CN116267998B - Compound preparation for resisting diseases and promoting growth and application thereof - Google Patents
Compound preparation for resisting diseases and promoting growth and application thereof Download PDFInfo
- Publication number
- CN116267998B CN116267998B CN202310007634.9A CN202310007634A CN116267998B CN 116267998 B CN116267998 B CN 116267998B CN 202310007634 A CN202310007634 A CN 202310007634A CN 116267998 B CN116267998 B CN 116267998B
- Authority
- CN
- China
- Prior art keywords
- metabolite
- trichoderma
- compound preparation
- trichoderma reesei
- growth
- 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
- 150000001875 compounds Chemical class 0.000 title claims abstract description 45
- 238000002360 preparation method Methods 0.000 title claims abstract description 40
- 201000010099 disease Diseases 0.000 title claims abstract description 39
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 title claims abstract description 39
- 230000012010 growth Effects 0.000 title claims abstract description 34
- 230000001737 promoting effect Effects 0.000 title claims abstract description 9
- 239000002207 metabolite Substances 0.000 claims abstract description 49
- 241000970937 Streptomyces mirabilis Species 0.000 claims abstract description 48
- 241000499912 Trichoderma reesei Species 0.000 claims abstract description 37
- 235000002566 Capsicum Nutrition 0.000 claims abstract description 17
- 238000004321 preservation Methods 0.000 claims abstract description 15
- 238000009629 microbiological culture Methods 0.000 claims abstract description 11
- 208000035240 Disease Resistance Diseases 0.000 claims abstract description 10
- 241000758706 Piperaceae Species 0.000 claims abstract 3
- 230000001580 bacterial effect Effects 0.000 claims description 28
- 241000223259 Trichoderma Species 0.000 claims description 26
- 239000000725 suspension Substances 0.000 claims description 24
- 235000016761 Piper aduncum Nutrition 0.000 claims description 16
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 claims description 15
- 241000223602 Alternaria alternata Species 0.000 claims description 13
- 241001345879 Valsa sordida Species 0.000 claims description 13
- 239000001963 growth medium Substances 0.000 claims description 13
- 238000000034 method Methods 0.000 claims description 13
- 241001529387 Colletotrichum gloeosporioides Species 0.000 claims description 11
- 241001221840 Neofusicoccum parvum Species 0.000 claims description 11
- 241000579198 Pestalotiopsis chamaeropis Species 0.000 claims description 11
- 241000218904 Pseudomonas oryzihabitans Species 0.000 claims description 11
- 241000131360 Pythium oligandrum Species 0.000 claims description 11
- 241000567083 Xanthomonas arboricola Species 0.000 claims description 11
- 239000000284 extract Substances 0.000 claims description 11
- 239000006228 supernatant Substances 0.000 claims description 10
- 238000010828 elution Methods 0.000 claims description 8
- 229940126902 Phlorizin Drugs 0.000 claims description 7
- IOUVKUPGCMBWBT-UHFFFAOYSA-N phloridzosid Natural products OC1C(O)C(O)C(CO)OC1OC1=CC(O)=CC(O)=C1C(=O)CCC1=CC=C(O)C=C1 IOUVKUPGCMBWBT-UHFFFAOYSA-N 0.000 claims description 7
- IOUVKUPGCMBWBT-GHRYLNIYSA-N phlorizin Chemical group O[C@@H]1[C@H](O)[C@H](O)[C@@H](CO)O[C@H]1OC1=CC(O)=CC(O)=C1C(=O)CCC1=CC=C(O)C=C1 IOUVKUPGCMBWBT-GHRYLNIYSA-N 0.000 claims description 7
- 235000019139 phlorizin Nutrition 0.000 claims description 7
- CSNNHWWHGAXBCP-UHFFFAOYSA-L Magnesium sulfate Chemical compound [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 claims description 6
- 229920002472 Starch Polymers 0.000 claims description 6
- 238000000605 extraction Methods 0.000 claims description 6
- 238000000855 fermentation Methods 0.000 claims description 6
- 230000004151 fermentation Effects 0.000 claims description 6
- 239000008107 starch Substances 0.000 claims description 6
- 235000019698 starch Nutrition 0.000 claims description 6
- 229930006000 Sucrose Natural products 0.000 claims description 5
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 claims description 5
- 238000012258 culturing Methods 0.000 claims description 5
- 239000005720 sucrose Substances 0.000 claims description 5
- 241000894110 Trichoderma spirale Species 0.000 claims description 4
- 238000004440 column chromatography Methods 0.000 claims description 4
- 230000008635 plant growth Effects 0.000 claims description 4
- 239000012137 tryptone Substances 0.000 claims description 4
- NEZONWMXZKDMKF-JTQLQIEISA-N Alkannin Chemical compound C1=CC(O)=C2C(=O)C([C@@H](O)CC=C(C)C)=CC(=O)C2=C1O NEZONWMXZKDMKF-JTQLQIEISA-N 0.000 claims description 3
- 241001071917 Lithospermum Species 0.000 claims description 3
- UNNKKUDWEASWDN-UHFFFAOYSA-N alkannin Natural products CC(=CCC(O)c1cc(O)c2C(=O)C=CC(=O)c2c1O)C UNNKKUDWEASWDN-UHFFFAOYSA-N 0.000 claims description 3
- 229910052943 magnesium sulfate Inorganic materials 0.000 claims description 3
- 235000019341 magnesium sulphate Nutrition 0.000 claims description 3
- 230000010355 oscillation Effects 0.000 claims description 2
- 244000302909 Piper aduncum Species 0.000 claims 2
- 230000000694 effects Effects 0.000 abstract description 48
- 241000187747 Streptomyces Species 0.000 abstract description 7
- 230000002888 effect on disease Effects 0.000 abstract description 3
- 235000009434 Actinidia chinensis Nutrition 0.000 abstract description 2
- 244000298697 Actinidia deliciosa Species 0.000 abstract description 2
- 235000009436 Actinidia deliciosa Nutrition 0.000 abstract description 2
- 238000012795 verification Methods 0.000 abstract 1
- 244000052616 bacterial pathogen Species 0.000 description 27
- 230000005764 inhibitory process Effects 0.000 description 25
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 21
- 239000002609 medium Substances 0.000 description 20
- 241000196324 Embryophyta Species 0.000 description 18
- 239000000203 mixture Substances 0.000 description 18
- 241000894006 Bacteria Species 0.000 description 16
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 15
- 239000006002 Pepper Substances 0.000 description 14
- 241000722363 Piper Species 0.000 description 14
- 235000017804 Piper guineense Nutrition 0.000 description 14
- 235000008184 Piper nigrum Nutrition 0.000 description 14
- 239000002689 soil Substances 0.000 description 13
- 229930002875 chlorophyll Natural products 0.000 description 12
- 235000019804 chlorophyll Nutrition 0.000 description 12
- ATNHDLDRLWWWCB-AENOIHSZSA-M chlorophyll a Chemical compound C1([C@@H](C(=O)OC)C(=O)C2=C3C)=C2N2C3=CC(C(CC)=C3C)=[N+]4C3=CC3=C(C=C)C(C)=C5N3[Mg-2]42[N+]2=C1[C@@H](CCC(=O)OC\C=C(/C)CCC[C@H](C)CCC[C@H](C)CCCC(C)C)[C@H](C)C2=C5 ATNHDLDRLWWWCB-AENOIHSZSA-M 0.000 description 12
- 239000007788 liquid Substances 0.000 description 12
- 230000000844 anti-bacterial effect Effects 0.000 description 11
- 239000003795 chemical substances by application Substances 0.000 description 11
- 239000003480 eluent Substances 0.000 description 11
- 238000009472 formulation Methods 0.000 description 11
- 239000000499 gel Substances 0.000 description 11
- 244000052769 pathogen Species 0.000 description 11
- 229920001817 Agar Polymers 0.000 description 10
- 239000008272 agar Substances 0.000 description 10
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 description 9
- 230000000443 biocontrol Effects 0.000 description 9
- 238000001514 detection method Methods 0.000 description 9
- 229920005654 Sephadex Polymers 0.000 description 8
- 239000012507 Sephadex™ Substances 0.000 description 8
- 244000089698 Zanthoxylum simulans Species 0.000 description 8
- 239000000243 solution Substances 0.000 description 8
- UHPMCKVQTMMPCG-UHFFFAOYSA-N 5,8-dihydroxy-2-methoxy-6-methyl-7-(2-oxopropyl)naphthalene-1,4-dione Chemical compound CC1=C(CC(C)=O)C(O)=C2C(=O)C(OC)=CC(=O)C2=C1O UHPMCKVQTMMPCG-UHFFFAOYSA-N 0.000 description 6
- 239000005747 Chlorothalonil Substances 0.000 description 6
- 241000223218 Fusarium Species 0.000 description 6
- CRQQGFGUEAVUIL-UHFFFAOYSA-N chlorothalonil Chemical compound ClC1=C(Cl)C(C#N)=C(Cl)C(C#N)=C1Cl CRQQGFGUEAVUIL-UHFFFAOYSA-N 0.000 description 6
- 238000011161 development Methods 0.000 description 6
- 230000002401 inhibitory effect Effects 0.000 description 6
- 239000000523 sample Substances 0.000 description 6
- 239000000126 substance Substances 0.000 description 6
- 101150035600 atpD gene Proteins 0.000 description 5
- 238000006243 chemical reaction Methods 0.000 description 5
- 230000018109 developmental process Effects 0.000 description 5
- 101150013736 gyrB gene Proteins 0.000 description 5
- 230000001976 improved effect Effects 0.000 description 5
- 230000001717 pathogenic effect Effects 0.000 description 5
- 239000000047 product Substances 0.000 description 5
- 101150079601 recA gene Proteins 0.000 description 5
- 101150090202 rpoB gene Proteins 0.000 description 5
- 239000008223 sterile water Substances 0.000 description 5
- 238000012360 testing method Methods 0.000 description 5
- 101150081616 trpB gene Proteins 0.000 description 5
- 235000007650 Aralia spinosa Nutrition 0.000 description 4
- 108090000790 Enzymes Proteins 0.000 description 4
- 102000004190 Enzymes Human genes 0.000 description 4
- 241000233866 Fungi Species 0.000 description 4
- 244000061456 Solanum tuberosum Species 0.000 description 4
- 235000002595 Solanum tuberosum Nutrition 0.000 description 4
- 241000949456 Zanthoxylum Species 0.000 description 4
- 230000003385 bacteriostatic effect Effects 0.000 description 4
- 210000004027 cell Anatomy 0.000 description 4
- 239000012141 concentrate Substances 0.000 description 4
- 230000006806 disease prevention Effects 0.000 description 4
- 239000012153 distilled water Substances 0.000 description 4
- 229940088598 enzyme Drugs 0.000 description 4
- 238000011835 investigation Methods 0.000 description 4
- 239000010410 layer Substances 0.000 description 4
- 238000012216 screening Methods 0.000 description 4
- 239000002904 solvent Substances 0.000 description 4
- 230000001954 sterilising effect Effects 0.000 description 4
- 238000004659 sterilization and disinfection Methods 0.000 description 4
- 235000017166 Bambusa arundinacea Nutrition 0.000 description 3
- 235000017491 Bambusa tulda Nutrition 0.000 description 3
- 108020004414 DNA Proteins 0.000 description 3
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 3
- 101100038261 Methanococcus vannielii (strain ATCC 35089 / DSM 1224 / JCM 13029 / OCM 148 / SB) rpo2C gene Proteins 0.000 description 3
- 101100261636 Methanothermobacter marburgensis (strain ATCC BAA-927 / DSM 2133 / JCM 14651 / NBRC 100331 / OCM 82 / Marburg) trpB2 gene Proteins 0.000 description 3
- 235000015334 Phyllostachys viridis Nutrition 0.000 description 3
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 3
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical class O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 3
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 3
- 238000010521 absorption reaction Methods 0.000 description 3
- 101150099875 atpE gene Proteins 0.000 description 3
- 101150048329 atpH gene Proteins 0.000 description 3
- 238000013329 compounding Methods 0.000 description 3
- 239000003814 drug Substances 0.000 description 3
- 238000002474 experimental method Methods 0.000 description 3
- 239000003337 fertilizer Substances 0.000 description 3
- 238000001914 filtration Methods 0.000 description 3
- 244000053095 fungal pathogen Species 0.000 description 3
- 229910017053 inorganic salt Inorganic materials 0.000 description 3
- 230000003993 interaction Effects 0.000 description 3
- 238000011068 loading method Methods 0.000 description 3
- 101150012629 parE gene Proteins 0.000 description 3
- 230000003071 parasitic effect Effects 0.000 description 3
- 239000000575 pesticide Substances 0.000 description 3
- 239000012071 phase Substances 0.000 description 3
- 238000004080 punching Methods 0.000 description 3
- 238000000746 purification Methods 0.000 description 3
- 101150085857 rpo2 gene Proteins 0.000 description 3
- 238000007789 sealing Methods 0.000 description 3
- 238000000926 separation method Methods 0.000 description 3
- 238000012163 sequencing technique Methods 0.000 description 3
- 230000007480 spreading Effects 0.000 description 3
- 238000003892 spreading Methods 0.000 description 3
- 238000004809 thin layer chromatography Methods 0.000 description 3
- 101150111232 trpB-1 gene Proteins 0.000 description 3
- 108020004465 16S ribosomal RNA Proteins 0.000 description 2
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 2
- 244000075850 Avena orientalis Species 0.000 description 2
- 241000209128 Bambusa Species 0.000 description 2
- 241000606161 Chlamydia Species 0.000 description 2
- 229920002307 Dextran Polymers 0.000 description 2
- 241000577837 Fusarium stilboides Species 0.000 description 2
- 229920001503 Glucan Polymers 0.000 description 2
- 239000006391 Luria-Bertani Medium Substances 0.000 description 2
- 108091028043 Nucleic acid sequence Proteins 0.000 description 2
- 239000001888 Peptone Substances 0.000 description 2
- 108010080698 Peptones Proteins 0.000 description 2
- 239000004480 active ingredient Substances 0.000 description 2
- 239000013543 active substance Substances 0.000 description 2
- 230000003321 amplification Effects 0.000 description 2
- 238000004458 analytical method Methods 0.000 description 2
- 239000003242 anti bacterial agent Substances 0.000 description 2
- 229940088710 antibiotic agent Drugs 0.000 description 2
- 235000015278 beef Nutrition 0.000 description 2
- 230000003115 biocidal effect Effects 0.000 description 2
- 210000002421 cell wall Anatomy 0.000 description 2
- 238000010276 construction Methods 0.000 description 2
- 238000010227 cup method (microbiological evaluation) Methods 0.000 description 2
- 238000007872 degassing Methods 0.000 description 2
- 229940079593 drug Drugs 0.000 description 2
- 238000001962 electrophoresis Methods 0.000 description 2
- 239000000706 filtrate Substances 0.000 description 2
- 239000007863 gel particle Substances 0.000 description 2
- 230000035784 germination Effects 0.000 description 2
- 150000002500 ions Chemical class 0.000 description 2
- 238000004811 liquid chromatography Methods 0.000 description 2
- 239000007791 liquid phase Substances 0.000 description 2
- 230000033001 locomotion Effects 0.000 description 2
- 230000007246 mechanism Effects 0.000 description 2
- 238000002156 mixing Methods 0.000 description 2
- 230000000877 morphologic effect Effects 0.000 description 2
- 238000003199 nucleic acid amplification method Methods 0.000 description 2
- 235000016709 nutrition Nutrition 0.000 description 2
- 230000035764 nutrition Effects 0.000 description 2
- 230000003287 optical effect Effects 0.000 description 2
- 235000019319 peptone Nutrition 0.000 description 2
- 108090000623 proteins and genes Proteins 0.000 description 2
- 239000012488 sample solution Substances 0.000 description 2
- 239000000741 silica gel Substances 0.000 description 2
- 229910002027 silica gel Inorganic materials 0.000 description 2
- 239000007787 solid Substances 0.000 description 2
- 230000002195 synergetic effect Effects 0.000 description 2
- 238000005406 washing Methods 0.000 description 2
- ZCYVEMRRCGMTRW-UHFFFAOYSA-N 7553-56-2 Chemical compound [I] ZCYVEMRRCGMTRW-UHFFFAOYSA-N 0.000 description 1
- 241000186361 Actinobacteria <class> Species 0.000 description 1
- 241000605422 Asparagus asparagoides Species 0.000 description 1
- 108020000946 Bacterial DNA Proteins 0.000 description 1
- 244000218454 Bambusa tulda Species 0.000 description 1
- 235000008534 Capsicum annuum var annuum Nutrition 0.000 description 1
- 240000008384 Capsicum annuum var. annuum Species 0.000 description 1
- 108010059892 Cellulase Proteins 0.000 description 1
- 102000012286 Chitinases Human genes 0.000 description 1
- 108010022172 Chitinases Proteins 0.000 description 1
- 229920000742 Cotton Polymers 0.000 description 1
- 238000007400 DNA extraction Methods 0.000 description 1
- 241001508802 Diaporthe Species 0.000 description 1
- 206010059866 Drug resistance Diseases 0.000 description 1
- 241000223221 Fusarium oxysporum Species 0.000 description 1
- 241000221779 Fusarium sambucinum Species 0.000 description 1
- 240000006053 Garcinia mangostana Species 0.000 description 1
- 235000017048 Garcinia mangostana Nutrition 0.000 description 1
- 241000238631 Hexapoda Species 0.000 description 1
- 239000012880 LB liquid culture medium Substances 0.000 description 1
- 241000606266 Nardostachys Species 0.000 description 1
- 240000007594 Oryza sativa Species 0.000 description 1
- 235000007164 Oryza sativa Nutrition 0.000 description 1
- 241000011500 Phyllostachys praecox Species 0.000 description 1
- 235000008545 Phyllostachys praecox Nutrition 0.000 description 1
- 241000589970 Spirochaetales Species 0.000 description 1
- 241001655322 Streptomycetales Species 0.000 description 1
- 241000223261 Trichoderma viride Species 0.000 description 1
- 241000607479 Yersinia pestis Species 0.000 description 1
- 241001079064 Zanthoxylum schinifolium Species 0.000 description 1
- 235000009932 Zanthoxylum simulans Nutrition 0.000 description 1
- 238000002835 absorbance Methods 0.000 description 1
- 230000003213 activating effect Effects 0.000 description 1
- 239000003463 adsorbent Substances 0.000 description 1
- 230000002411 adverse Effects 0.000 description 1
- 239000011543 agarose gel Substances 0.000 description 1
- 238000007605 air drying Methods 0.000 description 1
- 230000003042 antagnostic effect Effects 0.000 description 1
- 230000008485 antagonism Effects 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
- 230000001420 bacteriolytic effect Effects 0.000 description 1
- 239000011425 bamboo Substances 0.000 description 1
- 230000001680 brushing effect Effects 0.000 description 1
- 238000004364 calculation method Methods 0.000 description 1
- 229940041514 candida albicans extract Drugs 0.000 description 1
- 229940106157 cellulase Drugs 0.000 description 1
- 238000004587 chromatography analysis Methods 0.000 description 1
- 239000011248 coating agent Substances 0.000 description 1
- 238000000576 coating method Methods 0.000 description 1
- 230000034994 death Effects 0.000 description 1
- 230000000593 degrading effect Effects 0.000 description 1
- 238000013461 design Methods 0.000 description 1
- 239000003085 diluting agent Substances 0.000 description 1
- 235000021186 dishes Nutrition 0.000 description 1
- 238000004090 dissolution Methods 0.000 description 1
- 238000009826 distribution Methods 0.000 description 1
- 239000012149 elution buffer Substances 0.000 description 1
- 239000000469 ethanolic extract Substances 0.000 description 1
- 210000004709 eyebrow Anatomy 0.000 description 1
- 239000012634 fragment Substances 0.000 description 1
- 238000004108 freeze drying Methods 0.000 description 1
- 230000002538 fungal effect Effects 0.000 description 1
- 230000001408 fungistatic effect Effects 0.000 description 1
- 210000004602 germ cell Anatomy 0.000 description 1
- -1 glucanase Proteins 0.000 description 1
- 238000004128 high performance liquid chromatography Methods 0.000 description 1
- 239000005556 hormone Substances 0.000 description 1
- 229940088597 hormone Drugs 0.000 description 1
- 230000003301 hydrolyzing effect Effects 0.000 description 1
- 230000001939 inductive effect Effects 0.000 description 1
- 238000002347 injection Methods 0.000 description 1
- 239000007924 injection Substances 0.000 description 1
- 230000006799 invasive growth in response to glucose limitation Effects 0.000 description 1
- 229910052740 iodine Inorganic materials 0.000 description 1
- 239000011630 iodine Substances 0.000 description 1
- 238000002955 isolation Methods 0.000 description 1
- 230000002147 killing effect Effects 0.000 description 1
- 230000000670 limiting effect Effects 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 239000003550 marker Substances 0.000 description 1
- 238000004949 mass spectrometry Methods 0.000 description 1
- 239000012528 membrane Substances 0.000 description 1
- 230000004060 metabolic process Effects 0.000 description 1
- 244000005700 microbiome Species 0.000 description 1
- 239000011259 mixed solution Substances 0.000 description 1
- 239000012046 mixed solvent Substances 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 230000004899 motility Effects 0.000 description 1
- 229910052757 nitrogen Inorganic materials 0.000 description 1
- 238000012856 packing Methods 0.000 description 1
- 244000045947 parasite Species 0.000 description 1
- 239000012466 permeate Substances 0.000 description 1
- 238000005191 phase separation Methods 0.000 description 1
- 239000003375 plant hormone Substances 0.000 description 1
- 230000003234 polygenic effect Effects 0.000 description 1
- 239000003910 polypeptide antibiotic agent Substances 0.000 description 1
- 235000012015 potatoes Nutrition 0.000 description 1
- 239000000843 powder Substances 0.000 description 1
- 230000002265 prevention Effects 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 210000001938 protoplast Anatomy 0.000 description 1
- 230000002829 reductive effect Effects 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 230000002441 reversible effect Effects 0.000 description 1
- 235000009566 rice Nutrition 0.000 description 1
- 238000002390 rotary evaporation Methods 0.000 description 1
- 150000003839 salts Chemical class 0.000 description 1
- 238000005070 sampling Methods 0.000 description 1
- 229930000044 secondary metabolite Natural products 0.000 description 1
- 239000011780 sodium chloride Substances 0.000 description 1
- 238000005507 spraying Methods 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- 239000002344 surface layer Substances 0.000 description 1
- 230000033772 system development Effects 0.000 description 1
- 238000003828 vacuum filtration Methods 0.000 description 1
- 239000012138 yeast extract Substances 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
- A01N63/00—Biocides, pest repellants or attractants, or plant growth regulators containing microorganisms, viruses, microbial fungi, animals or substances produced by, or obtained from, microorganisms, viruses, microbial fungi or animals, e.g. enzymes or fermentates
- A01N63/20—Bacteria; Substances produced thereby or obtained therefrom
- A01N63/28—Streptomyces
-
- 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
- A01N35/00—Biocides, pest repellants or attractants, or plant growth regulators containing organic compounds containing a carbon atom having two bonds to hetero atoms with at the most one bond to halogen, e.g. aldehyde radical
- A01N35/06—Biocides, pest repellants or attractants, or plant growth regulators containing organic compounds containing a carbon atom having two bonds to hetero atoms with at the most one bond to halogen, e.g. aldehyde radical containing keto or thioketo groups as part of a ring, e.g. cyclohexanone, quinone; Derivatives thereof, e.g. ketals
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N43/00—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds
- A01N43/02—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds having rings with one or more oxygen or sulfur atoms as the only ring hetero atoms
- A01N43/04—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds having rings with one or more oxygen or sulfur atoms as the only ring hetero atoms with one hetero atom
- A01N43/14—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds having rings with one or more oxygen or sulfur atoms as the only ring hetero atoms with one hetero atom six-membered rings
- A01N43/16—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds having rings with one or more oxygen or sulfur atoms as the only ring hetero atoms with one hetero atom six-membered rings with oxygen as the ring hetero atom
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01P—BIOCIDAL, PEST REPELLANT, PEST ATTRACTANT OR PLANT GROWTH REGULATORY ACTIVITY OF CHEMICAL COMPOUNDS OR PREPARATIONS
- A01P21/00—Plant growth regulators
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
-
- 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
- C12P19/00—Preparation of compounds containing saccharide radicals
- C12P19/44—Preparation of O-glycosides, e.g. glucosides
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P7/00—Preparation of oxygen-containing organic compounds
- C12P7/24—Preparation of oxygen-containing organic compounds containing a carbonyl group
- C12P7/26—Ketones
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
- C12R2001/465—Streptomyces
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/645—Fungi ; Processes using fungi
- C12R2001/885—Trichoderma
-
- 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
- Y02A40/00—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production
- Y02A40/10—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production in agriculture
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Zoology (AREA)
- Chemical & Material Sciences (AREA)
- Wood Science & Technology (AREA)
- Health & Medical Sciences (AREA)
- Organic Chemistry (AREA)
- General Health & Medical Sciences (AREA)
- Biotechnology (AREA)
- Microbiology (AREA)
- Genetics & Genomics (AREA)
- Plant Pathology (AREA)
- Environmental Sciences (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Dentistry (AREA)
- Pest Control & Pesticides (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Agronomy & Crop Science (AREA)
- General Engineering & Computer Science (AREA)
- Biochemistry (AREA)
- Virology (AREA)
- Botany (AREA)
- Biomedical Technology (AREA)
- Tropical Medicine & Parasitology (AREA)
- Medicinal Chemistry (AREA)
- Agricultural Chemicals And Associated Chemicals (AREA)
Abstract
The invention discloses a compound preparation for disease resistance and growth promotion and application thereof. The compound preparation comprises a trichoderma reesei RS05 metabolite and streptomyces mirabilis BD2233, wherein the trichoderma reesei RS05 is preserved in 2021 in 12 months and 20 days, the preservation number is CGMCC No.40012, the streptomyces mirabilis BD2233 is preserved in 2022 in 9 months and 2 days, the preservation number is CGMCC No.25634, and both strains are preserved in the China general microbiological culture Collection center of the institute of microbiological culture of China academy of sciences. Proved by verification, the compound preparation prepared from the trichoderma reesei RS05 metabolite and the streptomyces kiwi BD2233 has the effects of resisting diseases and promoting growth of the peppers, and has remarkable effects on disease control and growth of the peppers.
Description
Technical Field
The invention relates to the technical field of medicines, in particular to a compound preparation for resisting diseases and promoting growth and application thereof.
Background
Trichoderma (Trichoderma) is a biocontrol fungus widely distributed in nature, the Trichoderma has strong adaptability and rapid growth and propagation, nutrition and occupation space can be rapidly utilized, and the biocontrol mechanism mainly comprises parasitic, competing, antibiotic and bacteriolytic effects. In the process of interaction of trichoderma with pathogenic bacteria, parasitic hyphae secrete substances to enable trichoderma to grow towards host fungi, and once the host is identified by trichoderma parasites, parasitic relations are established, so that nutrition can be directly absorbed from the host hyphae, and therefore the growth and development of the host are destroyed, and the host hyphae lose the integrity of protoplasts and break or only leave empty shells until death. In addition, trichoderma can secrete various antibiotic metabolites and hydrolytic enzymes for degrading cell walls of pathogenic bacteria, such as trichoderma, gliomycin, antibacterial peptide, trichoderma viride, chitinase, glucanase, cellulase and the like.
Actinomycetes can promote the growth of host plants or increase their ability to resist insect pests by a number of mechanisms. The streptomycete secondary metabolite is rich in types and various in structure types, comprises antibiotics, enzymes, hormones and other substances, and plays an important role in improving the disease resistance and stress resistance of plants. Some antibiotics control the occurrence and development of diseases by inhibiting the germination of conidia and the growth of hyphae of the germs. The enzyme metabolite can cause cell wall perforation, deformity and other phenomena through the dissolution of germ cell wall, thereby having the functions of inhibiting germination, growing and even killing spores and hyphae. The plant hormone metabolites improve the resistance of plants against rice blast by mainly inducing the defensive reaction of the plants.
The pathogenic bacteria Pseudomonas oryzihabitans, xanthomonas arboricola and the pathogenic fungi Alternaria alternata、Neofusicoccum parvum、Pestalotiopsis chamaeropis、Colletotrichum gloeosporioides、Cytospora chrysosperma、Pythium oligandrum are all 8 main pathogenic bacteria causing the branches of Sichuan pepper to be dried up, ulcerated, dried up, brown spots, black spots and rotten. The biocontrol bacteria can produce bacteriostatic active substances through metabolism, antagonize pathogenic bacteria and the like to achieve the effects of preventing diseases and promoting growth, and has wide application prospect due to the advantages of no pollution to the environment, no drug resistance and the like. However, the stability of the biocontrol bacteria in the field and the colonization capability of the strain are important factors for affecting the stable biocontrol effect. Therefore, it is necessary to further develop a compound preparation with good stress resistance and stable control effect.
Disclosure of Invention
The invention aims to provide a compound preparation for resisting diseases and promoting growth and application thereof, and the compound preparation has the disease-resisting effect of various diseases, can promote plant height, ground diameter and root length of Chinese prickly ash, and has remarkable effects in field disease prevention and growth promotion.
In order to achieve the aim, the invention provides a compound preparation for resisting diseases and promoting growth, which consists of a trichoderma spiral RS05 metabolite and streptomyces kiwi BD 2233.
The Streptomyces mirabilis BD2233 is preserved in China general microbiological culture Collection center (China general microbiological culture Collection center) on 9 th month of 2022, the preservation address is North Star Xway No.1, 3 in the Korean area of Beijing, the preservation number is CGMCC No.25634, latin name of the Streptomyces mirabilis BD2233 is Streptomyces mirabilis, and the Streptomyces mirabilis BD2233 is a bacterial suspension with the concentration of 1×10 5 cfu/mL.
The trichoderma spiralis RS05 metabolite is a substance obtained by extracting and separating fermentation supernatant of trichoderma spiralis RS05 with the preservation number of CGMCC No.40012, wherein the Latin name of the trichoderma spiralis RS05 is Trichoderma spirale, and the substance is phlorizin and shikonin.
The disease resistance includes anti Pseudomonas oryzihabitans、Xanthomonas arboricola、Alternaria alternata、Neofusicoccum parvum、Pestalotiopsis chamaeropis、Colletotrichum gloeosporioides、Cytospora chrysosperma and Pythium oligandrum.
The growth promotion comprises promotion of growth of plant height, ground diameter and root length of the Chinese prickly ash.
Preferably, the above-mentioned formulation is prepared from the trichoderma reesei RS05 metabolite: the volume ratio of the streptomyces mirabilis BD2233 bacterial suspension is 1:3.
The invention also provides streptomyces mirabilis BD2233 which is preserved in the China general microbiological culture Collection center of China Committee for culture Collection of microorganisms for 9 months and 2 days, wherein the preservation address is the West Song No. 1 and 3 of the Korean area North Star of Beijing city, the preservation number is CGMCC No.25634, and the streptomyces mirabilis has an inhibition effect on Fusarium binding.
The invention also provides a preparation method of the trichoderma reesei RS05 metabolite, which comprises the following steps:
Culturing Trichoderma reesei RS05 with a preservation number of CGMCC No.40012, and collecting fermentation supernatant;
Adding ethyl acetate into the obtained fermentation supernatant according to an equal volume for oscillation extraction;
Concentrating the obtained extract, separating by gel column chromatography, and collecting eluting peak sample;
and collecting the collected elution peak sample corresponding to the first characteristic peak at OD214, namely the trichoderma reesei RS05 metabolite.
Preferably, the culture in the above preparation method, the culture medium of which comprises sucrose in an amount of 20% by mass, soluble starch in an amount of 2% by mass, tryptone in an amount of 0.1% by mass and magnesium sulfate in an amount of 0.05% by mass.
The compound preparation provided by the invention can be applied to disease resistance and growth promotion of the Chinese prickly ash, wherein disease resistance comprises Pseudomonas oryzihabitans、Xanthomonas arboricola、Alternaria alternata、Neofusicoccum parvum、Pestalotiopsis chamaeropis、Colletotrichum gloeosporioides、Cytospora chrysosperma、Pythium oligandrum; resistance and growth promotion comprises promotion of growth of plant height, ground diameter and root length of the Chinese prickly ash.
The compound preparation provided by the invention solves the problems of poor stability and colonization capacity of single biocontrol bacteria, has good stress resistance and stable control effect, and has the following advantages:
The compound preparation of the trichoderma spiralis RS05 metabolite and the streptomyces mirabilis BD2233 provided by the invention has the effects of resisting diseases and promoting growth of pepper and has good inhibition effect on Pseudomonas oryzihabitans、Xanthomonas arboricola、Alternaria alternata、Neofusicoccum parvum、Pestalotiopsis chamaeropis、Colletotrichum gloeosporioides、Cytospora chrysosperma、Pythium oligandrum 8 pathogenic bacteria, so that the compound preparation has broad spectrum, has remarkable effects on disease control and growth of pepper, saves more cost than pesticide and fertilizer use, is suitable for commercial production, meets the strategic requirements of sustainable development, and has wide market prospect.
Drawings
FIG. 1 shows the results of the planar antagonism inhibition of pathogenic bacteria by Streptomyces mirabilis BD2233 of the present invention.
FIG. 2 shows colony growth of Streptomyces mirabilis BD2233 in different culture mediums.
FIG. 3 is a photomicrograph of Streptomyces mirabilis BD2233 of the present invention.
FIG. 4 shows the result of amplification electrophoresis of Streptomyces mirabilis BD2233 using 6 pairs of primers in the present invention.
FIG. 5 is a polygenic joined phylogenetic tree constructed based on gyrB, trpB, rpoB, recA and atpD gene sequences according to the invention.
FIG. 6 shows the results of thin layer chromatography of the Trichoderma reesei RS05 metabolites of the present invention.
FIG. 7 shows the detection result of OD 214 absorbance of the trichoderma spiral RS05 metabolite of the present invention after column chromatography separation.
FIG. 8 shows the results of liquid chromatography of component C in the present invention.
Detailed Description
The following description of the technical solutions in the embodiments of the present invention will be clear and complete, and it is obvious that the described embodiments are only some embodiments of the present invention, but not all embodiments. 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.
Experimental example 1 screening and identification of biocontrol bacteria
1. Isolation of strains
Sample collection was performed on healthy woodland of Hua Ning villages X green hybrid bamboos in the county of kernel shou of Sichuan, 7 months and 1 days of 2021, and rhizosphere soil was collected by a five-point sampling method. After removing dead branches and fallen leaves on the surface layer of the soil, collecting the root system with the soil in the depth of a soil layer of 0-20 cm in a circular range with a bamboo pole as the center and the diameter of 0.5m, gently shaking off a large block of soil without the root system, brushing off the soil (the distance is 0-5 mm) adhered around the root system by using a brush, taking the soil as rhizosphere soil, putting the rhizosphere soil into a sterile self-sealing bag, storing in an ice box, quickly taking the ice box back to a laboratory, and storing in a refrigerator at the temperature of 4 ℃.
10G of healthy plant rhizosphere soil is taken and placed in a 250mL conical flask filled with 90mL of sterile water, shaking is carried out uniformly, 10 -1 diluent is prepared, the soil sample solution is diluted to 10 -2、10-3、10-4、10-5、10-6、10-7 by the sterile water respectively, 100 mu L of the soil sample solution is sucked into a flat plate of beef extract peptone agar medium (NA medium: 3g of beef extract, 10g of peptone, 5g of sodium chloride, 20g of agar and 1000mL of distilled water) respectively, 121 ℃ high-pressure sterilization is carried out for 30min, a sterilization triangular bar is used for coating uniformly on the flat plate of the medium, the flat plate is placed in a 37 ℃ incubator in an inverted manner for constant temperature culture for 24h, and each treatment is repeated for 3 times. Single colonies with obvious colony morphology difference are picked up by a sterile inoculating loop, streak culture is carried out again on NA culture medium, 14 purified strains (named BD2230, BD2231, BD2232, BD2233, BD2234, BD2235, BD2236, BD2237, BD2238, BD2239, BD2240, BD2241, BD2242 and BD2243 respectively) are obtained, and the NA inclined plane is transferred to the purified strains for preservation at 4 ℃.
2. Flat plate inhibition effect
A6 mm-thick bacterial cake of pathogenic bacteria was placed on one side of a conventional PDA medium plate by an agar punching method using a sterile inoculating needle, punched with a 6 mm-thick sterile punch at a distance of 3cm from the bacterial cake of Fusarium roseum (Fusarium stilboides Wollenw.), and inoculated with 100. Mu.L of each purified strain culture broth cultured by shaking at 28℃for 24 hours with a 180r/min shaker at a concentration of 1X 10 5 cfu/mL, respectively. The blank was inoculated with a 6mm diameter cake of pathogenic fungi at the center of the circle and incubated at 25℃for 7 days, 3 replicates each, and the inhibition of each screened strain against Fusarium bouganii was as shown in Table 1 below.
Table 1 shows the inhibition ratios of the screening bacteria to Fusarium rosenbergii
Note that: inhibition ratio (%) = (control group pathogen diameter-treatment group pathogen diameter)/(control group pathogen diameter-cake diameter) ×100; data are mean ± standard error, different lower case letters after data represent significant differences (P < 0.05).
According to the result of the inhibition rate of each screening bacterium on fusarium wilt, the strain with the best effect is BD2233, and the strain has certain biocontrol capability. The inhibitory effect of the strain BD2233 on pathogenic bacteria on the plate is shown in FIG. 1, wherein pathogenic bacteria are on the left side of the plate, and the selected strain BD2233 is on the right side of the plate.
3. Identification of biocontrol bacteria
(1) Morphological identification
After the strain BD2233 subjected to screening is respectively cultured for 7 days at 28 ℃ on inorganic salt starch, gaoshi No. 1, yeast extract-malt extract, nardostachys, oat and potato extract culture mediums (different culture medium formulas are shown in table 2), the strain BD2233 is best grown on oat agar culture medium (optimal culture medium), and has regular colony, white color and rich aerial hyphae; the strain grows worst on a Chlamydia medium, the colony is round and off-white, the aerial hyphae are few, and the basal hyphae are abundant; the strain grows well on potato extract and yeast extract-malt extract agar medium, and grows well on inorganic salt starch and Gao's first medium, and the colony growth condition on each medium is shown in figure 2, wherein A in the figure is a soluble inorganic salt starch medium; b is Gao's first culture medium; c is yeast extract-malt extract agar medium; d is a Chlamydia medium; e is oatmeal medium; f is potato extract agar medium. The morphology of the growth of this strain on each medium is described in Table 3 below. The microscopic morphology of the strain BD2233 cultured in a culture medium No. 12d of Gao's first is shown in FIG. 3, wherein A in the figure is in a mycelium form, B in the figure is in a tightly spiraled spore wire form, and the scale bars at the lower right of the figure are all 10 μm. It was found that the spore form of the strain BD2233 was rod-shaped, and had no motility, which is one of the typical morphological features of Streptomyces mirabilis.
Wherein, the trace salt in Table 2 was prepared by dissolving 0.1g of FeSO 4·7H2 O, 0.1g of MnCl 2·4H2 O, and 0.1g of ZnSO 4·7H2 O in 100mL of distilled water.
TABLE 2 different Medium formulations
TABLE 3 morphology and culture characteristics of BD2233 strains on different media
(2) Molecular biological identification
Bacterial strain BD2233 genomic DNA was extracted using TIANGEN DNAKIT bacterial DNA extraction kit, and the products were subjected to PCR detection: the 16s rRNA, gyrB, rpoB, trpB, recA and atpD genes were amplified from the obtained genomic DNA with 2X EASYTAQ PCR Supermix (+dye) enzyme, and the primers used are shown in Table 4 below, and the reaction system and amplification procedure are shown in Table 5 below, respectively.
TABLE 4 amplified primer sequences and sequencing primers
Table 5 shows the PCR reaction system and the reaction procedure
The PCR product obtained was electrophoresed on a 1% agarose gel at a constant voltage of 110V for 25min to detect the PCR product. And (3) sending the PCR product to a Chengdu engine biotechnology limited company for sequencing, comparing the obtained sequencing results of the gene sequences in an NCBI (https:// www.ncbi.nlm.nih.gov /) database, determining the sequence used for tree construction according to the comparison result, constructing a multi-gene combined system development tree by adopting an adjacent method (M-E), and determining the system evolution status of antagonistic strains.
As shown in FIG. 4, there are 6 electrophoresis bands (two replicates for each target gene) of products obtained by amplifying 16s rRNA, gyrB, rpoB, trpB, recA and atpD genes of the strain BD2233, respectively, in which M is a marker of DL 2000. DNA fragments of 1129bp, 462bp, 810bp, 723bp, 818bp and 903bp in length were obtained, respectively. Each DNA sequence of BD2233 strain was submitted to the GenBank database for accession number (16S OP236556;gyrB OP413833;trpB OP413834;rpoB OP390164;recA OP390163;atpD OP390162).
BLAST comparison analysis is carried out in NCBI database, different DNA sequences of strains with highest homology are selected, phyloSuite is adopted for multi-gene combined tree construction, and a multi-gene combined phylogenetic tree constructed based on gyrB, rpoB, trpB, recA and atpD gene sequences of PhyloSuite strain BD2233 is shown in figure 5. From the developmental tree, BD2233 and Streptomyces mirabilis NRRL ISP-5553 support 1 branch with higher self-development value, and have the closest relationship with other Streptomyces strains and far relationship. Based on the characteristics, the strain BD2233 is classified and named as Streptomyces mirabilis (Streptomyces mirabilis) BD2233, and is preserved in China general microbiological culture Collection center (China general microbiological culture Collection center) of China academy of sciences of China, namely, the China general microbiological culture Collection center, with a preservation address of North Star Xiya No. 1, and a preservation number of CGMCC No.25634 in the Korean region of Beijing.
Experimental example 2 Trichoderma spiral RS05 metabolite extraction and active ingredient analysis
1. Extraction of trichoderma spirochete RS05 metabolites
Trichoderma reesei (Trichoderma spirale) RS05 strain is separated from healthy phyllostachys praecox rhizosphere soil in a cultivation area of the mangosteen of Sichuan eyebrow, and is preserved in China general microbiological culture Collection center (CGMCC No. 40012) of microbiological culture Collection center, china national institute of sciences of microbiology, national academy of sciences, north Star, of the Korean area of Beijing, 20 days in 2021.
Firstly, activating the strain, transferring the strain to a PSA culture medium (sucrose agar culture medium containing 200g of potatoes, 20g of agar powder and 20g of sucrose, and constant-temperature culturing for 3-5 days at 25 ℃ in distilled water with constant volume of 1000 mL). After the colony had grown sufficiently, it was inoculated into a liquid medium containing 20% by mass of sucrose, 2% by mass of soluble starch, 0.1% by mass of tryptone and 0.05% by mass of magnesium sulfate and cultured for 96 hours, leaving 1L of the original bacterial suspension for use in a control experiment. In addition, the remaining bacterial suspension was centrifuged at 25℃and 8000r/min for 10min to remove bacterial cells, and the bacterial suspension was filtered through a 0.22 μm sterile filter to obtain a sterile filtrate. 10L of sterile filtrate is taken, an equal volume of ethyl acetate is added for shaking extraction for 3 times, the extracts are combined, the mixture is concentrated to 100mL by rotary evaporation for 1h at 40 ℃, the metabolite is in a light yellow viscous liquid state, and the obtained concentrate is ready for use.
2. Thin layer chromatography
1) Selection of optimal developer
The eluent is a multipurpose mixed solvent, and different volume ratios of the mixed solution are adjusted according to dielectric constants and polarities of different solvents so as to achieve the purpose of separating the mixture. The solvent system in table 6 below was formulated. The optimal developing agents of the test samples are determined according to the optimal Rf values, wherein the optimal developing agents are developed in the developing agents, the developing distances are equal.
Table 6 formulation of the developing agent
2) Exhibition layer and detection
The Trichoderma reesei RS05 metabolite concentrate was spotted onto activated silica gel plates with capillaries, and spotted 3 times after air-drying, in line with the spots evenly 1cm from the bottom of the plates. A clean chromatographic cylinder is taken, a proper volume of spreading agent is added into the chromatographic cylinder, and the uniform mixing of the solvent is ensured, and the liquid level does not exceed the baseline of the sample application of the thin layer plate. And (3) placing the spotted thin layer plate into a chromatographic cylinder for spreading, taking out when the spreading agent goes up to the front 1cm, marking the front position, and naturally airing the solvent.
The thin silica gel plate is placed in iodine vapor for color development, the distribution of samples on the thin silica gel plate is observed, and the samples are photographed, so that the Rf value of the main component is calculated. Where rf=distance of solute movement/distance of solution movement.
The developing agent is ethyl acetate: water: the results of thin layer chromatography with methanol at a ratio of 12:8:2 are shown in FIG. 6, where lanes 1, 2, 3 are three replicates. It can be seen that when the developing agent is ethyl acetate: water: the methanol is separated into 4 components when the ratio is 12:8:2, the Rf values are 0.36, 0.53, 0.68 and 0.84 respectively, the components are more, and the Rf values are more reasonable, so that the developing agent is used as the optimal developing agent for separation and purification.
3. Gel column chromatography
1) Pretreatment of Sephadex G-20 gel
At room temperature, 25G of Sephadex G-20 gel particles were weighed into a beaker, and 250mL of eluent was added to completely swell the Sephadex. After a period of time, the excess fine gel particles suspended on the liquid surface were removed and the remaining gel was allowed to swell overnight in the eluent.
2) Column loading and balancing
The chromatographic column is vertically fixed on a support, PBS eluent with the height of 1/3 is added into the chromatographic column, the dextran Sephadex G-20 gel is uniformly and continuously poured into the chromatographic column, when the chromatographic column forms a column bed with the height of about 2cm, a water outlet below the chromatographic column is opened, along with the outflow of the eluent, the dextran Sephadex G-20 gel is uniformly and continuously supplemented at the inlet, the column loading is stopped when the gel surface is about 3cm away from the top end of the chromatographic column, and a proper amount of eluent is added on the gel surface. After completion of the column packing, the column was washed with 3 volumes of the adsorbent by eluting with 1.2mL/min until equilibrium was reached in the column.
3) Loading and elution
Opening Fang Gaizi on the chromatographic column to slowly drip out the liquid in the column, adding 5mL of the trichoderma reesei RS05 metabolite obtained in the step 1) of the experimental example 2 when the elution buffer PBS is tangential to the surface of the glucan Sephadex G-20 gel, washing the column wall 2-3 times by using a small amount of eluent when the trichoderma reesei RS05 metabolite completely permeates the glucan Sephadex G-20 gel, and adding the eluent with the column height of 3 cm. The flow rate was set at 1mL/min, one tube was collected every 4mL, and a total of 80 tubes were collected.
4) Combining and concentrating
The collected solutions were subjected to detection of OD 214 by an ultraviolet spectrophotometer, and the results are shown in FIG. 7. As can be seen from the figure, the concentrate obtained above has four distinct absorption peaks after Sephadex G-20 gel separation, wherein the No. 1 peak appears between No. 10 and No. 20 collecting pipes, the No. 2 peak appears between No. 20 and No. 32 collecting pipes, the No. 3 peak appears between No. 32 and No. 45 collecting pipes, and the No. 4 peak appears between No. 45 and No. 53 collecting pipes, wherein the peak heights of the No. 3 peak are highest, the No. 1 and No. 2 peaks are next, and the peak heights of the No. 4 peak are lowest. And combining the related main elution peaks, concentrating the main elution peaks by a rotary evaporator at 40 ℃, respectively combining all the tube collecting solutions of the related main elution peaks, freeze-drying, re-dissolving the main elution peaks in a proper amount of eluent, and preserving the main elution peaks at 4 ℃ for later use.
4. Detection of bacteriostatic Activity
1) Detection of antibacterial Activity
The oxford cup method is adopted to measure the antibacterial activity of the supernatant of the bacteria to be tested. By using Pseudomonas oryzihabitans and Xanthomonas arboricola as indicator bacteria, mixing 60 mu L of indicator bacteria with LB solid medium cooled to about 50 ℃, pouring the mixture into a flat plate, placing a sterile oxford cup with the diameter of 6mm in a culture dish, adding 150 mu L of supernatant into the oxford cup, and placing the mixture in a 37 ℃ incubator for culturing overnight (12 h). And (5) observing whether a transparent ring appears around the oxford cup, if so, indicating that the strain to be tested has antibacterial activity on the strain, and measuring and recording the diameter (mm) of the antibacterial ring. With the control of the addition of equal volumes of blank culture and original Trichoderma reesei RS05 suspension to the wells, each treatment was repeated 3 times and the statistical results are shown in Table 7 below.
2) Detection of fungistatic Activity
The fungus inhibition activity of the supernatant of the bacteria to be detected is measured by adopting an oxford cup method, Alternaria alternata、Neofusicoccumparvum、Pestalotiopsis chamaeropis、Colletotrichum gloeosporioides、Cytospora chrysosperma、Pythium oligandrum is taken as indicator bacteria respectively, a bacterial block is placed in the center of a PDA solid flat plate, a sterile oxford cup with the diameter of 6mm is placed at the position 3cm away from the pathogenic bacterial block on the periphery of the flat plate, 150 mu L of supernatant is added into the oxford cup, and the mixture is placed in a culture box at 25 ℃ for culturing for 3-7 days, and the diameter (mm) of a bacteria inhibition zone is observed and measured. The control was an equal volume of blank culture medium and original Trichoderma reesei RS05 suspension, each treatment was repeated 3 times, and the statistical results of the bacteriostatic activity are shown in Table 7 below.
TABLE 7 antibacterial Activity results of pathogenic bacteria
Note that: different lowercase letters in the same row indicate significant variability, P <0.05, and ck1 represents blank culture broth; CK2 represents the original bacterial suspension of Trichoderma reesei RS 05.
As can be seen from Table 7, the original bacterial suspension of Trichoderma reesei RS05 had only a weak inhibitory effect on 2 pathogenic bacteria and no inhibitory effect on the remaining 6 pathogenic bacteria. However, the antibacterial activity of the metabolite peak 1 of the trichoderma reesei RS05 on 8 pathogenic bacteria is obviously enhanced, which indicates that the trichoderma reesei RS05 has broad-spectrum disease resistance only after being extracted and purified. Substances contained in the peaks 2, 3 and 4 do not show obvious antibacterial activity, and the active substances with antibacterial activity are supposed to be mainly located in the peak 1, so that the eluent of the peak 1 is collected and eluted in a concentrated manner, and then concentrated by a rotary evaporator at 40 ℃ for the next high-performance liquid phase separation and purification experiment.
5. Preparation of high performance liquid chromatography
The conditions of the chromatography include: chromatographic column: c18 reverse phase column 0DS-2, 460 mm. Times.250 mm,5m; mobile phase: vacuum filtering with 100% methanol and 0.45 μm filter membrane, and ultrasonic degassing for 20min; flow rate: 0.4mL/min; the sample injection amount is 20 mu L; detection wavelength 280nm; room temperature. The collected fractions were dissolved in methanol and kept ready for use. The eluent methanol is subjected to vacuum filtration and then ultrasonic degassing, and is used as a mobile phase.
The concentrated peak 1 solution was subjected to preparative liquid phase purification to obtain a component C having antibacterial activity, whose analytical liquid chromatography detection results are shown in FIG. 8. It is known that component C contains more than one absorption peak, the peak-out time is within 0-4 minutes, and the component C has the activity of resisting various bacteria and fungi at the same time, and the structural identification of the component C is to be performed.
6. Structural identification of active ingredients
Further mass spectrometry was carried out on each absorption peak in the above component C, and the Mass Spectrometer (MS) was an MS-QP2010 Plus type mass Spectrometer (SHIMADZU) under the conditions of electron bombardment ion source (EI), ionization voltage of 70eV and ion source temperature of 250 ℃.
The identification results of the component C are shown in the following Table 8, and it is known that the antibacterial substances identified by the component C are phlorizin and shikonin.
TABLE 8 mass spectrometric identification of component C
Experimental example 3 preparation of the Complex formulation
1. Interaction of the Trichoderma spiral RS05 metabolite with Streptomyces kiwikiwiensis BD2233
The drug-containing plate experiment is adopted: the component C concentrate having bacteriostatic activity of Trichoderma reesei RS05 isolated in Experimental example 2 was diluted with water to a solution of 1 g/L. After filtration through a 0.22 μm sterile bacterial filter, 0.5mL, 1mL, 1.5mL, 2mL were added to the different dishes, respectively, and after rapid pouring into a sterilized Luria-Bertani medium (tryptone 10g, yeast extract 5g, naCl 10g, distilled water 1000mL, pH=7.4) cooled to about 45℃and mixed well, each treatment was repeated 3 times with sterile water as a control. 6mm Streptomyces mirabilis BD2233 bacterial cake is taken and connected to the center of the flat plate, the flat plate is placed in a constant temperature box at 25 ℃ for culture, the inhibition rate is measured and calculated every 5 days, the colony diameter is measured by a crisscross method, and the inhibition rate is calculated according to the following formula.
Inhibition ratio = (colony diameter of control group-colony diameter of treatment group)/(colony diameter of control group-original cake diameter).
The inhibition results are shown in the following table 9, and it is known that the isolated and purified RS05 metabolite (the mixture of phlorizin and phlorizin) has antibacterial activity but has no inhibition effect on streptomyces mirabilis BD2233, and the growth condition of streptomyces mirabilis is better along with the increase of the addition amount of the metabolite, which indicates that the RS05 metabolite (the mixture of phlorizin and phlorizin) may be helpful for the growth and propagation of streptomyces mirabilis BD 2233.
TABLE 9 interaction (inhibition) of Trichoderma spiral RS05 metabolites with Streptomyces mirabilis
Note that: different lowercase letters in the same column indicate significant variability, and the P <0.05,4 group treatment represents the per-dish addition of RS05 metabolite.
2. Inhibition effect of complex of trichoderma spiral RS05 metabolite and streptomyces kiwii BD2233 on pathogenic bacteria
1) Preparation of spiral trichoderma RS05 spore liquid
Trichoderma reesei was inoculated on PSA medium, incubated at 25℃for 7d, perforated at colony edges with a 6mm sterilization punch, and inoculated with a sterilization inoculating needle into new PSA medium, incubated at 25℃for 8-10d until a large number of spores were produced. Washing out spores in culture medium with sterile water, sealing with sealing film, shaking at 25deg.C and 180r/min for 30min, and filtering with sterilized absorbent cotton in ultra clean bench. mu.L of the drops were pipetted onto a hemocytometer and the number of spores was observed under an optical microscope and diluted with sterile water to prepare a spore suspension at a concentration of about 1X 10 10 cfu/mL.
2) Preparation of trichoderma spiral RS05 metabolite and streptomyces kiwikipedia BD2233 compound
Streptomyces mirabilis BD2233 was streaked onto a Luria-Bertani medium plate and cultured in a 28℃incubator for 24 hours to activate the strain. And (3) inoculating the activated streptomyces mirabilis BD2233 into an LB liquid culture medium conical flask, and placing the flask in a shaking table at 28 ℃ and 180r/min for shake culture for 48 hours to obtain the prepared seed liquid (1X 10 5 cfu/mL). And adding the prepared active component C of the trichoderma reesei into the streptomyces mirabilis suspension to obtain a compound.
3) Flat plate inhibition effect of compound
The inhibition effect of the compound on fusarium gracilis (Fusarium stilboides) and bamboos brown rot pathogen (Diaporthe guangxiensis) and two pepper disease pathogens Cytospora chrysosperma and ALTERNARIA ALTERNATA is measured according to an agar perforation method. The sterile puncher is used for punching a 6mm pathogenic bacteria cake, a sterile inoculating needle is used for placing the bacterial cake on one side of a conventional PDA culture medium plate, a 6mm sterile puncher is used for punching a position 3cm away from the pathogenic bacteria cake, and 100 mu L of the prepared 1X 10 5 cfu/mL Streptomyces mirabilis BD2233 seed liquid, 1X 10 10 cfu/mL Trichoderma reesei spore liquid, 1g/L trichoderma reesei RS05 metabolite and the prepared compound are respectively inoculated. The blank was inoculated with a 6mm diameter fungal cake of pathogenic fungi at the center of the PDA medium plate and incubated at 25℃for 7d, 3 replicates each, and the inhibition of the complex on the pathogenic fungi was as shown in Table 10 below.
Table 10 comparison of inhibitory Effect of Trichoderma spiral RS05 metabolite and Streptomyces kiwikii BD2233 Complex with Single factor
/>
Note that: different lowercase letters in the same column indicate significant variability, P <0.05.
As shown in Table 10, although the Trichoderma reesei RS05 and Streptomyces mirabilis BD2233 have inhibition effects on Fusarium gracile and Fusarium oxysporum brown rot pathogens, the inhibition rate on both pathogens is low and is not more than 30%, and the single treatment of the Trichoderma reesei RS05 metabolite on 4 pathogens improves the inhibition effect by 50% compared with single bacteria. Compared with the single bacterium or the metabolite thereof, the compound obtained after the trichoderma spiralis RS05 metabolite and the streptomyces mirabilis BD2233 are compounded has the advantages that the inhibition rate of two pathogens of the pepper is improved by 130-413%, the inhibition effect of the two pathogens is greatly improved by the compounding of the two pathogens, and the compound can be used as a compound preparation and has wide application prospect in the control of the pepper diseases.
Preparing Streptomyces mirabilis BD2233 bacterial suspension according to the method, adding the prepared diluted solution of the active component C of the trichoderma spiralis (solution with the concentration of 1 g/L) into the prepared Streptomyces mirabilis BD2233 bacterial suspension according to the volume ratio of 1:1, 1:2, 1:3, 1:4, 1:5 and 1:6, and researching the optimal compounding ratio.
Experimental example 4 investigation of the disease-resistant and growth-promoting Effect of the Compound formulation
The effect research test is carried out in 2022 in 8 disease natural occurrence areas of Zanthoxylum schinifolium planting area in Du city, sichuan province, and 1-2 annual Zanthoxylum bungeanum is selected as a tested plant for 8 pathogenic bacteria respectively for Pseudomonas oryzihabitans、Xanthomonas arboricola、Alternaria alternata、Neofusicoccum parvum、Pestalotiopsis chamaeropis、Colletotrichum gloeosporioides、Cytospora chrysosperma、Pythium oligandrum.. The test design is as follows:
The assay included the trichoderma reesei RS05 metabolite prepared as described above: the volume ratio of the streptomyces mirabilis BD2233 bacterial suspension is 1:1, 1:2, 1:3, 1:4, 1:5 and 1:6, the original bacterial suspension of the trichoderma reesei RS05 is 100 times liquid, the metabolite of the trichoderma reesei RS05 is 100 times liquid, the medicament of the streptomyces mirabilis suspension and clear water are used as controls, 50% of chlorothalonil 500 times liquid is disease prevention control, smilax Miao Liwang (28% of nitrogen) 1000 times is growth promotion control, and the total treatment is 12 groups, and each treatment is repeated for 3 times. 36 cells are arranged in the pepper-like place, the area of each cell is about 25m 2, and the cells are arranged randomly. The spraying of the pesticide is started before natural onset in the field, and the dosage is 500L/hm 2. The total application time is 3 times, the application time is 2022 years, 3 months, 15 days, 3 months, 30 days and 4 months, 15 days respectively, and the application is carried out in a rainfall-free time, so that the whole test period is free from adverse weather influence.
The investigation method comprises the following steps: the onset was investigated 30d after the last administration. And 5-point diagonal investigation is used for investigating 10 plants at each point, 50 plants are counted, the number of the disease plants and the number of the disease grade plants are recorded, and corresponding disease indexes and control effects are calculated. And after 30d and 60d of the last application, the plant height, root length and ground diameter (the diameter of 10cm from the seedling stem to the ground) of the pepper treated differently are measured, and samples are collected to measure the chlorophyll content of the pepper, wherein the disease index and the prevention and treatment effect are calculated as shown in the following formula.
Disease index=100×Σ (number of disease plants at each stage×representative value at each stage)/(total number of investigation×representative value at highest stage)
Control effect (%) = (disease index after administration in blank control area-disease index after administration in agent treatment area) ×100/disease index after administration in blank control area
Determination of chlorophyll content:
(1) Extraction of chlorophyll: 0.5g of leaf blades are weighed, sheared and placed into a test tube with 10mL of extract (acetone: absolute ethyl alcohol=1:1), extracted for 24 hours under the condition of normal temperature and darkness until the green pepper leaves turn white, then OD 645 and OD 663 are respectively measured by a spectrophotometer, the extract (acetone: absolute ethyl alcohol=1:1) is used as a blank control, and chlorophyll content Ct (mg/g) is calculated.
(2) The calculation formula is as follows: chlorophyll content Ct (mg/g) = (8.02×OD 663+20.2×OD645) ×V/1000×W
OD: an optical density reading of the chlorophyll extract at the specified wavelength;
v: final volume (mL) of chlorophyll acetone mixed with absolute ethanol extract;
W: fresh weight of sample (g).
The results of the disease prevention in the field are shown in tables 11 to 13 below. Wherein, table 11 is the influence of the compound preparation on the morbidity of 8 pathogenic bacteria, table 12 is the influence of the compound preparation on the disease index of 8 pathogenic bacteria, and table 13 is the control effect of the compound preparation on 8 pathogenic bacteria. It can be known that the incidence rate and the disease index of 8 kinds of pepper diseases are obviously reduced after the compound preparation is applied, the control effect is obviously improved, and compared with single-dose RS05 bacterial suspension, RS05 metabolite and streptomyces mirabilis bacterial suspension, the incidence rate and the disease index of 8 kinds of pepper diseases are obviously changed. The single dose has poorer effect than chlorothalonil, and has no obvious control effect on other 6 diseases except for a certain control effect on ALTERNARIA ALTERNATA and Cytospora chrysosperma diseases. The formulations are in particular prepared as RS05 metabolites: the compound preparation of the streptomyces mirabilis BD2233 bacterial suspension=1:3 has the optimal effect, the disease prevention effect of the compound preparation is greatly improved by comparing the chlorothalonil results, the disease incidence is generally controlled to be below 30%, the disease index is controlled to be below 3, the control effect is improved to be above 82%, the disease incidence of single-dose application is higher than 70%, the disease index is not obviously controlled except the two diseases, and the control effect is below 8%. The RS05 metabolite is proved to have synergistic reaction on disease control effect by compounding streptomyces mirabilis.
Table 11 Effect of the Complex formulation on the incidence of 8 pathogenic bacteria (%)
Note that: different lowercase letters in the same column indicate significant variability, P <0.05, ck 1 represents clear water; CK 2 represents 50% chlorothalonil. 1-8 represent 8 pathogenic bacteria, respectively in turn Pseudomonas oryzihabitans、Xanthomonas arboricola、Alternaria alternata、Neofusicoccum parvum、Pestalotiopsis chamaeropis、Colletotrichum gloeosporioides、Cytospora chrysosperma、Pythium oligandrum.
Table 12 Effect of the Complex formulation on the disease index of 8 pathogenic bacteria
Note that: different lowercase letters in the same column indicate significant variability, P <0.05, ck 1 represents clear water; CK 2 represents 50% chlorothalonil. 1-8 represent 8 pathogenic bacteria, respectively in turn Pseudomonas oryzihabitans、Xanthomonas arboricola、Alternaria alternata、Neofusicoccum parvum、Pestalotiopsis chamaeropis、Colletotrichum gloeosporioides、Cytospora chrysosperma、Pythium oligandrum.
Table 13 control effect of the Compound formulation on 8 pathogenic bacteria (%)
Note that: different lowercase letters in the same column indicate significant variability, P <0.05, ck represents 50% chlorothalonil; 1-8 represent 8 pathogenic bacteria, respectively in turn Pseudomonas oryzihabitans、Xanthomonas arboricola、Alternaria alternata、Neofusicoccumparvum、Pestalotiopsis chamaeropis、Colletotrichum gloeosporioides、Cytospora chrysosperma、Pythium oligandrum.
The effect of the compound preparation on the plant height, the ground diameter and the root length of the pricklyash peel is evaluated by the effect on the chlorophyll content of the pricklyash peel. The effect of the compound preparation on the plant height, ground diameter and root length of the pricklyash peel is shown in the following table 14, and the effect of the compound preparation on the chlorophyll content of the pricklyash peel is shown in the following table 15. It is known that the application of the compound preparation can improve the plant height, root length and ground diameter indexes of the pepper and promote the growth and development of the pepper, and the chlorophyll content of the pepper is obviously increased compared with that of the control and the smith danli Miao Liwang. However, the RS05 bacterial suspension, the RS05 metabolite and the streptomyces mirabilis bacterial suspension used alone have no significant pro-active effect, and the difference between each index and the clear water control is not significant. Wherein, RS05 metabolite: the compound preparation of Streptomyces mirabilis BD2233 bacterial suspension=1:3 has optimal promotion effect, has obvious difference with other compound preparations and Schdanli Miao Liwang, and after 60 days of application, the plant height, ground diameter and root length are respectively increased to 38.13cm, 0.79cm and 12.95cm, and the total chlorophyll net growth rate exceeds 45 percent, which indicates that the Trichoderma reesei RS05 metabolite and Streptomyces mirabilis compound preparation have stronger growth promotion effect.
Table 14 influence of the Compound formulation on plant height, ground diameter and root length of Zanthoxylum bungeanum
Note that: different lowercase letters in the same column indicate significant variability, P <0.05, ck 1 represents clear water; CK 2 represents smith Miao Liwang.
Table 15 effect of the compound formulation on chlorophyll content of zanthoxylum bungeanum
Note that: different lowercase letters in the same column indicate significant variability, P <0.05, ck 1 represents clear water; CK 2 represents the smith seedling stand-up fertilizer.
In conclusion, the trichoderma reesei RS05 metabolite and the streptomyces mirabilis BD2233 provided by the invention have a synergistic effect, the compound preparation prepared by the trichoderma reesei RS05 metabolite and the streptomyces mirabilis BD2233 has two purposes, and has the effects of disease resistance and growth promotion on the pepper, has obvious effects on disease control and growth of the pepper, and saves cost compared with the use of pesticides and fertilizers.
While the present invention has been described in detail through the foregoing description of the preferred embodiment, it should be understood that the foregoing description is not to be considered as limiting the invention. Many modifications and substitutions of the present invention will become apparent to those of ordinary skill in the art upon reading the foregoing. Accordingly, the scope of the invention should be limited only by the attached claims.
Claims (6)
1. The compound preparation for resisting diseases and promoting growth is characterized by comprising a trichoderma spiralis (Trichoderma spirale) RS05 metabolite and streptomyces mirabilis (Streptomyces mirabilis) BD 2233;
Wherein, the streptomyces mirabilis BD2233 is preserved in China general microbiological culture Collection center (China general microbiological culture Collection center) for 9 months of 2022, the preservation address is number 3 of West-road 1 of the Korean area North Star of Beijing city, the preservation number is CGMCC No.25634, and the Latin name of the streptomyces mirabilis BD2233 is Streptomyces mirabilis;
The trichoderma reesei RS05 metabolite is obtained by extracting and separating fermentation supernatant of the trichoderma reesei RS05 with the preservation number of CGMCC No.40012, the metabolite is phlorizin and shikonin, and the Latin name of the trichoderma reesei RS05 is Trichoderma spirale;
the disease resistance comprises any one or more than two of Pseudomonas oryzihabitans、Xanthomonas arboricola、Alternaria alternata、Neofusicoccum parvum、Pestalotiopsis chamaeropis、Colletotrichum gloeosporioides、Cytospora chrysosperma and Pythiumoligandrum;
The growth promotion comprises the promotion of the growth of plant height, ground diameter and root length of the wild pepper.
2. The compound preparation according to claim 1, wherein the streptomyces mirabilis BD2233 is a bacterial suspension with a concentration of 1 x 10 5 cfu/mL.
3. The compound preparation according to claim 1, wherein the compound preparation is prepared from the trichoderma spiralis RS05 metabolite and the streptomyces mirabilis BD2233 bacterial suspension in a volume ratio of 1:3.
4. A method for preparing a trichoderma reesei RS05 metabolite as claimed in claim 1, comprising:
Culturing Trichoderma reesei RS05 with a preservation number of CGMCC No.40012, and collecting fermentation supernatant;
Adding ethyl acetate into the obtained fermentation supernatant according to an equal volume for oscillation extraction;
Concentrating the obtained extract, separating by gel column chromatography, and collecting eluting peak sample;
And collecting the collected elution peak sample corresponding to the first characteristic peak at OD 214, namely the trichoderma reesei RS05 metabolite.
5. The method according to claim 4, wherein the culture medium comprises 20% sucrose, 2% soluble starch, 0.1% tryptone and 0.05% magnesium sulfate by mass fraction.
6. The use of the compound preparation according to any one of claims 1 to 3 in disease resistance and growth promotion of peppers, wherein the disease resistance comprises any one or more than two of Pseudomonas oryzihabitans、Xanthomonas arboricola、Alternaria alternata、Neofusicoccum parvum、Pestalotiopsis chamaeropis、Colletotrichum gloeosporioides、Cytospora chrysosperma and Pythiumoligandrum; the growth promotion comprises the promotion of the growth of plant height, ground diameter and root length of the wild pepper.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202310007634.9A CN116267998B (en) | 2023-01-04 | 2023-01-04 | Compound preparation for resisting diseases and promoting growth and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202310007634.9A CN116267998B (en) | 2023-01-04 | 2023-01-04 | Compound preparation for resisting diseases and promoting growth and application thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN116267998A CN116267998A (en) | 2023-06-23 |
| CN116267998B true CN116267998B (en) | 2024-06-04 |
Family
ID=86796713
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202310007634.9A Active CN116267998B (en) | 2023-01-04 | 2023-01-04 | Compound preparation for resisting diseases and promoting growth and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN116267998B (en) |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN116463239B (en) * | 2023-01-04 | 2024-05-14 | 四川农业大学 | Streptomyces mirabilis BD2233, oil suspending agent and application thereof |
Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPH03176454A (en) * | 1989-12-04 | 1991-07-31 | Mitsui Petrochem Ind Ltd | Production of shikonin |
| CN101421412A (en) * | 2004-12-17 | 2009-04-29 | 梅坦诺米克斯有限公司 | Process for the production of fine chemicals |
| CN103415286A (en) * | 2010-11-11 | 2013-11-27 | 阿克伦分子有限公司 | Compounds and methods for treating pain |
| TWI689349B (en) * | 2019-02-20 | 2020-04-01 | 農道科技股份有限公司 | Kitchen waste processor |
| CN114410526A (en) * | 2022-01-24 | 2022-04-29 | 山东农业大学 | Bacillus licheniformis XNRB-3 and application thereof |
| CN114958613A (en) * | 2022-04-02 | 2022-08-30 | 四川农业大学 | Trichoderma spirillum RS05 and application thereof in preventing and treating brown rot of dendrocalamus latiflorus |
| CN116463239A (en) * | 2023-01-04 | 2023-07-21 | 四川农业大学 | Streptomyces mirabilis BD2233, oil suspending agent and application thereof |
Family Cites Families (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2009151511A1 (en) * | 2008-04-29 | 2009-12-17 | Therasis, Inc. | Systems and methods for identifying combinations of compounds of therapeutic interest |
| JP2021512045A (en) * | 2017-11-14 | 2021-05-13 | シムライズ アーゲー | Mixture with antibacterial effect |
-
2023
- 2023-01-04 CN CN202310007634.9A patent/CN116267998B/en active Active
Patent Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPH03176454A (en) * | 1989-12-04 | 1991-07-31 | Mitsui Petrochem Ind Ltd | Production of shikonin |
| CN101421412A (en) * | 2004-12-17 | 2009-04-29 | 梅坦诺米克斯有限公司 | Process for the production of fine chemicals |
| CN103415286A (en) * | 2010-11-11 | 2013-11-27 | 阿克伦分子有限公司 | Compounds and methods for treating pain |
| TWI689349B (en) * | 2019-02-20 | 2020-04-01 | 農道科技股份有限公司 | Kitchen waste processor |
| CN114410526A (en) * | 2022-01-24 | 2022-04-29 | 山东农业大学 | Bacillus licheniformis XNRB-3 and application thereof |
| CN114958613A (en) * | 2022-04-02 | 2022-08-30 | 四川农业大学 | Trichoderma spirillum RS05 and application thereof in preventing and treating brown rot of dendrocalamus latiflorus |
| CN116463239A (en) * | 2023-01-04 | 2023-07-21 | 四川农业大学 | Streptomyces mirabilis BD2233, oil suspending agent and application thereof |
Non-Patent Citations (2)
| Title |
|---|
| Anti-inflammatory activity of aqueous extract of Mirabilis jalapaLinn. Leaves;Manjit Singh等;《Pharmacognosy Research》;20111201;第2卷(第6期);364-367 * |
| 真菌诱导子对植物次生代谢物的影响及应用;曾杨;文涛;喻晓;;中国野生植物资源;20070425(第01期);1-4, 28 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN116267998A (en) | 2023-06-23 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Banerjee et al. | Antibiotic activity of bryophytes | |
| CN103283786B (en) | The purposes of trichoderma strain in preparation control cucumber fusarium axysporum biopesticide | |
| CN114164135B (en) | Preparation method and application of banana vascular wilt resistant compound | |
| CN116267998B (en) | Compound preparation for resisting diseases and promoting growth and application thereof | |
| CN110157625B (en) | Composite microbial inoculum for inhibiting toxigenic aspergillus flavus, preparation method and application thereof | |
| CN108353906B (en) | Application of indole-3-formaldehyde and derivatives thereof in preventing and treating plant diseases caused by plant pathogenic fungi | |
| CN102337219B (en) | Saccharopolyspora spinosa strain, application of saccharopolyspora spinosa strain and method for preparing spinosad | |
| CN103740606A (en) | Streptomyces phytohabitans, method for producing new antibiotics Novonestmycin from Streptomyces phytohabitans, and application of Novonestmycin | |
| Navya et al. | Beneficial rhizospheric microorganisms mediated plant growth promotion and suppression of aflatoxigenic fungal and aflatoxin contamination in groundnut seeds | |
| CN110129212B (en) | Aspergillus flavus PEAS-10 without producing aflatoxin and application thereof | |
| CN115044487A (en) | Marine fungus strain with tobacco mosaic virus prevention and control effect | |
| Elsie et al. | Diversity of putatively toxigenic Aspergillus species in maize and soil samples in an aflatoxicosis hotspot in Eastern Kenya | |
| CN117402784A (en) | Paenibacillus polymyxa Mxdg-1, microbial inoculum and application thereof | |
| CN115851553B (en) | Streptomyces virginiae capable of preventing and treating clubroot and application thereof | |
| CN110679611A (en) | Application of fusarium proliferatum in prevention and control of invasive plant xanthium sibiricum | |
| CN105420119A (en) | Ginseng endophytic fungus and application thereof | |
| CN111440731B (en) | Chinese wolfberry endophytic fusarium strain and application thereof | |
| CN113913319B (en) | Saline-alkali-resistant acinetobacter calcoaceticus ANDA001 and application thereof | |
| CN110122508B (en) | Antagonistic antibacterial agent for producing toxic aspergillus flavus, preparation method and application thereof | |
| Holzmann-Wirth et al. | Anti-fungal substances extracted from Neotyphodium endophytes | |
| CN110885771A (en) | Biological agent for improving tobacco-planting soil micro-ecological environment level | |
| CN112680360A (en) | Aspergillus sydowii and application thereof in promoting plant growth and preventing and treating plant diseases | |
| CN117016549B (en) | Application of nucleoside compound and composition thereof in promoting plant growth | |
| CN117305388B (en) | Application of bacillus bailii SL-K2 in preparation of guanine extract, preparation method of guanine extract and application | |
| CN111440746B (en) | Ericerus pela capable of inhibiting fusarium pathogenic bacteria and application thereof |
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 |