, Zhang CS and Wu GX (2022) Phytotoxic Azaphilones From the Mangrove-Derived Fungus Penicilliumsclerotiorum HY5. Biological preservation information: Penicillium sclerotiorum Penicillium sclerotiorum HY5, deposited on August 31, 2020, at the China General Microbiological Culture Collection Center (CGMCC), specifically at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, with accession number CGMCC No. 20263.

[0053] The weed seeds used in the following embodiments of the present invention were all purchased from Taobao, and no specific source is specified.

[0054] Unless otherwise specified, the test methods used in the following examples are conventional test methods; the materials and reagents used are commercially available unless otherwise specified.

[0055] The technical solutions of the present invention will be clearly and completely described below with reference to the embodiments thereof.

[0056] Example 1 I. Source of the strain Penicillium sclerotiorum derived from leaves of Hainan mangroves Penicillium sclerotiorum HY5 has been deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 20263.

[0057] II. Fermentation Extraction 1. Fermentation culture and isolation / purification of strain HY5 1.1 Fermentation by strain Rice culture medium: 90 g rice, 110 mL sea salt (prepared by adding 3% crude sea salt by mass / volume ratio to 100 mL deionized water).

[0058] Corn culture medium: 90 g corn, 110 mL sea salt solution (prepared by adding 3% crude sea salt by mass / volume ratio to 100 mL deionized water).

[0059] The mycelia of the activated strain HY5 were inoculated into PDW medium and cultured on a shaker at 28℃ and 180 rpm for 3 days to obtain seed culture.

[0060] Add 5 mL of the above seed culture to each of the Erlenmeyer flasks containing the rice culture medium and corn culture medium (Erlenmeyer flask specifications: 1L, 100 flasks of rice culture medium and 100 flasks of corn culture medium), seal the flasks, and incubate at 28℃ for 30 days.

[0061] 1.2 Compound Extraction and Separation (1) Isolation and purification of rice fermentation products After fermentation, ethyl acetate was poured into 1L Erlenmeyer flasks containing rice culture medium fermentation products (400 mL ethyl acetate per flask) to submerge the cells, and allowed to stand at room temperature (25℃) for one day (24h). The filtrate was then filtered through gauze, and the ethyl acetate extract 1 obtained was collected. The extract was concentrated and evaporated to dryness using a rotary evaporator at 40℃ and 90 r / min to obtain fermentation broth extract 1.

[0062] The rice culture medium fermentation product soaked in ethyl acetate (the filtered residue) was then soaked in a dichloromethane-methanol mixed solvent (dichloromethane / methanol, v / v=1:1) (400 mL per bottle) at 25°C for 24 h. The mixture was then filtered again through two layers of gauze. The filtrate was collected and concentrated under reduced pressure to evaporate the organic solvent, yielding a concentrated product. Water (1:1 volume ratio) was then added to the concentrated product to prepare a concentrate. This concentrate was extracted three times with an equal volume of ethyl acetate, and the extracts were combined to obtain ethyl acetate extract 2. The solvent was evaporated using a rotary evaporator at 40°C and 90 r / min to obtain fermentation broth extract 2. The two fermentation broth extracts (fermentation broth extract 1 and fermentation broth extract 2) were combined and mixed thoroughly to obtain 178 g of crude extract.

[0063] The crude extract was dissolved in 300 mL of dichloromethane-methanol (v / v=1:1) solvent, and 500 mL of silica gel (silica gel particle size of 200~300 mesh) was added and mixed. After the solvent evaporated, the crude extract was separated by gradient elution chromatography using a vacuum silica gel column. The volume ratio of blank silica gel to mixed silica gel was 1.5:1. The gradient elution process sequentially uses elution systems 1 through 9, which are: PE-EtOAc with volume fractions of 90% / 10%, 80% / 20%, 70% / 30%, 50% / 50%, 30% / 70%, and 0 / 100%, and MeOH-EtOAc with volume fractions of 10% / 90%, 20% / 80%, and 30% / 70%, respectively; the circulation pump pressure reaches 0.03MPa~0.04MPa; the volume of PE-EtOAc with a volume fraction of 90% / 10% is 20 L, the volume of PE-EtOAc with a volume fraction of 80% / 20% is 15 L, the volume of PE-EtOAc with a volume fraction of 70% / 30% is 15 L, and the volume of PE-EtOAc with a volume fraction of 50% / 50% is 15 L. The volume fractions of PE-EtOAc (30% / 70%) and PE-EtOAc (0% / 100%) were 10 L each, along with 10 L of 10% / 90% EtOAc-MeOH, 5 L of 20% / 80% EtOAc-MeOH, and 5 L of 30% / 70% EtOAc-MeOH. Each elution system was collected in 500 mL bottles, concentrated to dryness, and then dissolved in a solvent mixture of ethyl acetate:methanol:dichloromethane (volume ratio = 1:1:1). The elution fractions of 90% / 10% PE-EtOAc were designated Fr.1, 80% / 20% and 70% / 30% PE-EtOAc as Fr.2, and 50% / 50% PE-EtOAc as Fr.3. The elution fractions of 30% / 70% PE-EtOAc are designated as Fr.4, 0 / 100% PE-EtOAc as Fr.5, 10% / 90% MeOH-EtOAc as Fr.6, 20% / 80% MeOH-EtOAc as Fr.7, and 30% / 70% MeOH-EtOAc as Fr.8.

[0064] Fr.2 was eluted by gradient chromatography on a C18 reversed silica gel column using a methanol-water solution with a volume fraction of 30%–100%. The average elution rate was 8 mL / min, and the volume of each volume fraction of methanol-water solution was 1500 mL. The eluent was collected in 150 mL portions and combined according to the volume fraction of the same elution system, designated as Fr.2.1–Fr.2.7. Specifically, the eluent with 30% methanol-water solution was designated Fr.2.1, 40% methanol-water solution Fr.2.2, 50% methanol-water solution Fr.2.3, 70% methanol-water solution Fr.2.4, 80% methanol-water solution Fr.2.5, 90% methanol-water solution Fr.2.6, and 100% methanol solution Fr.2.7.

[0065] For Fr.2.2, preliminary separation was first performed using a Sephadex LH-20 gel chromatography column (GE Healthcare, catalog number 117009002). An automatic collector was used to collect the fractions at a flow rate of 2.0 mL / min, collecting 10 mL from each tube, for a total of 40 tubes. Then, thin-layer chromatography (TLC) was performed using CH2Cl2-MeOH (10:1, v / v). Fractions with identical color development and Rf values ​​were grouped together as one fraction. Color development was performed using 12%–14% concentrated sulfuric acid reagent containing vanillin. The Rf value of the eluent was measured to be 0.4–0.5. Chromatographic solutions with identical spots were combined. Finally, semi-preparative high-performance liquid chromatography (HPLC) was used for further purification. The HPLC model was Waters e2695, the detector model was Waters 2998 PDA, and the column model was Waters X-Bridge C18, 10×250 mm, 5. μ m), injection volume 100 μL, column temperature 30±5℃, elution buffer MeOH:H2O solution, MeOH:H2O=65:35 (v / v), isocratic elution and purification, flow rate 2 min / mL, to obtain compound 8 (7.2 mg, t R =23.4-25.2 min).

[0066] Fr.3 (PE-EtOAc = 50% / 50% fraction) was subjected to gradient elution by C18 reversed silica gel column chromatography with elution systems of methanol-water solutions ranging from 30% to 100% by volume. The average elution rate was 8 mL / min, and the volume of each methanol-water solution was 1500 mL. The eluent was collected in 150 mL portions and combined according to the same volume fraction, and labeled as 8 subfractions (Fr.3.1-Fr.3.8); among them, the eluent of 30% methanol-water solution was labeled as Fr.3.1, the eluent of 40% methanol-water solution was labeled as Fr.3.2, the eluent of 50% methanol-water solution was labeled as Fr.3.3, the eluent of 70% methanol-water solution was labeled as Fr.3.4, the eluent of 80% methanol-water solution was labeled as Fr.3.5, the eluent of 90% methanol-water solution was labeled as Fr.3.6, the eluent of 95% methanol-water solution was labeled as Fr.3.7, and the eluent of 100% methanol solution was labeled as Fr.3.8.

[0067] Fraction Fr.3.4 was separated by Sephadex LH-20 gel column chromatography (GE Healthcare, catalog number 117009002). An automatic collector was used to collect the fractions at a flow rate of 2.0 mL / min, collecting 10 mL from each tube, for a total of 40 tubes. Thin-layer chromatography (TLC) was then performed using CH2Cl2-MeOH (20:1) as the developing system. Fractions showing the same color on the TLC plate were grouped together and analyzed using 12%-14% concentrated sulfuric acid reagent containing vanillin. The Rf value of the eluent was measured to be 0.4-0.5. Chromatographic solutions showing the same color were combined. Preparative TLC was then performed using CH2Cl2:MeOH = 20:1 to obtain compound 3 (35.3). (mg); after thin-layer chromatography separation using CH2Cl2:MeOH=20:1, further thin-layer chromatography separation using CH2Cl2:Acetone=20:1 was performed to obtain Fr.3.4.1.

[0068] Fr.3.4.1 Semi-preparative high-performance liquid chromatography (HPLC) separation was performed. The HPLC system used was Waters e2695, the detector was Waters 2998 PDA, and the column model was Waters X-Bridge C18 (10×250 mm, 5...). μ The injection volume was 100 μL, the column temperature was 30±5℃, and the eluent was CH3CN-H2O (CH3CN-H2O = 35:65 v / v). Isocratic elution and purification were performed at a flow rate of 2 min / mL to obtain compound 4 (9.6 mg, t). R =42.02-44.16min).

[0069] Fraction Fr.3.5 was separated by chromatography using a Sephadex LH-20 gel permeation column (GE Healthcare, catalog number 117009002). An automatic collector was used to collect the fractions at a flow rate of 2.0 mL / min, collecting 10 mL from each tube, for a total of 40 tubes. Thin-layer chromatography (TLC) was then performed using a PE:EtOAc (1:1, v / v) developing system. Fractions showing the same color on the TLC plate were grouped together and labeled as one fraction. The eluent was then developed with 12%-14% concentrated sulfuric acid containing vanillin. The Rf value of the eluent was measured to be 0.4-0.5. Chromatographic solutions showing the same color were combined and further purified using semi-preparative high-performance liquid chromatography (HPLC). The HPLC system used was a Waters e2695, the detector was a Waters 2998 PDA column, and the column model was a Waters X-Bridge C18, 10×250 mm, 5. μ m), injection volume 100 μL, column temperature 30±5℃, elution buffer MeOH:H2O solution, MeOH:H2O=50:50 (v / v), isocratic elution and purification, flow rate 2 min / mL, to obtain compound 1 (29.4 mg, t R =84.02-87.23 min), then separated and purified by semi-preparative liquid chromatography. The eluent was MeOH:H2O solution, MeOH:H2O=70:30, v / v, injection volume 100 μL, column temperature 30±5℃, liquid chromatography model: Waters e2695, detector model: Waters 2998 PDA column model: Waters X-Bridge C18, 10×250 mm, 5 μ Compound 2 (3.1 mg, t) was prepared by isocratic elution and purification at a flow rate of 2 min / mL. R =14.27-15.32 min).

[0070] Fr.4 (PE-EtOAc = 30% / 70% fraction) was subjected to gradient elution by C18 reversed silica column chromatography (Merck, Inc., catalog number 1.13900.1000). The elution system consisted of methanol-water solutions with volume fractions ranging from 30% to 90%. The average elution rate was 8 mL / min, and the volume of each methanol-water solution was 1500 mL. The eluent was collected in 150 mL portions and combined with the same volume fraction to separate five subfractions (Fr.4.1-Fr.4.5). The eluent with 30% methanol-water solution was designated Fr.4.1, the eluent with 40% methanol-water solution was designated Fr.4.2, the eluent with 50% methanol-water solution was designated Fr.4.3, the eluent with 70% methanol-water solution was designated Fr.4.4, and the eluent with 90% methanol-water solution was designated Fr.4.5.

[0071] Fr.4.2 After separation by Sephadex LH-20 gel chromatography column (GE, USA, catalog number 117009002), chromatographic separation was performed. The fractions were collected using an automatic collector at a flow rate of 2.0 mL / min, with 10 mL collected from each tube, for a total of 40 tubes. Thin-layer chromatography (TLC) was then performed using CH2Cl2:MeOH (10:1) as the developing system. The fractions with the same color development on the TLC plate were grouped together and referred to as one fraction. The fractions were then developed with 12%-14% concentrated sulfuric acid reagent containing vanillin. After development, the fractions with Rf=0.4 were combined and concentrated and dried using a rotary evaporator to obtain compound 5 (3.2 mg).

[0072] Fr.4.3 After separation by Sephadex LH-20 gel chromatography column (GE, USA, catalog number 117009002), the fraction was further separated by chromatography. The fraction was collected by an automatic collector at a flow rate of 2.0 mL / min, with 10 mL collected from each tube, for a total of 40 tubes. Then, thin-layer chromatography (TLC) was performed using CH2Cl2-MeOH (10:1) as the developing system. The fractions with the same color development on the TLC plate were grouped together and referred to as one fraction. The fractions were then developed with 12%-14% concentrated sulfuric acid reagent containing vanillin. The fractions with Rf=0.5 were combined and concentrated and dried by rotary evaporator to obtain compound 6 (8.0 mg).

[0073] Subfraction Fr.4.3 was further purified by semi-preparative high-performance liquid chromatography (HPLC) (CH3CN:H2O=45:55, v / v). The HPLC model was Waters e2695, the detector was Waters 2998 PDA column, and the column model was Waters X-Bridge C18 (10×250 mm, 5 μm). The injection volume was 100 μL, the column temperature was 30±5℃, and the eluent was CH3CN:H2O, CH3CN:H2O=45:55 (v / v) for isocratic elution at a flow rate of 2 min / mL, yielding compound 7 (10.5 mg, t). R =19.76-21.65min).

[0074] (2) Isolation and purification of fermentation products from maize culture medium After fermentation, the same extraction method was used as described above, followed by distillation and concentration to obtain 160 g of crude extract.

[0075] The crude extract was dissolved in 300 mL of dichloromethane-methanol (v / v=1:1) solvent, and 500 mL of silica gel (silica gel particle size of 200~300 mesh) was added and mixed. After the solvent evaporated, the crude extract was separated by gradient elution chromatography using a vacuum silica gel column. The volume ratio of blank silica gel to mixed silica gel was 1.5:1. The gradient elution process is performed sequentially using elution systems 1 to 9, which are: PE-EtOAc with volume fractions of 90% / 10%, 80% / 20%, 70% / 30%, 50% / 50%, 30% / 70%, and 0 / 100%, and MeOH-EtOAc with volume fractions of 10% / 90%, 20% / 80%, and 30% / 70%, respectively; the circulation pump pressure reaches 0.03MPa~0.04MPa; the volume of PE-EtOAc with a volume fraction of 90% / 10% is 20 L, the volume of PE-EtOAc with a volume fraction of 80% / 20% is 15 L, the volume of PE-EtOAc with a volume fraction of 70% / 30% is 15 L, and the volume of PE-EtOAc with a volume fraction of 50% / 50% is 15 L. The volume fractions of PE-EtOAc (30% / 70%) and PE-EtOAc (0% / 100%) were used in the following amounts: 10 L of 10% / 90% EtOAc-MeOH, 5 L of 20% / 80% EtOAc-MeOH, and 5 L of 30% / 70% EtOAc-MeOH. Each elution system was used at a rate of 500... The fractions were collected in mL / bottle, concentrated to dryness, and then dissolved in a solvent mixture of ethyl acetate, methanol, and dichloromethane (ethyl acetate:methanol:dichloromethane volume ratio 1:1:1) and transferred to test tubes to obtain 6 fractions (Hr.1-Hr.6). Among them, the eluent of 90% / 10% PE-EtOAc was designated as Hr.1, the eluent of 80% / 20% PE-EtOAc as Hr.2, the eluent of 70% / 30% PE-EtOAc as Hr.3, the eluent of 50% / 50% and 30% / 70% PE-EtOAc as Hr.4, the eluent of 0 / 100% PE-EtOAc and 10% / 90% MeOH-EtOAc as Hr.5, and the eluent of 20% / 80% and 30% / 70% MeOH-EtOAc as Hr.6.

[0076] Hr.3 (PE-EtOAc = 70% / 30% fraction) was subjected to gradient elution using a C18 reversed-phase silica column (Merck, catalog number 1.13900.1000). The elution system consisted of methanol-water solutions with volume fractions ranging from 30% to 80%. The average elution rate was 8 mL / min, and the volume of each methanol-water solution was 1500 mL. The eluent was collected in 150 mL portions and combined according to volume fraction to obtain four subfractions (Hr.3.1–Hr.3.5). The eluent with 30% methanol-water solution was designated Hr.3.1, the eluents with 40% and 50% methanol-water solutions were designated Hr.3.2, the eluent with 70% methanol-water solution was designated Hr.3.3, and the eluent with 80% methanol-water solution was designated Hr.3.4.

[0077] Hr.3.2 Chromatographic separation was performed using a Sephadex LH-20 gel permeation column (GE Healthcare, catalog number 117009002). An automatic collector was used to collect fractions at a flow rate of 2.0 mL / min, with 10 mL collected from each tube, for a total of 40 tubes. Thin-layer chromatography (TLC) was then performed using a CH2Cl2:MeOH (10:1) developing system. Spots showing the same color on the TLC plate were grouped together as one fraction and developed with 12%-14% concentrated sulfuric acid containing vanillin. After development, spots with Rf values ​​of 0.4-0.5 were combined and passed through a semi-preparative high-performance liquid chromatography (HPLC) system. The HPLC system model was Waters e2695, the detector model was Waters 2998 PDA, and the column model was Waters X-Bridge C18 (10×250 mm, 5...). μ The injection volume was 100 μL, the column temperature was 30±5℃, and the eluent was CH3CN-H2O, CH3CN-H2O = 40:60 (v / v). Isocratic elution and purification were performed at a flow rate of 2 min / mL, finally yielding compound 9 (10.0 mg, t). R =26.87-28.36 min).

[0078] Results and Analysis Structural identification of DMOA-type mixed terpenes in strain HY5 The DMOA-type mixed-source terpenoid compounds 1-9 obtained above were processed through the compound... 1 H, 13 Identified by C NMR and mass spectrometry, and compared with literature data, the structures of the isolated DMO-type mixed terpenoid compounds 1-9 are as follows: Figure 1As shown. Through the above identification, compound 1 was determined to be berkeleydione, compound 2 to be preaustinoid A3, compound 3 to be miniolutelide B, compound 4 to be 22-epoxyberkeleydione, compound 5 to be 22-deoxyminiolutelide B, compound 6 to be miniolutelide C, compound 7 to be berkeleyacteal, compound 8 to be preaustinoid A2, and compound 9 to be berkeleyacetal A.

[0079] Compound 1: White solid, molecular formula C 26 H 32 O7. 1 H-NMR (500 MHz, acetone- d 6): δ H 5.86 (1H, dd, J =8.5, 5.5 Hz, H-14), 5.33 (1H, s, H-23), 4.80 (1H, s, H-23), 3.67 (3H, s, 20-OCH3), 3.44 (1H, d, J =21.0 Hz, H-2 β ), 3.27 (1H, d, J =21.0 Hz, H-2 α ), 3.02 (1H, dd, J =14.0, 8.0 Hz, H-13 β ), 1.86-1.97 (4H, m, H-13 α , 5, 6), 1.74 (bs, 3H, H-25), 1.46 (3H, s, H-18), 1.44 (3H, s, H-24), 1.30 (3H, s, H-21), 1.28 (3H, s, H-17 or H-19), 1.27 (3H, s, H-17 or H-19); 13 C-NMR (125 MHz, DMSO- d 6): δ C209.2 (C-8), 206.0 (C-10), 170.6 (C-1), 169.4 (C-20), 145.7 (C-22), 141.3 (C-15), 136.5 (C-4), 129.8 (C-14), 127.2 (C-3), 112.1 (C-23), 81.7 (C-16), 79.0 (C-9), 72.0 (C-11), 67 .9 (C-12), 52.2 (C-20-OCH3), 51.6 (C-7), 45.6 (C-5), 42.8 (C-6), 37.1 (C-13), 34.6 (C-2) , 29.4 (C-17), 26.8 (C-18), 22.2 (C-24), 20.3 (C-19), 16.2 (C-25), 15.5 (C-21); ESIMS: m / z 457.2 [M + H] + After comparison with literature data [1] Compound 1 was identified as berkeleydione.

[0080] Compound 2: White solid, molecular formula C 26 H 32 O7. 1 H-NMR (500 MHz, CDCl3): δ H 6.36 (1H, d, J =10.0Hz, H-3), 6.03 (1H, d, J =10.0 Hz, H-2), 5.49 (1H, s, H-23), 4.99 (1H, s, H-23), 3.76 (3H, s, 20-OCH3), 2.95 (1H, d, J =15.0 Hz, H-6 β ), 2.88 (1H, br s, 9-OH), 2.66 (1H, td, J =14.0, 2.5Hz, H-13 β ), 2.21 (1H, dd, J =15.0, 7.0 Hz, H-6 α ), 2.04 (1H, m, H-13) α ), 1.62 (1H, m, H-14) β ), 1.53 (1H, m, H-14) α ), 1.50 (3H, s, H-24), 1.49 (3H, s, H-25), 1.48 (3H, s, H-19), 1.36 (3H, s, H-17), 1.22 (3H, s, H-18), 1.20 (3H, s, H-23); 13C-NMR (125 MHz, CDCl3): δ C 206.1 (C-8), 203.6 (C-10), 168.8 (C-20), 163.9 (C-1), 145.9 (C-3), 143.8 (C-21), 136 .1 (C-5), 131.9 (C-4), 119.7 (C-2), 113.9 (C-22), 85.7 (C-16), 78.0 (C-9), 71.7 (C-11 ), 52.7 (20-CH3), 51.0 (C-7), 47.0 (C-12), 45.5 (C-15), 26.1 (C-14), 25.9 (C-19), 25. 9 (C-17), 24.5 (C-13), 22.9 (C-18), 22.8 (C-24), 16.6 (C-23), 15.5 (C-25); ESIMS: m / z 479.2[M + Na] + 457.2 [M + H] + After comparison with literature data [2] Compound 2 was identified as preaustinoid A3.

[0081] Compound 3: White amorphous powder, molecular formula C 26 H 32 O 10 . 1 H-NMR (500 MHz, CDCl3): δ H 7.54 (1H, s, 10-OH), 5.85 (1H, br s, H-14), 5.33 (1H, s, H-2), 5.29 (1H, s, H-23), 4.55 (1H, d, J =3.0Hz, H-13), 3.89 (1H, q, J =6.5 Hz, H-9), 3.85 (3H, s, 20-OCH3), 2.75 (1H, m, H-3), 2.08 (1H, t, J =13.5 Hz, H-6 β ), 1.86 (1H, d, J =13.5 Hz, H-6 α ), 1.74 (1H, dd, J =13.5, 5.0 Hz, H-5), 1.63 (3H, s, H-17), 1.47 (3H, s, H-24), 1.40 (3H, d, J =6.5 Hz, H-21), 1.39 (1H, s, H-18), 1.14 (3H, d,J =8.0 Hz, H-25), 1.05 (3H, s, H-19),; 13 C-NMR (125 MHz, CDCl3): δ C 179.3 (C-8), 174.1 (C-20), 163.6 (C-1), 157.6 (C-3), 134.5 (C-15), 129.9 (C-14), 120.4 (C-2), 108.3 (C-23), 103.3 (C-10), 82.4 (C-16), 80.3 (C-13), 79.5 (C-22), 77.8 (C-9), 67 .6(C-11), 52.3(20-OCH3), 52.1(C-7), 50.1(C-12), 43.3(C-4), 41.8(C-5), 38.2(C-6), 27.1 (C-18), 26.0 (C-17), 19.3 (C-24), 18.4 (C-25), 17.8 (C-19), 13.5 (C-21); ESIMS: m / z 479.2[M + Na] + 457.2 [M + H] + After comparison with literature data [3] Compound 3 was identified as miniolutelide B.

[0082] Compound 4: White amorphous powder, molecular formula C 26 H 32 O8. 1 H-NMR (500 MHz, CDCl3): δ H 5.96 (1H, dd, J =8.5, 5.5 Hz, H-14), 3.71 (3H, s, 20-OCH3), 3.35 (1H, br d, J =21.0 Hz, H-2 β ), 3.24 (1H, br d, J =21.0 Hz, H-2 α ), 3.22 (1H, d, J =3.0 Hz, H-23b), 2.93 (1H, d, J =3.0 Hz, H-23a), 2.87 (1H, dd, J =14.5, 8.5 Hz, H-13 β ), 2.06 (1H, br d, J =13.5 Hz, H-5), 1.99 (1H, dd, J=13.0, 3.5 Hz, H-6 β ), 1.93 (1H, br d, J =13.0 Hz, H-6 α ), 1.82 (1H, dd, J =14.0, 5.0 Hz, H-13 α ), 1.70 (3H, s, H-21), 1.53 (3H, s, H-18), 1.47 (3H, s, H-19), 1.35 (3H, s, H-25), 1.30 (3H, s, H-17), 1.20 (3H, s, H-24); 13 C-NMR (125 MHz, CDCl3): δ C 207.9 (C-8), 203.5 (C-10), 169.7 (C-20), 168.0 (C-1), 139.7 (C-15), 134.4 (C-4), 129.5 (C-14), 126.8 (C-3), 82.3 (C-16), 78.0 (C-9), 68.3 (C-12), 67.7 (C-11), 59.5 (C-22), 52 .8 (20-OCH3), 50.5 (C-23), 49.3 (C-7), 43.8 (C-5), 39.8 (C-6), 37.8 (C-13), 34.3 (C-2), 29.4 (C-17), 26.7 (C-18), 21.0 (C-19), 18.1 (C-24), 15.7 (C-21), 15.5 (C-25); ESIMS: m / z 495.2[M + Na] + 473.2 [M + H] + After comparison with literature data [3] Compound 4 was identified as 22-epoxyberkeleydione.

[0083] Compound 5: White solid, molecular formula C 26 H 32 O9. 1 H-NMR (500 MHz, CDCl3): δ H 6.49 (1H, s, 10-OH), 6.13 (1H, br s, H-14), 5.89 (1H, br s, H-2), 5.53 (1H, d, J =7.5 Hz, H-23), 4.56 (1H, d, J =2.0 Hz, H-13), 3.84 (3H, s, 20-OCH3), 3.64 (1H, q, J=6.0 Hz, H-9), 3.02 (1H, d, J =7.5 Hz, H-22), 2.81 (1H, m, H-4), 1.82 (1H, d, J =13.0 Hz, H-6 β ), 1.74 (1H, dd, J =12.5, 5.0 Hz, H-5), 1.67 (1H, m, H-6 α ), 1.64 (3H, s, H-18), 1.50 (3H, s, H-24), 1.49 (3H, s, H-17), 1.38 (3H, d, J =6.5 Hz, H-21), 1.17 (3H, d, J =7.5 Hz, H-25), 1.00 (3H, s, H-19); 13 C-NMR (125 MHz, CDCl3): δ C 180.2 (C-8), 175.2 (C-20), 163.3 (C-1), 157.4 (C-3), 134.8 (C-15), 129.4 (C-14), 120.5 (C-2), 102.7 (C-10), 98.9 (C-23), 82.2 (C-16), 80.7 (C-13), 77.7 (C-9), 61.0 (C-11), 52 .7 (20-OCH3), 49.1 (C-12), 46.6 (C-7), 45.1 (C-22), 43.7 (C-5), 43.4 (C-4), 34.7 (C-6), 27.2 (C-17), 26.6 (C-24), 26.1 (C-18), 18.6 (C-25), 17.4 (C-19), 13.6 (C-21); ESIMS: m / z 489.2[M + H] + After comparison with literature data [4] Compound 5 was identified as 22-deoxyminiolutelide B.

[0084] Compound 6: White solid, molecular formula C 26 H 32 O9. 1 H-NMR (500 MHz, CDCl3): δ H 5.88 (1H, d, J =7.0Hz, H-23), 5.86 (1H, br s, 10-OH), 4.22 (1H, dd, J=10.5, 6.5 Hz, H-13), 3.87 (3H, s, 20-OCH3), 3.75 (1H, q, J =6.5 Hz, H-9), 3.36 (1H, m, H-2), 3.03 (1H, d, J =7.5 Hz, H-3), 3.02 (1H, d, J =20.0 Hz, H-2), 2.96 (1H, d, J =15.0 Hz, H-6), 2.82 (1H, m, H-14), 2.37-2.43 (2H, m, H-6, 14), 1.89 (3H, s, H-2 5), 1.50 (3H, s, H-18), 1.48 (3H, s, H-17), 1.44 (3H, s, H-24), 1.40 (3H, d, J =6.0 Hz, H-21), 1.13 (3H, s, H-19); 13 C-NMR (125 MHz, CDCl3): δ C 177.3 (C-8), 174.9 (C-20), 170.6 (C-1), 134.7 (C-5), 132.5 (C-15), 128.3 (C-4), 126.0 ( C-3), 100.6 (C-10), 98.3 (C-23), 84.3 (C-16), 79.3 (C-13), 73.1 (C-9), 62.0 (C-11), 53. 3 (20-OCH3), 50.5 (C-12), 46.2 (C-7), 44.7 (C-22), 34.9 (C-2), 34.0 (C-6), 33.9 (C-14), 26.8 (C-17), 26.3 (C-24), 26.2 (C-18), 19.1 (C-25), 14.9 (C-19), 13.7 (C-21); ESIMS: m / z 489.2[M + H] + After comparison with literature data [4,5] Compound 6 was identified as miniolutelide C.

[0085] Compound 7: White solid, molecular formula C 25 H 30 O8. 1 H-NMR (500 MHz, CDCl3): δ H 6.49 (1H, dd, J =7.5, 5.5 Hz, H-14), 4.44 (1H, d, J=10.0 Hz, H-23), 4.24 (1H, d, J =10.0 Hz, H-23), 4.06 (1H, q, J =6.5 Hz, H-9), 3.44 (1H, d, J =20.5 Hz, H-2), 3.36 (1H, dd, J =14.0, 8.0 Hz, H-13), 3.26 (1H, d, J =20.5 Hz, H-2), 3.04 (1H, br d, J =15.0 Hz, H-2), 1.77 (1H, t, J =14.5 Hz, H-6), 1.73 (1H, s, H-25), 1.65 (1H, dd, J =16.0, 1.0 Hz, H-6), 1.61 (3H, s, H-18), 1.55 (1H, dd, J =14.0, 5.0 Hz, H-13), 1.41 (3H, s, H-24), 1.30 (3H, s, H-17), 1.21 (3H, d, J =7.0 Hz, H-21), 0.96 (3H, s, H-19); 13 C-NMR (125 MHz, CDCl3): δ C 204.8 (C-10), 177.5 (C-8), 171.3 (C-20), 170.5 (C-1), 139.6 (C-15), 134.7 (C-4), 1 29.7 (C-14), 127.1 (C-3), 89.7 (C-22), 83.2 (C-16), 77.9 (C-9), 74.1 (C-23), 64.2 (C -11), 57.4 (C-12), 48.3 (C-7), 40.2 (C-5), 35.8 (C-13), 34.2 (C-2), 33.3 (C-6), 29.4 (C-17), 27.1 (C-18), 21.3 (C-19), 20.8 (C-21), 18.7 (C-24), 15.5 (C-25); ESIMS: m / z 459.2[M + H] + After comparison with literature data [6] Since the compound was unnamed, it was named berkeleyacteal X.

[0086] Compound 8: White solid, molecular formula C 25 H 31 O7. 1H-NMR (500 MHz, DMSO- d 6): δ H 6.37 (1H, br d, J =5.0 Hz, H-14), 6.03 (1H, dd, J =8.0, 5.5 Hz, H-2), 5.26 (1H, br s, H-23), 4.71 (1H, br s, H-23), 3.59 (3H, s, 20-OCH3), 2.06 (1H, m, H-13), 1.99 (1H, m, H-6), 1.80 (1H, m, H-13), 1.64 (3H, m, H-6, 14, 15), 1.51 (1H, m, H-14), 1.40 (3H, s, H-24), 1.33 (3H, s, H-17), 1.29 (3H, s, H-18), 1.27 (3H, s, H-21), 1.21 (3H, s, H-19), 1.08 (3H, s, H-25), 0.55 (1H, m, H-5); 13 C-NMR (125MHz, DMSO-) d 6): δ C 209.1 (C-8), 204.3 (C-10), 169.0 (C-20), 166.6 (C-1), 156.8 (C-3), 143.8 (C-22), 121.5 (C-2), 111.4 (C-23), 85.0 (C-16), 77.8 (C-9), 71.8 (C-11), 55.5 (C-15), 51.9 (C-7), 50. 4 (20-OCH3), 49.1 (C-5), 46.5 (C-12), 43.0 (C-4), 40.1 (C-6), 32.1 (C-18), 31.5 (C-13), 26.6 (C-17), 22.3 (C-14), 21.4 (C-24), 16.4 (C-19), 16.1 (C-21), 14.6 (C-25); ESIMS: m / z 459.2[M + H] + After comparison with literature data [7] Compound 8 was identified as preaustinoid A2.

[0087] Compound 9: White amorphous powder, molecular formula C 25 H 29 O8. 1 H-NMR (500 MHz, DMSO- d 6): δ H 6.37 (1H, d,J =5.0 Hz, H-23), 6.02 (1H, dd, J =8.0, 5.5 Hz, H-14), 4.64 (1H, q, J =6.5 Hz, H-14), 3.82 (3H, s, 20-OCH3), 3.81 (1H, d, J =5.0 Hz, H-23), 3.55 (1H, d, J =21.0 Hz, H-2), 3.33 (1H, d, J =21.0 Hz, H-2), 2.61 (1H, dd, J =13.5, 3.0 Hz, H-5), 2.47 (1H, dd, J =14.0, 8.0 Hz, H-13 β ), 1.82 (1H, dd, J =14.0, 3.0 Hz, H-6 β ), 1.76 (3H, s, H-25), 1.55 (3H, s, H-18), 1.49 (1H, dd, J =14.0, 4.5 Hz, H-6 α ), 1.40 (3H, s, H-17), 1.34 (3H, s, H-24), 1.26 (3H, d, J =7.0 Hz, H-21), 1.23 (1H, m, H-13 α ), 0.75 (1H, s, H-19); 13 C-NMR (125 MHz, DMSO- d 6): δ C 205.6 (C-10), 176.0 (C-8), 169.6 (C-1), 169.4 (C-20), 136.4 (C-15), 135.8 (C-4), 12 8.2 (C-14), 126.6 (C-3), 99.2 (C-23), 81.6 (C-16), 75.0 (C-9), 62.5 (C-11), 56.5 (C- 12), 53.4 (C-26), 51.5 (C-22), 43.1 (C-7), 38.3 (C-5), 35.4 (C-13), 34.5 (C-2), 29.5 (C-17), 28.8 (C-24), 26.3 (C-18), 20.9 (C-19), 19.3 (C-21), 15.7 (C-25); ESIMS: m / z 459.2[M + H] + After comparison with literature data [8]Compound 9 was identified as Berkeleyacetal A.

[0088] 1.3 Evaluation of the herbicidal activity of some compounds in strain HY5 1.3.1 Experimental Materials Monocotyledonous weed seeds: Goosegrass ( Eleusis is in the east. ), foxtail grass ( Green setaria (L.)Beauv).

[0089] Dicotyledonous weed seeds: Amaranthus retroflexus ( Amaranthus retroflexus L), clover ( Clover creeping L).

[0090] 1.3.2 Methods for evaluating herbicidal activity (1) Seed germination inhibition experiment at a concentration of 200 μg / mL Cut filter paper to the size of 12-well plates and sterilize it in an autoclave along with distilled water, gauze, and other necessary experimental materials. Select 40 plump seeds each of *Eleusine indica*, *Amaranthus retroflexus*, *Trifolium repens*, and *Setaria viridis*, and soak them in sterile water for 6 hours. After soaking, soak them in a 4% sodium hypochlorite solution for 10 minutes, and rinse repeatedly with sterile water. Weigh out compounds 1, 3, 4, 5, 6, and 7 with distinct structures and sufficient content, dissolve them in anhydrous methanol, and dissolve glyphosate and acetochlor in sterile water to prepare stock solutions with a concentration of 200 μg / mL. Place the sterilized filter paper into the 12-well plates, and use a pipette to add 333 μL of the stock solution to the treatment group wells. Let the methanol evaporate for 2.5–3 hours. Add 333 μL of sterile water to each well in the treatment and blank control groups, and add 333 μL of glyphosate and acetochlor stock solutions to each well in the positive control group. Four replicates were set up, with 10 weed seeds in each replicate. The seeds were sealed and incubated in an artificial incubator for 72 hours. The lengths of the radicle and plumule of each seed were measured, and the inhibition rates of the compound and glyphosate on the germination of the four weed seeds were calculated. The calculation formula is as follows: ………………Formula Ⅰ in: Luke The length (cm) of the radicle and plumule in the blank control group. It The length (cm) of the radicle and plumule in the treatment group.

[0091] This study used a seed germination inhibition assay to systematically evaluate the effects of the tested compound (at a concentration of 200 μg / mL) on the growth of the radicle and plumule of four weed seeds (see See...). Figure 2 The results showed that the tested compounds generally exhibited varying degrees of inhibitory effects on the elongation of the plumules or radicles of weed seeds (see...). Figure 3Notably, in the treatment of *Amaranthus retroflexus*, compounds 1, 3, 4, and 5 significantly promoted radicle growth, with promotion rates of 69.1%, 53.0%, 36.5%, and 24.6%, respectively. However, these compounds showed varying effects on the cotyledon: compounds 4 and 5 exhibited only weak promoting effects (promotion rates of 9.4% and 6.2%, respectively), while compounds 1 and 3 inhibited cotyledon elongation, with inhibition rates of 5.5% and 23.7%, respectively. In the treatment of clover, compound 4 showed a significant growth-promoting effect, with promotion rates of 30.5% and 55.1% for radicles and cotyledons, respectively, indicating that this compound may have the potential to be used as a plant growth promoter after structural optimization. In contrast, compound 6 showed the strongest herbicidal activity against clover cotyledon elongation, with an inhibition rate of 66.4%. In the treatment of foxtail millet seeds, compound 6 showed a particularly significant inhibitory effect on cotyledon elongation, with an inhibition rate as high as 95.3%. Furthermore, most compounds exhibited better inhibitory effects on radicle elongation than on plumule elongation. In the treatment of *Eleusine indica* seeds, compound 6 showed the most significant inhibitory effect on radicle and plumule elongation, with inhibition rates of 93.2% and 87.0%, respectively. This effect was superior to the positive controls glyphosate (79.8% and 84.7%) and acetochlor (85.1% and 86.3%). Meanwhile, compounds 3, 4, and 5 showed strong inhibitory activity on *Eleusine indica* radicle growth, with inhibition rates of 67.3%, 70.0%, and 68.1%, respectively.

[0092] (2) Experiment on the inhibition of seed germination of Eleusine indica by compounds 3, 4 and 6 Using the same procedures as described above, stock solutions were prepared for the target compound (methanol-dissolved system), acetochlor, and glyphosate (sterile water-dissolved system) at concentrations of 60, 50, 40, 30, 20, and 10 μg / mL. After completing the preset culture period, the lengths of the radicle and plumule of each seed were measured, and the IC50 values ​​for seed germination physiological parameters of each treatment group were calculated using nonlinear regression analysis. 50 value.

[0093] Given that compounds 3, 4, and 6 possess significant herbicidal activity and are present in sufficient quantities, this study further determined their EC50-95% inhibitory effect on the seed germination of *Eleusine indica*. 50 Values ​​are detailed in Table 1. The results show that compound 6 has an effect on the EC50 of plumule and radicle elongation. 50 The values ​​were 17.9 and 19.1 μg / mL, respectively. Although their inhibitory activity was not as high as that of acetochlor, it was superior to that of glyphosate (EC). 50 The values ​​were 66.2 and 30.9 μg / mL, respectively. These data indicate that compound 6 has the potential to be developed as a lead compound for green pesticides, and its herbicidal activity against monocotyledonous weeds is far superior to that against dicotyledonous weeds.

[0094] The results of the inhibitory activity of compound 6 on the germination of Eleusine indica seeds at different concentrations are shown in the figure. Figure 4 .

[0095] Table 1. Inhibition rates of compounds 3, 4, and 6 on seed germination of *Eleusine indica*.

[0096] (3) Transcriptomic and metabolomic analysis of the inhibition of seed germination in Eleusine indica Based on the herbicidal activity of the compounds obtained from the initial screening (the herbicidal activity evaluation results above), target compound 6 with the best biological activity was selected and applied at a concentration of 17.86 μg / mL (EC50). 50 Under the specified conditions, *Eleusine indica* seed samples were treated using a seed germination inhibition experimental system. Total RNA was extracted using TRIzol reagent according to the manufacturer's instructions, and RNA quality was determined using a NanoDrop 2000 micro-spectrophotometer and a 5300 nucleic acid fragment analyzer. Library construction and sequencing were performed by Shanghai MajorBio Technology Co., Ltd. Furthermore, untargeted metabolomics liquid chromatography-tandem mass spectrometry (LC-MS / MS) analysis of the test samples was performed using a UHPLC-Q Exactive mass spectrometry system. Raw data were imported into the metabolomics processing software ProgenesisQIv3.0 for peak extraction, registration, and retention time correction. MS / MS mass spectra were identified using the MJDBPM database established by Shanghai MajorBio Technology Co., Ltd. Transcriptomics and metabolomics data were analyzed on the MajorBio Cloud Platform (www.majorbio.com). Differentially expressed genes (DEGs) were analyzed using DESeq2, with significance levels... P <0.05, |log2FC|≥1. Pathway analysis was performed using the Kyoto Genome Encyclopedia (KEGG) database (http: / / www.genome.jp / kegg / ) and Python SciPy software. Differentially expressed metabolites (DEMs) between the treatment and control groups were mapped to biochemical pathways.

[0097] Transcriptomics analysis Based on existing research data, compound 6 exhibits the most outstanding herbicidal activity; therefore, its mechanism of action will be investigated. Based on previous experimental results, the EC50 of compound 6 was selected. 50The model plant *E. indica* was treated with a concentration of 17.9 μg / mL, followed by transcriptome analysis using RNA-seq technology. Sequencing results showed a total of 40.83 Gb of clean data, with Q30 bases exceeding 94.38% (Table 2). A total of 29,633 expressed genes were identified (including 28,644 annotated genes and 989 newly discovered genes), and 53,213 transcripts were detected (including 35,329 known transcripts and 17,884 new transcripts). Furthermore, 1,485 differentially expressed genes (DEGs) were screened from the treatment group, including 1,097 upregulated genes and 388 downregulated genes. Figure 5 A). All DEGs are annotated via the KEGG pathway. Figure 5 B). Notably, the plant hormone signaling pathway (ko04075) and the tryptophan metabolism pathway (ko00380) exhibited the most significant enrichment characteristics.

[0098] Analysis of plant signal transduction pathways revealed an upregulated trend in the JAZ protein-encoding gene within the jasmonic acid signaling pathway. As a repressor of this pathway, the JAZ protein plays a crucial regulatory role in plant defense responses and stress adaptation. Studies have shown that JAZ protein degradation can relieve the inhibition of ABA synthesis-related transcription factors, promote abscisic acid biosynthesis, and thus delay seed germination.

[0099] In the tryptophan metabolic pathway, the expression of the gene encoding aromatic L-amino acid decarboxylase (EC4.1.1.28) was upregulated, while the expression of the key enzyme gene catalyzing the conversion of serotonin (5-hydroxytryptamine) to melatonin (EC2.1.1.4) showed a decreasing trend. This bidirectional regulation led to a significant accumulation of serotonin in the treated plants. The study confirmed that serotonin, a widely distributed bioactive substance in the plant kingdom, similar to melatonin, has pleiotropic functions in plant growth and development regulation and stress response. Mechanistic studies showed that excessive serotonin can inhibit root growth through a dual action: on the one hand, it induces abnormal accumulation of hydrogen peroxide (H2O2) in roots, while simultaneously inhibiting superoxide anion (O2) production. 2- The distribution of auxin in meristems affects cell elongation and division activity; on the other hand, it interferes with the transport of auxin in the root tip, leading to an imbalance in root tip auxin homeostasis, which ultimately hinders seed germination.

[0100] LC-MS / MS metabolomics analysis In the in-depth study of the mechanism of action of compound 6 on *Eleusine indica*, a systematic analysis of differential metabolites (DEMs) was performed using the LC-MS / MS platform. The results showed that among the identified primary and secondary metabolites, 273 differential metabolites were upregulated, and 336 differential metabolites were downregulated. Figure 5 C). Through KEGG pathway enrichment analysis, these differentially metabolites were enriched in 53 metabolic pathways, including 10 key metabolic pathways (C). Figure 5 D). Principal component analysis (PCA) results showed significant differences among the different treatment groups, confirming the reliability of the analytical model. Figure 5 E). Further analysis revealed that the differentially metabolites were mainly enriched in α-linolenic acid metabolism (ko00592) and tryptophan metabolism (ko00380).

[0101] In the α-linolenic acid metabolic pathway, the metabolic level of jasmonic acid was significantly reduced, indicating that treatment with compound 6 led to the consumption of a large amount of jasmonic acid in *Eleusine indica* seeds. Combined with previous studies finding that jasmonic acid and abscisic acid synergistically delay seed germination, and that the jasmonic acid content in dormant seeds was significantly lower than in non-dormant seeds, this result further validates the above findings. In the tryptophan metabolic pathway, the metabolic level of serotonin was significantly increased. Combined with transcriptomic analysis, this suggests that serotonin accumulation during metabolic changes may have a significant impact on herbicidal activity.

[0102] Table 2. Summary of transcriptome readings for the blank and treatment groups of *Eleusine indica*.

[0103] Note: The blank group is CK. CK-1, CK-2 and CK-3 refer to three different replicates of treating *Eleusine indica* with water as the blank control. 6-1, 6-2 and 6-3 represent compounds 6-1, 6-2 and 6-3 respectively. Compounds 6-1, 6-2 and 6-3 refer to three different replicates of treating *Eleusine indica* with compound 6.

[0104] (4) Real-time PCR experiment The corresponding gene-specific primers were designed using the GenScript Biotech online PCR primer design tool. Primer sequences are detailed in Table 3. Primers were synthesized by Qingdao Weilai Biotechnology Co., Ltd. Total RNA from *Eleusine indica* seed samples was extracted by Shanghai Meiji Biotechnology Co., Ltd., and reverse transcribed into cDNA using the Nanjing Novizan reverse transcription kit and an S1000 gradient PCR instrument. Quantitative real-time PCR experiments were performed using the Nanjing Novizan qPCR kit, following the instructions of the real-time PCR instrument. cDNA was diluted 1:5 by volume. The cycling program was 95 ℃ for 2 minutes, followed by 40 cycles (95 ℃ 10 s, 60 ℃ 30 s, 72 ℃ 34 s). Internal reference genes were F (5′-TCCCTGGTATCGCTGACCGTA-3′) and R (5′-CCCTTTGAGATCCACATCTGTT-3′). 2 -ΔΔCT The relative expression levels of genes in the samples were calculated using a method. All treatments were configured with three biological replicates and three technical replicates.

[0105] Table 3 Primer sequences for real-time PCR

[0106] Analysis of Validation Results of Quantitative Real-Time PCR To verify the accuracy of transcriptomics data analysis, this invention selected four differentially expressed genes that were significantly enriched in plant hormone signaling pathways and tryptophan metabolism pathways, and validated them using quantitative real-time PCR. The experimental results showed that the expression levels of these specific genes exhibited up- and down-regulation trends highly consistent with the transcriptomics data. Figure 6 This discovery not only confirms the association between changes in the expression of related genes in *Eleusine indica* and the inhibition of its seed germination, but also lays an important foundation for further research into the regulatory mechanism of the jasmonic acid signaling pathway and key genes for serotonin accumulation in *Eleusine indica* seeds.

[0107] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A method for preparing DMOA-type mixed-source terpenoid compounds using Penicillium sclerotiorum HY5, characterized in that, The DMOA-type mixed-source terpenoid compound is compound 2 and / or compound 7; Compound 2 is: The compound 7 is: The preparation method of compound 2 and / or compound 7 includes the following steps: (1) The fermentation product was obtained by fermenting Penicillium HY5; the fermentation time was 20-40 days and the fermentation temperature was 25-30℃. (2) The fermentation product is soaked in ethyl acetate and filtered to obtain filtrate 1 and filter residue. Filtrate 1 is concentrated and dried to obtain fermentation broth extract 1. The filter residue is soaked in dichloromethane-methanol mixed solvent and filtered to collect filtrate 2. After concentration, water is added to obtain concentrated solution. The concentrated solution is extracted with ethyl acetate, concentrated and dried to obtain fermentation broth extract 2. (3) Fermentation broth extract 1 and fermentation broth extract 2 were combined to obtain crude extract; (4) The crude extract was subjected to silica gel column chromatography and C24 chromatography in sequence. 18 One or more of the following methods are used to separate and purify the compound: reverse silica gel column chromatography, Sephadex LH-20 column chromatography, thin-layer chromatography, and HPLC. In step (1), the culture medium for fermentation is rice culture medium; The rice culture medium is formulated with rice and sea salt water, wherein the mass-to-volume ratio of rice to sea salt water is 90 g: 110 mL.

2. The application of DMOA-type mixed-source terpenoids in regulating the growth of herbaceous plants, characterized by, The DMOA-type mixed-source terpene compound is compound 7 as described in claim 1, and any one of compound 1, compounds 3 to 6; Compound 1 and compounds 3-6 are: Any one of compounds 1, 3-7 inhibits the growth of goosegrass and foxtail grass; Compounds 1, 3, 4, or 5 promote the growth of reverse-branch amaranth; Compound 4 promotes clover growth; Compound 6 inhibits clover growth.

3. The application of DMOA-type mixed-source terpenoid compounds in the preparation of herbicides or plant growth regulators, characterized in that, The DMOA-type mixed-source terpene compound is compound 7 as described in claim 1, and any one of compound 1, compounds 3 to 6; Compound 1 and compounds 3-6 are: Any one of compounds 1, 3-7 inhibits the growth of goosegrass and foxtail grass; Compounds 1, 3, 4, or 5 promote the growth of reverse-branch amaranth; Compound 4 promotes clover growth; Compound 6 inhibits clover growth.

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