WO2021056936A1 - 具反式萘烷环的聚酮化合物及其制备方法和应用 - Google Patents

具反式萘烷环的聚酮化合物及其制备方法和应用 Download PDF

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WO2021056936A1
WO2021056936A1 PCT/CN2020/072772 CN2020072772W WO2021056936A1 WO 2021056936 A1 WO2021056936 A1 WO 2021056936A1 CN 2020072772 W CN2020072772 W CN 2020072772W WO 2021056936 A1 WO2021056936 A1 WO 2021056936A1
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compound
trans
polyketone
penicillium
water
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杜丰玉
肖�琳
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青岛农业大学
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C49/00—Ketones; Ketenes; Dimeric ketenes; Ketonic chelates
    • C07C49/29—Saturated compounds containing keto groups bound to rings
    • C07C49/337—Saturated compounds containing keto groups bound to rings containing hydroxy groups
    • C07C49/345—Saturated compounds containing keto groups bound to rings containing hydroxy groups polycyclic
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C69/00—Esters of carboxylic acids; Esters of carbonic or haloformic acids
    • C07C69/66—Esters of carboxylic acids having esterified carboxylic groups bound to acyclic carbon atoms and having any of the groups OH, O—metal, —CHO, keto, ether, acyloxy, groups, groups, or in the acid moiety
    • C07C69/73—Esters of carboxylic acids having esterified carboxylic groups bound to acyclic carbon atoms and having any of the groups OH, O—metal, —CHO, keto, ether, acyloxy, groups, groups, or in the acid moiety of unsaturated acids
    • C07C69/732—Esters of carboxylic acids having esterified carboxylic groups bound to acyclic carbon atoms and having any of the groups OH, O—metal, —CHO, keto, ether, acyloxy, groups, groups, or in the acid moiety of unsaturated acids of unsaturated hydroxy carboxylic acids
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/24—Preparation of oxygen-containing organic compounds containing a carbonyl group
    • C12P7/26—Ketones
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/62—Carboxylic acid esters
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C2602/00—Systems containing two condensed rings
    • C07C2602/02—Systems containing two condensed rings the rings having only two atoms in common
    • C07C2602/04—One of the condensed rings being a six-membered aromatic ring
    • C07C2602/10—One of the condensed rings being a six-membered aromatic ring the other ring being six-membered, e.g. tetraline

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  • the present invention relates to the technical field of microbial pesticides, in particular to a polyketide compound with trans-decalin ring obtained from a solid fermentation product of strain Penicillium sp.TR85, a separation and purification method thereof, and an application in weeding.
  • Echinochloa crusgalli is a worldwide malignant weed. Among the main weeds in rice fields in my country, Echinochloa crusgalli has the largest occurrence and damage area, accounting for about 43% of the total rice field area. For a long time, although chemical synthetic herbicides can effectively control weeds, they have caused environmental pollution and weed resistance. At the same time, long-term excessive use also causes excessive pesticide residues, endangering the safety of agricultural products and human health. Finding herbicidal active ingredients from biological resources such as microorganisms and plants has the advantages of low toxicity, good environmental compatibility and resistance to pesticides, and has become an important source for the development of "environmentally friendly" herbicides.
  • polyketides with trans-decalin ring have antibacterial, anti-tumor and immunomodulatory activities, and there are no reports about their herbicidal activity.
  • the two polyketone compounds with trans-decalin ring involved in the present invention are new compounds, and the polyketone compound with trans-decalin ring has been found for the first time to have herbicidal potential, and its structure is significantly different from known commercial herbicides. It provides guidelines for the further development of new biological herbicides.
  • the purpose of the present invention is to provide a polyketone compound with trans-decalin ring, its preparation method and its application in weeding.
  • the above-mentioned compound is obtained by fermentation of the above-mentioned Penicillium.
  • the preparation method of the above compound includes the steps of inoculating the above-mentioned Penicillium TR85 into a sterilized solid medium, standing at room temperature for fermentation, and obtaining the fermentation product through extraction and purification.
  • the solid medium is 100 g of rice and 0.6 g of peptone per 100 mL of natural seawater.
  • the fermentation product is repeatedly immersed and extracted with ethyl acetate, and the extracts are combined and concentrated to obtain a crude fermentation extract;
  • the polyketone compound with trans-decalin ring is at least one of the above-mentioned compounds.
  • the herbicidal composition contains at least one polyketone compound having a trans-decalin ring as an active ingredient.
  • the polyketone compound with trans-decalin ring in the herbicidal composition is at least one of the above compounds.
  • the weed is barnyardgrass.
  • the compound of the present invention can be combined with other drugs in the form of a composition; the composition contains the compound represented by formula (1) and a pesticide-acceptable salt and/or solvate as the active ingredient, and a pesticide-acceptable carrier.
  • the compound of the invention can be used in combination with other active ingredients as long as there is no antagonistic effect between them.
  • the herbicide of the present invention can also contain conventional pesticides acceptable auxiliary agents, which can be carriers, surfactants and safeners; according to the composition of the auxiliary agents, it can be prepared into any desired formulation form, such as emulsifiable concentrates, suspensions, and microemulsions. , Dispersible oil suspension, wettable powder, suspoemulsion, water emulsion or water dispersible granules. It may also contain other conventional components in pesticides, such as stabilizers, chelating agents, dyes, colorants, protective colloids, adhesives, thickeners, thixotropic agents, penetrants and polymers.
  • the amount of the herbicide contained in the composition of the present invention depends on the resistance of the weeds, the method of application, and the frequency of application; a person of ordinary skill in the art can determine the appropriate amount only by using conventional experimental methods.
  • the term "compound of the present invention” means a compound of formula (I) in any form, that is, any salt or non-salt form (for example, in free acid or free base form, or in its pharmaceutically acceptable salt form) and Any physical form (e.g., including non-solid form (e.g., liquid or semi-solid form), and solid form (e.g., amorphous or crystalline form, specific polymorphic form, solvate, including hydrate (e.g., single -, di- and hemi-hydrate)), and mixtures of its various forms.
  • any salt or non-salt form for example, in free acid or free base form, or in its pharmaceutically acceptable salt form
  • Any physical form e.g., including non-solid form (e.g., liquid or semi-solid form), and solid form (e.g., amorphous or crystalline form, specific polymorphic form, solvate, including hydrate (e.g., single -, di- and hemi-hydrate)), and mixtures
  • solvates for the solvate of the compound of the present invention or its salt in the form of a crystal, those skilled in the art will understand that a pesticide-acceptable solvate can be formed in which solvent molecules are incorporated into the crystal lattice during crystallization.
  • Solvates may contain non-aqueous solvents such as ethanol, isopropanol, DMSO, acetic acid, ethanolamine, and ethyl acetate, or they may contain water as a solvent (the solvent is incorporated into the crystal lattice).
  • Solvates in which water is the solvent (the solvent is incorporated into the crystal lattice) are commonly referred to as "hydrates.” Hydrates include stoichiometric amounts of hydrates and components that include variable amounts of water. The present invention includes such solvates.
  • the polyketone compound 1 and compound 2 with trans-decalin ring involved in the present invention are new compounds, and it was discovered for the first time that the polyketone compound with trans-decalin ring has herbicidal activity.
  • the growth inhibition rates of compound 1 and compound 2 on the radicle of barnyardgrass (Echinochloa crusgalli) seedlings were 81.5% and 79.6%, which were better than the commercial herbicide acetochlor and could be used as herbicidal activity.
  • Lead compounds or new pesticide ingredients are new compounds, and it was discovered for the first time that the polyketone compound with trans-decalin ring has herbicidal activity.
  • barnyardgrass Echinochloa crusgalli
  • the polyketide compound 1 and compound 2 with trans-decalin ring involved in the present invention are produced by fermentation of the strain Penicillium sp.TR85, which is convenient for large-scale fermentation production; secondly, compound 1 and compound 2 are natural products, compared Chemically synthesized pesticides have the advantages of good environmental compatibility and resistance to pesticides.
  • Figure 1 is the 1 H NMR spectrum of Compound 1.
  • Figure 2 is a 13 C NMR spectrum of Compound 1.
  • Figure 3 is the 1 H NMR spectrum of Compound 2.
  • Figure 4 is a 13 C NMR spectrum of Compound 2.
  • Figure 5 shows the inhibitory effect of compound 1 on the growth of barnyardgrass seedlings.
  • Figure 6 shows the inhibitory effect of compound 2 on the growth of barnyardgrass seedlings.
  • the present invention is isolated from the fermentation product of the strain Penicillium sp.TR85 (preserved at the General Microbiology Center of the Chinese Microbial Culture Collection Management Committee, preservation date: August 2, 2019, preservation number: CGMCC No. 18116) to obtain the following examples
  • the compounds referred to in, among them, the structures of compound 1 and compound 2 are as follows (the Arabic numerals in the structure are the standard positions of carbon atoms):
  • Example 1 Isolation and identification of strain Penicillium sp.TR85
  • the strain TR85 of the present invention is isolated from the roots of the saline-tolerant plant Suaeda salsa.
  • the formulation of the PDA double antibody medium 20% potato extract, 2% glucose, 1.5% agar, natural sea water, and adding penicillin potassium with a final concentration of 150 ⁇ g/mL and 120 ⁇ g/mL streptomycin sulfate.
  • the strain TR85 was inoculated on the surface of the PDA plate and observed after incubating at 28°C for 5-7 days.
  • the mycelium is white at the beginning, and then produces dark green spores, and can produce brown-yellow pigment.
  • ITS-5.8S-ITS2 region The rDNA gene sequence determination results of the strain (ITS-5.8S-ITS2 region) are as follows:
  • the strain was identified as Penicillium sp., and the strain has been deposited in the General Microbiology Center of the China Microbial Culture Collection Management Committee (CGMCC for short, address: No. 3, No. 1, Beichen West Road, Chaoyang District, Beijing, Chinese Academy of Sciences) Institute), the preservation date: August 2, 2019, the preservation number of the strain is CGMCC No. 18116.
  • CGMCC General Microbiology Center of the China Microbial Culture Collection Management Committee
  • the strain Penicillium sp.TR85 (size 2 ⁇ 2cm) growing on the surface of the PDA plate was cut and inoculated into a sterilized Erlenmeyer flask containing a solid medium, and allowed to stand at room temperature for 30 days.
  • the fermentation product is repeatedly immersed and extracted with ethyl acetate, and the extracts are combined and concentrated to obtain a crude fermentation extract.
  • the solid medium is 100 g of rice and 0.6 g of peptone per 100 mL of natural seawater.
  • the above fermentation crude extract was subjected to vacuum silica gel column chromatography (inner diameter 65mm, length 300mm, glass column with sand plate and suction nozzle), in order of increasing polarity of the eluent, with a gradient of 40:1 to 1.
  • the agar powder mixing method established by Luo Xiaoyong et al. (Journal of Qingdao Agricultural University, 2007, 24: 267-270) is used to quickly determine the herbicidal activity of compounds. It has the advantages of high sensitivity, simple operation, good repeatability, and convenient large-throughput screening. , And easy to observe the growth status of the indicator plants. Luo Xiaoyong et al. used this method to quickly determine the herbicidal activity of 40 garden plant leaves.
  • Zhang Lijuan Plant Protection, 2016, 42: 63-66) and Su Fansheng (Applied Chemistry, 2014, 31: 290-295) used barnyardgrass as the test weed, and used agar mixture method to quickly determine the corn stover and imidazole The herbicidal activity of similar compounds.
  • Zhang Yun Journal of Microbiology, 2015, 55:292-298
  • Li Shuai Journal of Agricultural and Food Chemistry, 2014, 62:8997-9001
  • Weed seeds were disinfected with 0.2% sodium hypochlorite for 15 minutes, then washed repeatedly with sterile distilled water, soaked for 4-6 hours, and germinated in a medical tray with sterile wet filter paper for 12 hours under dark conditions.
  • test compound was dissolved in dimethyl sulfoxide (DMSO), prepared as a 4 mg/mL solution, and diluted with a 50% DMSO aqueous solution to different concentrations for use.
  • DMSO dimethyl sulfoxide
  • the setting condition of the artificial climate box is an automatic cycle of 14 hours of light (25°C) and 10 hours of darkness (20°C), and the relative humidity in the box is 60%. Investigate the length of the radicle and hypocotyl after 2 days, and calculate the weed growth inhibition rate.
  • Growth amount the length of the radicle (hypocotyl) after treatment-the length of the radicle (hypocotyl) before treatment;
  • Inhibition rate (%) [(average growth of control-average growth of treatment)/average growth of control] ⁇ 100%.
  • test results are shown in Table 3.
  • the results show that at a concentration of 10 ⁇ g/mL, the inhibition rates of compound 1 (Figure 5) and compound 2 ( Figure 6) on barnyardgrass (E. crusgalli) seedling radicle growth were 81.5% and 79.6, respectively. %, significantly better than the commercial herbicide acetochlor, and can be used as a lead compound or new pesticide ingredient with herbicidal activity.
  • Brine shrimp is an aquatic animal that is highly sensitive to toxic substances. It can be used as a model organism to initially evaluate the toxicity of compounds to the environment. Masi, M. and others used brine shrimp to initially evaluate the toxicity of the herbicidal active compounds Colletochlorins E and F (Journal of Agricultural and Food Chemistry, 2017, 65: 1124-1130).
  • the compound to be tested was dissolved in DMSO, prepared as a mother liquor of 20 mg/mL, and diluted to a solution of 10,5 and 2.5 mg/mL successively for use.
  • the lethal activity of Artemia is expressed by the corrected mortality, and the calculation formula is as follows:
  • Corrected mortality (survival rate of the control group-survival rate of the treatment group)/survival rate of the control group ⁇ 100%, and calculate the LD 50 value of the mortality rate.
  • test results show that: even at 500 ⁇ g/mL, the compound represented by formula (1) does not show the lethal activity of Artemia.
  • the above results can preliminarily show that the compound represented by formula (1) has less toxic effect on the environment and has the potential for agricultural extension.

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Abstract

本发明涉及微生物农药技术领域，具体是一种从菌株Penicillium sp.TR85(保藏于中国微生物菌种保藏管理委员会普通微生物中心，保藏日：2019年8月2日，保藏号：CGMCC No.18116)固体发酵产物中得到具反式萘烷环的聚酮化合物及其分离纯化方法和在除草方面的应用。具反式萘烷环的聚酮化合物为式(一)所示化合物1和化合物2以及两者在农药上可接受的盐或溶剂合物。在10μg/mL浓度时，化合物1和化合物2对稗草[Echinochloa crusgalli]幼苗胚根的生长抑制率分别为81.5％与79.6％，优于商品化除草剂乙草胺，可作为具有除草活性的先导化合物或新农药成分。

Description

具反式萘烷环的聚酮化合物及其制备方法和应用 技术领域
本发明涉及微生物农药技术领域，具体的说是一种从菌株Penicillium sp.TR85固体发酵产物中得到具反式萘烷环的聚酮化合物及其分离纯化方法和在除草方面的应用。
背景技术
杂草与农作物争夺营养与生长空间，不仅引起农作物减产，也能够破坏田间生态平衡，因此杂草防治对农业生产意义重大。稗草[Echinochloa crusgalli]是世界性恶性杂草，在我国稻田的主要杂草中，稗草发生及危害面积最大，约占稻田总面积的43％。长期以来，化学合成除草剂虽然能够有效防治杂草，但是已经引起环境污染、杂草产生抗药性等问题；同时长期过量使用也造成农药残留超标，危及农产品安全及人类健康。从微生物、植物等生物资源中寻找除草活性成分，具有毒性低、环境兼容性好及不易产生抗药性等优点，已经成为开发“环境友好”除草剂的重要来源。
根据文献调研，具反式萘烷环的聚酮化合物具有抑菌、抗肿瘤及免疫调节活性，未见其具有除草活性的相关报道。而本发明涉及的两个具反式萘烷环的聚酮化合物为新化合物，且首次发现具反式萘烷环的聚酮化合物具有除草潜力，其结构显著区别于已知商品化除草剂，为进一步开发新型生物源除草剂提供了指引。
发明内容
本发明的目的是提供一种具反式萘烷环的聚酮化合物及其制备方法和在除草方面的应用。
为实现上述目的，本发明所采用的技术方案为：
式(一)所示化合物1和化合物2以及两者在农药上可接受的盐或溶剂合物：
Figure PCTCN2020072772-appb-000001
一株青霉菌，为青霉(Penicillium sp.)TR85，于2019年8月2日保藏于：中国微生物菌种保藏管理委员会普通微生物中心，保藏号为CGMCC No.18116，保藏地址为：北京市朝 阳区北辰西路1号院3号，请求保藏单位为青岛农业大学。
在上述方案的基础上，上述化合物由上述青霉菌发酵获得。
上述化合物的制备方法，步骤为：将上述青霉菌TR85接种至已灭菌的固体培养基中，室温静置发酵，发酵产物经提取纯化获得。
在上述方案的基础上，所述固体培养基为每100mL天然海水中加入大米100g，蛋白胨0.6g。
在上述方案的基础上，所述发酵产物的提取纯化步骤为：
(1)发酵产物经乙酸乙酯反复浸泡提取，合并提取液进行浓缩，获得发酵粗提物；
(2)将上述发酵粗提物进行减压硅胶柱层析，按照洗脱液极性递增顺序，以梯度为40:1至1:1(v/v)的石油醚-乙酸乙酯作为溶剂进行梯度洗脱；收集石油醚-乙酸乙酯＝1:1(v/v)洗脱组分，进行反相硅胶柱层析，以梯度为2:8至1:0(v/v)的甲醇-水作为溶剂进行梯度洗脱；
(3)收集甲醇-水＝6:4(v/v)反相硅胶洗脱组分，进行半制备高效液相色谱法纯化，流动相为50％甲醇-水，检测波长为220nm，流速为3mL/min，收集保留时间t R值为14.4min的组分，即得式(一)所示化合物1；
(4)收集甲醇-水＝8:2(v/v)反相硅胶洗脱组分，进行半制备高效液相色谱法纯化，流动相为76％甲醇-水，检测波长为220nm，流速为3mL/min，收集保留时间t R值为12.5min的组分，即得式(一)所示化合物2。
具反式萘烷环的聚酮化合物在杂草防治中的应用。
在上述方案的基础上，所述具反式萘烷环的聚酮化合物为上述化合物中的至少一种。
除草组合物，其有效成分包含具反式萘烷环的聚酮化合物中的至少一种。
在上述方案的基础上，所述除草组合物中所述具反式萘烷环的聚酮化合物为上述化合物中的至少一种。
在上述方案的基础上，所述杂草为稗草。
本发明的化合物可以与其他药物组成组合物形式；该组合物包含式(一)所示化合物及农药上可接受的盐和/或溶剂合物作为活性成分，以及农药上可接受的载体，本发明化合物可以与其他活性成分组合使用，只要它们之间不产生拮抗作用。
本发明的除草剂还可以包含农药上可接受的常规助剂，可以为载体、表面活性剂和安全剂；根据其助剂组成可以制备成任何需要的制剂形式，例如乳油、悬浮剂、微乳剂、可分散油悬浮剂、可湿性粉剂、悬乳剂、水乳剂或水分散粒剂。也可以含有农药中其他常规的组分，例如如稳定剂、螯合剂、染料、着色剂、保护胶体、粘合剂、增稠剂、触变剂、渗透剂和聚合物。
除草剂中含有本发明组合物的量依赖杂草的抗性强弱、施药方式和施药频率；本领域普通技术人员仅仅利用常规实验方法即可确定适当的用量。
本文所用术语“本发明化合物”表示以任何形式的式(一)化合物，即任何盐或非盐形式(例如，以游离酸或游离碱形式，或以其药学上可接受的盐形式)及其任何物理形式(例如，包括非固体形式(例如，液体或半固体形式)，和固体形式(例如，无定形或晶型，特定的多晶型形式，溶剂合物，包括水合物(例如，单-、二-和半-水合物))，及其多种形式的混合物。
溶剂合物
对于以晶体形式存在的本发明化合物或其盐的溶剂合物，本领域技术人员将理解可形成农药上可接受的溶剂合物，其中在结晶时溶剂分子被掺入晶格中。溶剂合物可包含非水溶剂例如乙醇、异丙醇、DMSO、乙酸、乙醇胺和乙酸乙酯，或者它们可包含水作为溶剂(该溶剂被掺入晶格中)。其中水为溶剂(该溶剂被掺入晶格中)的溶剂合物通常称为“水合物”。水合物包含化学计量的水合物以及包含可变量水的组分。本发明包括该类溶剂合物。
本发明所具有的优点：
(1)本发明所涉及的具反式萘烷环的聚酮化合物1与化合物2为新化合物，并首次发现具反式萘烷环的聚酮化合物具有除草活性。在10μg/mL浓度时，化合物1与化合物2对稗草(Echinochloa crusgalli)幼苗胚根的生长抑制率分别为81.5％与79.6％，优于商品化除草剂乙草胺，可作为具有除草活性的先导化合物或新农药成分。
(2)本发明所涉及的具反式萘烷环的聚酮化合物1与化合物2由菌株Penicillium sp.TR85发酵产生，便于进行规模发酵生产；其次，化合物1与化合物2为天然产物，相比较化学合成农药，具有环境兼容性好、不易产生抗药性等优点。
附图说明
图1为化合物1的 1H NMR谱图。
图2为化合物1的 13C NMR谱图。
图3为化合物2的 1H NMR谱图。
图4为化合物2的 13C NMR谱图。
图5为化合物1对稗草幼苗生长的抑制作用。
图6为化合物2对稗草幼苗生长的抑制作用。
具体实施方式
在本发明中所使用的术语，除非有另外说明，一般具有本领域普通技术人员通常理解的含义。
下面结合具体实施例，并参照数据进一步详细的描述本发明。以下实施例只是为了举例说明本发明，而非以任何方式限制本发明的范围。
本发明从菌株Penicillium sp.TR85(保藏于中国微生物菌种保藏管理委员会普通微生物中心，保藏日期：2019年8月2日，保藏号：CGMCC No.18116)的发酵产物中分离得到如下的实施例中所指的化合物，其中，化合物1和化合物2的结构如下(结构中的阿拉伯数字为碳原子的标位)：
Figure PCTCN2020072772-appb-000002
实施例1：菌株Penicillium sp.TR85的分离鉴定
(1)菌株分离
本发明所述菌株TR85是从耐盐碱植物碱蓬的根中分离得到。
样品采集：碱蓬样品采集自青岛市胶州湾潮间带湿地；
分离步骤：将碱蓬样品的根冲洗干净，75％乙醇浸泡2min，无菌水漂洗3～4次，无菌纱布蘸干水份；将碱蓬样品的根剪成0.5cm小段，植入PDA双抗培养基平板中，每皿植入4～5片，置于培养箱中，28℃倒置培养，每隔24h观察一次；挑取切面处的菌丝，采用平板划线法接种至PDA双抗培养基平板中，经反复纯化，得到菌落形态特征一致的碱蓬来源菌株，并编号保存。
所述PDA双抗培养基配方：20％土豆浸出液，2％葡萄糖，1.5％琼脂，天然海水配制，并加入终浓度为150μg/mL的青霉素钾和120μg/mL的硫酸链霉素。
(2)菌株鉴定
将菌株TR85接种至PDA平板表面，28℃培养5-7天后观察。菌丝初期为白色，后产生深绿色孢子，并能够产生棕黄色色素。
所述菌株的rDNA基因序列测定结果(ITS-5.8S-ITS2区)如下：
Figure PCTCN2020072772-appb-000003
Figure PCTCN2020072772-appb-000004
上述序列测定结果与Genbank数据库中已知菌株的相应序列信息比较，其与菌株Penicillium viridicatum(Accession number:MK583349)的相似度为99％。该序列已经提交Genbank数据库，序列号为：MN100452。
综上所述，菌株鉴定为Penicillium sp.，该菌株目前已保藏于中国微生物菌种保藏管理委员会普通微生物中心(简称CGMCC，地址：北京市朝阳区北辰西路1号院3号，中国科学院微生物研究所)，保藏日期：2019年08月02日，所述菌株的保藏编号为CGMCC No.18116。
实施例2：化合物1和化合物2的制备方法
(1)菌株发酵培养
切取生长于PDA平板表面的菌株Penicillium sp.TR85(大小2×2cm)，接种至已灭菌的、盛有固体培养基的锥形瓶中，室温静置培养30天。发酵产物经乙酸乙酯反复浸泡提取，合并提取液进行浓缩，获得发酵粗提物。
所述固体培养基为每100mL天然海水中加入大米100g，蛋白胨0.6g。
(2)化合物的制备
将上述发酵粗提物进行减压硅胶柱层析(内径65mm，长度300mm，具砂板及抽咀的玻璃层析柱)，按照洗脱液极性递增顺序，以梯度为40:1至1:1(v/v，下同)的石油醚-乙酸乙酯作为溶剂进行梯度洗脱；收集石油醚-乙酸乙酯＝1:1洗脱组分，进行反相硅胶柱层析(内径30mm，长度600mm，具标准四氟节门的玻璃层析柱)，以梯度为2:8至1:0的甲醇-水作为溶剂进行梯度洗脱。
收集甲醇-水＝6:4反相硅胶洗脱组分，进行半制备高效液相色谱法纯化，流动相为50％甲醇-水，检测波长为220nm，流速为3mL/min，收集保留时间t R值为14.4min的组分，即得化合物1。
收集甲醇-水＝8:2反相硅胶洗脱组分，进行半制备高效液相色谱法纯化，流动相为76％甲醇-水，检测波长为220nm，流速为3mL/min，收集保留时间t R值为12.5min的组分，即得化合物2。
(3)化合物的结构鉴定
表1.化合物1的 1H(500MHz)和 13C NMR(125MHz)谱图数据(NMR测试所用溶剂：氘代氯仿)
Figure PCTCN2020072772-appb-000005
表2.化合物2的 1H(500MHz)和 13C NMR(125MHz)谱图数据(NMR测试所用溶剂：氘代甲醇)
Figure PCTCN2020072772-appb-000006
化合物1，白色粉末，HR-ESI-MS m/z 289.1741[M+Na] +，提示分子式为C 16H 26O 3，其 1H-(附图1)和 13C-NMR(附图2)数据见表1。
化合物2，淡黄色粉末，HR-ESI-MS m/z 439.2107[M+Na] +，提示分子式为C 24H 32O 6，其 1H-(附图3)和 13C-NMR(附图4)数据见表2。
实施例3：除草活性试验
罗小勇等所建立的琼脂混粉法(青岛农业大学学报,2007,24:267-270)用于快速测定化合物的除草活性，具有灵敏度高、操作简便、重复性好、便于大通量筛选等优点，且易于观察指示植物的生长状况。罗小勇等利用该方法对40种园林植物叶片进行了快速除草活性测定。张丽娟(植物保护,2016,42:63-66)和苏范胜(应用化学,2014,31:290-295)以稗草为供试杂草，采用琼脂混粉法快速测定了玉米秸秆和咪唑类化合物的除草活性。张蕴(微生物学报,2015,55:292-298)和Li Shuai(Journal of Agricultural and Food Chemistry,2014,62:8997-9001)以反枝苋为供试杂草，研究了微生物次生代谢产物对反枝苋根生长的抑制作用。
(1)杂草种子的萌发
杂草种子先用0.2％次氯酸钠消毒处理15分钟，然后用无菌蒸馏水反复清洗，浸泡4-6小时，在黑暗条件下用无菌湿滤纸在医用托盘中发芽12小时。
(2)样品溶液的制备
待测化合物以二甲亚砜(DMSO)溶解，配制为4mg/mL溶液，以50％DMSO水溶液稀释成不同浓度，备用。
(3)试验方法
1mL的样品溶液和99mL无菌琼脂水溶液混合均匀，分别倒入3个25mL的小烧杯内冷凝。用尖嘴镊子在已冷凝的琼脂表面插5个小口，分别夹取胚根长达4～5mm的已发芽供试杂草幼苗，将胚根垂直轻轻插入小口中。每烧杯5粒，重复3次。而后将所有烧杯用锡纸封口并置于已消毒的小纸箱内，放人人工气候箱遮光培养2d。人工气候箱的设置条件为14h光照(25℃)和10h黑暗(20℃)的自动循环，箱内的相对湿度为60％。2d后调查胚根和胚轴的长度，计算杂草生长抑制率。
计算公式为(中国农学通报,2013,29:177-182)：
生长量＝处理后的胚根(胚轴)长度-处理前的胚根(胚轴)长度；
抑制率(％)＝[(对照平均生长量-处理平均生长量)/对照平均生长量]×100％。
表3.式(一)所示化合物1与化合物2对稗草幼苗胚根的生长抑制率(％)
Figure PCTCN2020072772-appb-000007
试验结果见表3，结果表明，在10μg/mL浓度时，化合物1(图5)与化合物2(图6) 对稗草(E.crusgalli)幼苗胚根的生长抑制率分别为81.5％与79.6％，显著优于商品化除草剂乙草胺，可作为具有除草活性的先导化合物或新农药成分。
实施例4：卤虫致死活性
卤虫(Brine shrimp)是一种对有毒物质高度敏感的水生动物，能够作为模式生物初步评价化合物对环境的毒性作用，Masi,M.等应用卤虫初步评价除草活性化合物Colletochlorins E与F的毒性(Journal of Agricultural and Food Chemistry,2017,65:1124-1130)。
1)卤虫卵的孵化
取卤虫卵100mg置于500mL烧杯中，加入人工海水400mL，用充气泵缓缓充气，室温孵化24h，除去卵壳及未孵化的卵，卤虫继续培养24h，备用。
2)样品溶液的制备
待测化合物以DMSO溶解，配制为20mg/mL的母液，并依次二倍稀释至10,5与2.5mg/mL溶液，备用。
3)试验方法
取96孔细胞培养板，每孔加195μL含10-15个卤虫的人工海水液，制成测试培养板。空白对照组和各浓度样品组各设三个平行孔，空白对照组加5μL人工海水，样品组加5μL不同浓度的样品液。室温培养24小时后，在双目解剖镜下计数卤虫死亡个体数目。
卤虫致死活性用校正死亡率表示，计算公式如下：
校正死亡率＝(对照组存活率－处理组存活率)/对照组存活率×100％，并计算半数致死率LD 50值。
试验结果表明：式(一)所示化合物即使在500μg/mL时，也不表现卤虫致死活性。上述结果能够初步表明，式(一)所示化合物对环境的毒性作用较小，具有农业推广的潜力。
以上所述的具体实施方式，对本发明的目的、技术方案和有益效果进行了进一步详细说明，所应理解的是，以上所述仅为本发明的具体实施方式而已，并不用于限定本发明的保护范围，凡在本发明的精神和原则之内，所做的任何修改、等同替换改进等，均应包含在本发明的保护范围之内。

Claims (10)

  1. 式(一)所示化合物1和化合物2以及两者在农药上可接受的盐或溶剂合物：
    Figure PCTCN2020072772-appb-100001
  2. 一株青霉菌，其特征在于，所述青霉菌为青霉(Penicillium sp.)TR85，于2019年8月2日保藏于：中国微生物菌种保藏管理委员会普通微生物中心，保藏号为CGMCC No.18116，保藏地址为：北京市朝阳区北辰西路1号院3号，请求保藏单位为青岛农业大学。
  3. 根据权利要求1所述化合物，其特征在于，由权利要求2所述青霉菌发酵获得。
  4. 权利要求1所述化合物的制备方法，其特征在于，步骤为：将权利要求2所述青霉菌TR85接种至已灭菌的固体培养基中，室温静置发酵，发酵产物经提取纯化获得。
  5. 根据权利要求4所述化合物的制备方法，其特征在于，所述固体培养基为每100mL天然海水中加入大米100g，蛋白胨0.6g。
  6. 根据权利要求4所述化合物的制备方法，其特征在于，所述发酵产物的提取纯化步骤为：
    (1)发酵产物经乙酸乙酯浸泡提取，合并提取液进行浓缩，获得发酵粗提物；
    (2)将上述发酵粗提物进行减压硅胶柱层析，按照洗脱液极性递增顺序，以梯度为40:1至1:1(v/v)的石油醚-乙酸乙酯作为溶剂进行梯度洗脱；收集石油醚-乙酸乙酯＝1:1洗脱组分，进行反相硅胶柱层析，以梯度为2:8至1:0(v/v)的甲醇-水作为溶剂进行梯度洗脱；
    (3)收集甲醇-水＝6:4(v/v)反相硅胶洗脱组分，进行半制备高效液相色谱法纯化，流动相为50％甲醇-水，检测波长为220nm，流速为3mL/min，收集保留时间t R值为14.4min的组分，即得式(一)所示化合物1；
    (4)收集甲醇-水＝8:2(v/v)反相硅胶洗脱组分，进行半制备高效液相色谱法纯化，流动相为76％甲醇-水，检测波长为220nm，流速为3mL/min，收集保留时间t R值为12.5min的组分，即得式(一)所示化合物2。
  7. 具反式萘烷环的聚酮化合物在杂草防治中的应用。
  8. 根据权利要求7所述应用，其特征在于，所述具反式萘烷环的聚酮化合物为权利要求1所述化合物中的至少一种。
  9. 除草组合物，其特征在于，其有效成分包含具反式萘烷环的聚酮化合物中的至少一种。
  10. 根据权利要求9所述除草组合物，其特征在于，所述具反式萘烷环的聚酮化合物为 权利要求1所述化合物中的至少一种。
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