CN115590023B - Application of decadienoic acid in preventing and treating phytophthora capsici of plants - Google Patents
Application of decadienoic acid in preventing and treating phytophthora capsici of plants Download PDFInfo
- Publication number
- CN115590023B CN115590023B CN202210404368.9A CN202210404368A CN115590023B CN 115590023 B CN115590023 B CN 115590023B CN 202210404368 A CN202210404368 A CN 202210404368A CN 115590023 B CN115590023 B CN 115590023B
- Authority
- CN
- China
- Prior art keywords
- decadienoic acid
- acid
- decadienoic
- water
- ethyl acetate
- Prior art date
- Legal status (The legal status 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 status listed.)
- Active
Links
- YKHVVNDSWHSBPA-BLHCBFLLSA-N (2E,4E)-deca-2,4-dienoic acid Chemical compound CCCCC\C=C\C=C\C(O)=O YKHVVNDSWHSBPA-BLHCBFLLSA-N 0.000 title claims abstract description 107
- XDQQQSFYCSYSCP-UHFFFAOYSA-N kaempferol 3-O-beta-D-glucopyranosyl(1-3)-alpha-L-rhamnopyranosyl(1-6)-beta-D-glucopyranoside Natural products CC=CCCC=CCCC(O)=O XDQQQSFYCSYSCP-UHFFFAOYSA-N 0.000 title claims abstract description 105
- 241000233616 Phytophthora capsici Species 0.000 title claims abstract description 18
- YKHVVNDSWHSBPA-ANYPYVPJSA-N (2E)-deca-2,4-dienoic acid Chemical compound CCCCCC=C\C=C\C(O)=O YKHVVNDSWHSBPA-ANYPYVPJSA-N 0.000 claims abstract description 36
- 238000000034 method Methods 0.000 claims abstract description 33
- 238000000855 fermentation Methods 0.000 claims abstract description 32
- 244000063299 Bacillus subtilis Species 0.000 claims abstract description 21
- 235000014469 Bacillus subtilis Nutrition 0.000 claims abstract description 21
- 238000000746 purification Methods 0.000 claims abstract description 20
- 241001460073 Trichoderma asperellum Species 0.000 claims abstract description 19
- 230000004151 fermentation Effects 0.000 claims abstract description 16
- 238000007254 oxidation reaction Methods 0.000 claims abstract description 8
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 claims description 111
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 62
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 48
- 239000007788 liquid Substances 0.000 claims description 42
- 230000001580 bacterial effect Effects 0.000 claims description 41
- 239000000284 extract Substances 0.000 claims description 30
- 239000001963 growth medium Substances 0.000 claims description 30
- 239000012046 mixed solvent Substances 0.000 claims description 24
- 238000004128 high performance liquid chromatography Methods 0.000 claims description 20
- YMWUJEATGCHHMB-UHFFFAOYSA-N Dichloromethane Chemical compound ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 claims description 18
- 239000003480 eluent Substances 0.000 claims description 18
- 238000010828 elution Methods 0.000 claims description 18
- 239000012043 crude product Substances 0.000 claims description 17
- 238000000926 separation method Methods 0.000 claims description 17
- 239000000741 silica gel Substances 0.000 claims description 15
- 229910002027 silica gel Inorganic materials 0.000 claims description 15
- DTQVDTLACAAQTR-UHFFFAOYSA-N Trifluoroacetic acid Chemical compound OC(=O)C(F)(F)F DTQVDTLACAAQTR-UHFFFAOYSA-N 0.000 claims description 14
- 238000002360 preparation method Methods 0.000 claims description 13
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 claims description 12
- WGLUMOCWFMKWIL-UHFFFAOYSA-N dichloromethane;methanol Chemical compound OC.ClCCl WGLUMOCWFMKWIL-UHFFFAOYSA-N 0.000 claims description 12
- 238000005406 washing Methods 0.000 claims description 12
- 238000011218 seed culture Methods 0.000 claims description 11
- 238000004440 column chromatography Methods 0.000 claims description 10
- 238000002156 mixing Methods 0.000 claims description 10
- JZQKTMZYLHNFPL-BLHCBFLLSA-N (2E,4E)-deca-2,4-dienal Chemical compound CCCCC\C=C\C=C\C=O JZQKTMZYLHNFPL-BLHCBFLLSA-N 0.000 claims description 9
- 238000001914 filtration Methods 0.000 claims description 9
- GBMDVOWEEQVZKZ-UHFFFAOYSA-N methanol;hydrate Chemical compound O.OC GBMDVOWEEQVZKZ-UHFFFAOYSA-N 0.000 claims description 9
- 239000001965 potato dextrose agar Substances 0.000 claims description 9
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical class [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 8
- 241001052560 Thallis Species 0.000 claims description 8
- 238000013375 chromatographic separation Methods 0.000 claims description 8
- 239000003810 Jones reagent Substances 0.000 claims description 7
- 238000012258 culturing Methods 0.000 claims description 6
- 239000000499 gel Substances 0.000 claims description 6
- 238000010898 silica gel chromatography Methods 0.000 claims description 5
- SRCZQMGIVIYBBJ-UHFFFAOYSA-N ethoxyethane;ethyl acetate Chemical compound CCOCC.CCOC(C)=O SRCZQMGIVIYBBJ-UHFFFAOYSA-N 0.000 claims description 4
- 239000003960 organic solvent Substances 0.000 claims description 4
- 239000003208 petroleum Substances 0.000 claims description 4
- PQLVXDKIJBQVDF-UHFFFAOYSA-N acetic acid;hydrate Chemical compound O.CC(O)=O PQLVXDKIJBQVDF-UHFFFAOYSA-N 0.000 claims description 3
- 239000012295 chemical reaction liquid Substances 0.000 claims description 3
- 238000001704 evaporation Methods 0.000 claims description 3
- 230000001105 regulatory effect Effects 0.000 claims description 3
- 230000000694 effects Effects 0.000 abstract description 54
- 238000011282 treatment Methods 0.000 abstract description 38
- 238000000605 extraction Methods 0.000 abstract description 13
- 230000000844 anti-bacterial effect Effects 0.000 abstract description 10
- 238000003786 synthesis reaction Methods 0.000 abstract description 7
- 239000002253 acid Substances 0.000 abstract description 6
- 241000233622 Phytophthora infestans Species 0.000 abstract description 4
- 230000003647 oxidation Effects 0.000 abstract description 3
- 230000002265 prevention Effects 0.000 abstract description 2
- 241000233647 Phytophthora nicotianae var. parasitica Species 0.000 description 49
- 239000012071 phase Substances 0.000 description 39
- 239000000243 solution Substances 0.000 description 34
- KRKNYBCHXYNGOX-UHFFFAOYSA-N citric acid Natural products OC(=O)CC(O)(C(O)=O)CC(O)=O KRKNYBCHXYNGOX-UHFFFAOYSA-N 0.000 description 22
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 21
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical group CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 description 18
- 210000004027 cell Anatomy 0.000 description 17
- MHAJPDPJQMAIIY-UHFFFAOYSA-N Hydrogen peroxide Chemical compound OO MHAJPDPJQMAIIY-UHFFFAOYSA-N 0.000 description 16
- ZKHQWZAMYRWXGA-KQYNXXCUSA-J ATP(4-) Chemical compound C1=NC=2C(N)=NC=NC=2N1[C@@H]1O[C@H](COP([O-])(=O)OP([O-])(=O)OP([O-])([O-])=O)[C@@H](O)[C@H]1O ZKHQWZAMYRWXGA-KQYNXXCUSA-J 0.000 description 14
- ZKHQWZAMYRWXGA-UHFFFAOYSA-N Adenosine triphosphate Natural products C1=NC=2C(N)=NC=NC=2N1C1OC(COP(O)(=O)OP(O)(=O)OP(O)(O)=O)C(O)C1O ZKHQWZAMYRWXGA-UHFFFAOYSA-N 0.000 description 14
- 239000002609 medium Substances 0.000 description 13
- 241000208125 Nicotiana Species 0.000 description 12
- 235000002637 Nicotiana tabacum Nutrition 0.000 description 12
- 102000019197 Superoxide Dismutase Human genes 0.000 description 11
- 108010012715 Superoxide dismutase Proteins 0.000 description 11
- 238000012360 testing method Methods 0.000 description 11
- 241000196324 Embryophyta Species 0.000 description 10
- WSMYVTOQOOLQHP-UHFFFAOYSA-N Malondialdehyde Chemical compound O=CCC=O WSMYVTOQOOLQHP-UHFFFAOYSA-N 0.000 description 9
- 201000010099 disease Diseases 0.000 description 9
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 9
- 229940118019 malondialdehyde Drugs 0.000 description 9
- 239000003963 antioxidant agent Substances 0.000 description 8
- 230000003078 antioxidant effect Effects 0.000 description 8
- 210000002421 cell wall Anatomy 0.000 description 8
- 230000001276 controlling effect Effects 0.000 description 8
- 239000000203 mixture Substances 0.000 description 8
- 239000001301 oxygen Substances 0.000 description 8
- 229910052760 oxygen Inorganic materials 0.000 description 8
- 239000002689 soil Substances 0.000 description 8
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 7
- 210000000170 cell membrane Anatomy 0.000 description 7
- 239000000047 product Substances 0.000 description 7
- 230000001681 protective effect Effects 0.000 description 7
- 102000016938 Catalase Human genes 0.000 description 6
- 108010053835 Catalase Proteins 0.000 description 6
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 6
- 238000002835 absorbance Methods 0.000 description 6
- 238000001514 detection method Methods 0.000 description 6
- 239000012153 distilled water Substances 0.000 description 6
- 102000039446 nucleic acids Human genes 0.000 description 6
- 108020004707 nucleic acids Proteins 0.000 description 6
- 150000007523 nucleic acids Chemical class 0.000 description 6
- 239000003642 reactive oxygen metabolite Substances 0.000 description 6
- 239000006228 supernatant Substances 0.000 description 6
- PXRKCOCTEMYUEG-UHFFFAOYSA-N 5-aminoisoindole-1,3-dione Chemical compound NC1=CC=C2C(=O)NC(=O)C2=C1 PXRKCOCTEMYUEG-UHFFFAOYSA-N 0.000 description 5
- 102000004190 Enzymes Human genes 0.000 description 5
- 108090000790 Enzymes Proteins 0.000 description 5
- 241000233614 Phytophthora Species 0.000 description 5
- 238000010306 acid treatment Methods 0.000 description 5
- 230000006378 damage Effects 0.000 description 5
- 230000002829 reductive effect Effects 0.000 description 5
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 4
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 4
- 244000061456 Solanum tuberosum Species 0.000 description 4
- 235000002595 Solanum tuberosum Nutrition 0.000 description 4
- 238000009835 boiling Methods 0.000 description 4
- 239000000872 buffer Substances 0.000 description 4
- 230000003247 decreasing effect Effects 0.000 description 4
- 239000008103 glucose Substances 0.000 description 4
- 239000012528 membrane Substances 0.000 description 4
- 210000003463 organelle Anatomy 0.000 description 4
- 239000000843 powder Substances 0.000 description 4
- 239000008223 sterile water Substances 0.000 description 4
- 239000003643 water by type Substances 0.000 description 4
- QAOWNCQODCNURD-UHFFFAOYSA-N Sulfuric acid Chemical compound OS(O)(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-N 0.000 description 3
- 235000013339 cereals Nutrition 0.000 description 3
- 230000008859 change Effects 0.000 description 3
- 239000013043 chemical agent Substances 0.000 description 3
- 238000006243 chemical reaction Methods 0.000 description 3
- 239000003153 chemical reaction reagent Substances 0.000 description 3
- 230000000875 corresponding effect Effects 0.000 description 3
- 238000002474 experimental method Methods 0.000 description 3
- 230000002401 inhibitory effect Effects 0.000 description 3
- 230000005764 inhibitory process Effects 0.000 description 3
- 150000002632 lipids Chemical class 0.000 description 3
- 238000004519 manufacturing process Methods 0.000 description 3
- 210000003470 mitochondria Anatomy 0.000 description 3
- 230000035699 permeability Effects 0.000 description 3
- 102000004169 proteins and genes Human genes 0.000 description 3
- 108090000623 proteins and genes Proteins 0.000 description 3
- 230000009467 reduction Effects 0.000 description 3
- 239000000725 suspension Substances 0.000 description 3
- YKHVVNDSWHSBPA-JPDBVBESSA-N (2z,4z)-deca-2,4-dienoic acid Chemical compound CCCCC\C=C/C=C\C(O)=O YKHVVNDSWHSBPA-JPDBVBESSA-N 0.000 description 2
- XDFNWJDGWJVGGN-UHFFFAOYSA-N 2-(2,7-dichloro-3,6-dihydroxy-9h-xanthen-9-yl)benzoic acid Chemical compound OC(=O)C1=CC=CC=C1C1C2=CC(Cl)=C(O)C=C2OC2=CC(O)=C(Cl)C=C21 XDFNWJDGWJVGGN-UHFFFAOYSA-N 0.000 description 2
- 229920001817 Agar Polymers 0.000 description 2
- 108010035563 Chloramphenicol O-acetyltransferase Proteins 0.000 description 2
- 240000008067 Cucumis sativus Species 0.000 description 2
- 235000010799 Cucumis sativus var sativus Nutrition 0.000 description 2
- CUKSFECWKQBVED-INIZCTEOSA-N Decursin Chemical compound C1=CC(=O)OC2=C1C=C1C[C@H](OC(=O)C=C(C)C)C(C)(C)OC1=C2 CUKSFECWKQBVED-INIZCTEOSA-N 0.000 description 2
- CUKSFECWKQBVED-UHFFFAOYSA-N Grandivittin Natural products C1=CC(=O)OC2=C1C=C1CC(OC(=O)C=C(C)C)C(C)(C)OC1=C2 CUKSFECWKQBVED-UHFFFAOYSA-N 0.000 description 2
- 206010061218 Inflammation Diseases 0.000 description 2
- 239000001888 Peptone Substances 0.000 description 2
- 108010080698 Peptones Proteins 0.000 description 2
- 244000062793 Sorghum vulgare Species 0.000 description 2
- 241000223259 Trichoderma Species 0.000 description 2
- 239000008272 agar Substances 0.000 description 2
- 235000015278 beef Nutrition 0.000 description 2
- 230000009286 beneficial effect Effects 0.000 description 2
- 230000005540 biological transmission Effects 0.000 description 2
- 239000007853 buffer solution Substances 0.000 description 2
- 229940117975 chromium trioxide Drugs 0.000 description 2
- WGLPBDUCMAPZCE-UHFFFAOYSA-N chromium trioxide Inorganic materials O=[Cr](=O)=O WGLPBDUCMAPZCE-UHFFFAOYSA-N 0.000 description 2
- GAMDZJFZMJECOS-UHFFFAOYSA-N chromium(6+);oxygen(2-) Chemical compound [O-2].[O-2].[O-2].[Cr+6] GAMDZJFZMJECOS-UHFFFAOYSA-N 0.000 description 2
- 150000001875 compounds Chemical class 0.000 description 2
- 230000002596 correlated effect Effects 0.000 description 2
- JXZWWIMXTVJNSF-UHFFFAOYSA-N decursin Natural products CC(=CC(=O)OC1Oc2cc3OC(=O)C=Cc3cc2CC1(C)C)C JXZWWIMXTVJNSF-UHFFFAOYSA-N 0.000 description 2
- 239000008367 deionised water Substances 0.000 description 2
- 229910021641 deionized water Inorganic materials 0.000 description 2
- 230000001419 dependent effect Effects 0.000 description 2
- 238000010586 diagram Methods 0.000 description 2
- 238000007865 diluting Methods 0.000 description 2
- 230000006806 disease prevention Effects 0.000 description 2
- 239000003814 drug Substances 0.000 description 2
- 239000012535 impurity Substances 0.000 description 2
- 230000004054 inflammatory process Effects 0.000 description 2
- 230000003859 lipid peroxidation Effects 0.000 description 2
- 235000019713 millet Nutrition 0.000 description 2
- 235000015097 nutrients Nutrition 0.000 description 2
- 235000019319 peptone Nutrition 0.000 description 2
- 238000004321 preservation Methods 0.000 description 2
- 230000002000 scavenging effect Effects 0.000 description 2
- 210000000582 semen Anatomy 0.000 description 2
- 239000011780 sodium chloride Substances 0.000 description 2
- 159000000000 sodium salts Chemical class 0.000 description 2
- 239000002904 solvent Substances 0.000 description 2
- 238000004809 thin layer chromatography Methods 0.000 description 2
- 230000004102 tricarboxylic acid cycle Effects 0.000 description 2
- 238000002137 ultrasound extraction Methods 0.000 description 2
- 230000001018 virulence Effects 0.000 description 2
- YKHVVNDSWHSBPA-MUIOLIGRSA-N (2z,4e)-deca-2,4-dienoic acid Chemical compound CCCCC\C=C\C=C/C(O)=O YKHVVNDSWHSBPA-MUIOLIGRSA-N 0.000 description 1
- TUSDEZXZIZRFGC-UHFFFAOYSA-N 1-O-galloyl-3,6-(R)-HHDP-beta-D-glucose Natural products OC1C(O2)COC(=O)C3=CC(O)=C(O)C(O)=C3C3=C(O)C(O)=C(O)C=C3C(=O)OC1C(O)C2OC(=O)C1=CC(O)=C(O)C(O)=C1 TUSDEZXZIZRFGC-UHFFFAOYSA-N 0.000 description 1
- VFNKZQNIXUFLBC-UHFFFAOYSA-N 2',7'-dichlorofluorescein Chemical compound O1C(=O)C2=CC=CC=C2C21C1=CC(Cl)=C(O)C=C1OC1=C2C=C(Cl)C(O)=C1 VFNKZQNIXUFLBC-UHFFFAOYSA-N 0.000 description 1
- 230000002407 ATP formation Effects 0.000 description 1
- 238000002970 ATP quantitation Methods 0.000 description 1
- 241000894006 Bacteria Species 0.000 description 1
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 description 1
- MYMOFIZGZYHOMD-UHFFFAOYSA-N Dioxygen Chemical compound O=O MYMOFIZGZYHOMD-UHFFFAOYSA-N 0.000 description 1
- 108090000371 Esterases Proteins 0.000 description 1
- 239000001263 FEMA 3042 Substances 0.000 description 1
- 241000720950 Gluta Species 0.000 description 1
- 239000005807 Metalaxyl Substances 0.000 description 1
- LRBQNJMCXXYXIU-PPKXGCFTSA-N Penta-digallate-beta-D-glucose Natural products OC1=C(O)C(O)=CC(C(=O)OC=2C(=C(O)C=C(C=2)C(=O)OC[C@@H]2[C@H]([C@H](OC(=O)C=3C=C(OC(=O)C=4C=C(O)C(O)=C(O)C=4)C(O)=C(O)C=3)[C@@H](OC(=O)C=3C=C(OC(=O)C=4C=C(O)C(O)=C(O)C=4)C(O)=C(O)C=3)[C@H](OC(=O)C=3C=C(OC(=O)C=4C=C(O)C(O)=C(O)C=4)C(O)=C(O)C=3)O2)OC(=O)C=2C=C(OC(=O)C=3C=C(O)C(O)=C(O)C=3)C(O)=C(O)C=2)O)=C1 LRBQNJMCXXYXIU-PPKXGCFTSA-N 0.000 description 1
- 229920005654 Sephadex Polymers 0.000 description 1
- 239000012507 Sephadex™ Substances 0.000 description 1
- 241000208292 Solanaceae Species 0.000 description 1
- YVNQAIFQFWTPLQ-UHFFFAOYSA-O [4-[[4-(4-ethoxyanilino)phenyl]-[4-[ethyl-[(3-sulfophenyl)methyl]amino]-2-methylphenyl]methylidene]-3-methylcyclohexa-2,5-dien-1-ylidene]-ethyl-[(3-sulfophenyl)methyl]azanium Chemical compound C1=CC(OCC)=CC=C1NC1=CC=C(C(=C2C(=CC(C=C2)=[N+](CC)CC=2C=C(C=CC=2)S(O)(=O)=O)C)C=2C(=CC(=CC=2)N(CC)CC=2C=C(C=CC=2)S(O)(=O)=O)C)C=C1 YVNQAIFQFWTPLQ-UHFFFAOYSA-O 0.000 description 1
- 238000009825 accumulation Methods 0.000 description 1
- 230000006518 acidic stress Effects 0.000 description 1
- 230000009471 action Effects 0.000 description 1
- 230000004913 activation Effects 0.000 description 1
- 230000006978 adaptation Effects 0.000 description 1
- 238000011276 addition treatment Methods 0.000 description 1
- -1 aliphatic diene compound Chemical class 0.000 description 1
- 239000003513 alkali Substances 0.000 description 1
- 239000008346 aqueous phase Substances 0.000 description 1
- 239000007864 aqueous solution Substances 0.000 description 1
- 230000000853 biopesticidal effect Effects 0.000 description 1
- 239000006227 byproduct Substances 0.000 description 1
- 230000030833 cell death Effects 0.000 description 1
- 230000003833 cell viability Effects 0.000 description 1
- 230000001413 cellular effect Effects 0.000 description 1
- 238000011097 chromatography purification Methods 0.000 description 1
- 239000012141 concentrate Substances 0.000 description 1
- IQFVPQOLBLOTPF-HKXUKFGYSA-L congo red Chemical compound [Na+].[Na+].C1=CC=CC2=C(N)C(/N=N/C3=CC=C(C=C3)C3=CC=C(C=C3)/N=N/C3=C(C4=CC=CC=C4C(=C3)S([O-])(=O)=O)N)=CC(S([O-])(=O)=O)=C21 IQFVPQOLBLOTPF-HKXUKFGYSA-L 0.000 description 1
- 238000001816 cooling Methods 0.000 description 1
- 239000013078 crystal Substances 0.000 description 1
- 210000000805 cytoplasm Anatomy 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 230000004069 differentiation Effects 0.000 description 1
- 230000004064 dysfunction Effects 0.000 description 1
- 239000000706 filtrate Substances 0.000 description 1
- 244000053095 fungal pathogen Species 0.000 description 1
- 239000011521 glass Substances 0.000 description 1
- 238000001727 in vivo Methods 0.000 description 1
- 230000002779 inactivation Effects 0.000 description 1
- 238000002347 injection Methods 0.000 description 1
- 239000007924 injection Substances 0.000 description 1
- 230000003834 intracellular effect Effects 0.000 description 1
- 238000003973 irrigation Methods 0.000 description 1
- 230000002262 irrigation Effects 0.000 description 1
- 230000031700 light absorption Effects 0.000 description 1
- 238000009630 liquid culture Methods 0.000 description 1
- 230000007774 longterm Effects 0.000 description 1
- 231100000053 low toxicity Toxicity 0.000 description 1
- 239000011159 matrix material Substances 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 230000010534 mechanism of action Effects 0.000 description 1
- 238000002844 melting Methods 0.000 description 1
- 230000008018 melting Effects 0.000 description 1
- 230000002503 metabolic effect Effects 0.000 description 1
- ZQEIXNIJLIKNTD-UHFFFAOYSA-N methyl N-(2,6-dimethylphenyl)-N-(methoxyacetyl)alaninate Chemical compound COCC(=O)N(C(C)C(=O)OC)C1=C(C)C=CC=C1C ZQEIXNIJLIKNTD-UHFFFAOYSA-N 0.000 description 1
- 230000000813 microbial effect Effects 0.000 description 1
- 244000005700 microbiome Species 0.000 description 1
- 238000001000 micrograph Methods 0.000 description 1
- 230000008811 mitochondrial respiratory chain Effects 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 239000005416 organic matter Substances 0.000 description 1
- 230000004792 oxidative damage Effects 0.000 description 1
- 230000001717 pathogenic effect Effects 0.000 description 1
- 238000005502 peroxidation Methods 0.000 description 1
- 239000013641 positive control Substances 0.000 description 1
- 238000004382 potting Methods 0.000 description 1
- 230000002035 prolonged effect Effects 0.000 description 1
- XJMOSONTPMZWPB-UHFFFAOYSA-M propidium iodide Chemical compound [I-].[I-].C12=CC(N)=CC=C2C2=CC=C(N)C=C2[N+](CCC[N+](C)(CC)CC)=C1C1=CC=CC=C1 XJMOSONTPMZWPB-UHFFFAOYSA-M 0.000 description 1
- 238000012342 propidium iodide staining Methods 0.000 description 1
- 239000012460 protein solution Substances 0.000 description 1
- 239000002516 radical scavenger Substances 0.000 description 1
- 239000011541 reaction mixture Substances 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 230000000630 rising effect Effects 0.000 description 1
- 238000004626 scanning electron microscopy Methods 0.000 description 1
- 239000000779 smoke Substances 0.000 description 1
- 238000002791 soaking Methods 0.000 description 1
- 238000010186 staining Methods 0.000 description 1
- 239000012192 staining solution Substances 0.000 description 1
- 230000001954 sterilising effect Effects 0.000 description 1
- 238000003756 stirring Methods 0.000 description 1
- 230000001502 supplementing effect Effects 0.000 description 1
- 230000004083 survival effect Effects 0.000 description 1
- LRBQNJMCXXYXIU-NRMVVENXSA-N tannic acid Chemical compound OC1=C(O)C(O)=CC(C(=O)OC=2C(=C(O)C=C(C=2)C(=O)OC[C@@H]2[C@H]([C@H](OC(=O)C=3C=C(OC(=O)C=4C=C(O)C(O)=C(O)C=4)C(O)=C(O)C=3)[C@@H](OC(=O)C=3C=C(OC(=O)C=4C=C(O)C(O)=C(O)C=4)C(O)=C(O)C=3)[C@@H](OC(=O)C=3C=C(OC(=O)C=4C=C(O)C(O)=C(O)C=4)C(O)=C(O)C=3)O2)OC(=O)C=2C=C(OC(=O)C=3C=C(O)C(O)=C(O)C=3)C(O)=C(O)C=2)O)=C1 LRBQNJMCXXYXIU-NRMVVENXSA-N 0.000 description 1
- 229940033123 tannic acid Drugs 0.000 description 1
- 235000015523 tannic acid Nutrition 0.000 description 1
- 229920002258 tannic acid Polymers 0.000 description 1
- 230000001988 toxicity Effects 0.000 description 1
- 231100000419 toxicity Toxicity 0.000 description 1
- 238000004627 transmission electron microscopy Methods 0.000 description 1
- 241000712461 unidentified influenza virus Species 0.000 description 1
- 235000021122 unsaturated fatty acids Nutrition 0.000 description 1
- 150000004670 unsaturated fatty acids Chemical class 0.000 description 1
- 210000003934 vacuole Anatomy 0.000 description 1
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
- A01N37/00—Biocides, pest repellants or attractants, or plant growth regulators containing organic compounds containing a carbon atom having three bonds to hetero atoms with at the most two bonds to halogen, e.g. carboxylic acids
- A01N37/02—Saturated carboxylic acids or thio analogues thereof; Derivatives thereof
- A01N37/04—Saturated carboxylic acids or thio analogues thereof; Derivatives thereof polybasic
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C51/00—Preparation of carboxylic acids or their salts, halides or anhydrides
- C07C51/42—Separation; Purification; Stabilisation; Use of additives
-
- 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
- C12P39/00—Processes involving microorganisms of different genera in the same process, simultaneously
-
- 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
- C12P7/40—Preparation of oxygen-containing organic compounds containing a carboxyl group including Peroxycarboxylic acids
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07B—GENERAL METHODS OF ORGANIC CHEMISTRY; APPARATUS THEREFOR
- C07B2200/00—Indexing scheme relating to specific properties of organic compounds
- C07B2200/09—Geometrical isomers
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Landscapes
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Wood Science & Technology (AREA)
- Zoology (AREA)
- Health & Medical Sciences (AREA)
- General Health & Medical Sciences (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Microbiology (AREA)
- Genetics & Genomics (AREA)
- General Engineering & Computer Science (AREA)
- Biotechnology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Biochemistry (AREA)
- Pest Control & Pesticides (AREA)
- Dentistry (AREA)
- Agronomy & Crop Science (AREA)
- Plant Pathology (AREA)
- Environmental Sciences (AREA)
- Oil, Petroleum & Natural Gas (AREA)
- Organic Low-Molecular-Weight Compounds And Preparation Thereof (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
The invention relates to the technical field of phytophthora capsici control, and provides application of decadienoic acid in controlling phytophthora capsici. The invention discovers the antibacterial activity of the decadienoic acid on the phytophthora infestans for the first time, and the application of the decadienoic acid in preventing and controlling the phytophthora infestans has good prevention and control effect and wide prospect; furthermore, the invention also provides two methods for preparing the decadienoic acid, which comprise a biological fermentation extraction method and a chemical synthesis method, wherein the biological fermentation extraction method is to culture trichoderma asperellum and bacillus subtilis together, and the decadienoic acid (including cis-decadienoic acid and trans-decadienoic acid) is separated and extracted from the obtained co-fermentation product, so that the method is environment-friendly, safe and efficient; the chemical synthesis method is to obtain the decdienoic acid through the oxidation of decdienal, and then obtain the cis-decdienoic acid and the trans-decdienoic acid through post-treatment and purification.
Description
Technical Field
The invention relates to the technical field of phytophthora capsici control, in particular to application of decadienoic acid in controlling phytophthora capsici.
Background
Phytophthora capsici is soil-borne pathogenic fungi which seriously damage crop quality, can infect various hosts such as cucumber, potato, solanaceae and the like, is influenced by factors such as variety replacement, cultivation system change, pathogenic variation differentiation, climate condition change and the like in recent years, and has obvious rising trend of disease occurrence, and is prominently represented by serious disease damage and enlarged disease area.
At present, the control method of phytophthora capsici is mainly based on chemical agents and disease-resistant varieties, but the chemical agents are only effective on individual special diseases at present, and long-term use of the chemical agents can cause the quality reduction of crops, cause the proportion imbalance of beneficial microorganisms in soil hardening, organic matter reduction and rhizosphere, further cause the destruction of a soil microbial ecological system and cause the inundation of soil-borne diseases. Therefore, the development of novel high-efficiency low-toxicity biopesticides is an urgent need for production and scientific research.
Decadienoic acid is a common aliphatic diene compound, has an effect of relieving inflammation of some human bodies, and for example, the application of (2Z, 4E) -2, 4-decadienoic acid in preparing medicaments for treating inflammation caused by influenza viruses is disclosed in patent CN 111743888A. However, there is no report on the activity of decadienoic acid in controlling phytophthora capsici.
Disclosure of Invention
In view of the above, the invention provides application of the decadienoic acid in controlling phytophthora capsici, and the invention discovers that the decadienoic acid has remarkable antibacterial activity on the phytophthora capsici for the first time, and the application of the decadienoic acid in controlling the phytophthora capsici has good control effect.
In order to achieve the above object, the present invention provides the following technical solutions:
application of decadienoic acid in preventing and treating phytophthora capsici.
Preferably, the decadienoic acid is one or two of trans-decadienoic acid and cis-decadienoic acid.
Preferably, the preparation method of the decadienoic acid comprises the following steps:
(1) Culturing the trichoderma asperellum spores, inoculating the obtained spore cultures into a shaking culture medium for shaking culture to obtain trichoderma asperellum shaking cultures; the culture medium for spore culture is a potato dextrose agar culture medium;
(2) Seed culture is carried out on the bacillus subtilis, and the obtained seed culture is inoculated into a shaking culture medium for shaking culture, so that the bacillus subtilis shaking culture is obtained;
(3) Mixing the bacillus subtilis shaking culture with the trichoderma asperellum shaking culture and then performing co-fermentation to obtain a co-fermentation liquid;
(4) Filtering the co-fermentation liquid to obtain bacterial liquid and bacterial cells, respectively extracting the bacterial liquid and the bacterial cells, and combining the obtained bacterial liquid extract and the bacterial cell extract to obtain a fermentation extract;
(5) Separating from the fermented extract to obtain trans-decadienoic acid and cis-decadienoic acid.
Preferably, the method for extracting the bacterial liquid comprises the following steps: extracting the bacterial liquid with ethyl acetate, and concentrating the ethyl acetate phase to dryness to obtain a bacterial liquid extract;
the method for extracting the thalli comprises the following steps: and ultrasonically extracting the thalli by using a dichloromethane-methanol mixed solvent, concentrating the obtained extracting solution until the extracting solution is dry, dissolving residues by using water to obtain a water phase, extracting the water phase by using ethyl acetate, and concentrating the water phase until the water phase is dry to obtain a thalli extract.
Preferably, the separation method comprises the following steps: subjecting the fermented extract to normal-phase pressure-reducing column chromatography separation, and performing gradient elution by using a petroleum ether-ethyl acetate mixed solvent to obtain 7 components, wherein the components are sequentially marked as A1-A7 according to the outflow sequence of the components;
separating the component A3 by gel column chromatography, eluting with dichloromethane-methanol mixed solvent to obtain 4 components, and sequentially marking as A3-1-A3-4 according to the outflow sequence of the components;
And (3) purifying the component A3-2 by high performance liquid chromatography to obtain trans-decadienoic acid and cis-decadienoic acid.
Preferably, the gradient elution is divided into 5 gradients, and the volume fraction of ethyl acetate in each gradient eluent is 5%, 10%, 30%, 50% and 100% in sequence.
Preferably, the volume ratio of the dichloromethane to the methanol in the dichloromethane-methanol mixed solvent is 1:1;
the mobile phase used for the high performance liquid chromatography purification is a mixed solvent of methanol, water and trifluoroacetic acid, the volume of the trifluoroacetic acid is 0.1% of the water volume, and the volume fraction of the methanol in the mixed solvent is 75%.
Preferably, the preparation method of the decadienoic acid comprises the following steps: mixing (2E, 4E) -2, 4-decadienal, a Jones reagent and an organic solvent for oxidation reaction, and sequentially filtering, washing and evaporating the obtained reaction liquid to obtain a crude product;
and (3) separating and purifying the crude product to obtain trans-decadienoic acid and cis-decadienoic acid.
Preferably, the separation and purification method comprises the following steps: dissolving the crude product in ethyl acetate to obtain a crude product solution; washing the crude product solution with water, regulating the pH value to 8-9, and extracting with water to obtain a water phase; adjusting the pH value of the water phase to 1-2, extracting with ethyl acetate to obtain an ethyl acetate phase, washing the ethyl acetate phase with saturated sodium chloride solution, and concentrating to obtain a concentrated residue; and sequentially carrying out silica gel column chromatographic separation and high performance liquid chromatography purification on the concentrated residues to obtain trans-decadienoic acid and cis-decadienoic acid.
Preferably, the silica gel column used in the silica gel column chromatographic separation is a trans-silica gel column, the eluent used in the silica gel column chromatographic separation is a methanol-water mixed solvent, the elution mode is gradient elution, the eluent is divided into 7 gradients, the volume fractions of methanol in each gradient eluent are 30%, 40%, 50%, 60%, 70%, 80% and 90% in sequence, and the components eluted by 70% methanol-water are collected for high performance liquid chromatography purification.
The invention provides application of decadienoic acid in preventing and treating phytophthora capsici. The invention discovers the antibacterial activity of the decadienoic acid on the phytophthora infestans for the first time, and the application of the decadienoic acid in preventing and controlling the phytophthora infestans has good prevention and control effect and wide prospect; furthermore, the invention also provides two methods for preparing the decadienoic acid, which comprise a biological fermentation extraction method and a chemical synthesis method, wherein the biological fermentation extraction method is to culture trichoderma asperellum and bacillus subtilis together, and the decadienoic acid (including cis-decadienoic acid and trans-decadienoic acid) is separated and extracted from the obtained co-fermentation product, so that the method is environment-friendly, safe and efficient; the chemical synthesis method is to obtain the decdienoic acid through the oxidation of decdienal, and then obtain the cis-decdienoic acid and the trans-decdienoic acid through post-treatment and purification. The results of the examples show that trans-decadienoic acid is capable of destroying the cell wall and cell membrane of the mycelium of Phytophthora nicotianae, deforming the mycelium, deforming the organelle, inhibiting the growth of Phytophthora nicotianae, and inhibiting the EC of Phytophthora nicotianae 50 The value is 34.59 mug/mL, and the result shows that the decadienoic acid has obvious phytophthora-inhibiting activity.
Drawings
FIG. 1 shows the results of a test for the antibacterial activity of decadienoic acid against Phytophthora nicotianae;
FIG. 2 is a graph showing the antibacterial activity of various concentrations of trans-decadienoic acid against Phytophthora nicotianae (bottom) and a regression curve of toxicity fitted thereto (top);
FIG. 3 shows the results of scanning electron microscopy (top) and transmission electron microscopy (bottom) observations of the effect of decadienoic acid on the morphology of Phytophthora nicotianae filaments;
FIG. 4 is a graph showing the effect of decadienoic acid on the walls of Phytophthora nicotianae cells;
FIG. 5 is a graph showing the effect of decadienoic acid on the permeability of the membrane of Phytophthora nicotianae silk;
FIG. 6 is a graph showing the effect of decadienoic acid on the extracellular relative conductivity of P.nicotianae;
FIG. 7 is a graph of the effect of concentration of decadienoic acid on reducing sugar content (right) fitted to the absorbance value at 540nm (left);
FIG. 8 is a fitted curve of protein content versus absorbance at 595nm (left) and the effect of decadienoic acid concentration on protein content (right);
FIG. 9 is a graph showing the effect of decadienoic acid concentration on nucleic acid content;
FIG. 10 is a graph showing the effect of decadienoic acid on the malondialdehyde content of Phytophthora nicotianae mycelia;
FIG. 11 is a graph showing the effect of decadienoic acid on active oxygen in Phytophthora nicotianae filaments;
FIG. 12 is a graph showing the effect of decadienoic acid on hydrogen peroxide content in Phytophthora nicotianae filaments;
FIG. 13 is a graph showing the effect of decadienoic acid on the activity of the antioxidant protective enzyme SOD in Phytophthora nicotianae silk;
FIG. 14 is the effect of decadienoic acid on the activity of the antioxidant protective enzyme CAT in Phytophthora nicotianae silks;
FIG. 15 is a graph showing the effect of decadienoic acid on ATP content of phytophthora nicotianae mycelium;
FIG. 16 is a graph showing the effect of decadienoic acid on the CA content of Phytophthora nicotianae mycelium;
fig. 17 is a physical diagram of a potted plant test for preventing and controlling tobacco black shank with decadienoic acid.
Detailed Description
The invention provides application of decadienoic acid in preventing and treating phytophthora capsici.
In the invention, the decadienoic acid is trans-decadienoic acid ((2E, 4E) -2, 4-decadienoic acid, the structural formula is shown as formula I) or cis-decadienoic acid ((2Z, 4Z) -2, 4-decadienoic acid, the structural formula is shown as formula II).
The invention is not particularly limited to the particular method of application described, but may be applied according to methods well known to those skilled in the art.
The invention has no special requirement on the types of host plants in the phytophthora capsici, and common plants which are easy to generate the phytophthora capsici in the field can be prevented and treated by adopting the method, such as tobacco, cucumber, potato and the like.
In the present invention, the decadienoic acid (including trans-decadienoic acid and cis-decadienoic acid) may be prepared by a biological fermentation extraction method or a chemical synthesis method, respectively, as described below.
In the present invention, when decursin is prepared by a biological fermentation extraction method, the preparation method of decursin preferably comprises the steps of:
(1) Culturing the trichoderma asperellum spores, inoculating the obtained spore cultures into a shaking culture medium for shaking culture to obtain trichoderma asperellum shaking cultures; the culture medium for spore culture is a potato dextrose agar culture medium;
(2) Seed culture is carried out on the bacillus subtilis, and the obtained seed culture is inoculated into a shaking culture medium for shaking culture, so that the bacillus subtilis shaking culture is obtained;
(3) Mixing the bacillus subtilis shaking culture with the trichoderma asperellum shaking culture and then performing co-fermentation to obtain a co-fermentation liquid;
(4) Filtering the co-fermentation liquid to obtain bacterial liquid and bacterial cells, respectively extracting the bacterial liquid and the bacterial cells, and combining the obtained bacterial liquid extract and the bacterial cell extract to obtain a fermentation extract;
(5) Separating from the fermented extract to obtain trans-decadienoic acid and cis-decadienoic acid.
The invention cultures the trichoderma asperellum spores, and inoculates the spore cultures obtained into a shaking culture medium for shaking culture, thus obtaining the trichoderma asperellum shaking culture. In the invention, the trichoderma asperellum is trichoderma asperellum HG1, and the preservation number of the trichoderma asperellum HG1 is CGMCCNO.19276; the culture medium for spore culture is a potato dextrose agar culture medium, and the potato dextrose agar culture medium comprises the following components: 6g/L of potato soaked powder, 20g/L of glucose, 20g/L of agar and the balance of deionized water; the temperature of the spore culture is preferably 25-28 ℃, and the time is preferably 5-7 days; after the spores are cultivated, the spores are rinsed, preferably with sterile water, and the resulting spore suspension is diluted to 1X 10 with sterile water 5 ～2×10 5 CFU/mL, and inoculating the diluted spore suspension into shaking culture medium. In the present invention, the shaking culture medium used in the step (1) is preferably NB medium, and the volume of the NB medium is preferably 250mL; the NB medium preferably comprises the following components: 10g/L of peptone, 10g/L of sodium chloride and 3g/L of beef powder; the temperature of the shaking culture in the step (1) is preferably 25-28 ℃, the time is preferably 24 hours, and the rotating speed is preferably 150-180 rpm.
The invention carries out seed culture on bacillus subtilis, and the obtained seed culture is inoculated into a shaking culture medium for shaking culture to obtain the bacillus subtilis shaking culture. In the invention, the bacillus subtilis is preferably bacillus subtilis Tpb55, and the preservation number of the bacillus subtilis Tpb55 is CGMCCNO.2843; the culture medium used for the seed culture is preferably an NA medium or an LB medium, and the components of the NA medium and the LB medium are not particularly limited in the present invention, and the above-mentioned culture medium known to those skilled in the art may be used. In the invention, the temperature of the seed culture is preferably 25-28 ℃, the time is preferably 24 hours, and after the seed culture is completed, single colony is selected and inoculated on a shaking culture medium for culture. In the present invention, the shaking culture medium used in the step (2) is preferably NB medium, and the temperature of the shaking culture in the step (2) is preferably 25 to 28℃for 24 hours, and the rotation speed is preferably 150 to 180rpm.
After the bacillus subtilis shaking culture and the trichoderma asperellum shaking culture are obtained, the bacillus subtilis shaking culture and the trichoderma asperellum shaking culture are mixed and then subjected to co-fermentation to obtain a co-fermentation liquid. The invention preferably draws 500 mu L of the bacillus subtilis after the completion of shaking culture and dilutes the bacillus subtilis to 1X 10 6 ～2×10 6 Inoculating the CFU/mL spore bacterial liquid into NB medium which is cultured for 24h of trichoderma; in the present invention, the temperature of the co-fermentation is preferably 25 to 28 ℃, the time is preferably 6d, and the rotation speed is preferably 150 to 180rpm.
After the co-fermentation liquid is obtained, the co-fermentation liquid is filtered to obtain bacterial liquid and bacterial cells, the bacterial liquid and the bacterial cells are respectively extracted, and the obtained bacterial liquid extract and the bacterial cell extract are combined to obtain the fermentation extract. In the present invention, the method for extracting the bacterial liquid preferably comprises: extracting the bacterial liquid with ethyl acetate, and concentrating the ethyl acetate phase to dryness to obtain a bacterial liquid extract; the volume ratio of the bacterial liquid to the ethyl acetate is preferably 1:1.5-2, the extraction times are preferably 2-3 times, and ethyl acetate phases obtained by multiple extraction are combined; the concentration conditions are not particularly limited in the present invention, and the concentration conditions well known to those skilled in the art can be used to concentrate the solution to dryness.
In the present invention, the method for extracting the bacterial cells comprises: and ultrasonically extracting the thalli by using a dichloromethane-methanol mixed solvent, concentrating the obtained extracting solution until the extracting solution is dry, dissolving residues by using water to obtain a water phase, extracting the water phase by using ethyl acetate, and concentrating the water phase until the water phase is dry to obtain a thalli extract. In the invention, the volume ratio of dichloromethane to methanol in the dichloromethane-methanol mixed solvent is preferably 1:1, the ultrasonic extraction time is preferably 24 hours, and the ultrasonic extraction power is preferably 200-300W; the volume ratio of the water phase to the ethyl acetate is preferably 1:2-1:3; the number of extractions is preferably 2 to 3, and the ethyl acetate phases obtained by the multiple extractions are combined and then concentrated to dryness.
After the fermentation extract is obtained, the invention separates from the fermentation extract to obtain trans-decadienoic acid and cis-decadienoic acid. In the present invention, the separation method is preferably:
subjecting the fermented extract to normal-phase pressure-reducing column chromatography separation, and performing gradient elution by using a petroleum ether-ethyl acetate mixed solvent to obtain 7 components, wherein the components are sequentially marked as A1-A7 according to the outflow sequence of the components;
separating the component A3 by gel column chromatography, eluting with dichloromethane-methanol mixed solvent to obtain 4 components, and sequentially marking as A3-1-A3-4 according to the outflow sequence of the components;
and (3) purifying the component A3-2 by high performance liquid chromatography to obtain trans-decadienoic acid and cis-decadienoic acid.
In the invention, the gradient elution in the normal phase pressure reduction column chromatographic separation is divided into 5 gradients, and the volume fraction of ethyl acetate in each gradient eluent is 5%, 10%, 30%, 50% and 100% in sequence; during elution, the eluted fractions were analyzed by thin layer chromatography and the same fractions were combined. The invention adopts a hypha growth rate method to verify the phytophthora nicotianae resistance activity of 7 components obtained by normal phase pressure-reducing column chromatography separation at the concentration of 0.5mg/mL, and the result shows that the antibacterial effect of the component A3 is best, so that the component A3 is adopted for further separation.
In the invention, the gel column type number adopted by the gel column chromatography separation is preferably Sephadex LH-20CC, and the volume ratio of dichloromethane to methanol in the dichloromethane-methanol mixed solvent is 1:1; the flow rate of the dichloromethane-methanol mixed solvent is preferably 2mL/min.
In the invention, the high performance liquid chromatography purification is preferably Waters high performance liquid chromatography purification, the chromatographic column used in the high performance liquid chromatography purification is preferably Waters ODSX-Bridge, and the specification of the chromatographic column is preferably 10X 250mm and 5 μm; the mobile phase used for the high performance liquid chromatography purification is a mixed solvent of methanol, water and trifluoroacetic acid, the volume of the trifluoroacetic acid is preferably 0.1% of the water volume, and the volume fraction of the methanol in the mixed solvent is preferably 75%; the flow rate of the mobile phase is preferably 2mL/min.
In the present invention, when the decadienoic acid is prepared by a chemical synthesis method, the method for preparing the decadienoic acid preferably comprises the steps of:
mixing (2E, 4E) -2, 4-decadienal, a Jones reagent and an organic solvent for oxidation reaction, and sequentially filtering, washing and evaporating the obtained reaction liquid to obtain a crude product;
and (3) separating and purifying the crude product to obtain trans-decadienoic acid and cis-decadienoic acid.
In the invention, the Jones reagent is preferably an aqueous solution prepared from chromium trioxide, sulfuric acid and water, more preferably, 2.67g of chromium trioxide is dissolved in 23mL of concentrated sulfuric acid and then diluted to 100mL with water; the volume ratio of the (2E, 4E) -2, 4-decadienal to the Jones reagent is preferably 2:8.5; the organic solvent is preferably acetone; the volume ratio of (2E, 4E) -2, 4-decadienal to acetone is preferably 2:25.
In the present invention, the temperature of the oxidation reaction is preferably room temperature, and the time is preferably 12 to 18 hours.
In the specific embodiment of the invention, the (2E, 4E) -2, 4-decadienal is preferably added into acetone to obtain (2E, 4E) -2, 4-decadienal solution, then a Jones reagent is dropwise added into the (2E, 4E) -2, 4-decadienal solution at the temperature of 0 ℃, and after the addition, the mixture is stirred and mixed for 10min at the temperature of 0 ℃, and then the mixture is heated to the room temperature for reaction.
After completion of the oxidation reaction, the present invention removes the residue by filtration and then washes with acetone, preferably 3 times.
In the present invention, the method for separating and purifying the crude product preferably comprises: dissolving the crude product in ethyl acetate to obtain a crude product solution; washing the crude product solution with water, regulating the pH value to 8-9, and extracting with water to obtain a water phase; adjusting the pH value of the water phase to 1-2, extracting with ethyl acetate to obtain an ethyl acetate phase, washing the ethyl acetate phase with saturated sodium chloride solution, and concentrating to obtain a concentrated residue; and sequentially carrying out silica gel column chromatographic separation and high performance liquid chromatographic purification on the concentrated residues to obtain trans-decadienoic acid and cis-decadienoic acid, wherein the trans-decadienoic acid is the main product.
In the invention, the reagent used for adjusting the pH value of the crude product after washing is preferably 4mol/L sodium hydroxide solution, and the invention adjusts the pH value to 8-9 to enable decadienoic acid obtained by the reaction to form sodium salt to enter into a water phase, and alkaline impurities are remained in an ethyl acetate phase, thereby realizing the separation of the product and the impurities. In the invention, the reagent used for adjusting the pH value of the water phase is preferably 4mol/L hydrochloric acid, and the pH value of the water phase is adjusted to be 1-2, so that the sodium salt formed by alkali adjustment in the previous step forms acid again, and the product is free from the water phase, thereby being beneficial to the subsequent extraction by ethyl acetate.
In the invention, a silica gel column adopted in the silica gel column chromatographic separation is a trans-silica gel column, an eluent adopted in the silica gel column chromatographic separation is a methanol-water mixed solvent, the elution mode is gradient elution, the eluent is divided into 7 gradients, the volume fraction of methanol in each gradient eluent is 30%, 40%, 50%, 60%, 70%, 80% and 90% in sequence, and the components eluted by 70% methanol-water are collected for high performance liquid chromatography purification; in the specific embodiment of the invention, thin layer chromatography is adopted for detection, and after the product is eluted under one gradient, the next gradient elution is carried out; the specific conditions of the high performance liquid chromatography purification are identical to those of the high performance liquid chromatography purification in the biological fermentation extraction method, and are not repeated here.
The technical solutions of the present invention will be clearly and completely described in the following in connection with the embodiments of the present invention. It will be apparent that the described embodiments are only some, but not all, embodiments of the invention. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
EXAMPLE 1 biological fermentation extraction method for the preparation of decadienoic acid
(1) Culturing Trichoderma asperellum HG1 on Potato Dextrose Agar (PDA) culture medium for 5 days at 25deg.C, washing spores with sterile water, diluting the obtained spore suspension with sterile water to 1×10 5 CFU/mL was inoculated into 250mL of Nutrient Broth (NB) medium (500 mL Erlenmeyer flask), and cultured with shaking at 28℃and 180rpm/min for 24h.
After culturing the bacillus subtilis Tbp55 on the NA culture medium at 28 ℃ for 24 hours, single colonies are inoculated into the NB liquid culture medium and cultured for 12 hours at 28 ℃ and 180 rpm/min. Absorbing 500 mu L and diluting to 1-2X 10 6 The spore bacteria liquid of CFU/mL is inoculated into NB medium which is cultivated for 24h of trichoderma, and the cultivation is continued for 6d under the conditions of 28 ℃ and 180 rpm/min.
Wherein, the Potato Dextrose Agar (PDA) culture medium comprises the following components: 6g/L of potato soaked powder, 20g/L of glucose, 20g/L of agar and the balance of deionized water.
The Nutrient Broth (NB) medium had the following composition: 10g/L peptone, 10g/L sodium chloride and 3g/L beef powder.
(2) After fermentation, the bacterial liquid and the bacterial cells are separated by filtration. Extracting the bacterial liquid with ethyl acetate with volume of 2 times for 2 times, and concentrating to dryness; the thallus adopts dichloromethane: methanol=1: 1 (volume ratio), soaking for 24 hours, concentrating to dryness, dissolving the remainder with water to obtain a water phase, extracting the water phase with ethyl acetate for 2 times, concentrating to dryness, and mixing with the bacterial liquid extract to obtain a fermentation extract.
(3) Separating the fermented extract by normal phase reduced pressure column chromatography, wherein the eluent is petroleum ether-ethyl acetate, the elution mode is gradient elution, and the volume fractions of the ethyl acetate in each gradient eluent are as follows: 5%, 10%, 30%, 50%, 100% of the eluent flow rate was 2mL/min, and 7 components were obtained in total, which were designated A1 to A7 in sequence. The hypha growth rate method is adopted to verify the phytophthora nicotianae resistance activity of each component at the concentration of 0.5mg/mL, wherein the component A3 obtained by eluting at the position of 10% ethyl acetate has the best antibacterial effect. Separating the component A3 by SephadexLH-20CC gel column chromatography, wherein the adopted mobile phase is dichloromethane-methanol, and the volume ratio of dichloromethane to methanol is 1:1, so as to obtain a component A3-2; component A3-2 was purified by Waters high performance liquid chromatography using Waters ODSX-Bridge10 x 250mm,5 μm mobile phase 75% methanol-water (0.1% trifluoroacetic acid) to give cis-and trans-decadienoic acids, respectively.
The nuclear magnetic data of the obtained cis-decadienoic acid are: 1 HNMR(CDCl 3 ,500MHz)δ7.70(1H,t,J＝13.0Hz),6.15(1H,t,J＝13.0Hz),5.87–5.95(1H,m),2.31(1H,q,J＝7.5Hz),1.43(2H,dt,J＝14.0,7.0Hz),1.31–1.34(4H,m),0.89(3H,t,J＝7.0Hz)； 13 CNMR(CDCl 3 ,125MHz)δ143.1(CH),141.8(CH),126.3(CH),120.1(CH),31.4(CH 2 ),29.0(CH 2 ),28.3(CH 2 ),22.5(CH 2 ),14.0(CH 3 ).
the nuclear magnetic data of the obtained trans-decadienoic acid are as follows: 1 HNMR(DMSO-d 6 ,500MHz)δ12.16(s),7.14(1H,dd,J＝15.5,10.0Hz),6.22(1H,dd,J＝15.5,10.0Hz),6.17–6.27(1H,m),5.78(1H,d,J＝15.5Hz),2.13(2H,q,J＝7.0Hz),1.39(2H,dt,J＝14.5,7.5Hz),1.23–1.30(4H,m),0.86(3H,t,J＝7.0Hz)； 13 CNMR(DMSO-d 6 ,125MHz)δ167.7(C),144.6(CH),144.2(CH),128.3(CH),120.1(CH),32.3(CH 2 ),30.9(CH 2 ),27.9(CH 2 ),21.9(CH 2 ),13.9(CH 3 )；ESIMSm/z169.11[M+H] + .
EXAMPLE 2 chemical Synthesis of decadienoic acid
(2E, 4E) -2, 4-decadienal (2 mL,1.0 eq) was added to 25mL of acetone solution, jones reagent (8.5 mL,1.5 eq) was added dropwise at 0deg.C, and the reaction mixture was stirred at 0deg.C for 10min, and then allowed to move to room temperature. After stirring overnight, the solution was filtered to remove the residue and washed with 3×5mL acetone. The filtrate was evaporated to dryness and redissolved with 20mL of ethyl acetate. The organic solution was washed with water (3X 10 mL) and then 4mol/L sodium hydroxide solution was added to the ethyl acetate phase until the pH was 8-9 and extracted with water (10 mL X3). 4M hydrochloric acid was added to the combined aqueous phase until the pH was 1-2. The solution was extracted with ethyl acetate (10 ml×3) and the combined organic extracts were washed with saturated sodium chloride solution (3×20 mL) and then concentrated in vacuo to give a residue. Sequentially performing silica gel column chromatography separation and high performance liquid chromatography purification on the residues to obtain (2E, 4E) -decadienoic acid as pale yellow oil (main product) and (2Z, 4Z) -decadienoic acid (by-product), wherein a silica gel column adopted in the silica gel column chromatography separation is a trans-silica gel column, an eluent adopted in the silica gel column chromatography separation is a methanol-water mixed solvent, the elution mode is gradient elution, the eluent is totally divided into 7 gradients, the volume fractions of methanol in each gradient eluent are sequentially 30%, 40%, 50%, 60%, 70%, 80% and 90%, and 70% of components eluted by methanol-water are collected for high performance liquid chromatography purification, wherein the conditions of the high performance liquid chromatography purification are consistent with those of the embodiment 1.
In the following, trans-decadienoic acid is taken as an example, and the activity against phytophthora nicotianae and the mechanism of action are studied.
EXAMPLE 3 Phytophthora nicotianae resistance Activity of decadienoic acid
Trans-decadienoic acid is melted in Oat Agar (OA) culture medium with a final concentration of 0.5mg/mL (0.5% DMSO is used as a solvent), 0.5% DMSO is used as a control, a phytophthora nicotianae cake is inoculated after uniformly mixing and pouring, colony growth diameter is measured after culturing for 3 days at 28 ℃, and the bacteriostasis rate is calculated, so that the bacteriostasis rate of an experimental group added with the trans-decadienoic acid reaches 100%. FIG. 1 is a graph showing the antibacterial activity of trans-decadienoic acid against Phytophthora nicotianae, wherein CK is a control group and 0.5mg/mLDA is an experimental group.
The inhibition rate of trans-decadienoic acid on phytophthora nicotianae was calculated by melting the plate at final concentrations of 0, 12.5, 25, 50, 100, 200, 400 μg/mL, respectively. Taking the lg value of the concentration as an abscissa and the biological probability statistical value corresponding to the bacteriostasis rate as an ordinate as a virulence regression equation, and obtaining the equation as follows: y=1.9365x+2.0197 (R 2 = 0.9923), wherein: x is the lg value of the concentration of the decadienoic acid, and y is the biological probability statistical value corresponding to the antibacterial rate of the decadienoic acid to the phytophthora nicotianae. Substituting the biological probability statistical value corresponding to 50% into an equation, and calculating to obtain the concentration EC in the inhibition of the decadienoic acid on the phytophthora nicotianae 50 ＝34.59μg/mL。
Fig. 2 is a graph showing the antibacterial activity of various concentrations of trans-decadienoic acid against phytophthora nicotianae (bottom) and a regression curve of virulence obtained by fitting (top).
Example 4 Effect of decadienoic acid on Phytophthora nicotianae physiological index
(1) Hypha sample preparation: 1.5g of phytophthora nicotianae filaments are suspended in PBS buffer solution (0.01 mol/L, pH 7.4), trans-decadienoic acid is added to make the final concentration be 35 mug/mL and 70 mug/mL respectively, 0.5% DMSO is used as a reference, and the mixture is respectively subjected to shaking culture for 4, 8 and 12 hours at 28 ℃ and 180rpm, and the mixture is washed for 3 times by the PBS buffer solution, so that a mycelium sample after decadienoic acid treatment is obtained. Hyphal samples were prepared according to this method in subsequent experiments.
(2) Effects on mycelium morphology
Trans-decadienoic acid is respectively dissolved in Oat Agar (OA) culture medium with final concentration of 35 and 70 mug/mL (0.5% DMSO is used as solvent), 0.5% DMSO is used as control, the mixture is poured into a plate, then a phytophthora nicotianae cake (5 mm) is connected, after the mixture is cultured for 72 hours at 28 ℃, hyphae are scraped off, and the mixture is fixed by using a Gluta fixing solution, and then Scanning Electron Microscope (SEM) and Transmission Electron Microscope (TEM) observation are carried out. The results are shown in FIG. 3, wherein the upper side of FIG. 3 is the result of scanning electron microscope observation, the scale of each figure is 10 μm, the magnification is 5K, the lower side is the result of transmission electron microscope observation, the magnification of the 0 μg/mL test group is 25K, the scale is 2 μm, the magnification of the 35 μg/mL test group is 15K, the scale is 2 μm, the magnification of the 70 μg/mL test group is 30K, and the scale is 1 μm. By observing the scanning electron microscope and the perspective electron microscope image (figure 3) of phytophthora nicotianae, the decylenic acid can be known to significantly influence the ultrastructure of phytophthora nicotianae filaments. The scanning electron microscope finds that the mycelium of the control group is normal in morphology, uniform in thickness, smooth, round and good in growth. The mycelium after decadienoic acid treatment is bent, contracted, collapsed and deformed. And mycelium deformation is more serious with increasing treatment concentration. The observation under the perspective electron microscope shows that all organelles of the control group are regularly arranged, the cell walls are clear, and the diaphragm is obvious. However, there were some changes in the decadienoic acid treatment group, including hyphal deformation, cell wall damage, vacuole size inequality, organelle deformation, etc.
(3) Effects on hyphal cell wall integrity
The mycelium sample after 12h treatment with decadienoic acid is picked and placed on a glass slide, naturally dried at room temperature, treated with 10% tannic acid solution for 1h, washed with distilled water, treated with 5% crystal violet solution for 5min, washed with distilled water, treated with 5% Congo red solution for 2min, washed with distilled water, dried at room temperature, and made into a smear, and the result is shown in FIG. 4. As can be seen from fig. 4, the cell wall structure of the mycelium in the control group is clearly visible, and the cytoplasm is stained light purple. After treatment of mycelia in the treatment group with 35. Mu.g/mL and 70. Mu.g/mL decadienoic acid for 12 hours, the cell wall staining in the mycelia became severely blurred, the whole cell wall could not be seen, and almost all mycelia were stained dark purple. The results indicate that decadienoic acid is able to destroy the cell wall of phytophthora nicotianae mycelium.
(4) Effects on hyphal cell membranes
1) Propidium Iodide (PI) staining
Taking a mycelium sample treated for 12 hours, adding PI staining solution with a final concentration of 40 mug/mL to enable the final concentration to be 40 mug/mL, incubating for 20 minutes in a dark place, observing under a laser confocal microscope, photographing and recording, and obtaining a result shown in figure 5. As can be seen from FIG. 5, after 12h of treatment of the mycelium, the control group showed little fluorescence, whereas the mycelium treated with decadienoic acid showed stronger fluorescence, and the higher the concentration of decadienoic acid, the stronger the fluorescence signal. Indicating that after treatment of phytophthora mycelium with decadienoic acid, the mycelium cell viability is reduced and the cell membrane integrity is destroyed.
2) Determination of relative conductivity of mycelium culture solution
1.5g of mycelium was added to 30mLPBS buffer (0.01 mol/L, pH 7.4), and as a control, 0.5% DMSO was used, trans-decadienoic acid was added to a final concentration of 35, 70. Mu.g/mL, and shaking culture was performed at 28℃and 180rpm, and the conductivity of the culture broth was immediately measured and recorded as L 0 . Taking samples every 4 hours, and measuring the conductivity of the culture solution and marking as L 1 . After the test is completed, the mycelia are killed by boiling water bath for 30min, and the measured conductivity is recorded as L 2 . The relative conductivity was calculated according to the following formula:
wherein: l (L) 0 Conductivity value measured for 0h, L 1 For a certain time conductivity value, L 2 Is the conductivity value measured after a boiling water bath.
The results are shown in FIG. 6. As can be seen from fig. 6, the extracellular relative conductivities of each of the treatment group and the control group gradually increased with increasing concentration of decadienoic acid treatment and prolonged treatment time, and tended to stabilize after 12 h. The relative conductivity of the 35, 70 μg/mL treated group was consistently significantly higher than the control group during the 24h treatment, and the higher the treatment concentration, the greater the relative conductivity. It is therefore speculated that decadienoic acid can disrupt the permeability of cell membranes, allowing leakage of cell contents, which in turn leads to an increase in extracellular relative conductivity.
3) Determination of mycelium content leakage
1.5g of mycelia was added to 30mL of buffer (0.01 mol/L, pH 7.4) containing 0.5% DMSO as a control, and decadienoic acid was added to a final concentration of 35, 70. Mu.g/mL, and cultured at 28℃under shaking at 180 rpm. Samples were taken every 4 hours.
Determination of reducing sugar content: accurately sucking 0, 0.2, 0.4, 0.6, 0.8 and 1.0mL glucose solution (1 mg/mL) respectively, adding 0.75mL LDNS reagent, mixing, and boiling water for 5min. After rapid cooling, distilled water was set to a volume of 10mL. The absorbance at 540nm of the solutions of different concentrations was measured separately and a glucose standard curve was fitted. The content of reducing sugar in 0.5mL of mycelium supernatant was measured by referring to the method for preparing a standard curve.
Protein content determination: accurately sucking 0, 0.2, 0.4, 0.6, 0.8 and 1.0mL protein solution (0.1 mg/mL), supplementing to 1.0mL with distilled water, adding 5mL coomassie brilliant blue G-250 reaction solution, mixing, standing for 5min to determine light absorption value at 595nm, and fitting protein standard curve. The protein content of 0.5mL mycelium supernatant was measured by reference to the method for preparing a standard curve.
Measuring the content of nucleic acid: the content of nucleic acid in the mycelium supernatant was determined at 260 nm.
The results are shown in FIGS. 7 to 9, and FIG. 7 shows a fitted curve of the reducing sugar content and the absorbance at 540nm (left) and the effect of the concentration of decadienoic acid on the reducing sugar content (right); FIG. 8 shows a fitted curve of protein content and absorbance at 595nm (left) and the effect of concentration of decadienoic acid on protein content (right), and FIG. 9 shows the effect of concentration of decadienoic acid on nucleic acid content.
Figures 7-9 show that the content of reducing sugar, soluble protein and nucleic acid in the mycelium culture solution of the treated group is significantly higher than that of the control group within 0-12 h after the treatment of the decadienoic acid, and the more serious the leakage of the cell content is along with the increase of the concentration of the decadienoic acid. After 12h of treatment, the absorbance at 260nm, soluble protein and reducing sugar contents of the 70. Mu.g/mL treatment group nucleic acid were 1.17, 1.07 and 1.01 times that of the control group, respectively. The above results indicate that decadienoic acid can disrupt phytophthora nicotianae silk cell membrane permeability, resulting in leakage of hyphal content, consistent with the above relative conductivity measurements.
4) Determination of the membrane peroxidation index (MDA content) of the cell membrane
Oxygen radicals act on unsaturated fatty acids of lipids to produce peroxidized lipids; the latter gradually breaks down into a complex series of compounds including Malondialdehyde (MDA). The level of lipid oxidation can be detected by detecting the level of MDA.
The method of treating the mycelia was the same as in the above "(1) preparation of mycelia samples". And (3) carrying out sample adding treatment on the mycelium supernatant according to a Malondialdehyde (MDA) content detection kit.
The detection results are shown in FIG. 10. As can be seen from fig. 10, the MDA content in the treated group was always higher than that in the control group, and the higher the decadienoic acid concentration, the higher the MDA content. It can thus be stated that decadienoic acid induces lipid peroxidation of the membrane of phytophthora mycelium, and that the degree of lipid peroxidation is positively correlated with the concentration.
(5) Influence on active oxygen and hydrogen peroxide content of mycelium and activity of antioxidant protective enzyme
1) Reactive Oxygen Species (ROS) level determination
After 2, 7-dihydro-dichloro-fluorescein diethyl ester (DCFH-DA) enters cells, 2, 7-dichloro-hydrogenated-fluorescein (DCFH) which cannot pass through cell membranes is generated through the action of relevant esterase. DCFH binds ROS and can produce autofluorescent 2, 7-Dichlorofluorescein (DCF). Intracellular ROS levels can be reflected by detection of DCF.
The mycelium was prepared and treated in the same manner as in the above "(1) mycelium sample preparation". The treated mycelia were washed 3 times with PBS buffer, DCFH-DA (final concentration: 10. Mu. Mol/L) was added, incubated for 30 minutes in the dark, and after washing with PBS buffer, they were observed by a laser confocal microscope, and the results are shown in FIG. 11. The main endogenous source of reactive oxygen species is the mitochondrial respiratory chain, which plays an important role in cell survival, oxidative damage of cellular compounds, enzyme inactivation and membrane rupture, ultimately leading to cell dysfunction or cell death. DCFH-DA is widely used to detect the production of endogenous reactive oxygen species. As can be seen from fig. 11, the mycelium fluorescence density was significantly increased in the decadienoic acid-treated group, and the higher the decadienoic acid concentration, the stronger the fluorescence signal was detected. Whereas only a few fluorescence were observed in the control group. The results show that decadienoic acid causes the production and accumulation of active oxygen in the phytophthora nicotianae filaments.
2) Hydrogen peroxide (H) 2 O 2 ) Content determination
Hydrogen peroxide is the most common active oxygen molecule in living beings and is also the hub for active oxygen interconversion. The method of treating the mycelia was the same as in the above "(1) preparation of mycelia samples". According to hydrogen peroxide (H) 2 O 2 ) The mycelium supernatant was subjected to sample addition treatment in the content detection kit, and the test results are shown in FIG. 12. As can be seen from FIG. 12, during 12H of treatment, H in the control group 2 O 2 The content is basically stable after being increased. The hydrogen peroxide content in the 35 μg/mL decadienoic acid treated mycelium was always in an elevated state during the treatment, and at 12 the content was at the highest point. The hydrogen peroxide content in the mycelia of the 70. Mu.g/mL treatment group was already at a higher level at 4 hours and was always in a high content state thereafter. Throughout the treatment, H in the treatment group 2 O 2 The content is always obviously higher than that of the control group, thereby indicating that the decadienoic acid stress hypha generates H 2 O 2 And is positively correlated to its concentration.
3) Determination of antioxidant protective enzyme (SOD, CAT) Activity
SOD is a metalloenzyme widely existing in living bodies, is an important oxygen radical scavenger, and plays an important role in biological antioxidant systems. CAT is an important hydrogen peroxide scavenging enzyme and plays an important role in the active oxygen scavenging system.
The mycelium treatment method was the same as in "(1) mycelium sample preparation" above, and the activities of superoxide dismutase (SOD) and Catalase (CAT) in the mycelium supernatant were examined using a kit.
The results are shown in FIGS. 13-14, wherein FIG. 13 shows the effect of decadienoic acid on the activity of antioxidant protective enzyme SOD in Phytophthora nicotianae silk, and FIG. 14 shows the effect of decadienoic acid on the activity of antioxidant protective enzyme CAT in Phytophthora nicotianae silk. As can be seen from FIG. 13, the SOD enzyme activity of the control group was in a trend of decreasing followed by increasing within 4 to 12 hours, while the SOD enzyme activity of the treatment group showed a trend of decreasing all the time. The SOD enzyme activity of the treated group is always lower than that of the control group, and the higher the concentration of the decadienoic acid is, the lower the SOD enzyme activity is, so that the decadienoic acid can inhibit the SOD enzyme activity in mycelium.
As can be seen from FIG. 14, CAT enzyme activities of the control group and the treatment group were increased and then decreased during the treatment for 4 to 12 hours. At each time point, the CAT enzyme activity of the treatment group is always obviously higher than that of the control group, so that the mycelium can be presumed to activate in-vivo antioxidant protective enzyme to act after being stressed by decadienoic acid, and the damage of active oxygen to the mycelium is eliminated.
(6) Effects on Adenosine Triphosphate (ATP) content and Citric Acid (CA) content
Mitochondria are the major organelles that produce Adenosine Triphosphate (ATP), which reflects the energy metabolic state of organisms. Citric acid is the product of the first step of the tricarboxylic acid cycle, which occurs at the mitochondria. Thus, the effect of decadienoic acid on mitochondria can be reflected by measuring the changes in ATP and CA content.
The method of treating the mycelia was the same as in the above "(1) preparation of mycelia samples". The content of Adenosine Triphosphate (ATP) and Citric Acid (CA) in mycelium was detected according to the kit, and the detection results are shown in FIGS. 15-16, wherein FIG. 15 shows the effect of decadienoic acid on the ATP content of phytophthora nicotianae mycelium, and FIG. 16 shows the effect of decadienoic acid on the CA content of phytophthora nicotianae mycelium.
As can be seen from fig. 15, the trend of ATP change in the treated group and the control group was substantially consistent after decadienoic acid treatment, i.e., the ATP content was gradually decreased with the increase of the treatment time and the increase of the treatment concentration. After 12h of treatment, the ATP content of the decadienoic acid-treated groups at 35 μg/mL and 70 μg/mL was reduced by 19.86% and 30.69% compared with the control group, significantly reducing the adenosine triphosphate content in the mycelium, and the higher the decadienoic acid concentration, the lower the ATP content in the mycelium, indicating that decadienoic acid inhibits ATP production by the mycelium.
As can be seen from fig. 16, decadienoic acid reduces the citric acid content in a concentration-dependent manner. The ATP content of the 35 μg/mL and 70 μg/mL decadienoic acid treated groups was reduced by 10.43% and 40% compared to the control group. All these results together indicate that decadienoic acid may inhibit phytophthora nicotianae growth by affecting the TCA cycle.
EXAMPLE 5 potted disease prevention test of decadienoic acid on tobacco black shank
The preparation method of the phytophthora nicotianae cereal comprises the following steps: boiling semen Setariae with distilled water for about 30min until 2/3 of semen Setariae bloom, taking out, filtering with gauze, and sterilizing under high pressure (115 deg.C for 30 min) in a conical flask. After the phytophthora nicotianae is inoculated on an oat medium (OA) for 4d of activation, a phytophthora cake is taken by a puncher. 3-5 bacterial cakes are placed in each bottle of sterilized millet culture medium, and the culture is carried out for 14d at 25 ℃ to obtain the phytophthora nicotianae millet.
Soil was mixed with phytophthora grain (4 g grain: 1kg soil) as the disease soil, and the test soil was derived from the tobacco-derived, i.e., the ink-based, sterilized matrix soil. And selecting tobacco seedlings with consistent growth vigor, transplanting the tobacco seedlings into diseased soil, and immediately applying treatment. Potting experiments total 4 treatments: CK (clear water treatment); positive control (metalaxyl 800-fold liquid), decadienoic acid 1600-fold liquid, and decadienoic acid 800-fold liquid. And (3) injection: 1600 times liquid 1g medicine is dissolved in 1600mL water, and the adopted decadienoic acid is trans-decadienoic acid.
Each pot was 1 plant of smoke, 15 plants per treatment, and repeated 3 times. According to different treatments, adding 20mL of treatment liquid into each pot of tobacco seedlings for root irrigation treatment, and investigating the disease condition of black shank of each treated tobacco by taking plants as units after transplanting for 4d and 7d, and counting the disease index and the disease prevention effect.
The results obtained are shown in Table 1;
table 1 potted plant test of decadienoic acid for preventing and controlling tobacco black shank
FIG. 17 is a physical diagram of a potted plant test for preventing and controlling tobacco black shank with decadienoic acid (7 d).
Potted plant experiments prove that the decadienoic acid has obvious control effect on tobacco black shank. As shown in table 1, the incidence of the treatment was reduced in both treatments 4d and 7d compared with the control group, and it can be seen from table 1 that the control effect of decadienoic acid high-power solution (61.05%) was superior to that of low-power solution (47.57%), indicating that decadienoic acid has a concentration-dependent inhibition effect on tobacco black shank.
The foregoing is merely a preferred embodiment of the present invention and it should be noted that modifications and adaptations to those skilled in the art may be made without departing from the principles of the present invention, which are intended to be comprehended within the scope of the present invention.
Claims (10)
1. Application of decadienoic acid in preventing and treating phytophthora capsici.
2. The use according to claim 1, wherein said decadienoic acid is one or both of trans-decadienoic acid and cis-decadienoic acid.
3. The use according to claim 2, characterized in that the preparation method of decadienoic acid comprises the following steps:
(1) Culturing the trichoderma asperellum spores, inoculating the obtained spore cultures into a shaking culture medium for shaking culture to obtain trichoderma asperellum shaking cultures; the culture medium for spore culture is a potato dextrose agar culture medium;
(2) Seed culture is carried out on the bacillus subtilis, and the obtained seed culture is inoculated into a shaking culture medium for shaking culture, so that the bacillus subtilis shaking culture is obtained;
(3) Mixing the bacillus subtilis shaking culture with the trichoderma asperellum shaking culture and then performing co-fermentation to obtain a co-fermentation liquid;
(4) Filtering the co-fermentation liquid to obtain bacterial liquid and bacterial cells, respectively extracting the bacterial liquid and the bacterial cells, and combining the obtained bacterial liquid extract and the bacterial cell extract to obtain a fermentation extract;
(5) Separating from the fermented extract to obtain trans-decadienoic acid and cis-decadienoic acid.
4. The use according to claim 3, wherein the method for extracting the bacterial liquid comprises the following steps: extracting the bacterial liquid with ethyl acetate, and concentrating the ethyl acetate phase to dryness to obtain a bacterial liquid extract;
the method for extracting the thalli comprises the following steps: and ultrasonically extracting the thalli by using a dichloromethane-methanol mixed solvent, concentrating the obtained extracting solution until the extracting solution is dry, dissolving residues by using water to obtain a water phase, extracting the water phase by using ethyl acetate, and concentrating the water phase until the water phase is dry to obtain a thalli extract.
5. The use according to claim 3, wherein the separation method is: subjecting the fermented extract to normal-phase pressure-reducing column chromatography separation, and performing gradient elution by using a petroleum ether-ethyl acetate mixed solvent to obtain 7 components, wherein the components are sequentially marked as A1-A7 according to the outflow sequence of the components;
separating the component A3 by gel column chromatography, eluting with dichloromethane-methanol mixed solvent to obtain 4 components, and sequentially marking as A3-1-A3-4 according to the outflow sequence of the components;
and (3) purifying the component A3-2 by high performance liquid chromatography to obtain trans-decadienoic acid and cis-decadienoic acid.
6. The use according to claim 5, wherein the gradient elution is divided into 5 gradients, the volume fraction of ethyl acetate in each gradient eluent being in turn 5%, 10%, 30%, 50%, 100%.
7. The use according to claim 5, wherein the volume ratio of dichloromethane to methanol in the dichloromethane-methanol mixed solvent is 1:1;
the mobile phase used for the high performance liquid chromatography purification is a mixed solvent of methanol, water and trifluoroacetic acid, the volume of the trifluoroacetic acid is 0.1% of the water volume, and the volume fraction of the methanol in the mixed solvent is 75%.
8. The use according to claim 2, characterized in that the preparation method of decadienoic acid comprises the following steps: mixing (2E, 4E) -2, 4-decadienal, a Jones reagent and an organic solvent for oxidation reaction, and sequentially filtering, washing and evaporating the obtained reaction liquid to obtain a crude product;
and (3) separating and purifying the crude product to obtain trans-decadienoic acid and cis-decadienoic acid.
9. The use according to claim 8, wherein the method of separation and purification comprises: dissolving the crude product in ethyl acetate to obtain a crude product solution; washing the crude product solution with water, regulating the pH value to 8-9, and extracting with water to obtain a water phase; adjusting the pH value of the water phase to 1-2, extracting with ethyl acetate to obtain an ethyl acetate phase, washing the ethyl acetate phase with saturated sodium chloride solution, and concentrating to obtain a concentrated residue; and sequentially carrying out silica gel column chromatographic separation and high performance liquid chromatography purification on the concentrated residues to obtain trans-decadienoic acid and cis-decadienoic acid.
10. The use according to claim 9, wherein the silica gel column used for the silica gel column chromatography separation is a trans silica gel column, the eluent used for the silica gel column chromatography separation is a methanol-water mixed solvent, the elution mode is gradient elution, the eluent is divided into 7 gradients, the volume fractions of methanol in each gradient eluent are 30%, 40%, 50%, 60%, 70%, 80% and 90% in sequence, and the components eluted by 70% methanol-water are collected for high performance liquid chromatography purification.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202210404368.9A CN115590023B (en) | 2022-04-18 | 2022-04-18 | Application of decadienoic acid in preventing and treating phytophthora capsici of plants |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202210404368.9A CN115590023B (en) | 2022-04-18 | 2022-04-18 | Application of decadienoic acid in preventing and treating phytophthora capsici of plants |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN115590023A CN115590023A (en) | 2023-01-13 |
| CN115590023B true CN115590023B (en) | 2024-02-09 |
Family
ID=84841724
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202210404368.9A Active CN115590023B (en) | 2022-04-18 | 2022-04-18 | Application of decadienoic acid in preventing and treating phytophthora capsici of plants |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN115590023B (en) |
Families Citing this family (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN116891821B (en) * | 2023-08-21 | 2024-06-07 | 中国农业科学院烟草研究所 | Culture medium for co-culture of trichoderma asperellum HG1 and bacillus subtilis Tpb55 and biocontrol agent thereof |
| CN117511749A (en) * | 2023-10-31 | 2024-02-06 | 中国农业科学院烟草研究所(中国烟草总公司青州烟草研究所) | Solid state fermentation method for trichoderma asperellum HG1 and bacillus subtilis Tpb55 and application thereof |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5376541A (en) * | 1991-09-30 | 1994-12-27 | Suntory Limited | Process for production of 8,11-eicosadienoic acid using Mortierella alpina |
| CN102220352A (en) * | 2011-04-04 | 2011-10-19 | 山东农业大学 | Function identification and application of phytophthora infestans Delta 12 desaturase gene PinD12 of potato |
| CN111500644A (en) * | 2020-04-28 | 2020-08-07 | 中国农业科学院烟草研究所 | Preparation method of composite bacteria fermentation product for preventing and treating phytophthora root rot of plants, composite bacteria fermentation product and application of composite bacteria fermentation product |
| CN112106774A (en) * | 2019-06-21 | 2020-12-22 | 中国农业大学 | Application of dicarboxylic acid compound in preventing and treating plant diseases |
| CN112106775A (en) * | 2019-06-21 | 2020-12-22 | 中国农业大学 | Application of hydroxycarboxylic acid compound in preventing and treating plant diseases |
-
2022
- 2022-04-18 CN CN202210404368.9A patent/CN115590023B/en active Active
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5376541A (en) * | 1991-09-30 | 1994-12-27 | Suntory Limited | Process for production of 8,11-eicosadienoic acid using Mortierella alpina |
| CN102220352A (en) * | 2011-04-04 | 2011-10-19 | 山东农业大学 | Function identification and application of phytophthora infestans Delta 12 desaturase gene PinD12 of potato |
| CN112106774A (en) * | 2019-06-21 | 2020-12-22 | 中国农业大学 | Application of dicarboxylic acid compound in preventing and treating plant diseases |
| CN112106775A (en) * | 2019-06-21 | 2020-12-22 | 中国农业大学 | Application of hydroxycarboxylic acid compound in preventing and treating plant diseases |
| CN111500644A (en) * | 2020-04-28 | 2020-08-07 | 中国农业科学院烟草研究所 | Preparation method of composite bacteria fermentation product for preventing and treating phytophthora root rot of plants, composite bacteria fermentation product and application of composite bacteria fermentation product |
Non-Patent Citations (2)
| Title |
|---|
| Metabolites from the Endophytic Fungus Curvularia sp. M12 Act as Motility Inhibitors against Phytophthora capsici Zoospores;Mondol, Muhammad Abdul Mojid;Journal of Natural Products;第80卷(第2期);第347-355页 * |
| 棘孢木霉T-6对烟草促生及对黑胫病和根黑腐病的抗病作用;宋玉娟;韩雪莲;张安盛;王玉军;高克祥;梁元存;;中国烟草科学;41(1);第50-55页 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN115590023A (en) | 2023-01-13 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN115590023B (en) | Application of decadienoic acid in preventing and treating phytophthora capsici of plants | |
| CN108165500A (en) | One plant of marine fungi Aspergillus terreus C23-3, zymotic fluid activity extract and its preparation method and application | |
| CN114164135B (en) | Preparation method and application of banana vascular wilt resistant compound | |
| Panaccione et al. | Conidial dimorphism in Colletotrichum graminicola | |
| CN108033905B (en) | The preparation method and application of compound pencolide | |
| CN105543108B (en) | Purple stain mould QL-9204 and conversion phloridzin prepare the application in phloretin | |
| CN112602717B (en) | Medicine for preventing and controlling rice blast germs | |
| CN110776518A (en) | Azaphilone spiro compounds and preparation method and application thereof | |
| CN102875551A (en) | Chloro alkaloid compound and preparation and application thereof | |
| CN114369051B (en) | Pyrrolidinol compound and preparation method and application thereof | |
| CN110408547A (en) | A kind of green trichoderma for preventing and treating phytoph-thora capsici leonian, application and pepper cultivation method | |
| Uchiyama et al. | Disappearance of the cuticle and wax in outermost layer of callus cultures and decrease of protective ability against microorganisms | |
| CN102786528B (en) | Polyoxybiotic alkali compound as well as preparation method and application thereof | |
| CN109706082B (en) | Biocontrol giant-cavity phoma strain P2 and application thereof | |
| CN101830868B (en) | Oligosporol derivatives and application thereof | |
| CN102031277B (en) | Aurovertin metabolins and application thereof | |
| CN104893986B (en) | Dragonfly enterobacteriaceae Aspergillus terreus QT122 and its metabolite and application | |
| KR100477890B1 (en) | A new Streptomyces sp. 8E12 and its use as a herbicidal agent | |
| CN110563683A (en) | benzopyranone compound and preparation method and application thereof | |
| CN115505538B (en) | Metarhizium anisopliae strain CIPPMA0941, application thereof in preventing and treating solenopsis invicta and microbial inoculum | |
| CN114717119B (en) | Sarcandra glabra endophytic fungus and application thereof | |
| CN116473060B (en) | Medicine for preventing and treating rice blast | |
| CN115812744B (en) | Tung tree extract and application thereof in inhibiting anthracnose of oil tea | |
| Reddy et al. | Bio-control efficiency of Pseudomonas fluorescens against stem rot of tuberose caused by Sclerotium rolfsii. | |
| CN117904227A (en) | Method for improving fusarium oxysporum fusarium acid yield |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |