WO2018166158A1 - 真菌三糖酯化合物及其在制备防治植物真菌病害药物中的应用 - Google Patents

真菌三糖酯化合物及其在制备防治植物真菌病害药物中的应用 Download PDF

Info

Publication number
WO2018166158A1
WO2018166158A1 PCT/CN2017/102047 CN2017102047W WO2018166158A1 WO 2018166158 A1 WO2018166158 A1 WO 2018166158A1 CN 2017102047 W CN2017102047 W CN 2017102047W WO 2018166158 A1 WO2018166158 A1 WO 2018166158A1
Authority
WO
WIPO (PCT)
Prior art keywords
fungus
genus
compound
formula
medicament
Prior art date
Application number
PCT/CN2017/102047
Other languages
English (en)
French (fr)
Inventor
徐良雄
段学武
魏孝义
薛璟花
冯琳燕
吴萍
Original Assignee
中国科学院华南植物园
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 中国科学院华南植物园 filed Critical 中国科学院华南植物园
Priority to US16/322,947 priority Critical patent/US20190166835A1/en
Publication of WO2018166158A1 publication Critical patent/WO2018166158A1/zh

Links

Images

Classifications

    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N43/00—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds
    • A01N43/02—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds having rings with one or more oxygen or sulfur atoms as the only ring hetero atoms
    • A01N43/04—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds having rings with one or more oxygen or sulfur atoms as the only ring hetero atoms with one hetero atom
    • A01N43/14—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds having rings with one or more oxygen or sulfur atoms as the only ring hetero atoms with one hetero atom six-membered rings
    • A01N43/16—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds having rings with one or more oxygen or sulfur atoms as the only ring hetero atoms with one hetero atom six-membered rings with oxygen as the ring hetero atom
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23B—PRESERVING, e.g. BY CANNING, MEAT, FISH, EGGS, FRUIT, VEGETABLES, EDIBLE SEEDS; CHEMICAL RIPENING OF FRUIT OR VEGETABLES; THE PRESERVED, RIPENED, OR CANNED PRODUCTS
    • A23B7/00—Preservation or chemical ripening of fruit or vegetables
    • A23B7/14—Preserving or ripening with chemicals not covered by groups A23B7/08 or A23B7/10
    • A23B7/153—Preserving or ripening with chemicals not covered by groups A23B7/08 or A23B7/10 in the form of liquids or solids
    • A23B7/154—Organic compounds; Microorganisms; Enzymes
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L3/00—Preservation of foods or foodstuffs, in general, e.g. pasteurising, sterilising, specially adapted for foods or foodstuffs
    • A23L3/34—Preservation of foods or foodstuffs, in general, e.g. pasteurising, sterilising, specially adapted for foods or foodstuffs by treatment with chemicals
    • A23L3/3454—Preservation of foods or foodstuffs, in general, e.g. pasteurising, sterilising, specially adapted for foods or foodstuffs by treatment with chemicals in the form of liquids or solids
    • A23L3/3463—Organic compounds; Microorganisms; Enzymes
    • A23L3/3562—Sugars; Derivatives thereof
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H13/00—Compounds containing saccharide radicals esterified by carbonic acid or derivatives thereof, or by organic acids, e.g. phosphonic acids
    • C07H13/02—Compounds containing saccharide radicals esterified by carbonic acid or derivatives thereof, or by organic acids, e.g. phosphonic acids by carboxylic acids
    • C07H13/04—Compounds containing saccharide radicals esterified by carbonic acid or derivatives thereof, or by organic acids, e.g. phosphonic acids by carboxylic acids having the esterifying carboxyl radicals attached to acyclic carbon atoms
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H13/00—Compounds containing saccharide radicals esterified by carbonic acid or derivatives thereof, or by organic acids, e.g. phosphonic acids
    • C07H13/02—Compounds containing saccharide radicals esterified by carbonic acid or derivatives thereof, or by organic acids, e.g. phosphonic acids by carboxylic acids
    • C07H13/04—Compounds containing saccharide radicals esterified by carbonic acid or derivatives thereof, or by organic acids, e.g. phosphonic acids by carboxylic acids having the esterifying carboxyl radicals attached to acyclic carbon atoms
    • C07H13/06—Fatty acids
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
    • A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs

Definitions

  • the invention belongs to the field of natural products, in particular to fungal trisaccharide compounds and their use in the preparation of medicaments for controlling plant fungal diseases.
  • Plant diseases have always been one of the important constraints to the high quality and high yield of crops. In plant diseases, 70% to 80% of the diseases are caused by infection by pathogenic fungi. Plant fungal diseases not only directly cause the decline of crop yield, but also cause a lot of decay and deterioration during the storage and transportation of agricultural products, and some pathogenic fungi can secrete a variety of mycotoxins and harmful metabolism harmful to humans and animals during the process of infecting crops and agricultural products. Things pose a great threat to the safety of agricultural products. At present, the control of crop fungal diseases often relies on chemical control. The use of fungicides not only increases production costs, but also its repeated application inevitably brings environmental pollution and pesticide residues in agricultural products.
  • a first object of the present invention is to provide three trisaccharide compounds which have significant inhibitory activity against common pathogenic fungi of 12 plants and have a preservative effect on fruits.
  • a trisaccharide ester compound of the present invention which is Compound 1, 2, 3, 4 or 5, of said Compound 1
  • the structural formula is as shown in Formula 1
  • the structural formula of Compound 2 is as shown in Formula 2
  • the structural formula of Compound 3 is as shown in Formula 3
  • the structural formula of Compound 4 is shown in Formula 4
  • the structural formula of Compound 5 is shown in Formula 5
  • a second object of the present invention is to provide the use of the above compound 1, 2, 3, 4 or 5 for the preparation of a medicament for controlling fungal diseases.
  • the medicament for controlling fungal diseases is a medicament for controlling plant fungal diseases.
  • a medicament for controlling plant fungal diseases comprising the compound 1, 2, 3, 4 or 5 of claim 1 as an active ingredient.
  • the medicament for controlling plant fungal diseases is a fungus of the genus Alternaria, a fungus of the genus Botryospuaeria, a fungus of the genus Botrytis, a fungus of the genus Botrytis, a fungus of the genus Colletotrichum, a fungus of the genus Curvularia, a fungus of the genus Fusarium, a fungus of the genus Helminthosporium, a fungus of the genus Penicillium, and a genus of the genus Peronophythora.
  • the plant for controlling plant fungal diseases is for controlling tomato early blight, apple ring disease, tomato gray mold, mango anthracnose, banana crescent, banana, watermelon wilt, citrus white mold, corn leaf spot , citrus blue mold, lychee Phytophthora, rice sheath blight or rice false disease drugs.
  • a third object of the invention is to provide the use of compound 1, 2, 3, 4 or 5 in the preparation of a fruit preservative.
  • a fruit preservative comprising the compound 1, 2, 3, 4 or 5 of claim 1 as an active ingredient.
  • the fruit preservative is a citrus preservative.
  • the present invention isolates S. sphaeroides SC1337 from endophytes of the cedar, which is capable of producing five compounds having significant inhibitory activity against common pathogenic fungi of 12 plants and having a fresh-keeping effect on fruits, and thus can be prepared therefrom. These five compounds have obvious inhibitory activity against common pathogenic fungi of 12 plants and have a fresh-keeping effect on fruits. They are used for the control of phytopathogenic fungi and the preservation of fruits, and have broad application prospects.
  • GDMCC Guangdong Provincial Museum of Microbial Cultures on February 15, 2017 (referred to as GDMCC, Address: Guangdong Institute of Microbiology, Building 59, No. 100, Xianlie Middle Road, Guangzhou, China) Zip code: 510070), the deposit number is GDMCC No: 60144.
  • Figure 1 shows the colony morphology of Pezicula sp. SC1337.
  • Figure 2 is a mycelium and spore morphology of Pezicula sp. SC1337.
  • the Pezicula sp. SC1337 of the present invention is from the branches of the cedar branches of the South China Botanical Garden. Separated by the bacteria, the separation method is as follows: the collected samples of the cedar branches are sterilized by 75% ethanol for 30 s, then sterilized by mass fraction of 10% sodium hypochlorite for 3 min, rinsed with sterile water for 3 times, dried, and inoculated to PDA culture. The substrate was placed in a 28 ° C incubator for 5 days, and the target colony was picked up with a sterile toothpick.
  • the PDA medium contained 6 g of potato extract per liter, 20 g of glucose, 20 g of agar, and the rest was water, pH 6.0- 6.5, whereby Pezicula sp. SC1337 was isolated.
  • Taxonomic characteristics of Pezicula sp. SC1337 Pezicula sp. SC1337 has a hyphae width of 2 to 3 ⁇ m; spores are oblong, with a size of 14 to 22 ⁇ 4 to 6 ⁇ m, colony morphology The morphology of the hyphae (including spores) is shown in Figures 1 and 2.
  • the mycelium was extracted by classical CTAB method and passed as ITS4(5'-3'):TCC TCC GCT TAT TGA TAT GC and ITS5(5'-3'):GGA AGT AAA AGT CGT AAC AAG G as primers.
  • ITS sequence fragment 917bp
  • Pezicula sp. SC1337 which was deposited on February 15, 2017 in the Guangdong Provincial Collection of Microbial Cultures (GDMCC, Address: No. 100, Xianlie Middle Road, Guangzhou, 59) Guangdong Institute of Microbiology, No. 510070, the deposit number is GDMCC No: 60144.
  • Pezicula sp. SC1337 was cultured on a wheat solid medium (mixed with wheat and water at a mass ratio of 1:1.5), and statically cultured at 28 ° C for 12 days in the dark to obtain a solid fermentation culture.
  • the solid fermentation culture (3.6L) was soaked three times with an equal volume of 95% ethanol aqueous solution for 48 hours, the soaking solution was combined and filtered, then concentrated under reduced pressure to remove ethanol, and the volume was adjusted to 1 L with water, and then equal volume was used.
  • the ethyl acetate extract was combined three times, and the obtained ethyl acetate extracts were combined, and then concentrated to obtain 17.6 g of ethyl acetate extract.
  • the ethyl acetate extract was applied to a silica gel column (silica gel 100-200 mesh, 300 g), and eluted with a gradient of 90:10 to 70:30 in a chloroform-methanol mixed solvent, and detected by thin layer chromatography (silica gel). Merge similar For the fractions, the fraction of chloroform-methanol at a volume ratio of 85:15 on the thin layer was collected and the fraction was shifted from 0.3 to 0.4, and concentrated under reduced pressure to obtain a fraction A 1.69 g and a fraction with a specific shift of 0.2 to 0.3. Concentrated under reduced pressure to obtain a fraction B of 8.50 g;
  • Fraction A was subjected to reversed-phase silica gel column chromatography (Develosil ODS, 75 ⁇ m, 150 g, product of Fuji Chemical Co., Japan), and eluted with a volume fraction of 88% methanol aqueous solution to collect the fractions in the main point concentration, and after combining, High performance liquid chromatography (LC-6AD semi-preparative high performance liquid chromatography, RID-10A detector, manufactured by Shimadzu Corporation, Japan; Shim-pack PRC-ODS, 10 ⁇ 250 mm, 4.5 ⁇ m) with a volume fraction of 88
  • Compound 1 a white powder, having the formula C 41 H 72 O 19 , a positive ion HRESIMS m/z 891.4552 [M+Na] + (calcd for C 41 H 72 O 19 Na, 891.4560), a positive ion ESIMS m/z 891 [ M+Na] + , 907[M+K] + ; negative ion ESIMS m/z 867[M-H] - , 903[M+Cl] - .
  • Table 1 The data of 1 H NMR and 13 C NMR (solvent: deuterated methanol) are shown in Table 1.
  • Compound 1 was identified as 6-O- ⁇ -L-mannosyl-3-O-(2-methylbutyryl)-4-O-(8-methyloxime) by the above spectral data and two-dimensional nuclear magnetic resonance.
  • Acyl)-2-O-(4-methylhexanoyl) trehalose having the structural formula shown in Formula 1.
  • compound 2 white powder, molecular formula is C 40 H 70 O 19 , positive ion HRESIMS m/z 877.4411 [M+Na] + (calcd for C 40 H 70 O 19 Na, 877.4404), positive ion ESIMS m/z 877 [M+Na] + ; Negative ion ESIMS m/z 853 [M-H] - , 889 [M+Cl] - .
  • Table 1 H NMR and 13 C NMR solvent: deuterated methanol
  • Compound 2 was identified as 4-O-decanoyl-6-O- ⁇ -L-mannosyl-3-O-(2-methylbutyryl)-2-O by the above spectral data and two-dimensional nuclear magnetic resonance.
  • -(4-Methylhexanoyl) trehalose having the structural formula shown in Formula 2.
  • compound 3 white powder, having the formula C 39 H 68 O 19 , cation HRESIMS m/z 863.4255 [M+Na] + (calcd for C 39 H 68 O 19 Na, 863.4247), positive ion ESIMS m/z 863 [M+Na] + ; Negative ion ESIMS m/z 839 [M-H] - , 875 [M+Cl] - .
  • Table 1 H NMR and 13 C NMR solvent: deuterated methanol
  • the compound 3 was identified as 6-O- ⁇ -L-mannosyl-3-O-(2-methylbutyryl)-2-O-(4-methylhexyl) by the above spectral data and two-dimensional nuclear magnetic resonance.
  • Compound 4 was identified as 6-O- ⁇ -L-mannosyl-3-O-(2-methylbutyryl)-2-O-(4-methylhexyl) by the above spectral data and two-dimensional nuclear magnetic resonance.
  • Acyl)-4-O-octanoyl trehalose having the structural formula shown in Formula 4.
  • Example 3 In vitro inhibitory activity test of the trisaccharide esters (Compounds 1-5) obtained in Example 2 against 12 common pathogenic fungi of plants
  • test strains used included: 1) Alternaria solani tomato early blight; 2) Botryospuaeria Berengeriana apple ringworm; 3) Botrytis cinerea, Botrytis cinerea; 4) Colletotrichum gloeosporioides, mango anthracnose; 5) Curvularia lunata, banana, Curvularia; 6) Fusarium oxysporium banana, watermelon Fusarium; 7) Geotrichum citri-aurantii Phytophthora citrifolia; 8) Helminthosporium maydis Phytophthora sphaeroides; 9) Penicillium italicum citrus Penicillium; 10) Peronophythora litchii Phytophthora capsici; 11) Rhizoctonia solani, Rhizoctonia solani; 12) Ustilaginoidea virens.
  • the antifungal activity of the compounds 1 to 5 was determined by a filter paper agar diffusion method: the activated test bacteria were separately inoculated into a petri dish containing a PDA solid medium, and uniformly coated with a T-type applicator; the sample to be tested was Methanol was dissolved and formulated into 50 mg/mL. 10 ⁇ L of the solution was added to a filter paper (6 mm in diameter) and dried on a clean bench. Each filter paper finally contained 500 ⁇ g of the sample, and then the filter paper was placed on the filter paper. In the above culture dish, the inhibition width (the inhibition width is the average diameter of the inhibition zone) was measured after incubation at 28 ° C for 72 or 96 hours, and a filter paper of an equal volume of methanol was added as a negative control.
  • the results showed that the trisaccharide extract and the compounds 1 to 5 all inhibited the growth of all the above fungi, and the inhibition zone width was 10 to 22 mm, showing a broad spectrum of fungal inhibitory activity, and the results are shown in Table 3.
  • the trisaccharide extract is a mixture containing compounds 1 to 4 in which the compound ratio of 1, 2, 3, and 4 is about 60:35:4:1.
  • Example 4 Preservation experiment of trisaccharide extract obtained in Example 2 on postharvest citrus storage
  • Test sample The trisaccharide extract obtained in Example 2 (containing compound 1, 2, 3, 4 by mass ratio of about 60:35:4:1) was dissolved in a small amount of ethanol and diluted with water to 200, 400, 600 mg / L is used as a treatment solution.
  • the citrus fruit was soaked in the treatment solution for 5 min, dried, and then placed in a plastic basket which was sterilized by drying on a 75% alcohol surface, sealed in a 0.02 mm polyethylene film bag, and stored at 25 ° C for 15 days. Observe and record the disease index.
  • Test fruit Sugar orange is collected from the orchard.
  • the experimental results are shown in Table 4.
  • the trisaccharide extract has a significant inhibitory effect on the development of fungi during postharvest citrus storage, and is superior to the commonly used chemical antifungal agent thiram, which is equivalent to Tekdo and has obvious antiseptic and fresh-keeping effects.
  • the antibacterial and fresh-keeping effect of the trisaccharide extract is slightly worse than that of the chemical antifungal agent, but Baco is banned in the EU due to its high toxicity in 2004.

Landscapes

  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Organic Chemistry (AREA)
  • Health & Medical Sciences (AREA)
  • General Health & Medical Sciences (AREA)
  • Wood Science & Technology (AREA)
  • Zoology (AREA)
  • Biochemistry (AREA)
  • Molecular Biology (AREA)
  • Genetics & Genomics (AREA)
  • Biotechnology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Microbiology (AREA)
  • General Chemical & Material Sciences (AREA)
  • Food Science & Technology (AREA)
  • Polymers & Plastics (AREA)
  • Environmental Sciences (AREA)
  • Dentistry (AREA)
  • Plant Pathology (AREA)
  • Pest Control & Pesticides (AREA)
  • Agronomy & Crop Science (AREA)
  • Nutrition Science (AREA)
  • Agricultural Chemicals And Associated Chemicals (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)

Abstract

公开了真菌三糖酯化合物及其在制备防治植物真菌病害药物中的应用。从落羽杉枝条内生菌中分离得到无柄盘菌(Pezicula sp.)SC1337，其能够产生五个对12种植物常见致病真菌有明显的抑制活性以及对水果具有保鲜效果的化合物，因此可以用其制备这五个对12种植物常见致病真菌有明显的抑制活性以及对水果具有保鲜效果的化合物，用于植物致病真菌的防治和水果的保鲜。

Description

真菌三糖酯化合物及其在制备防治植物真菌病害药物中的应用 技术领域
本发明属于天然产物领域，具体涉及真菌三糖酯化合物及其在制备防治植物真菌病害药物中的应用。
背景技术
植物病害一直是农作物优质高产的重要制约因素之一。在植物病害中，70％～80％的病害是病原真菌侵染所引致的。植物真菌病害不仅直接造成农作物产量下降，农产品贮藏和运输期间大量腐烂变质，造成重大损失，而且部分病原真菌在侵染农作物和农产品过程中，可分泌产生多种对人畜有害的真菌毒素与有害代谢物，对农产品的安全性构成极大威胁。目前，农作物真菌病害的控制往往依赖化学防治，杀菌剂的使用不仅增大生产成本，而且其反复施用不可避免地带来环境污染与农产品农药残留问题。我国主要农作物重大真菌病害的化学防治主要依赖少数杀菌剂，病原真菌生产抗药性而导致杀菌剂丧失防治效果的风险性较高。因此，研发新型、高效的真菌抑制剂意义重大。
传统的化学合成的真菌抑制剂由于长期大量使用，病原菌产生部分抗药性，造成药效减弱，且在环境中不易分解，残留药剂会流失到周围环境中，对农林业生态系统及人类健康造成危害。而天然产物真菌抑制剂对防治对象多有较高选择性，且易于降解，残留较低，对人、畜和有益生物相对安全，环境污染较小，是近年植物病害防治研究的重点之一。目前常用的农用真菌抑制剂如井冈霉素、春雷霉素、多抗霉素、中生霉素、宁南霉素和申嗪霉素等，均来源于环境微生物(主要是植物根际土壤防线菌)次生代谢产物，来自真菌的较少。然而，植物内生真菌次生代谢产物种类丰富、结构多样，也是研发微生物源新型、高效的真菌抑制剂的重要资源。
发明内容
本发明的第一个目的是提供五个对12种植物常见致病真菌有明显的抑制活性以及对水果具有保鲜效果的三糖酯化合物。
本发明的三糖酯化合物，其为化合物1、2、3、4或5，所述的化合物1的 结构式如式1所示，化合物2的结构式如式2所示，化合物3的结构式如式3所示，化合物4的结构式如式4所示，化合物5的结构式如式5所示，
Figure PCTCN2017102047-appb-000001
本发明第二个目的是提供了上述化合物1、2、3、4或5在制备防治真菌病害药物中的应用。
所述的防治真菌病害药物是防治植物真菌病害药物。
一种防治植物真菌病害药物，含有权利要求1中的化合物1、2、3、4或5作为活性成分。
优选，所述的防治植物真菌病害药物是防治Alternaria属真菌、Botryospuaeria属真菌、Botrytis属真菌、Colletotrichum属真菌、Curvularia属真菌、Fusarium属真菌、Geotrichum属真菌、Helminthosporium属真菌、Penicillium属真菌、Peronophythora属真菌、Rhizoctonia属真菌、Ustilaginoidea属真菌的药物。
所述的防治植物真菌病害药物是防治番茄早疫病、苹果轮纹病、番茄灰霉病、芒果炭疽病、香蕉新月弯孢霉、香蕉、西瓜枯萎病、柑橘白地霉病、玉米小斑病、柑橘青霉病、荔枝霜疫霉、水稻纹枯病或稻曲病的药物。
本发明第三个目的是提供化合物1、2、3、4或5在制备水果保鲜剂中的应用。
一种水果保鲜剂，含有权利要求1中的化合物1、2、3、4或5作为活性成分。
优选，所述的水果保鲜剂为柑橘保鲜剂。
本发明从落羽杉枝条内生菌中分离得到无柄盘菌SC1337，其能够产生五个对12种植物常见致病真菌有明显的抑制活性以及对水果具有保鲜效果的化合物，因此可以用其制备这五个对12种植物常见致病真菌有明显的抑制活性以及对水果具有保鲜效果的化合物，用于植物致病真菌的防治和水果的保鲜，具有广阔的应用前景。
无柄盘菌(Pezicula sp.)SC1337于2017年2月15日保藏在广东省微生物菌种保藏中心(简称为GDMCC，地址：广州市先烈中路100号大院59号楼广东省微生物研究所，邮编：510070)，保藏号为GDMCC No：60144。
附图说明：
图1是无柄盘菌(Pezicula sp.)SC1337的菌落形态。
图2是无柄盘菌(Pezicula sp.)SC1337菌丝和孢子形态。
具体实施方式：
以下实施例是对本发明的进一步说明，而不是对本发明的限制。
实施例1：
本发明的无柄盘菌(Pezicula sp.)SC1337是从华南植物园的落羽杉枝条内 生菌中分离获得，分离方法为：将采集好的落羽杉枝条样品经75％乙醇表面消毒30s，再经质量分数10％次氯酸钠消毒3min，无菌水冲洗3遍后晾干，接种至PDA培养基上，放至28℃培养箱培养5d，用无菌牙签挑取目标菌落即得，所述PDA培养基每升含马铃薯浸提物6g，葡萄糖20g，琼脂20g，其余为水，pH 6.0～6.5，由此分离得到无柄盘菌(Pezicula sp.)SC1337。
无柄盘菌(Pezicula sp.)SC1337的分类学特征：无柄盘菌(Pezicula sp.)SC1337的菌丝宽2～3μm；孢子长椭圆形，大小为14～22×4～6μm，菌落形态和菌丝(含孢子)的形态如图1和图2所示。菌丝体以经典的CTAB法提取得到DNA，通过以ITS4(5’-3’)：TCC TCC GCT TAT TGA TAT GC和ITS5(5’-3’)：GGA AGT AAA AGT CGT AAC AAG G为引物，在10×PCR buffer 5μL，Mg2+(25mM)4μL，dNTPs(10mM)1μL，Primers(10μM/each)2μL，Taq酶(5U/μL)0.3μL，模板DNA(10ng/μL)5μL，ddH2O 32.7μL的体系中，在94℃5min，94℃40s，57-52℃1min，72℃1min，cycles 35，72℃6min程序下进行PCR扩增后，经测序得到的DNA核苷酸序列如SEQ ID NO.1所示。测序结果在Genbank数据库用Blast进行检索和同源性比较，其ITS序列片段(共917bp)同源性与Pezicula cinnamomea的同源性最高，ITS相似性为96％。因此将此菌命名为：无柄盘菌(Pezicula sp.)SC1337，于2017年2月15日保藏在广东省微生物菌种保藏中心(简称为GDMCC，地址：广州市先烈中路100号大院59号楼广东省微生物研究所，邮编510070)，保藏号为GDMCC No：60144。
实施例2：5种三糖酯衍生物的制备及其理化和波谱数据：
将无柄盘菌(Pezicula sp.)SC1337在小麦固体培养基(质量比1:1.5的小麦和水混合组成)上，黑暗中于28℃静置培养12d，得到固体发酵培养物。
将固体发酵培养物(3.6L)用等体积的体积分数95％乙醇水溶液浸泡三次，每次48h，浸泡液合并后过滤，然后减压浓缩除去乙醇，并加水定容至1L，再用等体积乙酸乙酯萃取3次，将得到的乙酸乙酯萃取物合并后，再浓缩得到乙酸乙酯提取物17.6g。
乙酸乙酯提取物上硅胶柱(硅胶为100～200目，300g)，以体积比90:10～70:30的氯仿-甲醇混合溶剂进行梯度洗脱，用薄层层析(硅胶板)检测合并相似 的流份，收集薄层板上氯仿-甲醇体积比85:15展开时比移值0.3～0.4的流份，减压浓缩得流份A 1.69g，和比移值0.2～0.3的流份，减压浓缩得流份B 8.50g；
将流份A进行反相硅胶柱层析(Develosil ODS，75μm，150g，日本富士化学公司产品)，以体积分数88％的甲醇水溶液等度洗脱，收集主点集中的流份，合并后在高效液相色谱(LC-6AD型半制备高效液相色谱仪，RID-10A检测器，日本Shimadzu公司生产；层析柱为Shim-pack PRC-ODS，10×250mm，4.5μm)以体积分数88％的甲醇水溶液为流动相，流速为5ml/min，纯化得到白色粉末化合物5(0.030g，tR＝60min)。
取流份B 1.0g进行反相硅胶柱层析(Develosil ODS，75μm，150g)，以体积分数65％～85％的甲醇水溶液梯度洗脱，收集85％洗脱部分浓缩后经高效液相色谱(仪器和层析柱同前)，以体积分数87％的甲醇水溶液为流动相，流速为5ml/min，分离纯化得到四个白色粉末物质，分别为化合物1(0.122g，tR＝55min)，化合物2(0.120g，tR＝45min)，化合物3(0.010g，tR＝33min)和化合物4(0.122g，tR＝28min)。
化合物1，白色粉末，分子式为C41H72O19，正离子HRESIMS m/z 891.4552[M+Na]+(calcd for C41H72O19Na,891.4560)，正离子ESIMS m/z 891[M+Na]+，907[M+K]+；负离子ESIMS m/z 867[M－H]－，903[M+Cl]－。1H NMR和13C NMR(溶剂：氘代甲醇)的数据见表1。通过以上光谱数据和二维核磁共振等方法鉴定化合物1为6-O-β-L-甘露糖基-3-O-(2-甲基丁酰)-4-O-(8-甲基癸酰)-2-O-(4-甲基己酰)海藻糖，其结构式如式1所示。
其中化合物2，白色粉末，分子式为C40H70O19，正离子HRESIMS m/z 877.4411[M+Na]+(calcd for C40H70O19Na,877.4404)，正离子ESIMS m/z 877[M+Na]+；负离子ESIMS m/z 853[M－H]－，889[M+Cl]－。1H NMR和13C NMR(溶剂：氘代甲醇)的数据见表1。通过以上光谱数据和二维核磁共振等方法鉴定化合物2为4-O-癸酰-6-O-β-L-甘露糖基-3-O-(2-甲基丁酰)-2-O-(4-甲基己酰)海藻糖，其结构式如式2所示。
其中化合物3，白色粉末，分子式为C39H68O19，正离子HRESIMS m/z 863.4255[M+Na]+(calcd for C39H68O19Na,863.4247)，正离子ESIMS m/z 863[M+Na]+；负离子ESIMS m/z 839[M－H]－，875[M+Cl]－。1H NMR和13C NMR(溶剂： 氘代甲醇)的数据见表1。通过以上光谱数据和二维核磁共振等方法鉴定化合物3为6-O-β-L-甘露糖基-3-O-(2-甲基丁酰)-2-O-(4-甲基己酰)-4-O-(6-甲基辛酰)海藻糖，其结构式如式3所示。
其中化合物4，白色粉末，分子式为C38H66O19，正离子HRESIMS m/z 849.4088[M+Na]+(calcd for C38H66O19Na,849.4091)，正离子ESIMS m/z 849[M+Na]+；负离子ESIMS m/z 825[M－H]－。1H NMR和13C NMR(溶剂：氘代甲醇)的数据见表2。通过以上光谱数据和二维核磁共振等方法鉴定化合物4为6-O-β-L-甘露糖基-3-O-(2-甲基丁酰)-2-O-(4-甲基己酰)-4-O-辛酰海藻糖，其结构式如式4所示。
其中化合物5，白色粉末，分子式为C43H74O20，正离子ESIMS m/z 933[M+Na]+，949[M+K]+；负离子ESIMS m/z 909[M－H]－，945[M+Cl]－。1H NMR和13C NMR(溶剂：氘代甲醇)的数据见表2。通过以上光谱数据和二维核磁共振等方法鉴定化合物5为6-O-β-L-甘露糖基-3-O-(2-甲基丁酰)-4-O-(8-甲基癸酰)-2-O-(4-甲基己酰)-6'-O-乙酰基海藻糖，其结构式如式5所示。
表1 化合物1～3的1H和13C NMR数据
Figure PCTCN2017102047-appb-000002
Figure PCTCN2017102047-appb-000003
Figure PCTCN2017102047-appb-000004
*相应位置的暂定信号可以互换
表2 化合物4和5的1H和13C NMR数据
Figure PCTCN2017102047-appb-000005
Figure PCTCN2017102047-appb-000006
Figure PCTCN2017102047-appb-000007
*相应位置的暂定信号可以互换
Figure PCTCN2017102047-appb-000008
实施例3：实施例2得到的三糖酯(化合物1～5)的对植物常见12种科属的致病真菌的体外抑制活性试验
所用测试菌种包括：1)Alternaria solani番茄早疫病菌；2)Botryospuaeria  berengeriana苹果轮纹病菌；3)Botrytis cinerea番茄灰霉病菌；4)Colletotrichum gloeosporioides芒果炭疽病菌；5)Curvularia lunata香蕉新月弯孢霉；6)Fusarium oxysporium香蕉、西瓜枯萎病菌；7)Geotrichum citri-aurantii柑橘白地霉病菌；8)Helminthosporium maydis玉米小斑病菌；9)Penicillium italicum柑橘青霉病菌；10)Peronophythora litchii荔枝霜疫霉菌；11)Rhizoctonia solani水稻纹枯病菌；12)Ustilaginoidea virens稻曲病菌。
以滤纸片琼脂扩散法测定化合物1～5的抗真菌活性：将活化好的测试菌分别接种到装有PDA固体培养基的培养皿中，用T型涂布器涂均匀备用；待测试样品以甲醇溶解，配成50mg/mL，实验时取10μL滴加到滤纸片(直径为6mm)上，于超净工作台上晾干，使每滤纸片最终含有样品500μg，然后将滤纸片贴放于上述培养皿中，28℃黑暗条件下培养72或96h后测量抑菌宽度(抑菌宽度为抑菌圈平均直径)，以加入等体积甲醇的滤纸片作阴性对照。
结果显示三糖酯提取物和化合物1～5均可以抑制以上所有真菌的生长，抑菌圈宽度为10～22mm，显示出广谱的真菌抑制活性，结果见表3。所述三糖酯提取物为含有化合物1～4的混合物，其中化合物1、2、3、4质量比约为60:35:4:1。
表3 三糖酯提取物和化合物1～5对12种植物致病真菌的体外抑制活性
Figure PCTCN2017102047-appb-000009
实施例4：实施例2得到的三糖酯提取物对采后柑橘贮藏中保鲜实验
供试样品：实施例2得到的三糖酯提取物(含化合物1、2、3、4质量比约为60:35:4:1)以少量乙醇溶解，用水稀释为200、400、600mg/L作为处理溶液。
将柑橘果实在处理溶液中浸泡5min，晾干后，将果实置于经75％酒精表面灭菌晾干的塑料筐中，用0.02mm的聚乙烯薄膜袋包装密封，于25℃贮藏15天，观察并记录其感病指数，感病指数计算方法为：0为无明显感染；1为轻微感染；2为感染面积为果实的≤1/4；3为感染面积为果实的1/4-1/2；4为感染面积≥1/2，感病指数＝∑(感病等级×相应果实数量)/(5×果实总数量)×100％。每处理设3个重复，每重复用果约60个。
供试水果：砂糖橘采自从化果园。
实验结果如表4所示。该三糖酯提取物对采后柑橘贮藏过程中的真菌发生发展有明显的抑制作用，优于常用化学抗真菌剂福美双，与特克多相当，具有明显的防腐保鲜作用。该三糖酯提取物的抑菌保鲜效果略差于化学抗真菌剂百可得，但百可得由于毒性较高，2004年已被欧盟禁止使用。
表4 三糖酯提取物对采后柑橘果实贮藏中对致病真菌的抑制活性
Figure PCTCN2017102047-appb-000010
Figure PCTCN2017102047-appb-000011

Claims (9)

  1. 三糖酯化合物，其为化合物1、2、3、4或5，所述的化合物1的结构式如式1所示，化合物2的结构式如式2所示，化合物3的结构式如式3所示，化合物4的结构式如式4所示，化合物5的结构式如式5所示，
    Figure PCTCN2017102047-appb-100001
  2. 权利要求1中的化合物1、2、3、4或5在制备防治真菌病害药物中的应用。
  3. 根据权利要求2所述的应用，其特征在于，所述的防治真菌病害药物是 防治植物真菌病害药物。
  4. 根据权利要求3所述的应用，其特征在于，所述的防治植物真菌病害药物是防治Alternaria属真菌、Botryospuaeria属真菌、Botrytis属真菌、Colletotrichum属真菌、Curvularia属真菌、Fusarium属真菌、Geotrichum属真菌、Helminthosporium属真菌、Penicillium属真菌、Peronophythora属真菌、Rhizoctonia属真菌或Ustilaginoidea属真菌的药物。
  5. 一种防治植物真菌病害药物，其特征在于，含有权利要求1中的化合物1、2、3、4或5作为活性成分。
  6. 根据权利要求5所述的防治植物真菌病害药物，其特征在于，所述的防治植物真菌病害药物是防治Alternaria属真菌、Botryospuaeria属真菌、Botrytis属真菌、Colletotrichum属真菌、Curvularia属真菌、Fusarium属真菌、Geotrichum属真菌、Helminthosporium属真菌、Penicillium属真菌、Peronophythora属真菌、Rhizoctonia属真菌或Ustilaginoidea属真菌的药物。
  7. 根据权利要求5所述的防治植物真菌病害药物，其特征在于，所述的防治植物真菌病害药物是防治番茄早疫病、苹果轮纹病、番茄灰霉病、芒果炭疽病、香蕉新月弯孢霉、香蕉、西瓜枯萎病、柑橘白地霉病、玉米小斑病、柑橘青霉病、荔枝霜疫霉、水稻纹枯病或稻曲病的药物。
  8. 权利要求1中的化合物1、2、3、4或5在制备水果保鲜剂中的应用。
  9. 根据权利要求8所述的应用，其特征在于，所述的水果保鲜剂为柑橘保鲜剂。
PCT/CN2017/102047 2017-03-15 2017-09-18 真菌三糖酯化合物及其在制备防治植物真菌病害药物中的应用 WO2018166158A1 (zh)

Priority Applications (1)

Application Number Priority Date Filing Date Title
US16/322,947 US20190166835A1 (en) 2017-03-15 2017-09-18 Fungal Trisaccharide Ester Compounds and Use Thereof in Preparing Agents for Preventing and Controlling Plant Fungal Diseases

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN201710154256.1 2017-03-15
CN201710154256.1A CN106946955B (zh) 2017-03-15 2017-03-15 真菌三糖酯化合物及其在制备防治植物真菌病害药物中的应用

Publications (1)

Publication Number Publication Date
WO2018166158A1 true WO2018166158A1 (zh) 2018-09-20

Family

ID=59471988

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2017/102047 WO2018166158A1 (zh) 2017-03-15 2017-09-18 真菌三糖酯化合物及其在制备防治植物真菌病害药物中的应用

Country Status (3)

Country Link
US (1) US20190166835A1 (zh)
CN (1) CN106946955B (zh)
WO (1) WO2018166158A1 (zh)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN110038019A (zh) * 2019-05-27 2019-07-23 惠州学院 一种三糖酯类化合物在制备抗细胞增殖性紊乱或抗癌药物中的应用

Families Citing this family (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN106946955B (zh) * 2017-03-15 2019-06-21 中国科学院华南植物园 真菌三糖酯化合物及其在制备防治植物真菌病害药物中的应用
CN106867917B (zh) * 2017-03-15 2019-06-21 中国科学院华南植物园 无柄盘菌sc1337菌株和利用它制备三糖酯衍生物的方法
CN110511878B (zh) * 2019-07-31 2021-06-11 中国林业科学研究院亚热带林业研究所 具抑菌能力和木质纤维素降解活性的新孢无柄盘菌及用途
CN111887056B (zh) * 2020-06-30 2022-11-01 阿克苏优能农业科技股份有限公司 促糖心防虫害的苹果果树调控剂及应用

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1269361A (zh) * 2000-04-28 2000-10-11 吴忠 葡聚烯糖化合物及其合成方法与应用
CN105506029A (zh) * 2016-01-06 2016-04-20 西南大学柑桔研究所 用柑橘皮制备抗真菌果胶低聚糖的方法及其应用和柑橘保鲜方法
CN106867917A (zh) * 2017-03-15 2017-06-20 中国科学院华南植物园 无柄盘菌sc1337菌株和利用它制备三糖酯衍生物的方法
CN106946955A (zh) * 2017-03-15 2017-07-14 中国科学院华南植物园 真菌三糖酯化合物及其在制备防治植物真菌病害药物中的应用

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
ATE387457T1 (de) * 2000-08-25 2008-03-15 Eastman Chem Co Verfahren zur herstellung von disaccharid und trisaccharid c6-c12 fettsäure-estern mit hohem alpha gehalt und materialien davon

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1269361A (zh) * 2000-04-28 2000-10-11 吴忠 葡聚烯糖化合物及其合成方法与应用
CN105506029A (zh) * 2016-01-06 2016-04-20 西南大学柑桔研究所 用柑橘皮制备抗真菌果胶低聚糖的方法及其应用和柑橘保鲜方法
CN106867917A (zh) * 2017-03-15 2017-06-20 中国科学院华南植物园 无柄盘菌sc1337菌株和利用它制备三糖酯衍生物的方法
CN106946955A (zh) * 2017-03-15 2017-07-14 中国科学院华南植物园 真菌三糖酯化合物及其在制备防治植物真菌病害药物中的应用

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN110038019A (zh) * 2019-05-27 2019-07-23 惠州学院 一种三糖酯类化合物在制备抗细胞增殖性紊乱或抗癌药物中的应用

Also Published As

Publication number Publication date
CN106946955B (zh) 2019-06-21
CN106946955A (zh) 2017-07-14
US20190166835A1 (en) 2019-06-06

Similar Documents

Publication Publication Date Title
WO2018166158A1 (zh) 真菌三糖酯化合物及其在制备防治植物真菌病害药物中的应用
Tian et al. Biocontrol and the mechanisms of Bacillus sp. w176 against postharvest green mold in citrus
WO2018166159A1 (zh) 无柄盘菌sc1337菌株和利用它制备三糖酯衍生物的方法
Shakeel et al. Optimization of the cultural medium and conditions for production of antifungal substances by Streptomyces platensis 3-10 and evaluation of its efficacy in suppression of clubroot disease (Plasmodiophora brassicae) of oilseed rape
Ghany Eco-friendly and safe role of Juniperus procera in controlling of fungal growth and secondary metabolites
Kim et al. Isolation of antimicrobial substances from Hericium erinaceum
CN114891670A (zh) 一株具有广谱抑菌性的暹罗芽孢杆菌ZJh及其应用
Maung et al. Antifungal compound, methyl hippurate from Bacillus velezensis CE 100 and its inhibitory effect on growth of Botrytis cinerea
CN102742605A (zh) 一株银杏内生球毛壳菌防治植物病原真菌的应用
CN110622975B (zh) 咪康唑在制备用于防治植物病原菌的杀菌剂中的应用
Abdel-Aziz et al. Ethyl acetate extract of Streptomyces spp. isolated from Egyptian soil for management of Fusarium oxysporum: The causing agent of wilt disease of tomato
de Carvalho et al. Diversity and antimicrobial activity of culturable endophytic fungi associated with the neotropical ethnomedicinal plants Copaifera langsdorffii and Copaifera pubiflora
Wang et al. Bacillus amyloliquefaciens FG14 as a potential biocontrol strain against rusty root rot of Panax ginseng, and its impact on the rhizosphere microbial community
Elzein et al. Effects of inoculum type and propagule concentration on shelf life of Pesta formulations containing Fusarium oxysporum Foxy 2, a potential mycoherbicide agent for Striga spp.
Palazzini et al. Osmotic stress adaptation, compatible solutes accumulation and biocontrol efficacy of two potential biocontrol agents on Fusarium head blight in wheat
Gao et al. Actinomycins produced by endophyte Streptomyces sp. GLL‐9 from navel orange plant exhibit high antimicrobial effect against Xanthomonas citri susp. citri and Penicillium italicum
Haggag et al. Improving the biological control of Botryodiplodia disease on some Annona cultivars using single or multi-bioagents in Egypt
Leung et al. Brown Root Rot Disease of Phyllanthus myrtifolius: The causal agent and two potential biological control agents
CN109456900B (zh) 一种复合生物制剂及其用途
Ntemafack et al. Chemical profile, biological potential, bioprospection and biotechnological application of endophytes of Rumex: a systematic review
Sandhu et al. Endophytic fungi: eco-friendly future resource for novel bioactive compounds
CN102742606A (zh) 一株银杏内生球毛壳菌防治植物病原真菌的应用
Chen et al. Control of plant diseases with secondary metabolite of Clitocybe nuda
US8592344B2 (en) Pesticidal compositions comprising 4,5-dihydroxyindan-1-one
Meenambiga et al. Endophytic Fungi, A Novel source in the treatment of Oral infections

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 17900306

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 17900306

Country of ref document: EP

Kind code of ref document: A1